WorldWideScience

Sample records for 3d cell microenvironments

  1. Regulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactors.

    Sart, Sébastien; Agathos, Spiros N; Li, Yan; Ma, Teng

    2016-01-01

    Human mesenchymal stem cells (hMSCs) have emerged as an important cell type in cell therapy and tissue engineering. In these applications, maintaining the therapeutic properties of hMSCs requires tight control of the culture environments and the structural cell organizations. Bioreactor systems are essential tools to achieve these goals in the clinical-scale expansion and tissue engineering applications. This review summarizes how different bioreactors provide cues to regulate the structure and the chemico-mechanical microenvironment of hMSCs with a focus on 3D organization. In addition to conventional bioreactors, recent advances in microfluidic bioreactors as a novel approach to better control the hMSC microenvironment are also discussed. These advancements highlight the key role of bioreactor systems in preserving hMSC's functional properties by providing dynamic and temporal regulation of in vitro cellular microenvironment.

  2. Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

    Kurniawan, Nicholas Agung; Chaudhuri, Parthiv Kant; Lim, Chwee Teck

    2015-01-01

    The ability of cells to migrate is crucial in a wide variety of cell functions throughout life from embryonic development and wound healing to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell’s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration. PMID:25867104

  3. The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures

    Soto Ana M

    2010-06-01

    Full Text Available Abstract Background Stromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination. Methods The human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks, the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square. Results Most of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures. Conclusions Our results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to

  4. Limbal melanocytes support limbal epithelial stem cells in 2D and 3D microenvironments.

    Dziasko, Marc A; Tuft, Stephen J; Daniels, Julie T

    2015-09-01

    Human limbal epithelial stem cells (LESCs) are essential for the maintenance of the corneal epithelium of the ocular surface. LESCs are located within limbal crypts between the palisades of Vogt in the limbus; the interface between the peripheral cornea and conjunctiva. The limbal crypts have been proposed as a LESC niche owing to their support of epithelial cells, which can form holoclone colonies in vitro. Closely associated with the limbal crypts is a concentrated population of melanocytes. The anatomical location and close proximity to putative LESC suggests that melanocytes might play a role in maintenance of these stem cells in the niche. The aim of this study was to assess the ability of human limbal melanocytes (hLM) to support the expansion of human limbal epithelial cells (LECs) in vitro as an indicator of functional cell-cell interaction. After observing that hLM co-localize with clusters of compact epithelial cells in the native limbal crypts, hLM were isolated from crypt-rich cadaveric limbal biopsies and used as feeders for the culture of LECs. Interestingly, LECs grown on mitotically active hLM were able to generate large epithelial colonies that contained small and compact cells with morphological stem cell characteristics. Immunocytochemistry revealed that LECs expanded on hLM were positive for the expression of the putative stem cell markers CK15, Bmi-1 and p63α and negative for the marker of terminal cell differentiation CK3. LECs and hLM were finally co-cultured on RAFT (real architecture for 3D tissue) collagen tissue equivalents. In 3D co-cultures, hLM promoted multi-layering of the epithelial sheet in which basal cells were maintained in an undifferentiated state. Taken together, these observations suggest melanocytes could play an important role in the maintenance of LESCs in the native human limbal stem cell niche.

  5. The Synergistic Effects of Matrix Stiffness and Composition on the Response of Chondroprogenitor Cells in a 3D Precondensation Microenvironment.

    Carrion, Bita; Souzanchi, Mohammad F; Wang, Victor T; Tiruchinapally, Gopinath; Shikanov, Ariella; Putnam, Andrew J; Coleman, Rhima M

    2016-05-01

    Improve functional quality of cartilage tissue engineered from stem cells requires a better understanding of the functional evolution of native cartilage tissue. Therefore, a biosynthetic hydrogel was developed containing RGD, hyaluronic acid and/or type-I collagen conjugated to poly(ethylene glycol) acrylate to recapitulate the precondensation microenvironment of the developing limb. Conjugation of any combination of the three ligands did not alter the shear moduli or diffusion properties of the PEG hydrogels; thus, the influence of ligand composition on chondrogenesis could be investigated in the context of varying matrix stiffness. Gene expression of ligand receptors (CD44 and the b1-integrin) as well as markers of condensation (cell clustering and N-cadherin gene expression) and chondrogenesis (Col2a1 gene expression and sGAG production) by chondroprogenitor cells in this system were modulated by both matrix stiffness and ligand composition, with the highest gene expression occurring in softer hydrogels containing all three ligands. Cell proliferation in these 3D matrices for 7 d prior to chondrogenic induction increased the rate of sGAG production in a stiffness-dependent manner. This biosynthetic hydrogel supports the features of early limb-bud condensation and chondrogenesis and is a novel platform in which the influence of the matrix physicochemical properties on these processes can be elucidated.

  6. Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.

    Anna C Luca

    Full Text Available Three-dimensional (3D tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC cell lines. LrECM on-top (3D culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular

  7. HER2 signaling pathway activation and response of breast cancer cells to HER2-targeting agents is dependent strongly on the 3D microenvironment

    Weigelt, Britta; Lo, Alvin T; Park, Catherine C; Gray, Joe W; Bissell, Mina J

    2009-07-27

    Development of effective and durable breast cancer treatment strategies requires a mechanistic understanding of the influence of the microenvironment on response. Previous work has shown that cellular signaling pathways and cell morphology are dramatically influenced by three-dimensional (3D) cultures as opposed to traditional two-dimensional (2D) monolayers. Here, we compared 2D and 3D culture models to determine the impact of 3D architecture and extracellular matrix (ECM) on HER2 signaling and on the response of HER2-amplified breast cancer cell lines to the HER2-targeting agents Trastuzumab, Pertuzumab and Lapatinib. We show that the response of the HER2-amplified AU565, SKBR3 and HCC1569 cells to these anti-HER2 agents was highly dependent on whether the cells were cultured in 2D monolayer or 3D laminin-rich ECM gels. Inhibition of {beta}1 integrin, a major cell-ECM receptor subunit, significantly increased the sensitivity of the HER2-amplified breast cancer cell lines to the humanized monoclonal antibodies Trastuzumab and Pertuzumab when grown in a 3D environment. Finally, in the absence of inhibitors, 3D cultures had substantial impact on HER2 downstream signaling and induced a switch between PI3K-AKT- and RAS-MAPKpathway activation in all cell lines studied, including cells lacking HER2 amplification and overexpression. Our data provide direct evidence that breast cancer cells are able to rapidly adapt to different environments and signaling cues by activating alternative pathways that regulate proliferation and cell survival, events that may play a significant role in the acquisition of resistance to targeted therapies.

  8. Novel Morphologic and Genetic Analysis of Cancer Cells in a 3D Microenvironment Identifies STAT3 as a Regulator of Tumor Permeability Barrier Function.

    Park, Min Chul; Jeong, Hyobin; Son, Sung Hwa; Kim, YounHa; Han, Daeyoung; Goughnour, Peter C; Kang, Taehee; Kwon, Nam Hoon; Moon, Hyo Eun; Paek, Sun Ha; Hwang, Daehee; Seol, Ho Jun; Nam, Do-Hyun; Kim, Sunghoon

    2016-03-01

    Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK-STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient-derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance.

  9. Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment.

    Rinker, Torri E; Hammoudi, Taymour M; Kemp, Melissa L; Lu, Hang; Temenoff, Johnna S

    2014-03-01

    Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess their colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on the neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts at high glucose levels. In contrast, MSCs showed no reduction of viability or clonogenicity when cultured with adipocytes under high glucose conditions, and the adipogenic gene expression indicates that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided a novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis.

  10. Altering the Microenvironment to Promote Dormancy of Metastatic Breast Cancer Cell in a 3D Bone Culture System

    2015-04-01

    postmenopausal women. If we obtain future funding, we plan to continue to characterize the matrix. Physical methods such as atomic force...metastasis tumor stage already contain DTC in their bone marrow [10]. Dormant cells apparently survive chemotherapy, radiation and adjuvant therapy , and may...drug for the treatment of osteoporosis . J Bone Miner Res 21(3):354–365 22. Mundy GR et al (2008) Cytokines and bone remodeling. In: Marus R et al (eds

  11. Rapid fabrication of complex 3D extracellular microenvironments by dynamic optical projection stereolithography.

    Zhang, A Ping; Qu, Xin; Soman, Pranav; Hribar, Kolin C; Lee, Jin W; Chen, Shaochen; He, Sailing

    2012-08-16

    The topographic features of the extracelluar matrix (ECM) lay the foundation for cellular behavior. A novel biofabrication method using a digital-mirror device (DMD), called dynamic optical projection stereolithography (DOPsL) is demonstrated. This robust and versatile platform can generate complex biomimetic scaffolds within seconds. Such 3D scaffolds have promising potentials for studying cell interactions with microenvironments in vitro and in vivo.

  12. Microtissues in Cardiovascular Medicine: Regenerative Potential Based on a 3D Microenvironment

    Julia Günter

    2016-01-01

    Full Text Available More people die annually from cardiovascular diseases than from any other cause. In particular, patients who suffer from myocardial infarction may be affected by ongoing adverse remodeling processes of the heart that may ultimately lead to heart failure. The introduction of stem and progenitor cell-based applications has raised substantial hope for reversing these processes and inducing cardiac regeneration. However, current stem cell therapies using single-cell suspensions have failed to demonstrate long-lasting efficacy due to the overall low retention rate after cell delivery to the myocardium. To overcome this obstacle, the concept of 3D cell culture techniques has been proposed to enhance therapeutic efficacy and cell engraftment based on the simulation of an in vivo-like microenvironment. Of great interest is the use of so-called microtissues or spheroids, which have evolved from their traditional role as in vitro models to their novel role as therapeutic agents. This review will provide an overview of the therapeutic potential of microtissues by addressing primarily cardiovascular regeneration. It will accentuate their advantages compared to other regenerative approaches and summarize the methods for generating clinically applicable microtissues. In addition, this review will illustrate the unique properties of the microenvironment within microtissues that makes them a promising next-generation therapeutic approach.

  13. 3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments

    Jacob L. Albritton

    2017-01-01

    Full Text Available Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo. Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies.

  14. 3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments

    Albritton, Jacob L.

    2017-01-01

    ABSTRACT Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo. Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies. PMID:28067628

  15. Engineering three-dimensional cell mechanical microenvironment with hydrogels.

    Huang, Guoyou; Wang, Lin; Wang, Shuqi; Han, Yulong; Wu, Jinhui; Zhang, Qiancheng; Xu, Feng; Lu, Tian Jian

    2012-12-01

    Cell mechanical microenvironment (CMM) significantly affects cell behaviors such as spreading, migration, proliferation and differentiation. However, most studies on cell response to mechanical stimulation are based on two-dimensional (2D) planar substrates, which cannot mimic native three-dimensional (3D) CMM. Accumulating evidence has shown that there is a significant difference in cell behavior in 2D and 3D microenvironments. Among the materials used for engineering 3D CMM, hydrogels have gained increasing attention due to their tunable properties (e.g. chemical and mechanical properties). In this paper, we provide an overview of recent advances in engineering hydrogel-based 3D CMM. Effects of mechanical cues (e.g. hydrogel stiffness and externally induced stress/strain in hydrogels) on cell behaviors are described. A variety of approaches to load mechanical stimuli in 3D hydrogel-based constructs are also discussed.

  16. Calcite Biohybrids as Microenvironment for Stem Cells

    Razi Vago

    2012-04-01

    Full Text Available A new type of composite 3D biomaterial that provides extracellular cues that govern the differentiation processes of mesenchymal stem cells (MSCs has been developed. In the present study, we evaluated the chondrogenecity of a biohybrid composed of a calcium carbonate scaffold in its calcite polymorph and hyaluronic acid (HA. The source of the calcite scaffolding is an exoskeleton of a sea barnacle Tetraclita rifotincta (T. rifotincta, Pilsbry (1916. The combination of a calcium carbonate-based bioactive scaffold with a natural polymeric hydrogel is designed to mimic the organic-mineral composite of developing bone by providing a fine-tuned microenvironment. The results indicate that the calcite-HA interface creates a suitable microenvironment for the chondrogenic differentiation of MSCs, and therefore, the biohybrid may provide a tool for tissue-engineered cartilage.

  17. 3D Cell Culture in Alginate Hydrogels

    Therese Andersen

    2015-03-01

    Full Text Available This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent, and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

  18. 3D culture for cardiac cells.

    Zuppinger, Christian

    2016-07-01

    This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical “wiring”, a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  19. Engineering biomolecular microenvironments for cell instructive biomaterials.

    Custódio, Catarina A; Reis, Rui L; Mano, João F

    2014-06-01

    Engineered cell instructive microenvironments with the ability to stimulate specific cellular responses are a topic of high interest in the fabrication and development of biomaterials for application in tissue engineering. Cells are inherently sensitive to the in vivo microenvironment that is often designed as the cell "niche." The cell "niche" comprising the extracellular matrix and adjacent cells, influences not only cell architecture and mechanics, but also cell polarity and function. Extensive research has been performed to establish new tools to fabricate biomimetic advanced materials for tissue engineering that incorporate structural, mechanical, and biochemical signals that interact with cells in a controlled manner and to recapitulate the in vivo dynamic microenvironment. Bioactive tunable microenvironments using micro and nanofabrication have been successfully developed and proven to be extremely powerful to control intracellular signaling and cell function. This Review is focused in the assortment of biochemical signals that have been explored to fabricate bioactive cell microenvironments and the main technologies and chemical strategies to encode them in engineered biomaterials with biological information.

  20. A microfluidic device for 2D to 3D and 3D to 3D cell navigation

    Shamloo, Amir; Amirifar, Leyla

    2016-01-01

    Microfluidic devices have received wide attention and shown great potential in the field of tissue engineering and regenerative medicine. Investigating cell response to various stimulations is much more accurate and comprehensive with the aid of microfluidic devices. In this study, we introduced a microfluidic device by which the matrix density as a mechanical property and the concentration profile of a biochemical factor as a chemical property could be altered. Our microfluidic device has a cell tank and a cell culture chamber to mimic both 2D to 3D and 3D to 3D migration of three types of cells. Fluid shear stress is negligible on the cells and a stable concentration gradient can be obtained by diffusion. The device was designed by a numerical simulation so that the uniformity of the concentration gradients throughout the cell culture chamber was obtained. Adult neural cells were cultured within this device and they showed different branching and axonal navigation phenotypes within varying nerve growth factor (NGF) concentration profiles. Neural stem cells were also cultured within varying collagen matrix densities while exposed to NGF concentrations and they experienced 3D to 3D collective migration. By generating vascular endothelial growth factor concentration gradients, adult human dermal microvascular endothelial cells also migrated in a 2D to 3D manner and formed a stable lumen within a specific collagen matrix density. It was observed that a minimum absolute concentration and concentration gradient were required to stimulate migration of all types of the cells. This device has the advantage of changing multiple parameters simultaneously and is expected to have wide applicability in cell studies.

  1. Biomedical-grade, high mannuronic acid content (BioMVM) alginate enhances the proteoglycan production of primary human meniscal fibrochondrocytes in a 3-D microenvironment

    Rey-Rico, Ana; Klich, Angelique; Cucchiarini, Magali; Madry, Henning

    2016-01-01

    Alginates are important hydrogels for meniscus tissue engineering as they support the meniscal fibrochondrocyte phenotype and proteoglycan production, the extracellular matrix (ECM) component chiefly responsible for its viscoelastic properties. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to provide the best three-dimensional (3-D) microenvironment and to support proteoglycan synthesis of encapsulated human meniscal fibrochondrocytes in vitro. Biomedical-grade, high mannuronic acid alginate spheres (BioLVM, BioMVM) were the most uniform in size, indicating an effect of the purity of alginate on the shape of the spheres. Interestingly, the purity of alginates did not affect cell viability. Of note, only fibrochondrocytes encapsulated in BioMVM alginate produced and retained significant amounts of proteoglycans. Following transplantation in an explant culture model, the alginate spheres containing fibrochondrocytes remained in close proximity with the meniscal tissue adjacent to the defect. The results reveal a promising role of BioMVM alginate to enhance the proteoglycan production of primary human meniscal fibrochondrocytes in a 3-D hydrogel microenvironment. These findings have significant implications for cell-based translational studies aiming at restoring lost meniscal tissue in regions containing high amounts of proteoglycans. PMID:27302206

  2. Microenvironment promotes tumor cell reprogramming in human breast cancer cell lines.

    Fabrizio D'Anselmi

    Full Text Available The microenvironment drives mammary gland development and function, and may influence significantly both malignant behavior and cell growth of mammary cancer cells. By restoring context, and forcing cells to properly interpret native signals from the microenvironment, the cancer cell aberrant behavior can be quelled, and organization re-established. In order to restore functional and morphological differentiation, human mammary MCF-7 and MDA-MB-231 cancer cells were allowed to grow in a culture medium filled with a 10% of the albumen (EW, Egg White from unfertilized chicken egg. That unique microenvironment behaves akin a 3D culture and induces MCF-7 cells to produce acini and branching duct-like structures, distinctive of mammary gland differentiation. EW-treated MDA-MB-231 cells developed buds of acini and duct-like structures. Both MCF-7 and MDA-MB-231 cells produced β-casein, a key milk component. Furthermore, E-cadherin expression was reactivated in MDA-MB-231 cells, as a consequence of the increased cdh1 expression; meanwhile β-catenin - a key cytoskeleton component - was displaced behind the inner cell membrane. Such modification hinders the epithelial-mesenchymal transition in MDA-MB-231 cells. This differentiating pathway is supported by the contemporary down-regulation of canonical pluripotency markers (Klf4, Nanog. Given that egg-conditioned medium behaves as a 3D-medium, it is likely that cancer phenotype reversion could be ascribed to the changed interactions between cells and their microenvironment.

  3. Cell Interactions within Biomimetic Apatite Microenvironments

    Tsang, Eric

    2014-01-01

    Bioactive ceramics, such as calcium phosphate-based materials, have been studied extensively for the regeneration of bone tissue. Accelerated apatite coatings prepared from biomimetic methods is one approach that has had a history of success in both in vitro and in vivo studies for bone regeneration [1]-[4]. However, how cells interact within the apatite microenvironment remains largely unclear, despite the vast literature available today. In response, this thesis evaluates the in vitro i...

  4. A High-Throughput Screening Model of the Tumor Microenvironment for Ovarian Cancer Cell Growth.

    Lal-Nag, Madhu; McGee, Lauren; Guha, Rajarshi; Lengyel, Ernst; Kenny, Hilary A; Ferrer, Marc

    2017-01-01

    The tumor microenvironment plays an important role in the processes of tumor growth, metastasis, and drug resistance. We have used a multilayered 3D primary cell culture model that reproduces the human ovarian cancer metastatic microenvironment to study the effect of the microenvironment on the pharmacological responses of different classes of drugs on cancer cell proliferation. A collection of oncology drugs was screened to identify compounds that inhibited the proliferation of ovarian cancer cells growing as monolayers or forming spheroids, on plastic and on a 3D microenvironment culture model of the omentum metastatic site, and also cells already in preformed spheroids. Target-based analysis of the pharmacological responses revealed that several classes of targets were more efficacious in cancer cells growing in the absence of the metastatic microenvironment, and other target classes were less efficacious in cancer cells in preformed spheres compared to forming spheroid cultures. These findings show that both the cellular context of the tumor microenvironment and cell adhesion mode have an essential role in cancer cell drug resistance. Therefore, it is important to perform screens for new drugs using model systems that more faithfully recapitulate the tissue composition at the site of tumor growth and metastasis.

  5. Cancer Cell Colonisation in the Bone Microenvironment

    Casina Kan

    2016-10-01

    Full Text Available Bone metastases are a common complication of epithelial cancers, of which breast, prostate and lung carcinomas are the most common. The establishment of cancer cells to distant sites such as the bone microenvironment requires multiple steps. Tumour cells can acquire properties to allow epithelial-to-mesenchymal transition, extravasation and migration. Within the bone metastatic niche, disseminated tumour cells may enter a dormancy stage or proliferate to adapt and survive, interacting with bone cells such as hematopoietic stem cells, osteoblasts and osteoclasts. Cross-talk with the bone may alter tumour cell properties and, conversely, tumour cells may also acquire characteristics of the surrounding microenvironment, in a process known as osteomimicry. Alternatively, these cells may also express osteomimetic genes that allow cell survival or favour seeding to the bone marrow. The seeding of tumour cells in the bone disrupts bone-forming and bone-resorbing activities, which can lead to macrometastasis in bone. At present, bone macrometastases are incurable with only palliative treatment available. A better understanding of how these processes influence the early onset of bone metastasis may give insight into potential therapies. This review will focus on the early steps of bone colonisation, once disseminated tumour cells enter the bone marrow.

  6. Cancer Cell Colonisation in the Bone Microenvironment

    Kan, Casina; Vargas, Geoffrey; Le Pape, François; Clézardin, Philippe

    2016-01-01

    Bone metastases are a common complication of epithelial cancers, of which breast, prostate and lung carcinomas are the most common. The establishment of cancer cells to distant sites such as the bone microenvironment requires multiple steps. Tumour cells can acquire properties to allow epithelial-to-mesenchymal transition, extravasation and migration. Within the bone metastatic niche, disseminated tumour cells may enter a dormancy stage or proliferate to adapt and survive, interacting with bone cells such as hematopoietic stem cells, osteoblasts and osteoclasts. Cross-talk with the bone may alter tumour cell properties and, conversely, tumour cells may also acquire characteristics of the surrounding microenvironment, in a process known as osteomimicry. Alternatively, these cells may also express osteomimetic genes that allow cell survival or favour seeding to the bone marrow. The seeding of tumour cells in the bone disrupts bone-forming and bone-resorbing activities, which can lead to macrometastasis in bone. At present, bone macrometastases are incurable with only palliative treatment available. A better understanding of how these processes influence the early onset of bone metastasis may give insight into potential therapies. This review will focus on the early steps of bone colonisation, once disseminated tumour cells enter the bone marrow. PMID:27782035

  7. Collagen scaffold microenvironments modulate cell lineage commitment for differentiation of bone marrow cells into regulatory dendritic cells

    Fang, Yongxiang; Wang, Bin; Zhao, Yannan; Xiao, Zhifeng; Li, Jing; Cui, Yi; Han, Sufang; Wei, Jianshu; Chen, Bing; Han, Jin; Meng, Qingyuan; Hou, Xianglin; Luo, Jianxun; Dai, Jianwu; Jing, Zhizhong

    2017-01-01

    The microenvironment plays a pivotal role for cell survival and functional regulation, and directs the cell fate determination. The biological functions of DCs have been extensively investigated to date. However, the influences of the microenvironment on the differentiation of bone marrow cells (BMCs) into dendritic cells (DCs) are not well defined. Here, we established a 3D collagen scaffold microenvironment to investigate whether such 3D collagen scaffolds could provide a favourable niche for BMCs to differentiate into specialised DCs. We found that BMCs embedded in the 3D collagen scaffold differentiated into a distinct subset of DC, exhibiting high expression of CD11b and low expression of CD11c, co-stimulator (CD40, CD80, CD83, and CD86) and MHC-II molecules compared to those grown in 2D culture. DCs cultured in the 3D collagen scaffold possessed weak antigen uptake ability and inhibited T-cell proliferation in vitro; in addition, they exhibited potent immunoregulatory function to alleviate allo-delay type hypersensitivity when transferred in vivo. Thus, DCs differentiated in the 3D collagen scaffold were defined as regulatory DCs, indicating that collagen scaffold microenvironments probably play an important role in modulating the lineage commitment of DCs and therefore might be applied as a promising tool for generation of specialised DCs. PMID:28169322

  8. Of Microenvironments and Mammary Stem Cells

    LaBarge, Mark A; Petersen, Ole W; Bissell, Mina J

    2007-06-01

    In most adult tissues there reside pools of stem and progenitor cells inside specialized microenvironments referred to as niches. The niche protects the stem cells from inappropriate expansion and directs their critical functions. Thus guided, stem cells are able to maintain tissue homeostasis throughout the ebb and flow of metabolic and physical demands encountered over a lifetime. Indeed, a pool of stem cells maintains mammary gland structure throughout development, and responds to the physiological demands associated with pregnancy. This review discusses how stem cells were identified in both human and mouse mammary glands; each requiring different techniques that were determined by differing biological needs and ethical constraints. These studies together create a robust portrait of mammary gland biology and identify the location of the stem cell niche, elucidate a developmental hierarchy, and suggest how the niche might be manipulated for therapeutic benefit.

  9. Segmentation of vascular structures and hematopoietic cells in 3D microscopy images and quantitative analysis

    Mu, Jian; Yang, Lin; Kamocka, Malgorzata M.; Zollman, Amy L.; Carlesso, Nadia; Chen, Danny Z.

    2015-03-01

    In this paper, we present image processing methods for quantitative study of how the bone marrow microenvironment changes (characterized by altered vascular structure and hematopoietic cell distribution) caused by diseases or various factors. We develop algorithms that automatically segment vascular structures and hematopoietic cells in 3-D microscopy images, perform quantitative analysis of the properties of the segmented vascular structures and cells, and examine how such properties change. In processing images, we apply local thresholding to segment vessels, and add post-processing steps to deal with imaging artifacts. We propose an improved watershed algorithm that relies on both intensity and shape information and can separate multiple overlapping cells better than common watershed methods. We then quantitatively compute various features of the vascular structures and hematopoietic cells, such as the branches and sizes of vessels and the distribution of cells. In analyzing vascular properties, we provide algorithms for pruning fake vessel segments and branches based on vessel skeletons. Our algorithms can segment vascular structures and hematopoietic cells with good quality. We use our methods to quantitatively examine the changes in the bone marrow microenvironment caused by the deletion of Notch pathway. Our quantitative analysis reveals property changes in samples with deleted Notch pathway. Our tool is useful for biologists to quantitatively measure changes in the bone marrow microenvironment, for developing possible therapeutic strategies to help the bone marrow microenvironment recovery.

  10. Fabrication of Nanostructured Poly-ε-caprolactone 3D Scaffolds for 3D Cell Culture Technology

    Schipani, Rossana

    2015-04-21

    Tissue engineering is receiving tremendous attention due to the necessity to overcome the limitations related to injured or diseased tissues or organs. It is the perfect combination of cells and biomimetic-engineered materials. With the appropriate biochemical factors, it is possible to develop new effective bio-devices that are capable to improve or replace biological functions. Latest developments in microfabrication methods, employing mostly synthetic biomaterials, allow the production of three-dimensional (3D) scaffolds that are able to direct cell-to-cell interactions and specific cellular functions in order to drive tissue regeneration or cell transplantation. The presented work offers a rapid and efficient method of 3D scaffolds fabrication by using optical lithography and micro-molding techniques. Bioresorbable polymer poly-ε-caprolactone (PCL) was the material used thanks to its high biocompatibility and ability to naturally degrade in tissues. 3D PCL substrates show a particular combination in the designed length scale: cylindrical shaped pillars with 10μm diameter, 10μm height, arranged in a hexagonal lattice with spacing of 20μm were obtained. The sidewalls of the pillars were nanostructured by attributing a 3D architecture to the scaffold. The suitability of these devices as cell culture technology supports was evaluated by plating NIH/3T3 mouse embryonic fibroblasts and human Neural Stem Cells (hNSC) on them. Scanning Electron Microscopy (SEM) analysis was carried out in order to examine the micro- and nano-patterns on the surface of the supports. In addition, after seeding of cells, SEM and immunofluorescence characterization of the fabricated systems were performed to check adhesion, growth and proliferation. It was observed that cells grow and develop healthy on the bio-polymeric devices by giving rise to well-interconnected networks. 3D PCL nano-patterned pillared scaffold therefore may have considerable potential as effective tool for

  11. Development of a 3D cell printed construct considering angiogenesis for liver tissue engineering.

    Lee, Jin Woo; Choi, Yeong-Jin; Yong, Woon-Jae; Pati, Falguni; Shim, Jin-Hyung; Kang, Kyung Shin; Kang, In-Hye; Park, Jaesung; Cho, Dong-Woo

    2016-01-12

    Several studies have focused on the regeneration of liver tissue in a two-dimensional (2D) planar environment, whereas actual liver tissue is three-dimensional (3D). Cell printing technology has been successfully utilized for building 3D structures; however, the poor mechanical properties of cell-laden hydrogels are a major concern. Here, we demonstrate the printing of a 3D cell-laden construct and its application to liver tissue engineering using 3D cell printing technology through a multi-head tissue/organ building system. Polycaprolactone (PCL) was used as a framework material because of its excellent mechanical properties. Collagen bioink containing three different types of cells-hepatocytes (HCs), human umbilical vein endothelial cells , and human lung fibroblasts--was infused into the canals of a PCL framework to induce the formation of capillary--like networks and liver cell growth. A co-cultured 3D microenvironment of the three types of cells was successfully established and maintained. The vascular formation and functional abilities of HCs (i.e., albumin secretion and urea synthesis) demonstrated that the heterotypic interaction among HCs and nonparenchymal cells increased the survivability and functionality of HCs within the collagen gel. Therefore, our results demonstrate the prospect of using cell printing technology for the creation of heterotypic cellular interaction within a structure for liver tissue engineering.

  12. Sensitivity of Dendritic Cells to Microenvironment Signals

    Motta, Juliana Maria; Rumjanek, Vivian Mary

    2016-01-01

    Dendritic cells are antigen-presenting cells capable of either activating the immune response or inducing and maintaining immune tolerance. They do this by integrating stimuli from the environment and changing their functional status as a result of plasticity. The modifications suffered by these cells have consequences in the way the organism may respond. In the present work two opposing situations known to affect dendritic cells are analyzed: tumor growth, leading to a microenvironment that favors the induction of a tolerogenic profile, and organ transplantation, which leads to a proinflammatory profile. Lessons learned from these situations may help to understand the mechanisms of modulation resulting not only from the above circumstances, but also from other pathologies. PMID:27088097

  13. Sensitivity of Dendritic Cells to Microenvironment Signals

    Juliana Maria Motta

    2016-01-01

    Full Text Available Dendritic cells are antigen-presenting cells capable of either activating the immune response or inducing and maintaining immune tolerance. They do this by integrating stimuli from the environment and changing their functional status as a result of plasticity. The modifications suffered by these cells have consequences in the way the organism may respond. In the present work two opposing situations known to affect dendritic cells are analyzed: tumor growth, leading to a microenvironment that favors the induction of a tolerogenic profile, and organ transplantation, which leads to a proinflammatory profile. Lessons learned from these situations may help to understand the mechanisms of modulation resulting not only from the above circumstances, but also from other pathologies.

  14. "Constructing" the Cell Cycle in 3D

    Koc, Isil; Turan, Merve

    2012-01-01

    The cycle of duplication and division, known as the "cell cycle," is the essential mechanism by which all living organisms reproduce. This activity allows students to develop an understanding of the main events that occur during the typical eukaryotic cell cycle mostly in the process of mitotic phase that divides the duplicated genetic material…

  15. Cyto-3D-print to attach mitotic cells.

    Castroagudin, Michelle R; Zhai, Yujia; Li, Zhi; Marnell, Michael G; Glavy, Joseph S

    2016-08-01

    The Cyto-3D-print is an adapter that adds cytospin capability to a standard centrifuge. Like standard cytospinning, Cyto-3D-print increases the surface attachment of mitotic cells while giving a higher degree of adaptability to other slide chambers than available commercial devices. The use of Cyto-3D-print is cost effective, safe, and applicable to many slide designs. It is durable enough for repeated use and made of biodegradable materials for environment-friendly disposal.

  16. Phenotypic characterization of prostate cancer LNCaP cells cultured within a bioengineered microenvironment.

    Shirly Sieh

    Full Text Available Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to

  17. Cell type-specific adaptation of cellular and nuclear volume in micro-engineered 3D environments.

    Greiner, Alexandra M; Klein, Franziska; Gudzenko, Tetyana; Richter, Benjamin; Striebel, Thomas; Wundari, Bayu G; Autenrieth, Tatjana J; Wegener, Martin; Franz, Clemens M; Bastmeyer, Martin

    2015-11-01

    Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are structurally and mechanically well-defined and ideal for systematically investigating the influence of three-dimensionality and substrate stiffness on cell behavior. Here, we show that different fibroblast-like and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in DLW-fabricated 3D scaffolds of different mechanics and geometry. Furthermore, the molecular composition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D culture conditions is identical, based on the analysis of several marker proteins (paxillin, phospho-paxillin, phospho-focal adhesion kinase, vinculin, β1-integrin). However, fibroblast-like and epithelial cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments. While fibroblast-like cell lines display significantly increased cell and nuclear volumes in 3D substrates compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D environments. Despite differential cell volume regulation between fibroblasts and epithelial cells in 3D environments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio regardless of culture conditions.

  18. An Optically Controlled 3D Cell Culturing System

    Kelly S. Ishii

    2011-01-01

    Full Text Available A novel 3D cell culture system was developed and tested. The cell culture device consists of a microfluidic chamber on an optically absorbing substrate. Cells are suspended in a thermoresponsive hydrogel solution, and optical patterns are utilized to heat the solution, producing localized hydrogel formation around cells of interest. The hydrogel traps only the desired cells in place while also serving as a biocompatible scaffold for supporting the cultivation of cells in 3D. This is demonstrated with the trapping of MDCK II and HeLa cells. The light intensity from the optically induced hydrogel formation does not significantly affect cell viability.

  19. Peptide hydrogels – versatile matrices for 3D cell culture in cancer medicine

    Peter eWorthington

    2015-04-01

    Full Text Available Traditional two-dimensional (2D cell culture systems have contributed tremendously to our understanding of cancer biology but have significant limitations in mimicking in vivo conditions such as the tumor microenvironment. In vitro, three-dimensional (3D cell culture models represent a more accurate, intermediate platform between simplified 2D culture models and complex and expensive in vivo models. 3D in vitro models can overcome 2D in vitro limitations caused by the oversupply of nutrients, and unphysiological cell-cell and cell-material interactions, and allow for dynamic interactions between cells, stroma, and extracellular matrix. In addition, 3D cultures allow for the development of concentration gradients, including oxygen, metabolites and growth factors, with chemical gradients playing an integral role in many cellular functions ranging from development to signaling in normal epithelia and cancer environments in vivo. Currently, the most common matrices used for 3D culture are biologically derived materials such as matrigel and collagen. However, in recent years, more defined, synthetic materials have become available as scaffolds for 3D culture with the advantage of forming well-defined, designed, tunable materials to control matrix charge, stiffness, porosity, nanostructure, degradability and adhesion properties, in addition to other material and biological properties. One important area of synthetic materials currently available for 3D cell culture are short sequence, self-assembling peptide hydrogels. In addition to the review of recent work towards the control of material, structure, and mechanical properties, we will also discuss the biochemical functionalization of peptide hydrogels and how this functionalization, coupled with desired hydrogel material characteristics, affects tumor cell behavior in 3D culture.

  20. Laser printing of cells into 3D scaffolds

    Ovsianikov, A; Gruene, M; Koch, L; Maiorana, F; Chichkov, B [Nanotechnology Department, Laser Zentrum Hannover eV, Hollerithallee 8, 30419 Hannover (Germany); Pflaum, M; Wilhelmi, M; Haverich, A, E-mail: a.ovsianikov@lzh.d [Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover (Germany)

    2010-03-15

    One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are required. The two-photon polymerization (2PP) technique, used in this report for the fabrication of scaffolds, allows the realization of arbitrary 3D structures with submicron spatial resolution. Highly porous 3D scaffolds, produced by 2PP of acrylated poly(ethylene glycol), are seeded with cells by means of laser-induced forward transfer (LIFT). In this laser printing approach, a propulsive force, resulting from laser-induced shock wave, is used to propel individual cells or cell groups from a donor substrate towards the receiver substrate. We demonstrate that with this technique printing of multiple cell types into 3D scaffolds is possible. Combination of LIFT and 2PP provides a route for the realization of 3D multicellular tissue constructs and artificial ECM engineered on the microscale.

  1. 3D printing of biomimetic microstructures for cancer cell migration

    Huang, Tina Qing; Qu, Xin; Liu, Justin; Chen, Shaochen

    2013-01-01

    To understand the physical behavior and migration of cancer cells, a 3D in vitro micro-chip in hydrogel was created using 3D projection printing. The micro-chip has a honeycomb branched structure, aiming to mimic 3D vascular morphology to test, monitor, and analyze differences in the behavior of cancer cells (i.e. HeLa) vs. non-cancerous cell lines (i.e. 10T1/2). The 3D Projection Printing system can fabricate complex structures in seconds from user-created designs. The fabricated microstructures have three different channel widths of 25, 45, and 120 microns wide to reflect a range of blood vessel diameters. HeLa and 10T1/2 cells seeded within the micro-chip were then analyzed for morphology and cell migration speed. 10T1/2 cells exhibited greater changes in morphology due to channel size width than HeLa cells; however, channel width had a limited effect on 10T1/2 cell migration while HeLa cancer cell migration increased as channel width decreased. This physiologically relevant 3D cancer tissue model has the potential to be a powerful tool for future drug discoveries and cancer migration studies PMID:24150602

  2. Crystalline calcium carbonate and hydrogels as microenvironment for stem cells.

    Astachov, Liliana; Nevo, Zvi; Aviv, Moran; Vago, Razi

    2011-01-01

    Stem cell development and fate decisions are dictated by the microenvironment in which the stem cell is embedded. Among the advanced goals of tissue engineering is the creation of a microenvironment that will support the maintenance and differentiation of the stem cell--based on embryonic and adult stem cells as potent, cellular sources--for a variety of clinical applications. This review discusses some of the approaches used to create regulatory and instructive microenvironments for the directed differentiation of mesenchymal stem cells (MSCs) using three-dimensional crystalline calcium carbonate biomaterials of marine origin combined with a hydrated gel based on hyaluronan.

  3. 3D culture broadly regulates tumor cell hypoxia response and angiogenesis via pro-inflammatory pathways.

    DelNero, Peter; Lane, Maureen; Verbridge, Scott S; Kwee, Brian; Kermani, Pouneh; Hempstead, Barbara; Stroock, Abraham; Fischbach, Claudia

    2015-07-01

    Oxygen status and tissue dimensionality are critical determinants of tumor angiogenesis, a hallmark of cancer and an enduring target for therapeutic intervention. However, it is unclear how these microenvironmental conditions interact to promote neovascularization, due in part to a lack of comprehensive, unbiased data sets describing tumor cell gene expression as a function of oxygen levels within three-dimensional (3D) culture. Here, we utilized alginate-based, oxygen-controlled 3D tumor models to study the interdependence of culture context and the hypoxia response. Microarray gene expression analysis of tumor cells cultured in 2D versus 3D under ambient or hypoxic conditions revealed striking interdependence between culture dimensionality and hypoxia response, which was mediated in part by pro-inflammatory signaling pathways. In particular, interleukin-8 (IL-8) emerged as a major player in the microenvironmental regulation of the hypoxia program. Notably, this interaction between dimensionality and oxygen status via IL-8 increased angiogenic sprouting in a 3D endothelial invasion assay. Taken together, our data suggest that pro-inflammatory pathways are critical regulators of tumor hypoxia response within 3D environments that ultimately impact tumor angiogenesis, potentially providing important therapeutic targets. Furthermore, these results highlight the importance of pathologically relevant tissue culture models to study the complex physical and chemical processes by which the cancer microenvironment mediates new vessel formation.

  4. Microenvironment-Centred Dynamics in Aggressive B-Cell Lymphomas

    Matilde Cacciatore

    2012-01-01

    Full Text Available Aggressive B-cell lymphomas share high proliferative and invasive attitudes and dismal prognosis despite heterogeneous biological features. In the interchained sequence of events leading to cancer progression, neoplastic clone-intrinsic molecular events play a major role. Nevertheless, microenvironment-related cues have progressively come into focus as true determinants for this process. The cancer-associated microenvironment is a complex network of nonneoplastic immune and stromal cells embedded in extracellular components, giving rise to a multifarious crosstalk with neoplastic cells towards the induction of a supportive milieu. The immunological and stromal microenvironments have been classically regarded as essential partners of indolent lymphomas, while considered mainly negligible in the setting of aggressive B-cell lymphomas that, by their nature, are less reliant on external stimuli. By this paper we try to delineate the cardinal microenvironment-centred dynamics exerting an influence over lymphoid clone progression in aggressive B-cell lymphomas.

  5. Molecular predictors of 3D morphogenesis by breast cancer cell lines in 3D culture.

    Ju Han

    2010-02-01

    Full Text Available Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype. Next, associations with molecular features were realized through (i differential analysis within each morphological cluster, and (ii regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPARgamma has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARgamma has been validated through two supporting biological assays.

  6. Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture

    Han, Ju; Chang, Hang; Giricz, Orsi; Lee, Genee; Baehner, Frederick; Gray, Joe; Bissell, Mina; Kenny, Paraic; Parvin, Bahram

    2010-02-01

    Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPAR? has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPAR? has been validated through two supporting biological assays.

  7. Polymer-based mesh as supports for multi-layered 3D cell culture and assays.

    Simon, Karen A; Park, Kyeng Min; Mosadegh, Bobak; Subramaniam, Anand Bala; Mazzeo, Aaron D; Ngo, Philip M; Whitesides, George M

    2014-01-01

    Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system-Cells-in-Gels-in-Mesh (CiGiM)-that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells-layer-by-layer-within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis-(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format.

  8. Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer

    Chitcholtan, Kenny, E-mail: kenny.chitcholtan@otago.ac.nz [Department of Obstetrics and Gynaecology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand); Asselin, Eric, E-mail: Eric.Asselin@uqtr.ca [Department of Chemistry and Biology, University of Quebec, at Trois-Rivières, C.P. 500, Trois-Rivières, Quebec, Canada G9A 5H7 (Canada); Parent, Sophie, E-mail: Sophie.Parent@uqtr.ca [Department of Chemistry and Biology, University of Quebec, at Trois-Rivières, C.P. 500, Trois-Rivières, Quebec, Canada G9A 5H7 (Canada); Sykes, Peter H., E-mail: peter.sykes@otago.ac.nz [Department of Obstetrics and Gynaecology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand); Evans, John J., E-mail: john.evans@otago.ac.nz [Department of Obstetrics and Gynaecology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand); Centre of Neuroendocrinology and The MacDiarmid Institute of Advanced Materials and Nanotechnology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand)

    2013-01-01

    Three-dimensional (3D) in vitro models have an invaluable role in understanding the behaviour of tumour cells in a well defined microenvironment. This is because some aspects of tumour characteristics cannot be fully recapitulated in a cell monolayer (2D). In the present study, we compared growth patterns, expression of signalling molecules, and metabolism-associated proteins of endometrial cancer cell lines in 3D and 2D cell cultures. Cancer cells formed spherical structures in 3D reconstituted basement membrane (3D rBM), and the morphological appearance was cell line dependent. Cell differentiation was observed after 8 days in the 3D rBM. There was reduced proliferation, detected by less expression of PCNA in 3D rBM than in 2D cell monolayers. The addition of exogenous epidermal growth factor (EGF) to cancer cells induced phosphorylation of EGFR and Akt in both cell culture conditions. The uptake of glucose was selectively altered in the 3D rBM, but there was a lack of association with Glut-1 expression. The secretion of vascular endothelial growth factor (VEGF) and prostaglandin E{sub 2} (PGE{sub 2}) was selectively altered in 3D rBM, and it was cell line dependent. Our data demonstrated that 3D rBM as an in vitro model can influence proliferation and metabolism of endometrial cancer cell behaviour compared to 2D cell monolayer. Changes are specific to individual cell types. The use of 3D rBM is, therefore, important in the in vitro study of targeted anticancer therapies.

  9. Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.

    Pati, Falguni; Song, Tae-Ha; Rijal, Girdhari; Jang, Jinah; Kim, Sung Won; Cho, Dong-Woo

    2015-01-01

    3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals.

  10. A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions

    Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace

    2016-08-01

    Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.

  11. A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion

    Neufeld Gera

    2009-11-01

    Full Text Available Abstract Background The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of in-vivo invasion assays, there is need for quantitative in-vitro invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled. Methods We have established a novel asymmetric 3D in-vitro invasion assay by embedding a monolayer of tumor cells between two layers of collagen. The cells were then allowed to invade the upper and lower layers of collagen. To visualize invading cells the gels were sectioned perpendicular to the monolayer so that after seeding the monolayer appears as a thin line precisely defining the origin of invasion. The number of invading tumor cells, their proliferation rate, the distance they traverse and the direction of invasion could then be determined quantitatively. Results The assay was used to compare the invasive properties of several tumor cell types and the results compare well with those obtained by previously described assays. Lysyl-oxidase like protein-2 (Loxl2 is a potent inducer of invasiveness. Using our assay we show for the first time that inhibition of endogenous Loxl2 expression in several types of tumor cells strongly inhibits their invasiveness. We also took advantage of the asymmetric nature of the assay in order to show that fibronectin enhances the invasiveness of breast cancer cells more potently than laminin. The asymmetric properties of the assay were also used to demonstrate that soluble factors derived from fibroblasts can preferentially attract invading breast cancer cells. Conclusion Our assay displays several advantages over previous invasion assays as it is allows the quantitative analysis of directional invasive behavior of tumor cells in a 3D environment mimicking the tumor microenvironment. It should be particularly useful for the study of the effects of components of the tumor microenvironment on

  12. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  13. Influence of scaffold design on 3D printed cell constructs.

    Souness, Auryn; Zamboni, Fernanda; Walker, Gavin M; Collins, Maurice N

    2017-02-14

    Additive manufacturing is currently receiving significant attention in the field of tissue engineering and biomaterial science. The development of precise, affordable 3D printing technologies has provided a new platform for novel research to be undertaken in 3D scaffold design and fabrication. In the past, a number of 3D scaffold designs have been fabricated to investigate the potential of a 3D printed scaffold as a construct which could support cellular life. These studies have shown promising results; however, few studies have utilized a low-cost desktop 3D printing technology as a potential rapid manufacturing route for different scaffold designs. Here six scaffold designs were manufactured using a Fused deposition modeling, a "bottom-up" solid freeform fabrication approach, to determine optimal scaffold architecture for three-dimensional cell growth. The scaffolds, produced from PLA, are coated using pullulan and hyaluronic acid to assess the coating influence on cell proliferation and metabolic rate. Scaffolds are characterized both pre- and postprocessing using water uptake analysis, mechanical testing, and morphological evaluation to study the inter-relationships between the printing process, scaffold design, and scaffold properties. It was found that there were key differences between each scaffold design in terms of porosity, diffusivity, swellability, and compressive strength. An optimal design was chosen based on these physical measurements which were then weighted in accordance to design importance based on literature and utilizing a design matrix technique. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017.

  14. 3D Reconstruction of Coronary Artery Vascular Smooth Muscle Cells.

    Tong Luo

    Full Text Available The 3D geometry of individual vascular smooth muscle cells (VSMCs, which are essential for understanding the mechanical function of blood vessels, are currently not available. This paper introduces a new 3D segmentation algorithm to determine VSMC morphology and orientation.A total of 112 VSMCs from six porcine coronary arteries were used in the analysis. A 3D semi-automatic segmentation method was developed to reconstruct individual VSMCs from cell clumps as well as to extract the 3D geometry of VSMCs. A new edge blocking model was introduced to recognize cell boundary while an edge growing was developed for optimal interpolation and edge verification. The proposed methods were designed based on Region of Interest (ROI selected by user and interactive responses of limited key edges. Enhanced cell boundary features were used to construct the cell's initial boundary for further edge growing. A unified framework of morphological parameters (dimensions and orientations was proposed for the 3D volume data. Virtual phantom was designed to validate the tilt angle measurements, while other parameters extracted from 3D segmentations were compared with manual measurements to assess the accuracy of the algorithm. The length, width and thickness of VSMCs were 62.9±14.9 μm, 4.6±0.6 μm and 6.2±1.8 μm (mean±SD. In longitudinal-circumferential plane of blood vessel, VSMCs align off the circumferential direction with two mean angles of -19.4±9.3° and 10.9±4.7°, while an out-of-plane angle (i.e., radial tilt angle was found to be 8±7.6° with median as 5.7°.A 3D segmentation algorithm was developed to reconstruct individual VSMCs of blood vessel walls based on optical image stacks. The results were validated by a virtual phantom and manual measurement. The obtained 3D geometries can be utilized in mathematical models and leads a better understanding of vascular mechanical properties and function.

  15. Induction of mitochondrial dysfunction as a strategy for targeting tumour cells in metabolically compromised microenvironments.

    Zhang, Xiaonan; Fryknäs, Mårten; Hernlund, Emma; Fayad, Walid; De Milito, Angelo; Olofsson, Maria Hägg; Gogvadze, Vladimir; Dang, Long; Påhlman, Sven; Schughart, Leoni A Kunz; Rickardson, Linda; D'Arcy, Padraig; Gullbo, Joachim; Nygren, Peter; Larsson, Rolf; Linder, Stig

    2014-01-01

    Abnormal vascularization of solid tumours results in the development of microenvironments deprived of oxygen and nutrients that harbour slowly growing and metabolically stressed cells. Such cells display enhanced resistance to standard chemotherapeutic agents and repopulate tumours after therapy. Here we identify the small molecule VLX600 as a drug that is preferentially active against quiescent cells in colon cancer 3-D microtissues. The anticancer activity is associated with reduced mitochondrial respiration, leading to bioenergetic catastrophe and tumour cell death. VLX600 shows enhanced cytotoxic activity under conditions of nutrient starvation. Importantly, VLX600 displays tumour growth inhibition in vivo. Our findings suggest that tumour cells in metabolically compromised microenvironments have a limited ability to respond to decreased mitochondrial function, and suggest a strategy for targeting the quiescent populations of tumour cells for improved cancer treatment.

  16. Matrix mechanics and fluid shear stress control stem cells fate in three dimensional microenvironment.

    Chen, Guobao; Lv, Yonggang; Guo, Pan; Lin, Chongwen; Zhang, Xiaomei; Yang, Li; Xu, Zhiling

    2013-07-01

    Stem cells have the ability to self-renew and to differentiate into multiple mature cell types during early life and growth. Stem cells adhesion, proliferation, migration and differentiation are affected by biochemical, mechanical and physical surface properties of the surrounding matrix in which stem cells reside and stem cells can sensitively feel and respond to the microenvironment of this matrix. More and more researches have proven that three dimensional (3D) culture can reduce the gap between cell culture and physiological environment where cells always live in vivo. This review summarized recent findings on the studies of matrix mechanics that control stem cells (primarily mesenchymal stem cells (MSCs)) fate in 3D environment, including matrix stiffness and extracellular matrix (ECM) stiffness. Considering the exchange of oxygen and nutrients in 3D culture, the effect of fluid shear stress (FSS) on fate decision of stem cells was also discussed in detail. Further, the difference of MSCs response to matrix stiffness between two dimensional (2D) and 3D conditions was compared. Finally, the mechanism of mechanotransduction of stem cells activated by matrix mechanics and FSS in 3D culture was briefly pointed out.

  17. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  18. Single molecule microscopy in 3D cell cultures and tissues.

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

  19. Multiparametric classification links tumor microenvironments with tumor cell phenotype.

    Bojana Gligorijevic

    2014-11-01

    Full Text Available While it has been established that a number of microenvironment components can affect the likelihood of metastasis, the link between microenvironment and tumor cell phenotypes is poorly understood. Here we have examined microenvironment control over two different tumor cell motility phenotypes required for metastasis. By high-resolution multiphoton microscopy of mammary carcinoma in mice, we detected two phenotypes of motile tumor cells, different in locomotion speed. Only slower tumor cells exhibited protrusions with molecular, morphological, and functional characteristics associated with invadopodia. Each region in the primary tumor exhibited either fast- or slow-locomotion. To understand how the tumor microenvironment controls invadopodium formation and tumor cell locomotion, we systematically analyzed components of the microenvironment previously associated with cell invasion and migration. No single microenvironmental property was able to predict the locations of tumor cell phenotypes in the tumor if used in isolation or combined linearly. To solve this, we utilized the support vector machine (SVM algorithm to classify phenotypes in a nonlinear fashion. This approach identified conditions that promoted either motility phenotype. We then demonstrated that varying one of the conditions may change tumor cell behavior only in a context-dependent manner. In addition, to establish the link between phenotypes and cell fates, we photoconverted and monitored the fate of tumor cells in different microenvironments, finding that only tumor cells in the invadopodium-rich microenvironments degraded extracellular matrix (ECM and disseminated. The number of invadopodia positively correlated with degradation, while the inhibiting metalloproteases eliminated degradation and lung metastasis, consistent with a direct link among invadopodia, ECM degradation, and metastasis. We have detected and characterized two phenotypes of motile tumor cells in vivo, which

  20. Functional live cell imaging of the pulmonary neuroepithelial body microenvironment

    De Proost, Ian; Pintelon, Isabel; Brouns, Inge; Kroese, A; Riccardi, Daniela; Kemp, Paul J.; Timmermans, Jean-Pierre; Adriaensen, Dirk

    2008-01-01

    Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells invariably accompanied by Clara-like cells. Together with NEBs, Clara-like cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem cell niche. Conclusive

  1. 3D visualization of membrane failures in fuel cells

    Singh, Yadvinder; Orfino, Francesco P.; Dutta, Monica; Kjeang, Erik

    2017-03-01

    Durability issues in fuel cells, due to chemical and mechanical degradation, are potential impediments in their commercialization. Hydrogen leak development across degraded fuel cell membranes is deemed a lifetime-limiting failure mode and potential safety issue that requires thorough characterization for devising effective mitigation strategies. The scope and depth of failure analysis has, however, been limited by the 2D nature of conventional imaging. In the present work, X-ray computed tomography is introduced as a novel, non-destructive technique for 3D failure analysis. Its capability to acquire true 3D images of membrane damage is demonstrated for the very first time. This approach has enabled unique and in-depth analysis resulting in novel findings regarding the membrane degradation mechanism; these are: significant, exclusive membrane fracture development independent of catalyst layers, localized thinning at crack sites, and demonstration of the critical impact of cracks on fuel cell durability. Evidence of crack initiation within the membrane is demonstrated, and a possible new failure mode different from typical mechanical crack development is identified. X-ray computed tomography is hereby established as a breakthrough approach for comprehensive 3D characterization and reliable failure analysis of fuel cell membranes, and could readily be extended to electrolyzers and flow batteries having similar structure.

  2. Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

    Radtke, Andrea L; Herbst-Kralovetz, Melissa M

    2012-04-03

    Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties

  3. Fundamentals of microfluidic cell culture in controlled microenvironments.

    Young, Edmond W K; Beebe, David J

    2010-03-01

    Microfluidics has the potential to revolutionize the way we approach cell biology research. The dimensions of microfluidic channels are well suited to the physical scale of biological cells, and the many advantages of microfluidics make it an attractive platform for new techniques in biology. One of the key benefits of microfluidics for basic biology is the ability to control parameters of the cell microenvironment at relevant length and time scales. Considerable progress has been made in the design and use of novel microfluidic devices for culturing cells and for subsequent treatment and analysis. With the recent pace of scientific discovery, it is becoming increasingly important to evaluate existing tools and techniques, and to synthesize fundamental concepts that would further improve the efficiency of biological research at the microscale. This tutorial review integrates fundamental principles from cell biology and local microenvironments with cell culture techniques and concepts in microfluidics. Culturing cells in microscale environments requires knowledge of multiple disciplines including physics, biochemistry, and engineering. We discuss basic concepts related to the physical and biochemical microenvironments of the cell, physicochemical properties of that microenvironment, cell culture techniques, and practical knowledge of microfluidic device design and operation. We also discuss the most recent advances in microfluidic cell culture and their implications on the future of the field. The goal is to guide new and interested researchers to the important areas and challenges facing the scientific community as we strive toward full integration of microfluidics with biology.

  4. 2D- and 3D-culture of cell

    Khoruzhenko A. I.

    2011-02-01

    Full Text Available The cultivation of mammalian cells in three-dimensional conditions acquires a priority in a variety of biomedical applications. In the areas of toxicology and anticancer drug development it concerns a significant difference of responses to proapoptotic factors of the cells cultured in 2D versus 3D environment. Besides, the clear-cut differences have been found in cell polarity, cytoskeleton structure, distribution of receptors to wide range of hormones, growth factors, etc. in mammalian cells depending on culture conditions. It is resulted in different response of cultured cells to extracellular stimuli. Multicellular spheroids are regarded presently as the most convenient model of solid tumour growth in vitro. The cultivation of thyroid follicles, mammary acini and other structure units, maintaining initial tissue organization, allows studying the behavior, biochemical features and gene profile of differentiated cells. On the other hand, 3D cultures have some limitations in comparison with a well established monolayer culture. The advantages and disadvantages of each type of cultures and their application in biological and medical researches will be discussed in this review

  5. 3D surface topology guides stem cell adhesion and differentiation.

    Viswanathan, Priyalakshmi; Ondeck, Matthew G; Chirasatitsin, Somyot; Ngamkham, Kamolchanok; Reilly, Gwendolen C; Engler, Adam J; Battaglia, Giuseppe

    2015-06-01

    Polymerized high internal phase emulsion (polyHIPE) foams are extremely versatile materials for investigating cell-substrate interactions in vitro. Foam morphologies can be controlled by polymerization conditions to result in either open or closed pore structures with different levels of connectivity, consequently enabling the comparison between 2D and 3D matrices using the same substrate with identical surface chemistry conditions. Additionally, here we achieve the control of pore surface topology (i.e. how different ligands are clustered together) using amphiphilic block copolymers as emulsion stabilizers. We demonstrate that adhesion of human mesenchymal progenitor (hES-MP) cells cultured on polyHIPE foams is dependent on foam surface topology and chemistry but is independent of porosity and interconnectivity. We also demonstrate that the interconnectivity, architecture and surface topology of the foams has an effect on the osteogenic differentiation potential of hES-MP cells. Together these data demonstrate that the adhesive heterogeneity of a 3D scaffold could regulate not only mesenchymal stem cell attachment but also cell behavior in the absence of soluble growth factors.

  6. 3D-printed external light trap for solar cells.

    van Dijk, Lourens; Paetzold, Ulrich W; Blab, Gerhard A; Schropp, Ruud E I; di Vece, Marcel

    2016-05-01

    We present a universally applicable 3D-printed external light trap for enhanced absorption in solar cells. The macroscopic external light trap is placed at the sun-facing surface of the solar cell and retro-reflects the light that would otherwise escape. The light trap consists of a reflective parabolic concentrator placed on top of a reflective cage. Upon placement of the light trap, an improvement of 15% of both the photocurrent and the power conversion efficiency in a thin-film nanocrystalline silicon (nc-Si:H) solar cell is measured. The trapped light traverses the solar cell several times within the reflective cage thereby increasing the total absorption in the cell. Consequently, the trap reduces optical losses and enhances the absorption over the entire spectrum. The components of the light trap are 3D printed and made of smoothened, silver-coated thermoplastic. In contrast to conventional light trapping methods, external light trapping leaves the material quality and the electrical properties of the solar cell unaffected. To explain the theoretical operation of the external light trap, we introduce a model that predicts the absorption enhancement in the solar cell by the external light trap. The corresponding calculated path length enhancement shows good agreement with the empirically derived value from the opto-electrical data of the solar cell. Moreover, we analyze the influence of the angle of incidence on the parasitic absorptance to obtain full understanding of the trap performance. © 2015 The Authors. Progress in Photovoltaics: Research and Applications published by John Wiley & Sons, Ltd.

  7. Surface modified alginate microcapsules for 3D cell culture

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

    2016-06-01

    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  8. Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism

    Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate tha...

  9. Assessing Drug Efficacy in a Miniaturized Pancreatic Cancer In Vitro 3D Cell Culture Model.

    Shelper, Todd B; Lovitt, Carrie J; Avery, Vicky M

    2016-09-01

    Pancreatic cancer continues to have one of the poorest prognoses among all cancers. The drug discovery efforts for this disease have largely failed, with no significant improvement in survival outcomes for advanced pancreatic cancer patients over the past 20 years. Traditional in vitro cell culture techniques have been used extensively in both basic and early drug discovery; however, these systems offer poor models to assess emerging therapeutics. More predictive cell-based models, which better capture the cellular heterogeneity and complexities of solid pancreatic tumors, are urgently needed not only to improve drug discovery success but also to provide insight into the tumor biology. Pancreatic tumors are characterized by a unique micro-environment that is surrounded by a dense stroma. A complex network of interactions between extracellular matrix (ECM) components and the effects of cell-to-cell contacts may enhance survival pathways within in vivo tumors. This biological and physical complexity is lost in traditional cell monolayer models. To explore the predictive potential of a more complex cellular system, a three-dimensional (3D) micro-tumor assay was evaluated. Efficacy of six current chemotherapeutics was determined against a panel of primary and metastatic pancreatic tumor cell lines in a miniaturized ECM-based 3D cell culture system. Suitability for potential use in high-throughput screening applications was assessed, including ascertaining the effects that miniaturization and automation had on assay robustness. Cellular health was determined by utilizing an indirect population-based metabolic activity assay and a direct imaging-based cell viability assay.

  10. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

  11. Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

    Gartia, Manas Ranjan [Department of Nuclear, Plasma and Radiological Engineering, University of Illinois, Urbana, IL 61801 (United States); Hsiao, Austin; Logan Liu, G [Department of Bioengineering, University of Illinois, Urbana, IL 61801 (United States); Sivaguru, Mayandi [Institute for Genomic Biology, University of Illinois, Urbana, IL 61801 (United States); Chen Yi, E-mail: loganliu@illinois.edu [Department of Electrical and Computer Engineering, University of Illinois, Urbana, IL 61801 (United States)

    2011-09-07

    We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.

  12. SECs (Sinusoidal Endothelial Cells), Liver Microenvironment, and Fibrosis

    Natarajan, Vaishaali; Harris, Edward N.

    2017-01-01

    Liver fibrosis is a wound-healing response to chronic liver injury such as alcoholic/nonalcoholic fatty liver disease and viral hepatitis with no FDA-approved treatments. Liver fibrosis results in a continual accumulation of extracellular matrix (ECM) proteins and paves the way for replacement of parenchyma with nonfunctional scar tissue. The fibrotic condition results in drastic changes in the local mechanical, chemical, and biological microenvironment of the tissue. Liver parenchyma is supported by an efficient network of vasculature lined by liver sinusoidal endothelial cells (LSECs). These nonparenchymal cells are highly specialized resident endothelial cell type with characteristic morphological and functional features. Alterations in LSECs phenotype including lack of LSEC fenestration, capillarization, and formation of an organized basement membrane have been shown to precede fibrosis and promote hepatic stellate cell activation. Here, we review the interplay of LSECs with the dynamic changes in the fibrotic liver microenvironment such as matrix rigidity, altered ECM protein profile, and cell-cell interactions to provide insight into the pivotal changes in LSEC physiology and the extent to which it mediates the progression of liver fibrosis. Establishing the molecular aspects of LSECs in the light of fibrotic microenvironment is valuable towards development of novel therapeutic and diagnostic targets of liver fibrosis. PMID:28293634

  13. Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging.

    Annibale, Paolo; Gratton, Enrico

    2015-03-19

    Multi-cell biochemical assays and single cell fluorescence measurements revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Here by employing high-speed 3D fluorescence nanoimaging techniques we resolve and track at the single cell level multiple, distinct regions of mRNA synthesis within the model system of a large transgene array. We demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm. Using fluctuation spectroscopy and the phasor analysis approach we were able to extract the local PolII elongation rate at each site as a function of time. We measured a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell, demonstrating a correlation between local transcription kinetics and the movement of the transcription site. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability.

  14. 3D photospheric velocity field of a Supergranular cell

    Del Moro, Dario; Berrilli, Francesco

    2007-01-01

    We investigate the plasma flow properties inside a Supergranular (SG) cell, in particular its interaction with small scale magnetic field structures. The SG cell has been identified using the magnetic network (CaII wing brightness) as proxy, applying the TST to high spatial, spectral and temporal resolution observations obtained by IBIS. The full 3D velocity vector field for the SG has been reconstructed at two different photospheric heights. In order to strengthen our findings, we also computed the mean radial flow of the SG by means of cork tracing. We also studied the behaviour of the horizontal and Line of Sight plasma flow cospatial with cluster of bright CaII structures of magnetic origin to better understand the interaction between photospheric convection and small scale magnetic features. The SG cell we investigated seems to be organized with an almost radial flow from its centre to the border. The large scale divergence structure is probably created by a compact region of costant up-flow close to the...

  15. Dendritic Cells in the Cancer Microenvironment

    Yang Ma, Galina V. Shurin, Zhu Peiyuan, Michael R. Shurin

    2013-01-01

    Full Text Available The complexity of the tumor immunoenvironment is underscored by the emergence and discovery of different subsets of immune effectors and regulatory cells. Tumor-induced polarization of immune cell differentiation and function makes this unique environment even more intricate and variable. Dendritic cells (DCs represent a special group of cells that display different phenotype and activity at the tumor site and exhibit differential pro-tumorigenic and anti-tumorigenic functions. DCs play a key role in inducing and maintaining the antitumor immunity, but in the tumor environment their antigen-presenting function may be lost or inefficient. DCs might be also polarized into immunosuppressive/tolerogenic regulatory DCs, which limit activity of effector T cells and support tumor growth and progression. Although various factors and signaling pathways have been described to be responsible for abnormal functioning of DCs in cancer, there are still no feasible therapeutic modalities available for preventing or reversing DC malfunction in tumor-bearing hosts. Thus, better understanding of DC immunobiology in cancer is pivotal for designing novel or improved therapeutic approaches that will allow proper functioning of DCs in patients with cancer.

  16. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Ansari, Nariman [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Esner, Milan; Bickle, Marc [Max Planck Institute of Molecular Cell Biology and Genetics, High-Throughput Technology Development Studio (TDS), Dresden (Germany); Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H. [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Parczyk, Karsten; Prechtl, Stefan [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Steigemann, Patrick, E-mail: Patrick.Steigemann@bayer.com [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany)

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

  17. Human embryonic stem cells and microenvironment

    Banu İskender

    2014-09-01

    Full Text Available Human embryonic stem cells (hESCs possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less labor-intensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3: 486-495

  18. Engineering anisotropic biomimetic fibrocartilage microenvironment by bioprinting mesenchymal stem cells in nanoliter gel droplets.

    Gurkan, Umut A; El Assal, Rami; Yildiz, Simin E; Sung, Yuree; Trachtenberg, Alexander J; Kuo, Winston P; Demirci, Utkan

    2014-07-07

    Over the past decade, bioprinting has emerged as a promising patterning strategy to organize cells and extracellular components both in two and three dimensions (2D and 3D) to engineer functional tissue mimicking constructs. So far, tissue printing has neither been used for 3D patterning of mesenchymal stem cells (MSCs) in multiphase growth factor embedded 3D hydrogels nor been investigated phenotypically in terms of simultaneous differentiation into different cell types within the same micropatterned 3D tissue constructs. Accordingly, we demonstrated a biochemical gradient by bioprinting nanoliter droplets encapsulating human MSCs, bone morphogenetic protein 2 (BMP-2), and transforming growth factor β1 (TGF- β1), engineering an anisotropic biomimetic fibrocartilage microenvironment. Assessment of the model tissue construct displayed multiphasic anisotropy of the incorporated biochemical factors after patterning. Quantitative real time polymerase chain reaction (qRT-PCR) results suggested genomic expression patterns leading to simultaneous differentiation of MSC populations into osteogenic and chondrogenic phenotype within the multiphasic construct, evidenced by upregulation of osteogenesis and condrogenesis related genes during in vitro culture. Comprehensive phenotypic network and pathway analysis results, which were based on genomic expression data, indicated activation of differentiation related mechanisms, via signaling pathways, including TGF, BMP, and vascular endothelial growth factor.

  19. Mesenchymal stem cells enhance ovarian cancer cell infiltration through IL6 secretion in an amniochorionic membrane based 3D model

    Touboul Cyril

    2013-01-01

    Full Text Available Abstract Background The early peritoneal invasion of epithelial ovarian cancer (EOC by tumoral aggregates presents in ascites is a major concern. The role of the microenvironment seems to be important in this process but the lack of adequate models to study cellular interactions between cancer cells and stromal cells does not allow to uncover the molecular pathways involved. Our goal was to study the interactions between ovarian cancer cells (OCC and mesenchymal stem cells (MSC using a 3D model. Methods We used millimetric pieces of amniochorionic membrane - referred to as amniotic membrane scaffold (AMS - to create 3D peritoneal nodules mimicking EOC early invasion. We were able to measure the distribution and the depth of infiltration using confocal microsopy. We extracted MSC from the amniochorionic membrane using the markers CD34-, CD45-, CD73+, CD90+, CD105+ and CD29+ at the Fluorescence Activated Cell Sorting (FACS analysis. We used transwell and wound healing tests to test OCC migration and invasion in vitro. Results Here we show that OCC tumors were located in regions rich in MSC (70%. The tumors infiltrated deeper within AMS in regions rich in MSC (p Conclusions The use of tridimensional models using AMS could be a useful tool to decipher early molecular events in ovarian cancer metastasis. Cytokine inhibitors interrupting the cross-talk between OCCs and MSCs such as IL6 should be investigated as a new therapeutic approach in ovarian cancer.

  20. File list: Pol.ALL.50.Polr3d.AllCell [Chip-atlas[Archive

    Full Text Available Pol.ALL.50.Polr3d.AllCell mm9 RNA polymerase Polr3d All cell types SRX301459,SRX373...04147 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.50.Polr3d.AllCell.bed ...

  1. File list: Pol.ALL.05.Polr3d.AllCell [Chip-atlas[Archive

    Full Text Available Pol.ALL.05.Polr3d.AllCell mm9 RNA polymerase Polr3d All cell types SRX373040,SRX373...04148 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.05.Polr3d.AllCell.bed ...

  2. File list: Pol.ALL.10.Polr3d.AllCell [Chip-atlas[Archive

    Full Text Available Pol.ALL.10.Polr3d.AllCell mm9 RNA polymerase Polr3d All cell types SRX373040,SRX301...04147 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.10.Polr3d.AllCell.bed ...

  3. File list: Pol.ALL.20.Polr3d.AllCell [Chip-atlas[Archive

    Full Text Available Pol.ALL.20.Polr3d.AllCell mm9 RNA polymerase Polr3d All cell types SRX301459,SRX373...73041 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.20.Polr3d.AllCell.bed ...

  4. Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

    Ouyang, Liliang; Yao, Rui; Zhao, Yu; Sun, Wei

    2016-09-16

    3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and

  5. Differentiation Induction of Mouse Neural Stem Cells in Hydrogel Tubular Microenvironments with Controlled Tube Dimensions.

    Onoe, Hiroaki; Kato-Negishi, Midori; Itou, Akane; Takeuchi, Shoji

    2016-05-01

    In this paper, a tubular 3D microenvironment created in a calcium alginate hydrogel microtube with respect to the effect of scaffold dimensions on the differentiation of mouse neuronal stem cells (mNSCs) is evaluated. Five types of hydrogel microtubes with different core diameters (≈65-200 μm) and shell thicknesses (≈30-110 μm) are fabricated by using a double coaxial microfluidic device, and differentiation of encapsulated mNSCs is induced by changing the growth medium to the differentiation medium. The influence of the microtube geometries is examined by using quantitative real-time polymerase chain reaction and fluorescent immunocytochemistry. The analyses reveal that differences in microtube thickness within 30-110 μm affected the relative Tuj1 expression but do not affect the morphology of encapsulated mNSCs. The diameters of cores influence both the relative Tuj1 expression and morphology of the differentiated neurons. It is found that the tubular microenvironment with a core diameter of less than ≈100 μm contributes to forming highly viable and aligned neural tissue. The tubular microenvironment can provide an effective method for constructing microfiber-shaped neural tissues with geometrically controlled differentiation induction.

  6. Carcinoma Cell Hyaluronan as a "Portable" Cancerized Prometastatic Microenvironment.

    Turley, Eva A; Wood, David K; McCarthy, James B

    2016-05-01

    Hyaluronan (HA) is a structurally simple polysaccharide, but its ability to act as a template for organizing pericellular matrices and its regulated synthesis and degradation are key to initiating repair responses. Importantly, these HA functions are usurped by tumor cells to facilitate progression and metastasis. Recent advances have identified the functional complexities associated with the synthesis and degradation of HA-rich matrices. Three enzymes synthesize large HA polymers while multiple hyaluronidases or tissue free radicals degrade these into smaller bioactive fragments. A family of extracellular and cell-associated HA-binding proteins/receptors translates the bioinformation encrypted in this complex polymer mixture to activate signaling networks required for cell survival, proliferation, and migration in an actively remodeling microenvironment. Changes in HA metabolism within both the peritumor stroma and parenchyma are linked to tumor initiation, progression, and poor clinical outcome. We review evidence that metastatic tumor cells must acquire the capability to autonomously synthesize, assemble, and process their own "portable" HA-rich microenvironments to survive in the circulation, metastasize to ectopic sites, and escape therapeutic intervention. Strategies to disrupt the HA machinery of primary tumor and circulating tumor cells may enhance the effectiveness of current conventional and targeted therapies. Cancer Res; 76(9); 2507-12. ©2016 AACR.

  7. Gel de plaquetas: arcabouço 3D para cultura celular Platelet gel: 3D scaffold for cell culture

    Andrei Moroz

    2009-01-01

    Full Text Available INTRODUÇÃO: O reparo tissular é o objetivo final da cirurgia. A cultura celular requer arcabouço mecânico que dê suporte ao crescimento celular e difusão dos nutrientes. O uso do plasma rico em plaquetas (PRP como um arcabouço 3D possui diversas vantagens: é material biológico, de fácil absorção pós-transplante, rico em fatores de crescimento, em especial PDGF- ββ e TGF-β que estimula síntese de matriz extracelular na cartilagem. OBJETIVO: Desenvolver arcabouço 3D à base de PRP. MATERIAIS E MÉTODOS: Duas formas foram idealizadas: Sphere e Carpet. Condições estéreis foram utilizadas. O gel de plaquetas permaneceu em cultura celular, observado diariamente em microscópio invertido. RESULTADOS: Ambos arcabouços obtiveram sucesso, com aspectos positivos e negativos. DISCUSSÃO: A forma Sphere não aderiu ao plástico. Observou-se retração do gel e investigação ao microscópio dificultada devido às áreas opacas no campo visual. A forma Carpet não aderiu ao plástico e apresentou-se translúcida. O tempo de estudo foi de 20 dias. CONCLUSÕES: A produção de um arcabouço 3D PRP foi um sucesso, e trata-se de uma alternativa que necessita ser mais utilizado e investigado para que se consolide em uma rota eficiente e confiável na tecnologia de engenharia tissular, particularmente em cultura de tecido cartilaginoso.INTRODUCTION: Tissue repair has been the ultimate goal of surgery. Cell culture requires a mechanical scaffold that supports cell growth and nutrient diffusion. Using platelet-rich plasma (PRP as a 3D scaffold presents various advantages: it is a biological material, easily absorbed after transplantation, rich in growth factors, in particular, PDGF-ββ and TGF-β that stimulate extracellular matrix synthesis in cartilage culture. OBJECTIVE: To develop a PRP 3D scaffold. Material and METHODS: Two forms were idealized: Sphere and Carpet. Sterile conditions were used. The platelet gel remained in culture

  8. Platelet gel: 3D scaffold for cell culture

    Andrei Moroz; Renata Aparecida de Camargo Bittencourt; Sérgio Luis Felisbino; Hamilton da Rosa Pereira; Rosana Rossi-Ferreira; Elenice Deffune

    2009-01-01

    INTRODUÇÃO: O reparo tissular é o objetivo final da cirurgia. A cultura celular requer arcabouço mecânico que dê suporte ao crescimento celular e difusão dos nutrientes. O uso do plasma rico em plaquetas (PRP) como um arcabouço 3D possui diversas vantagens: é material biológico, de fácil absorção pós-transplante, rico em fatores de crescimento, em especial PDGF- ββ e TGF-β que estimula síntese de matriz extracelular na cartilagem. OBJETIVO: Desenvolver arcabouço 3D à base de PR...

  9. 3D-Printing Crystallographic Unit Cells for Learning Materials Science and Engineering

    Rodenbough, Philip P.; Vanti, William B.; Chan, Siu-Wai

    2015-01-01

    Introductory materials science and engineering courses universally include the study of crystal structure and unit cells, which are by their nature highly visual 3D concepts. Traditionally, such topics are explored with 2D drawings or perhaps a limited set of difficult-to-construct 3D models. The rise of 3D printing, coupled with the wealth of…

  10. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    Knowlton, Stephanie; Yu, Chu Hsiang; Ersoy, Fulya; Emadi, Sharareh; Khademhosseini, Ali; Tasoglu, Savas

    2016-06-20

    Three-dimensional (3D) printing offers potential to fabricate high-throughput and low-cost fabrication of microfluidic devices as a promising alternative to traditional techniques which enables efficient design iterations in the development stage. In this study, we demonstrate a single-step fabrication of a 3D transparent microfluidic chip using two alternative techniques: a stereolithography-based desktop 3D printer and a two-step fabrication using an industrial 3D printer based on polyjet technology. This method, compared to conventional fabrication using relatively expensive materials and labor-intensive processes, presents a low-cost, rapid prototyping technique to print functional 3D microfluidic chips. We enhance the capabilities of 3D-printed microfluidic devices by coupling 3D cell encapsulation and spatial patterning within photocrosslinkable gelatin methacryloyl (GelMA). The platform presented here serves as a 3D culture environment for long-term cell culture and growth. Furthermore, we have demonstrated the ability to print complex 3D microfluidic channels to create predictable and controllable fluid flow regimes. Here, we demonstrate the novel use of 3D-printed microfluidic chips as controllable 3D cell culture environments, advancing the applicability of 3D printing to engineering physiological systems for future applications in bioengineering.

  11. Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment.

    Palchesko, Rachelle N; Szymanski, John M; Sahu, Amrita; Feinberg, Adam W

    2014-09-01

    Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments.

  12. Sliding Hydrogels with Mobile Molecular Ligands and Crosslinks as 3D Stem Cell Niche.

    Tong, Xinming; Yang, Fan

    2016-09-01

    The development of a sliding hydrogel with mobile crosslinks and biochemical ligands as a 3D stem cell niche is reported. The molecular mobility of this sliding hydrogel allows stem cells to reorganize the surrounding ligands and change their morphology in 3D. Without changing matrix stiffness, sliding hydrogels support efficient stem cell differentiation toward multiple lineages including adipogenesis, chondrogenesis, and osteogenesis.

  13. Mean deformation metrics for quantifying 3D cell-matrix interactions without requiring information about matrix material properties.

    Stout, David A; Bar-Kochba, Eyal; Estrada, Jonathan B; Toyjanova, Jennet; Kesari, Haneesh; Reichner, Jonathan S; Franck, Christian

    2016-03-15

    Mechanobiology relates cellular processes to mechanical signals, such as determining the effect of variations in matrix stiffness with cell tractions. Cell traction recorded via traction force microscopy (TFM) commonly takes place on materials such as polyacrylamide- and polyethylene glycol-based gels. Such experiments remain limited in physiological relevance because cells natively migrate within complex tissue microenvironments that are spatially heterogeneous and hierarchical. Yet, TFM requires determination of the matrix constitutive law (stress-strain relationship), which is not always readily available. In addition, the currently achievable displacement resolution limits the accuracy of TFM for relatively small cells. To overcome these limitations, and increase the physiological relevance of in vitro experimental design, we present a new approach and a set of associated biomechanical signatures that are based purely on measurements of the matrix's displacements without requiring any knowledge of its constitutive laws. We show that our mean deformation metrics (MDM) approach can provide significant biophysical information without the need to explicitly determine cell tractions. In the process of demonstrating the use of our MDM approach, we succeeded in expanding the capability of our displacement measurement technique such that it can now measure the 3D deformations around relatively small cells (∼10 micrometers), such as neutrophils. Furthermore, we also report previously unseen deformation patterns generated by motile neutrophils in 3D collagen gels.

  14. Identification of Senescent Cells in the Bone Microenvironment

    Farr, Joshua N; Fraser, Daniel G; Wang, Haitao; Jaehn, Katharina; Ogrodnik, Mikolaj B; Weivoda, Megan M; Drake, Matthew T; Tchkonia, Tamara; LeBrasseur, Nathan K; Kirkland, James L; Bonewald, Lynda F; Pignolo, Robert J; Monroe, David G; Khosla, Sundeep

    2017-01-01

    Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a, profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells

  15. A 3D Hydrodynamic Model for Cytokinesis of Eukaryotic Cells

    2014-08-01

    division or binary fission, which is the primary method for reproduction. For eukaryotic cells, it’s called cell mitotic process or mitosis . At the late...of cytokinesis in animal, yeast and plant cells. Experimental observations have provide us with a basic picture of cell mitosis . For eu- karyotic... mitosis and cytokinesis in mammalian cells. 2014 Special Focus on Rho GTPases, page e29770, 2014. [7] Ulrike S. Eggert, Timothy J. Mitchison, and

  16. Dynamic microenvironments: the fourth dimension.

    Tibbitt, Mark W; Anseth, Kristi S

    2012-11-14

    The extracellular space, or cell microenvironment, choreographs cell behavior through myriad controlled signals, and aberrant cues can result in dysfunction and disease. For functional studies of human cell biology or expansion and delivery of cells for therapeutic purposes, scientists must decipher this intricate map of microenvironment biology and develop ways to mimic these functions in vitro. In this Perspective, we describe technologies for four-dimensional (4D) biology: cell-laden matrices engineered to recapitulate tissue and organ function in 3D space and over time.

  17. Using 3D Super-Resolution Microscopy to Probe Breast Cancer Stem Cells and Their Microenvironment

    2014-05-01

    Medicine and Biology , Marina Del Ray, CA, April 10-12, 2013. 5. Mustafa Mir, Tiina Jokela, Marc LaBarge, and Lydia L. Sohn, “Quantifying the effects...Cancer Microenvironments”, Poster presentation, 7th International Conference on Microtechnologies in Medicine and Biology , Marina Del Ray, CA, April 10...will help elucidate the mechanisms for, and biology of, breast cancer. In particular, quantitative phase imaging allows us to go beyond current end

  18. Using 3-D Super-Resolution Microscopy to Probe Breast Cancer Stem Cells and Their Microenvironment

    2013-03-01

    Microenvironments”, Poster presentation, 7th International Conference on Microtechnologies in Medicine and Biology , Marina Del Ray, CA, April 10-12, 2013. List...7th International Conference on Microtechnologies in Medicine and Biology , Marina Del Ray, CA, April 10-12, 2013 Poster: Tunable Micropost Arrays

  19. 3D in vitro cell culture models of tube formation

    Zegers, M.M.P.

    2014-01-01

    Building the complex architecture of tubular organs is a highly dynamic process that involves cell migration, polarization, shape changes, adhesion to neighboring cells and the extracellular matrix, physicochemical characteristics of the extracellular matrix and reciprocal signaling with the mesench

  20. Impedance Spectroscopic Characterisation of Porosity in 3D Cell Culture Scaffolds with Different Channel Networks

    Canali, Chiara; Mohanty, Soumyaranjan; Heiskanen, Arto

    2015-01-01

    We present the application of electrochemical impedance spectroscopy (EIS) as a method for discriminating between different polydimethylsiloxane (PDMS) scaffolds for three-dimensional (3D) cell cultures. The validity of EIS characterisation for scaffolds having different degree of porosity...... serve as means of single-frequency measurements for fast scaffold characterization combined with in vitro monitoring of 3D cell cultures....

  1. Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

    Ahlf Wheatcraft, Dorothy R.; Liu, Xin; Hummon, Amanda B.

    2014-01-01

    Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribu...

  2. AlgiMatrix™ based 3D cell culture system as an in-vitro tumor model for anticancer studies.

    Chandraiah Godugu

    Full Text Available BACKGROUND: Three-dimensional (3D in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening. METHODS: Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100-300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin and nanoparticle (NLC were done using spheroids. RESULTS: IC(50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. CONCLUSION: The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.

  3. Bipotent mammary stem cells: now in amazing 3D

    van Amerongen, R.

    2014-01-01

    For many decades, developmental biologists and cancer researchers alike have been trying to understand the relationship between the basal and luminal cell compartments in the mouse mammary epithelium. Delineating the mammary stem and progenitor cell hierarchy will provide fundamental knowledge of ho

  4. Biofabrication of cell-loaded 3D spider silk constructs.

    Schacht, Kristin; Jüngst, Tomasz; Schweinlin, Matthias; Ewald, Andrea; Groll, Jürgen; Scheibel, Thomas

    2015-02-23

    Biofabrication is an emerging and rapidly expanding field of research in which additive manufacturing techniques in combination with cell printing are exploited to generate hierarchical tissue-like structures. Materials that combine printability with cytocompatibility, so called bioinks, are currently the biggest bottleneck. Since recombinant spider silk proteins are non-immunogenic, cytocompatible, and exhibit physical crosslinking, their potential as a new bioink system was evaluated. Cell-loaded spider silk constructs can be printed by robotic dispensing without the need for crosslinking additives or thickeners for mechanical stabilization. Cells are able to adhere and proliferate with good viability over at least one week in such spider silk scaffolds. Introduction of a cell-binding motif to the spider silk protein further enables fine-tuned control over cell-material interactions. Spider silk hydrogels are thus a highly attractive novel bioink for biofabrication.

  5. Development of 3-D Hydrogel Culture Systems With On-Demand Cell Separation

    Hamilton, Sharon K.; Bloodworth, Nathaniel C.; Massad, Christopher S.; Hammoudi, Taymour M.; Suri, Shalu; Yang, Peter J.; Lu, Hang; Temenoff, Johnna S

    2013-01-01

    Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3-D co-culture methods lack the ability to effectively separate 2 cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3-D hydrogel co-culture system...

  6. Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an In Vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture.

    Miyamoto, Yoshitaka; Ikeuchi, Masashi; Noguchi, Hirofumi; Yagi, Tohru; Hayashi, Shuji

    2017-01-08

    The application of stem cells for cell therapy has been extensively studied in recent years. Among the various types of stem cells, human adipose tissue-derived stem cells (ASCs) can be obtained in large quantities with relatively few passages, and they possess a stable quality. ASCs can differentiate into a number of cell types, such as adipose cells and ectodermal cells. We therefore focused on the in vitro microenvironment required for such differentiation and attempted to induce the differentiation of human stem cells into microtissues using a microelectromechanical system. We first evaluated the adipogenic differentiation of human ASC spheroids in a three-dimensional (3D) culture. We then created the in vitro microenvironment using a 3D combinatorial TASCL device and attempted to induce the adipogenic differentiation of human ASCs. The differentiation of human ASC spheroids cultured in maintenance medium and those cultured in adipocyte differentiation medium was evaluated via Oil red O staining using lipid droplets based on the quantity of accumulated triglycerides. The differentiation was confirmed in both media, but the human ASCs in the 3D cultures contained higher amounts of triglycerides than those in the 2D cultures. In the short culture period, greater adipogenic differentiation was observed in the 3D cultures than in the 2D cultures. The 3D culture using the TASCL device with adipogenic differentiation medium promoted greater differentiation of human ASCs into adipogenic lineages than either a 2D culture or a culture using a maintenance medium. In summary, the TASCL device created a hospitable in vitro microenvironment and may therefore be a useful tool for the induction of differentiation in 3D culture. The resultant human ASC spheroids were "adipose-like microtissues" that formed spherical aggregation perfectly and are expected to be applicable in regenerative medicine as well as cell transplantation.

  7. Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an In Vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture

    Miyamoto, Yoshitaka; Ikeuchi, Masashi; Noguchi, Hirofumi; Yagi, Tohru; Hayashi, Shuji

    2017-01-01

    The application of stem cells for cell therapy has been extensively studied in recent years. Among the various types of stem cells, human adipose tissue-derived stem cells (ASCs) can be obtained in large quantities with relatively few passages, and they possess a stable quality. ASCs can differentiate into a number of cell types, such as adipose cells and ectodermal cells. We therefore focused on the in vitro microenvironment required for such differentiation and attempted to induce the differentiation of human stem cells into microtissues using a microelectromechanical system. We first evaluated the adipogenic differentiation of human ASC spheroids in a three-dimensional (3D) culture. We then created the in vitro microenvironment using a 3D combinatorial TASCL device and attempted to induce the adipogenic differentiation of human ASCs. The differentiation of human ASC spheroids cultured in maintenance medium and those cultured in adipocyte differentiation medium was evaluated via Oil red O staining using lipid droplets based on the quantity of accumulated triglycerides. The differentiation was confirmed in both media, but the human ASCs in the 3D cultures contained higher amounts of triglycerides than those in the 2D cultures. In the short culture period, greater adipogenic differentiation was observed in the 3D cultures than in the 2D cultures. The 3D culture using the TASCL device with adipogenic differentiation medium promoted greater differentiation of human ASCs into adipogenic lineages than either a 2D culture or a culture using a maintenance medium. In summary, the TASCL device created a hospitable in vitro microenvironment and may therefore be a useful tool for the induction of differentiation in 3D culture. The resultant human ASC spheroids were “adipose-like microtissues” that formed spherical aggregation perfectly and are expected to be applicable in regenerative medicine as well as cell transplantation.

  8. Generation of compartmentalized pressure by a nuclear piston governs cell motility in a 3D matrix.

    Petrie, Ryan J; Koo, Hyun; Yamada, Kenneth M

    2014-08-29

    Cells use actomyosin contractility to move through three-dimensional (3D) extracellular matrices. Contractility affects the type of protrusions cells use to migrate in 3D, but the mechanisms are unclear. In this work, we found that contractility generated high-pressure lobopodial protrusions in human cells migrating in a 3D matrix. In these cells, the nucleus physically divided the cytoplasm into forward and rear compartments. Actomyosin contractility with the nucleoskeleton-intermediate filament linker protein nesprin-3 pulled the nucleus forward and pressurized the front of the cell. Reducing expression of nesprin-3 decreased and equalized the intracellular pressure. Thus, the nucleus can act as a piston that physically compartmentalizes the cytoplasm and increases the hydrostatic pressure between the nucleus and the leading edge of the cell to drive lamellipodia-independent 3D cell migration.

  9. Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

    Smith, W. A.; Lam, K.-P.; Dempsey, K. P.; Mazzocchi-Jones, D.; Richardson, J. B.; Yang, Y.

    2014-02-01

    Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.

  10. Treatment with Y-27632, a ROCK Inhibitor, Increases the Proinvasive Nature of SW620 Cells on 3D Collagen Type 1 Matrix

    Ramana Vishnubhotla

    2012-01-01

    Full Text Available The concept of using tissue density as a mechanism to diagnose a tumor has been around for centuries. However, this concept has not been sufficiently explored in a laboratory setting. Therefore, in this paper, we observed the effects of cell density and extracellular matrix (ECM density on colon cancer invasion and proliferation using SW620 cells. We also attempted to inhibit ROCK-I to determine its effect on cell invasion and proliferation using standard molecular biology techniques and advanced imaging. Increasing cell seeding density resulted in a 2-fold increase in cell invasion as well as cell proliferation independent of treatment with Y-27632. Increasing collagen I scaffold density resulted in a 2.5-fold increase in cell proliferation while treatment with Y-27632 attenuated this effect although 1.5 fold increase in cell invasion was observed in ROCK inhibited samples. Intriguingly, ROCK inhibition also resulted in a 3.5-fold increase in cell invasion within 3D collagen scaffolds for cells seeded at lower densities. We show in this paper that ROCK-I inhibition leads to increased invasion within 3D collagen I microenvironments. This data suggests that although ROCK inhibitors have been used clinically to treat several medical conditions, its effect largely depends on the surrounding microenvironment.

  11. Local 3D matrix confinement determines division axis through cell shape.

    He, Lijuan; Chen, Weitong; Wu, Pei-Hsun; Jimenez, Angela; Wong, Bin Sheng; San, Angela; Konstantopoulos, Konstantinos; Wirtz, Denis

    2016-02-01

    How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via β1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of β1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype.

  12. Spontaneous Electroless Galvanic Cell Deposition of 3D Hierarchical and Interlaced S-M-S Heterostructures.

    Tan, Chuan Fu; Azmansah, Siti Aishah Bte; Zhu, Hai; Xu, Qing-Hua; Ho, Ghim Wei

    2017-01-01

    One-pot electroless galvanic cell deposition of a 3D hierarchical semiconductor-metal-semiconductor interlaced nanoarray is demonstrated. The fabricated 3D photoanode deviates from the typical planar geometry, and aims to optimize the effective surface area for light harvesting and long-range charge transfer-collection pathways.

  13. 3D tissue formation : the kinetics of human mesenchymal stem cells

    Higuera Sierra, Gustavo Andrés

    2010-01-01

    The main thesis in this book proposes that physical phenomena underlies the formation of three-dimensional (3D) tissue. In this thesis, tissue regeneration with mesenchymal stem cells was studied through the law of conservation of mass. MSCs proliferation and 3D tissue formation were explored from 2

  14. Fluid and cell behaviors along a 3D printed alginate/gelatin/fibrin channel.

    Xu, Yufan; Wang, Xiaohong

    2015-08-01

    Three-dimensional (3D) cell manipulation is available with the integration of microfluidic technology and rapid prototyping techniques. High-Fidelity (Hi-Fi) constructs hold enormous therapeutic potential for organ manufacturing and regenerative medicine. In the present paper we introduced a quasi-three-dimensional (Q3D) model with parallel biocompatible alginate/gelatin/fibrin hurdles. The behaviors of fluids and cells along the microfluidic channels with various widths were studied. Cells inside the newly designed microfluidic channels attached and grew well. Morphological changes of adipose-derived stem cells (ADSCs) in both two-dimensional (2D) and 3D milieu were found on the printed constructs. Endothelialization occurred with the co-cultures of ADSCs and hepatocytes. This study provides insights into the interactions among fluids, cells and biomaterials, the behaviors of fluids and cells along the microfluidic channels, and the applications of Q3D techniques.

  15. Nonpolarized signaling reveals two distinct modes of 3D cell migration.

    Petrie, Ryan J; Gavara, Núria; Chadwick, Richard S; Yamada, Kenneth M

    2012-04-30

    We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized. Reducing RhoA, Rho-associated protein kinase (ROCK), or myosin II activity switched the cells to lamellipodia-based 3D migration. These modes of 3D migration were regulated by matrix physical properties. Specifically, experimentally modifying the elasticity of the CDM or collagen gels established that nonlinear elasticity supported lamellipodia-based migration, whereas linear elasticity switched cells to lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by RhoA, ROCK, and myosin II and extrinsically by the elastic behavior of the 3D extracellular matrix.

  16. Design of 3D printed insert for hanging culture of Caco-2 cells.

    Shen, Chong; Meng, Qin; Zhang, Guoliang

    2014-12-17

    A Caco-2 cell culture on Transwell, an alternative testing to animal or human testing used in evaluating drug intestinal permeability, incorrectly estimated the absorption of actively transported drugs due to the low expression of membrane transporters. Similarly, three-dimensional (3D) cultures of Caco-2 cells, which have been recommended to be more physiological relevant, were not superior to the Transwell culture in either accuracy or convenience in drug permeability testing. Using rapid 3D printing prototyping techniques, this study proposed a hanging culture of Caco-2 cells that performed with high accuracy in predicting drug permeability in humans. As found, hanging cultured Caco-2 cells formed a confluent monolayer and maintained high cell viability on the 3D printed insert. Compared with the normal culture on Transwell, the Caco-2 cells on the 3D printed insert presented ∼30-100% higher brush border enzyme activity and ∼2-7 folds higher activity of P-glycoprotein/multidrug resistance-associated protein 2 during 21 days of incubation. For the eight membrane transporter substrates, the predictive curve of the 3D printing culture exhibited better linearity (R(2) = 0.92) to the human oral adsorption than that of the Transwell culture (R(2) = 0.84), indicating better prediction by the 3D printing culture. In this regard, the 3D printed insert for hanging culture could be potentially developed as a convenient and low-cost tool for testing drug oral absorption.

  17. Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture

    Frampton, J P; Hynd, M R; Shain, W [Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12210 (United States); Shuler, M L, E-mail: jf7674@albany.edu [Department of Biomedical Engineering, 270 Olin Hall, Cornell University, Ithaca, NY 14850 (United States)

    2011-02-15

    Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cell-cell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models.

  18. Bioimpedance monitoring of 3D cell culturing--complementary electrode configurations for enhanced spatial sensitivity.

    Canali, Chiara; Heiskanen, Arto; Muhammad, Haseena Bashir; Høyum, Per; Pettersen, Fred-Johan; Hemmingsen, Mette; Wolff, Anders; Dufva, Martin; Martinsen, Ørjan Grøttem; Emnéus, Jenny

    2015-01-15

    A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of the entire process of three-dimensional (3D) cell culturing in a hydrogel scaffold. In this study, the dynamics involved in the whole process of 3D cell culturing, starting from polymerisation of a bare 3D gelatin scaffold, to human mesenchymal stem cell (MSC) encapsulation and proliferation, was monitored over time. The platform consists of a large rectangular culture chamber with four embedded vertical gold plate electrodes that were exploited in two- and three terminal (2T and 3T) measurement configurations. By switching between the different combinations of electrode couples, it was possible to generate a multiplexing-like approach, which allowed for collecting spatially distributed information within the 3D space. Computational finite element (FE) analysis and electrochemical impedance spectroscopic (EIS) characterisation were used to determine the configurations' sensitivity field localisation. The 2T setup gives insight into the interfacial phenomena at both electrode surfaces and covers the central part of the 3D cell culture volume, while the four 3T modes provide focus on the dynamics at the corners of the 3D culture chamber. By combining a number of electrode configurations, complementary spatially distributed information on a large 3D cell culture can be obtained with maximised sensitivity in the entire 3D space. The experimental results show that cell proliferation can be monitored within the tested biomimetic environment, paving the way to further developments in bioimpedance tracking of 3D cell cultures and tissue engineering.

  19. Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells.

    Postovit, Lynne-Marie; Margaryan, Naira V; Seftor, Elisabeth A; Kirschmann, Dawn A; Lipavsky, Alina; Wheaton, William W; Abbott, Daniel E; Seftor, Richard E B; Hendrix, Mary J C

    2008-03-18

    Embryonic stem cells sustain a microenvironment that facilitates a balance of self-renewal and differentiation. Aggressive cancer cells, expressing a multipotent, embryonic cell-like phenotype, engage in a dynamic reciprocity with a microenvironment that promotes plasticity and tumorigenicity. However, the cancer-associated milieu lacks the appropriate regulatory mechanisms to maintain a normal cellular phenotype. Previous work from our laboratory reported that aggressive melanoma and breast carcinoma express the embryonic morphogen Nodal, which is essential for human embryonic stem cell (hESC) pluripotency. Based on the aberrant expression of this embryonic plasticity gene by tumor cells, this current study tested whether these cells could respond to regulatory cues controlling the Nodal signaling pathway, which might be sequestered within the microenvironment of hESCs, resulting in the suppression of the tumorigenic phenotype. Specifically, we discovered that metastatic tumor cells do not express the inhibitor to Nodal, Lefty, allowing them to overexpress this embryonic morphogen in an unregulated manner. However, exposure of the tumor cells to a hESC microenvironment (containing Lefty) leads to a dramatic down-regulation in their Nodal expression concomitant with a reduction in clonogenicity and tumorigenesis accompanied by an increase in apoptosis. Furthermore, this ability to suppress the tumorigenic phenotype is directly associated with the secretion of Lefty, exclusive to hESCs, because it is not detected in other stem cell types, normal cell types, or trophoblasts. The tumor-suppressive effects of the hESC microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored therapeutic modalities for cancer treatment.

  20. Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

    Yinzhi Lai

    Full Text Available Cells cultured in three dimensional (3D scaffolds as opposed to traditional two-dimensional (2D substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensionality biomarkers. With this goal in mind, we conducted a transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate. Up-regulation of cytokines as a group in 3D and neurospheres was observed. A group of 13 cytokines were commonly up-regulated in cells cultured in polystyrene scaffolds and neurospheres, suggesting potential for any or a combination from this list to serve as three-dimensionality biomarkers. These results are supportive of further cytokine identification and validation studies with cells from non-neural tissue.

  1. BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models.

    Cemal Cagatay Bilgin

    Full Text Available BioSig3D is a computational platform for high-content screening of three-dimensional (3D cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i morphogenesis of a panel of human mammary epithelial cell lines (HMEC, and (ii heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation.

  2. 3D printing – a key technology for tailored biomedical cell culture lab ware

    Schmieder Florian

    2016-09-01

    Full Text Available Today’s 3D printing technologies offer great possibilities for biomedical researchers to create their own specific laboratory equipment. With respect to the generation of ex vivo vascular perfusion systems this will enable new types of products that will embed complex 3D structures possibly coupled with cell loaded scaffolds closely reflecting the in-vivo environment. Moreover this could lead to microfluidic devices that should be available in small numbers of pieces at moderate prices. Here, we will present first results of such 3D printed cell culture systems made from plastics and show their use for scaffold based applications.

  3. Examination of 1D Solar Cell Model Limitations Using 3D SPICE Modeling: Preprint

    McMahon, W. E.; Olson, J. M.; Geisz, J. F.; Friedman, D. J.

    2012-06-01

    To examine the limitations of one-dimensional (1D) solar cell modeling, 3D SPICE-based modeling is used to examine in detail the validity of the 1D assumptions as a function of sheet resistance for a model cell. The internal voltages and current densities produced by this modeling give additional insight into the differences between the 1D and 3D models.

  4. 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs.

    Kolesky, David B; Truby, Ryan L; Gladman, A Sydney; Busbee, Travis A; Homan, Kimberly A; Lewis, Jennifer A

    2014-05-21

    A new bioprinting method is reported for fabricating 3D tissue constructs replete with vasculature, multiple types of cells, and extracellular matrix. These intricate, heterogeneous structures are created by precisely co-printing multiple materials, known as bioinks, in three dimensions. These 3D micro-engineered environments open new -avenues for drug screening and fundamental studies of wound healing, angiogenesis, and stem-cell niches.

  5. Effect of tumor cells and tumor microenvironment on NK-cell function.

    Vitale, Massimo; Cantoni, Claudia; Pietra, Gabriella; Mingari, Maria Cristina; Moretta, Lorenzo

    2014-06-01

    The ability of tumors to manage an immune-mediated attack has been recently included in the "next generation" of cancer hallmarks. In solid tumors, the microenvironment that is generated during the first steps of tumor development has a pivotal role in immune regulation. An intricate net of cross-interactions occurring between tumor components, stromal cells, and resident or recruited immune cells skews the possible acute inflammatory response toward an aberrant ineffective chronic inflammatory status that favors the evasion from the host's defenses. Natural killer (NK) cells have powerful cytotoxic activity, but their activity may be eluded by the tumor microenvironment. Immunosubversion, immunoediting or immunoselection of poorly immunogenic tumor cells and interference with tumor infiltration play a major role in evading NK-cell responses to tumors. Tumor cells, tumor-associated fibroblasts and tumor-induced aberrant immune cells (i.e. tolerogenic or suppressive macrophages, dendritic cells (DCs) and T cells) can interfere with NK-cell activation pathways or the complex receptor array that regulate NK-cell activation and antitumor activity. Thus, the definition of tumor microenvironment-related immunosuppressive factors, along with the identification of new classes of tissue-residing NK-like innate lymphoid cells, represent key issues to design effective NK-cell-based therapies of solid tumors.

  6. Bioimpedance monitoring of 3D cell culturing-Complementary electrode configurations for enhanced spatial sensitivity

    Canali, Chiara; Heiskanen, Arto; Muhammad, Haseena Bashir

    2015-01-01

    configurations. By switching between the different combinations of electrode couples, it was possible to generate a multiplexing-like approach, which allowed for collecting spatially distributed information within the 3D space. Computational finite element (FE) analysis and electrochemical impedance......A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of the entire process of three-dimensional (3D) cell culturing in a hydrogel scaffold. In this study, the dynamics involved in the whole process of 3D cell culturing, starting from polymerisation...... of a bare 3D gelatin scaffold, to human mesenchymal stem cell (MSC) encapsulation and proliferation, was monitored over time. The platform consists of a large rectangular culture chamber with four embedded vertical gold plate electrodes that were exploited in two- and three terminal (2T and 3T) measurement...

  7. Osteomimicry of mammary adenocarcinoma cells in vitro; increased expression of bone matrix proteins and proliferation within a 3D collagen environment.

    Rachel F Cox

    Full Text Available Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.

  8. Jawbone microenvironment promotes periodontium regeneration by regulating the function of periodontal ligament stem cells

    Zhu, Bin; Liu, Wenjia; Liu, Yihan; Zhao, Xicong; Zhang, Hao; Luo, Zhuojing; Jin, Yan

    2017-01-01

    During tooth development, the jawbone interacts with dental germ and provides the development microenvironment. Jawbone-derived mesenchymal stem cells (JBMSCs) maintain this microenvironment for root and periodontium development. However, the effect of the jawbone microenvironment on periodontium tissue regeneration is largely elusive. Our previous study showed that cell aggregates (CAs) of bone marrow mesenchymal stem cells promoted periodontium regeneration on the treated dentin scaffold. Here, we found that JBMSCs enhanced not only the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) but also their adhesion to titanium (Ti) material surface. Importantly, the compound CAs of PDLSCs and JBMSCs regenerated periodontal ligament-like fibers and mineralized matrix on the Ti scaffold surface, both in nude mice ectopic and minipig orthotopic transplantations. Our data revealed that an effective regenerative microenvironment, reconstructed by JBMSCs, promoted periodontium regeneration by regulating PDLSCs function on the Ti material. PMID:28053317

  9. Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy.

    Jiang, Huaidong; Song, Changyong; Chen, Chien-Chun; Xu, Rui; Raines, Kevin S; Fahimian, Benjamin P; Lu, Chien-Hung; Lee, Ting-Kuo; Nakashima, Akio; Urano, Jun; Ishikawa, Tetsuya; Tamanoi, Fuyuhiko; Miao, Jianwei

    2010-06-22

    Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.

  10. Label-free characterization of white blood cells by measuring 3D refractive index maps

    Yoon, Jonghee; Park, HyunJoo; Choi, Chulhee; Jang, Seongsoo; Park, YongKeun

    2015-01-01

    The characterization of white blood cells (WBCs) is crucial for blood analyses and disease diagnoses. However, current standard techniques rely on cell labeling, a process which imposes significant limitations. Here we present three-dimensional (3D) optical measurements and the label-free characterization of mouse WBCs using optical diffraction tomography. 3D refractive index (RI) tomograms of individual WBCs are constructed from multiple two-dimensional quantitative phase images of samples illuminated at various angles of incidence. Measurements of the 3D RI tomogram of WBCs enable the separation of heterogeneous populations of WBCs using quantitative morphological and biochemical information. Time-lapse tomographic measurements also provide the 3D trajectory of micrometer-sized beads ingested by WBCs. These results demonstrate that optical diffraction tomography can be a useful and versatile tool for the study of WBCs.

  11. Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels

    Lai, Janice H.; Kajiyama, Glen; Smith, Robert Lane; Maloney, William; Yang, Fan

    2013-12-01

    Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

  12. High sensitivity plasmonic biosensor based on nanoimprinted quasi 3D nanosquares for cell detection

    Zhu, Shuyan; Li, Hualin; Yang, Mengsu; Pang, Stella W.

    2016-07-01

    Quasi three-dimensional (3D) plasmonic nanostructures consisting of Au nanosquares on top of SU-8 nanopillars and Au nanoholes on the bottom were developed and fabricated using nanoimprint lithography with simultaneous thermal and UV exposure. These 3D plasmonic nanostructures were used to detect cell concentration of lung cancer A549 cells, retinal pigment epithelial (RPE) cells, and breast cancer MCF-7 cells. Nanoimprint technology has the advantage of producing high uniformity plasmonic nanostructures for such biosensors. Multiple resonance modes were observed in these quasi 3D plasmonic nanostructures. The hybrid coupling of localized surface plasmon resonances and Fabry-Perot cavity modes in the quasi 3D nanostructures resulted in high sensitivity of 496 nm/refractive index unit. The plasmonic resonance peak wavelength and sensitivity could be tuned by varying the Au thickness. Resonance peak shifts for different cells at the same concentration were distinct due to their different cell area and confluency. The cell concentration detection limit covered a large range of 5 × 102 to 1 × 107 cells ml-1 with these new plasmonic nanostructures. They also provide a large resonance peak shift of 51 nm for as little as 0.08 cells mm-2 of RPE cells for high sensitivity cell detection.

  13. Assembly of cell-laden hydrogel fiber into non-liquefied and liquefied 3D spiral constructs by perfusion-based layer-by-layer technique.

    Sher, Praveen; Oliveira, Sara M; Borges, João; Mano, João F

    2015-01-06

    In this work, three-dimensional (3D) self-sustaining, spiral-shaped constructs were produced through a combination of ionotropic gelation, to form cell-encapsulated alginate fibers, and a perfusion-based layer-by-layer (LbL) technique. Single fibers were assembled over cylindrical molds by reeling to form spiral shapes, both having different geometries and sizes. An uninterrupted nanometric multilayer coating produced by a perfusion-based LbL technique, using alginate and chitosan, generated stable 3D spiral-shaped macrostructures by gripping and affixing the threads together without using any crosslinking/binding agent. The chelation process altered the internal microenvironment of the 3D construct from the solid to the liquefied state while preserving the external geometry. L929 cell viability by MTS and dsDNA quantification favor liquefied 3D constructs more than non-liquefied ones. The proposed technique setup helps us to generate complex polyelectrolyte-based 3D constructs for tissue engineering applications and organ printing.

  14. 3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan.

    Bédouin, Y; Pellen Mussi, P; Tricot-Doleux, S; Chauvel-Lebret, D; Auroy, P; Ravalec, X; Oudadesse, H; Perez, F

    2015-01-01

    This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS2. The 46S6 and CH composite (46S6-CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6-CH-immersed media. After six days, 46S6-CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6-CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differentiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6-CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes' properties.

  15. Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds

    Chen, Chih-Hao [Department of Electrical Engineering, I-Shou University, Taiwan, ROC (China); Neurosurgery, Department of Surgery, Kaohsiung Veterans General Hospital, Taiwan, ROC (China); Department of Biomedical Engineering, I-Shou University, Taiwan, ROC (China); Kuo, Shyh Ming [Department of Biomedical Engineering, I-Shou University, Taiwan, ROC (China); Liu, Guei-Sheung [Centre for Eye Research Australia, University of Melbourne (Australia); Chen, Wan-Nan U. [Department of Biological Science and Technology, I-Shou University, Taiwan, ROC (China); Chuang, Chin-Wen [Department of Electrical Engineering, I-Shou University, Taiwan, ROC (China); Liu, Li-Feng, E-mail: liulf@isu.edu.tw [Department of Biological Science and Technology, I-Shou University, Taiwan, ROC (China)

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. Black-Right-Pointing-Pointer Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. Black-Right-Pointing-Pointer 3-D collagen culture of NCSCs shows an advance differentiation than 2-D culture. -- Abstract: Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 {mu}m porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

  16. Hypoxia Created Human Mesenchymal Stem Cell Sheet for Prevascularized 3D Tissue Construction.

    Zhang, Lijun; Xing, Qi; Qian, Zichen; Tahtinen, Mitchell; Zhang, Zhaoqiang; Shearier, Emily; Qi, Shaohai; Zhao, Feng

    2016-02-01

    3D tissue based on human mesenchymal stem cell (hMSC) sheets offers many interesting opportunities for regenerating multiple types of connective tissues. Prevascularizing hMSC sheets with endothelial cells (ECs) will improve 3D tissue performance by supporting cell survival and accelerating integration with host tissue. It is hypothesized that hypoxia cultured hMSC sheets can promote microvessel network formation and preserve stemness of hMSCs. This study investigates the vascularization of hMSC sheets under different oxygen tensions. It is found that the HN condition, in which hMSC sheets formed under physiological hypoxia (2% O2 ) and then cocultured with ECs under normoxia (20% O2 ), enables longer and more branched microvessel network formation. The observation is corroborated by higher levels of angiogenic factors in coculture medium. Additionally, the hypoxic hMSC sheet is more uniform and less defective, which facilitates fabrication of 3D prevascularized tissue construct by layering the prevascularized hMSC sheets and maturing in rotating wall vessel bioreactor. The hMSCs in the 3D construct still maintain multilineage differentiation ability, which indicates the possible application of the 3D construct for various connective tissues regeneration. These results demonstrate that hypoxia created hMSC sheets benefit the microvessel growth and it is feasible to construct 3D prevascularized tissue construct using the prevascularized hMSC sheets.

  17. 3D texture analysis in renal cell carcinoma tissue image grading.

    Kim, Tae-Yun; Cho, Nam-Hoon; Jeong, Goo-Bo; Bengtsson, Ewert; Choi, Heung-Kook

    2014-01-01

    One of the most significant processes in cancer cell and tissue image analysis is the efficient extraction of features for grading purposes. This research applied two types of three-dimensional texture analysis methods to the extraction of feature values from renal cell carcinoma tissue images, and then evaluated the validity of the methods statistically through grade classification. First, we used a confocal laser scanning microscope to obtain image slices of four grades of renal cell carcinoma, which were then reconstructed into 3D volumes. Next, we extracted quantitative values using a 3D gray level cooccurrence matrix (GLCM) and a 3D wavelet based on two types of basis functions. To evaluate their validity, we predefined 6 different statistical classifiers and applied these to the extracted feature sets. In the grade classification results, 3D Haar wavelet texture features combined with principal component analysis showed the best discrimination results. Classification using 3D wavelet texture features was significantly better than 3D GLCM, suggesting that the former has potential for use in a computer-based grading system.

  18. 3D Texture Analysis in Renal Cell Carcinoma Tissue Image Grading

    Tae-Yun Kim

    2014-01-01

    Full Text Available One of the most significant processes in cancer cell and tissue image analysis is the efficient extraction of features for grading purposes. This research applied two types of three-dimensional texture analysis methods to the extraction of feature values from renal cell carcinoma tissue images, and then evaluated the validity of the methods statistically through grade classification. First, we used a confocal laser scanning microscope to obtain image slices of four grades of renal cell carcinoma, which were then reconstructed into 3D volumes. Next, we extracted quantitative values using a 3D gray level cooccurrence matrix (GLCM and a 3D wavelet based on two types of basis functions. To evaluate their validity, we predefined 6 different statistical classifiers and applied these to the extracted feature sets. In the grade classification results, 3D Haar wavelet texture features combined with principal component analysis showed the best discrimination results. Classification using 3D wavelet texture features was significantly better than 3D GLCM, suggesting that the former has potential for use in a computer-based grading system.

  19. Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

    Lewis, Katherine Jean Reeder

    The alveolar epithelium consists of two cell phenotypes, elongated alveolar type I cells (AT1) and rounded alveolar type II cells (ATII), and exists in a complex three-dimensional environment as a polarized cell layer attached to a thin basement membrane and enclosing a roughly spherical lumen. Closely surrounding the alveolar cysts are capillary endothelial cells as well as interstitial pulmonary fibroblasts. Many factors are thought to influence alveolar epithelial cell differentiation during lung development and wound repair, including physical and biochemical signals from the extracellular matrix (ECM), and paracrine signals from the surrounding mesenchyme. In particular, disrupted signaling between the alveolar epithelium and local fibroblasts has been implicated in the progression of several pulmonary diseases. However, given the complexity of alveolar tissue architecture and the multitude of signaling pathways involved, designing appropriate experimental platforms for this biological system has been difficult. In order to isolate key factors regulating cellular behavior, the researcher ideally should have control over biophysical properties of the ECM, as well as the ability to organize multiple cell types within the scaffold. This thesis aimed to develop a 3D synthetic hydrogel platform to control alveolar epithelial cyst formation, which could then be used to explore how extracellular cues influence cell behavior in a tissue-relevant cellular arrangement. To accomplish this, a poly(ethylene glycol) (PEG) hydrogel network containing enzymatically-degradable crosslinks and bioadhesive pendant peptides was employed as a base material for encapsulating primary alveolar epithelial cells. First, an array of microwells of various cross-sectional shapes was photopatterned into a PEG gel containing photo-labile crosslinks, and primary ATII cells were seeded into the wells to examine the role of geometric confinement on differentiation and multicellular arrangement

  20. Importance of the stem cell microenvironment forophthalmological cell-based therapy

    Peng-Xia Wan; Bo-Wen Wang; Zhi-Chong Wang

    2015-01-01

    Cell therapy is a promising treatment for diseasesthat are caused by cell degeneration or death. Thecells for clinical transplantation are usually obtainedby culturing healthy allogeneic or exogenous tissue invitro . However, for diseases of the eye, obtaining theadequate number of cells for clinical transplantationis difficult due to the small size of tissue donors andthe frequent needs of long-term amplification ofcells in vitro , which results in low cell viability aftertransplantation. In addition, the transplanted cells oftendevelop fibrosis or degrade and have very low survival.Embryonic stem cells (ESCs) and induced pluripotentstem cells (iPS) are also promising candidates for celltherapy. Unfortunately, the differentiation of ESCs canbring immune rejection, tumorigenicity and undesireddifferentiated cells, limiting its clinical application.Although iPS cells can avoid the risk of immune rejectioncaused by ES cell differentiation post-transplantation,the low conversion rate, the risk of tumor formationand the potentially unpredictable biological changesthat could occur through genetic manipulation hinderits clinical application. Thus, the desired clinical effectof cell therapy is impaired by these factors. Recentresearch findings recognize that the reason for lowsurvival of the implanted cells not only depends on theseeded cells, but also on the cell microenvironment,which determines the cell survival, proliferation andeven reverse differentiation. When used for cell therapy,the transplanted cells need a specific three-dimensionalstructure to anchor and specific extra cellular matrixcomponents in addition to relevant cytokine signalingto transfer the required information to support theirgrowth. These structures present in the matrix inwhich the stem cells reside are known as the stem cellmicroenvironment. The microenvironment interactionwith the stem cells provides the necessary homeostasisfor cell maintenance and growth. A large number ofstudies

  1. Differential effects of MAPK pathway inhibitors on migration and invasiveness of BRAF(V600E) mutant thyroid cancer cells in 2D and 3D culture.

    Ingeson-Carlsson, Camilla; Martinez-Monleon, Angela; Nilsson, Mikael

    2015-11-01

    Tumor microenvironment influences targeted drug therapy. In this study we compared drug responses to RAF and MEK inhibitors on tumor cell migration in 2D and 3D culture of BRAF(V600E) mutant cell lines derived from human papillary (BCPAP) and anaplastic (SW1736) thyroid carcinomas. Scratch wounding was compared to a double-layered collagen gel model developed for analysis of directed tumor cell invasion during prolonged culture. In BCPAP both PLX4720 and U0126 inhibited growth and migration in 2D and decreased tumor cell survival in 3D. In SW1736 drugs had no effect on migration in 2D but decreased invasion in 3D, however this related to reduced growth. Dual inhibition of BRAF(V600E) and MEK reduced but did not prevent SW1736 invasion although rebound phosphorylation of ERK in response to PLX4720 was blocked by U0126. These findings indicate that anti-tumor drug effects in vitro differ depending on culture conditions (2D vs. 3D) and that the invasive features of anaplastic thyroid cancer depend on non-MEK mechanism(s).

  2. 3D Cell Printing of Functional Skeletal Muscle Constructs Using Skeletal Muscle-Derived Bioink.

    Choi, Yeong-Jin; Kim, Taek Gyoung; Jeong, Jonghyeon; Yi, Hee-Gyeong; Park, Ji Won; Hwang, Woonbong; Cho, Dong-Woo

    2016-10-01

    Engineered skeletal muscle tissues that mimic the structure and function of native muscle have been considered as an alternative strategy for the treatment of various muscular diseases and injuries. Here, it is demonstrated that 3D cell-printing of decellularized skeletal muscle extracellular matrix (mdECM)-based bioink facilitates the fabrication of functional skeletal muscle constructs. The cellular alignment and the shape of the tissue constructs are controlled by 3D cell-printing technology. mdECM bioink provides the 3D cell-printed muscle constructs with a myogenic environment that supports high viability and contractility as well as myotube formation, differentiation, and maturation. More interestingly, the preservation of agrin is confirmed in the mdECM, and significant increases in the formation of acetylcholine receptor clusters are exhibited in the 3D cell-printed muscle constructs. In conclusion, mdECM bioink and 3D cell-printing technology facilitate the mimicking of both the structural and functional properties of native muscle and hold great promise for producing clinically relevant engineered muscle for the treatment of muscular injuries.

  3. Quantification by SIFT-MS of acetaldehyde released by lung cells in a 3D model.

    Rutter, Abigail V; Chippendale, Thomas W E; Yang, Ying; Španěl, Patrik; Smith, David; Sulé-Suso, Josep

    2013-01-07

    Our previous studies have shown that both lung cancer cells and non-malignant lung cells release acetaldehyde in vitro. However, data from other laboratories have produced conflicting results. Furthermore, all these studies have been carried out in 2D models which are less physiological cell growth systems when compared to 3D models. Therefore, we have carried out further work on the release of acetaldehyde by lung cells in 3D collagen hydrogels. Lung cancer cells CALU-1 and non-malignant lung cells NL20 were seeded in these hydrogels at different cell concentrations and the release of acetaldehyde was measured with the Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) technique. The data obtained showed that the amount of acetaldehyde released by both cell types grown in a 3D model is higher when compared to that of the same cells grown in 2D models. More importantly, acetaldehyde from the headspace of lung cancer cells could be measured even at a low cell concentration (10(5) cells per hydrogel). The differential of acetaldehyde release could be, depending on the cell concentration, more than 3 fold higher for cancer cells when compared to non-malignant lung cells. This pilot study is the first to study acetaldehyde emission from albeit only two cell types cultured in 3D scaffolds. Clearly, from such limited data the behaviour of other cell types and of tumour cells in vivo cannot be predicted with confidence. Nevertheless, this work represents another step in the search for volatile biomarkers of tumour cells, the ultimate goal of which is to exploit volatile compounds in exhaled breath and other biological fluids as biomarkers of tumours in vivo.

  4. 3D Nanochannel Array Platform for High-throughput Cell Manipulation and Nano-electroporation

    Chang, Lingqian

    Electroporation is one of the most common non-viral methods for gene delivery. Recent progress in gene therapy has offered special opportunities to electroporation for in vitro and in vivo applications. However, conventional bulk electroporation (BEP) inevitably causes serious cell damage and stochastic transfection between cells. Microfluidic electroporation (MEP) has been claimed to provide benign single cell transfection for the last decade. Nevertheless, the intracellular transport in both MEP and BEP systems is highly diffusion-dominant, which prevents precise dose control and high uniformity. In this Ph.D. research, we developed a 3D nanochannel-electroporation (3D NEP) platform for mass cell transfection. A silicon-based nanochannel array (3D NEP) chip was designed and fabricated for cell manipulation and electroporation. The chip, designed as Z-directional microchannel - nanochannel array, was fabricated by clean room techniques including projection photolithography and deep reactive-ion etching (DRIE). The fabricated 3D NEP chip is capable of handling 40,000 cells per 1 cm2, up to 1 million per wafer (100 mm diameter). High-throughput cell manipulation technologies were investigated for precise alignment of individual cells to the nanochannel array, a key step for NEP to achieve dose control. We developed three techniques for cell trapping in this work. (1) Magnetic tweezers (MTs) were integrated on the chip to remotely control cells under a programmed magnetic field. (2) A positive dielectrophoresis (pDEP) power system was built as an alternative to trap cells onto the nanochannel array using DEP force. (3) A novel yet simple 'dipping-trap' method was used to rapidly trap cells onto a nanochannel array, aligned by a micro-cap array pattern on the 3D NEP chip, which eventually offered 70 - 90 % trapping efficiency and 90 % specificity. 3D NEP platforms were assembled for cell transfection based on the Si-based nanochannel array chip and cell manipulation

  5. 3D tumor models: history, advances and future perspectives.

    Benien, Parul; Swami, Archana

    2014-05-01

    Evaluation of cancer therapeutics by utilizing 3D tumor models, before clinical studies, could be more advantageous than conventional 2D tumor models (monolayer cultures). The 3D systems mimic the tumor microenvironment more closely than 2D systems. The following review discusses the various 3D tumor models present today with the advantages and limitations of each. 3D tumor models replicate the elements of a tumor microenvironment such as hypoxia, necrosis, angiogenesis and cell adhesion. The review introduces application of techniques such as microfluidics, imaging and tissue engineering to improve the 3D tumor models. Despite their tremendous potential to better screen chemotherapeutics, 3D tumor models still have a long way to go before they are used commonly as in vitro tumor models in pharmaceutical industrial research.

  6. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    Machado, C B [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Ventura, J M G [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Lemos, A F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Ferreira, J M F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Leite, M F [Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Goes, A M [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil)

    2007-06-01

    A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

  7. 3D-printed concentrator arrays for external light trapping on thin film solar cells

    van Dijk, Lourens; Marcus, E. A. Pepijn; Oostra, A. Jolt; Schropp, Ruud E. I.; Di Vece, Marcel

    2015-01-01

    After our recent demonstration of a 3D-printed external light trap on a small solar cell, we now consider its potential for large solar panels. An external light trap consists of a parabolic concentrator and a spacer that redirects the photons that are reflected by the solar cell back towards the so

  8. Cell volume and geometric parameters determination in living cells using confocal microscopy and 3D reconstruction

    sprotocols

    2015-01-01

    Authors: David Hevia, Aida Rodriguez-Garcia, Marta Alonso-Gervós, Isabel Quirós-González, Henar M Cimadevilla, Carmen Gómez-Cordovés, Rosa M Sainz & Juan C Mayo ### Abstract The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO...

  9. Comparison of the transcriptomic profile of hepatic human induced pluripotent stem like cells cultured in plates and in a 3D microscale dynamic environment.

    Leclerc, Eric; Kimura, Keiichi; Shinohara, Marie; Danoy, Mathieu; Le Gall, Morgane; Kido, Taketomo; Miyajima, Atsushi; Fujii, Teruo; Sakai, Yasuyuki

    2017-01-01

    We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore, the results of the transcriptomic profile, coupled with immunostaining, and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells, hepatocytes like cells, and endothelial like cells. However, the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless, the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented.

  10. 3D reconstitution of the patterned neural tube from embryonic stem cells.

    Meinhardt, Andrea; Eberle, Dominic; Tazaki, Akira; Ranga, Adrian; Niesche, Marco; Wilsch-Bräuninger, Michaela; Stec, Agnieszka; Schackert, Gabriele; Lutolf, Matthias; Tanaka, Elly M

    2014-12-09

    Inducing organogenesis in 3D culture is an important aspect of stem cell research. Anterior neural structures have been produced from large embryonic stem cell (ESC) aggregates, but the steps involved in patterning such complex structures have been ill defined, as embryoid bodies typically contained many cell types. Here we show that single mouse ESCs directly embedded in Matrigel or defined synthetic matrices under neural induction conditions can clonally form neuroepithelial cysts containing a single lumen in 3D. Untreated cysts were uniformly dorsal and could be ventralized to floor plate (FP). Retinoic acid posteriorized cysts to cervical levels and induced localize FP formation yielding full patterning along the dorsal/ventral (DV) axis. Correct spatial organization of motor neurons, interneurons, and dorsal interneurons along the DV axis was observed. This system serves as a valuable tool for studying morphogen action in 3D and as a source of patterned spinal cord tissue.

  11. CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

    Hodneland, Erlend; Kögel, Tanja; Frei, Dominik Michael; Gerdes, Hans-Hermann; Lundervold, Arvid

    2013-08-09

    : The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening.

  12. An Innovative Cell Microincubator for Drug Discovery Based on 3D Silicon Structures

    Francesca Aredia

    2016-01-01

    Full Text Available We recently employed three-dimensional (3D silicon microstructures (SMSs consisting in arrays of 3 μm-thick silicon walls separated by 50 μm-deep, 5 μm-wide gaps, as microincubators for monitoring the biomechanical properties of tumor cells. They were here applied to investigate the in vitro behavior of HT1080 human fibrosarcoma cells driven to apoptosis by the chemotherapeutic drug Bleomycin. Our results, obtained by fluorescence microscopy, demonstrated that HT1080 cells exhibited a great ability to colonize the narrow gaps. Remarkably, HT1080 cells grown on 3D-SMS, when treated with the DNA damaging agent Bleomycin under conditions leading to apoptosis, tended to shrink, reducing their volume and mimicking the normal behavior of apoptotic cells, and were prone to leave the gaps. Finally, we performed label-free detection of cells adherent to the vertical silicon wall, inside the gap of 3D-SMS, by exploiting optical low coherence reflectometry using infrared, low power radiation. This kind of approach may become a new tool for increasing automation in the drug discovery area. Our results open new perspectives in view of future applications of the 3D-SMS as the core element of a lab-on-a-chip suitable for screening the effect of new molecules potentially able to kill tumor cells.

  13. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

  14. AI-05IMPACT OF GBM MICROENVIRONMENT ON EXPRESSION PROFILE OF BONE MARROW DERIVED PROGENITOR CELLS

    Burrell, Kelly; Singh, Sanjay; Agnihotri, Sameer; Hill, Richard; Aldape, Kenneth; Zadeh, Gelareh

    2014-01-01

    We have recently shown that bone marrow derived cells (BMDC) provide a distinct tumor region dependent contribution to glioblastoma multiforme (GBM) neovascularization. The influence of GBM microenvironment on differentiation and modulation of expression factors by BMDC however remains unknown. In this study we establish the differential expression profile of BMDC as a consequence of recruitment and interaction with the GBM microenvironment and in response to radiation (RTx) and anti-angiogen...

  15. Endostatin induces proliferation of oral carcinoma cells but its effect on invasion is modified by the tumor microenvironment

    Alahuhta, Ilkka [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland); Aikio, Mari [Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu (Finland); Oulu Center for Cell-Matrix Research, University of Oulu (Finland); Väyrynen, Otto; Nurmenniemi, Sini [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland); Suojanen, Juho [Department of Oral and Maxillo-facial Diseases, University of Helsinki, Helsinki University Central Hospital (Finland); Teppo, Susanna [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland); Pihlajaniemi, Taina; Heljasvaara, Ritva [Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu (Finland); Oulu Center for Cell-Matrix Research, University of Oulu (Finland); Salo, Tuula [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland); Department of Oral and Maxillo-facial Diseases, University of Helsinki, Helsinki University Central Hospital (Finland); Department of Oral Diagnosis, School of Dentistry, State University of Campinas, Piracicaba, Sao Paolo (Brazil); Nyberg, Pia, E-mail: pia.nyberg@oulu.fi [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland)

    2015-08-01

    The turnover of extracellular matrix liberates various cryptic molecules with novel biological activities. Endostatin is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of collagen XVIII. Although there are a large number of studies on its anti-tumor effects, the molecular mechanisms are not yet completely understood, and the reasons why endostatin has not been successful in clinical trials are unclear. Research has mostly focused on its anti-angiogenic effect in tumors. Here, we aimed to elucidate how endostatin affects the behavior of aggressive tongue HSC-3 carcinoma cells that were transfected to overproduce endostatin. Endostatin inhibited the invasion of HSC-3 cells in a 3D collagen–fibroblast model. However, it had no effect on invasion in a human myoma organotypic model, which lacks vital fibroblasts. Recombinant endostatin was able to reduce the Transwell migration of normal fibroblasts, but had no effect on carcinoma associated fibroblasts. Surprisingly, endostatin increased the proliferation and decreased the apoptosis of cancer cells in organotypic models. Also subcutaneous tumors overproducing endostatin grew bigger, but showed less local invasion in nude mice xenografts. We conclude that endostatin affects directly to HSC-3 cells increasing their proliferation, but its net effect on cancer invasion seem to depend on the cellular composition and interactions of tumor microenvironment. - Highlights: • Endostatin affects not only angiogenesis, but also carcinoma cells and fibroblasts. • Endostatin increased carcinoma cell proliferation, but decreased 3D invasion. • The invasion inhibitory effect was sensitive to the microenvironment composition. • Fibroblasts may be a factor regulating the fluctuating roles of endostatin.

  16. Modeling spatial distribution of oxygen in 3d culture of islet beta-cells.

    McReynolds, John; Wen, Yu; Li, Xiaofei; Guan, Jianjun; Jin, Sha

    2017-01-01

    Three-dimensional (3D) scaffold culture of pancreatic β-cell has been proven to be able to better mimic physiological conditions in the body. However, one critical issue with culturing pancreatic β-cells is that β-cells consume large amounts of oxygen, and hence insufficient oxygen supply in the culture leads to loss of β-cell mass and functions. This becomes more significant when cells are cultured in a 3D scaffold. In this study, in order to understand the effect of oxygen tension inside a cell-laden collagen culture on β-cell proliferation, a culture model with encapsulation of an oxygen-generator was established. The oxygen-generator was made by embedding hydrogen peroxide into nontoxic polydimethylsiloxane to avoid the toxicity of a chemical reaction in the β-cell culture. To examine the effectiveness of the oxygenation enabled 3D culture, the spatial-temporal distribution of oxygen tension inside a scaffold was evaluated by a mathematical modeling approach. Our simulation results indicated that an oxygenation-aided 3D culture would augment the oxygen supply required for the β-cells. Furthermore, we identified that cell seeding density and the capacity of the oxygenator are two critical parameters in the optimization of the culture. Notably, cell-laden scaffold cultures with an in situ oxygen supply significantly improved the β-cells' biological function. These β-cells possess high insulin secretion capacity. The results obtained in this work would provide valuable information for optimizing and encouraging functional β-cell cultures. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:221-228, 2017.

  17. Bioengineered 3D Glial Cell Culture Systems and Applications for Neurodegeneration and Neuroinflammation.

    Watson, P Marc D; Kavanagh, Edel; Allenby, Gary; Vassey, Matthew

    2017-02-01

    Neurodegeneration and neuroinflammation are key features in a range of chronic central nervous system (CNS) diseases such as Alzheimer's and Parkinson's disease, as well as acute conditions like stroke and traumatic brain injury, for which there remains significant unmet clinical need. It is now well recognized that current cell culture methodologies are limited in their ability to recapitulate the cellular environment that is present in vivo, and there is a growing body of evidence to show that three-dimensional (3D) culture systems represent a more physiologically accurate model than traditional two-dimensional (2D) cultures. Given the complexity of the environment from which cells originate, and their various cell-cell and cell-matrix interactions, it is important to develop models that can be controlled and reproducible for drug discovery. 3D cell models have now been developed for almost all CNS cell types, including neurons, astrocytes, microglia, and oligodendrocyte cells. This review will highlight a number of current and emerging techniques for the culture of astrocytes and microglia, glial cell types with a critical role in neurodegenerative and neuroinflammatory conditions. We describe recent advances in glial cell culture using electrospun polymers and hydrogel macromolecules, and highlight how these novel culture environments influence astrocyte and microglial phenotypes in vitro, as compared to traditional 2D systems. These models will be explored to illuminate current trends in the techniques used to create 3D environments for application in research and drug discovery focused on astrocytes and microglial cells.

  18. Study of a Microfluidic Chip Integrating Single Cell Trap and 3D Stable Rotation Manipulation

    Liang Huang

    2016-08-01

    Full Text Available Single cell manipulation technology has been widely applied in biological fields, such as cell injection/enucleation, cell physiological measurement, and cell imaging. Recently, a biochip platform with a novel configuration of electrodes for cell 3D rotation has been successfully developed by generating rotating electric fields. However, the rotation platform still has two major shortcomings that need to be improved. The primary problem is that there is no on-chip module to facilitate the placement of a single cell into the rotation chamber, which causes very low efficiency in experiment to manually pipette single 10-micron-scale cells into rotation position. Secondly, the cell in the chamber may suffer from unstable rotation, which includes gravity-induced sinking down to the chamber bottom or electric-force-induced on-plane movement. To solve the two problems, in this paper we propose a new microfluidic chip with manipulation capabilities of single cell trap and single cell 3D stable rotation, both on one chip. The new microfluidic chip consists of two parts. The top capture part is based on the least flow resistance principle and is used to capture a single cell and to transport it to the rotation chamber. The bottom rotation part is based on dielectrophoresis (DEP and is used to 3D rotate the single cell in the rotation chamber with enhanced stability. The two parts are aligned and bonded together to form closed channels for microfluidic handling. Using COMSOL simulation and preliminary experiments, we have verified, in principle, the concept of on-chip single cell traps and 3D stable rotation, and identified key parameters for chip structures, microfluidic handling, and electrode configurations. The work has laid a solid foundation for on-going chip fabrication and experiment validation.

  19. 3D Cell Culture in a Self-Assembled Nanofiber Environment

    Gubbe, John D.; Brekke, John H.

    2016-01-01

    The development and utilization of three-dimensional cell culture platforms has been gaining more traction. Three-dimensional culture platforms are capable of mimicking in vivo microenvironments, which provide greater physiological relevance in comparison to conventional two-dimensional cultures. The majority of three-dimensional culture platforms are challenged by the lack of cell attachment, long polymerization times, and inclusion of undefined xenobiotics, and cytotoxic cross-linkers. In this study, we review the use of a highly defined material composed of naturally occurring compounds, hyaluronic acid and chitosan, known as Cell-Mate3DTM. Moreover, we provide an original measurement of Young’s modulus using a uniaxial unconfined compression method to elucidate the difference in microenvironment rigidity for acellular and cellular conditions. When hydrated into a tissue-like hybrid hydrocolloid/hydrogel, Cell-Mate3DTM is a highly versatile three-dimensional culture platform that enables downstream applications such as flow cytometry, immunostaining, histological staining, and functional studies to be applied with relative ease. PMID:27632425

  20. New data-driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume.

    Roselli, L; Paparella, F; Stanca, E; Basset, A

    2015-06-01

    Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex-shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex-shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data-driven formula-based approach to the calculation of biovolume of this complex-shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.

  1. Primed 3D injectable microniches enabling low-dosage cell therapy for critical limb ischemia.

    Li, Yaqian; Liu, Wei; Liu, Fei; Zeng, Yang; Zuo, Simin; Feng, Siyu; Qi, Chunxiao; Wang, Bingjie; Yan, Xiaojun; Khademhosseini, Ali; Bai, Jing; Du, Yanan

    2014-09-16

    The promise of cell therapy for repair and restoration of damaged tissues or organs relies on administration of large dose of cells whose healing benefits are still limited and sometimes irreproducible due to uncontrollable cell loss and death at lesion sites. Using a large amount of therapeutic cells increases the costs for cell processing and the risks of side effects. Optimal cell delivery strategies are therefore in urgent need to enhance the specificity, efficacy, and reproducibility of cell therapy leading to minimized cell dosage and side effects. Here, we addressed this unmet need by developing injectable 3D microscale cellular niches (microniches) based on biodegradable gelatin microcryogels (GMs). The microniches are constituted by in vitro priming human adipose-derived mesenchymal stem cells (hMSCs) seeded within GMs resulting in tissue-like ensembles with enriched extracellular matrices and enhanced cell-cell interactions. The primed 3D microniches facilitated cell protection from mechanical insults during injection and in vivo cell retention, survival, and ultimate therapeutic functions in treatment of critical limb ischemia (CLI) in mouse models compared with free cell-based therapy. In particular, 3D microniche-based therapy with 10(5) hMSCs realized better ischemic limb salvage than treatment with 10(6) free-injected hMSCs, the minimum dosage with therapeutic effects for treating CLI in literature. To the best of our knowledge, this is the first convincing demonstration of injectable and primed cell delivery strategy realizing superior therapeutic efficacy for treating CLI with the lowest cell dosage in mouse models. This study offers a widely applicable cell delivery platform technology to boost the healing power of cell regenerative therapy.

  2. Microrheology and ROCK signaling of human endothelial cells embedded in a 3D matrix.

    Panorchan, Porntula; Lee, Jerry S H; Kole, Thomas P; Tseng, Yiider; Wirtz, Denis

    2006-11-01

    Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

  3. Fabrication of 3D cell-laden hydrogel microstructures through photo-mold patterning.

    Occhetta, P; Sadr, N; Piraino, F; Redaelli, A; Moretti, M; Rasponi, M

    2013-09-01

    Native tissues are characterized by spatially organized three-dimensional (3D) microscaled units which functionally define cells-cells and cells-extracellular matrix interactions. The ability to engineer biomimetic constructs mimicking these 3D microarchitectures is subject to the control over cell distribution and organization. In the present study we introduce a novel protocol to generate 3D cell laden hydrogel micropatterns with defined size and shape. The method, named photo-mold patterning (PMP), combines hydrogel micromolding within polydimethylsiloxane (PDMS) stamps and photopolymerization through a recently introduced biocompatible ultraviolet (UVA) activated photoinitiator (VA-086). Exploiting PDMS micromolds as geometrical constraints for two methacrylated prepolymers (polyethylene glycol diacrylate and gelatin methacrylate), micrometrically resolved structures were obtained within a 3 min exposure to a low cost and commercially available UVA LED. The PMP was validated both on a continuous cell line (human umbilical vein endothelial cells expressing green fluorescent protein, HUVEC GFP) and on primary human bone marrow stromal cells (BMSCs). HUVEC GFP and BMSCs were exposed to 1.5% w/v VA-086 and UVA light (1 W, 385 nm, distance from sample = 5 cm). Photocrosslinking conditions applied during the PMP did not negatively affect cells viability or specific metabolic activity. Quantitative analyses demonstrated the potentiality of PMP to uniformly embed viable cells within 3D microgels, creating biocompatible and favorable environments for cell proliferation and spreading during a seven days' culture. PMP can thus be considered as a promising and cost effective tool for designing spatially accurate in vitro models and, in perspective, functional constructs.

  4. Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods.

    Gildea, John J; McGrath, Helen E; Van Sciver, Robert E; Wang, Dora Bigler; Felder, Robin A

    2013-01-01

    The kidney is a highly heterogeneous organ that is responsible for fluid and electrolyte balance. Much interest is focused on determining the function of specific renal epithelial cells in humans, which can only be accomplished through the isolation and growth of nephron segment-specific epithelial cells. However, human renal epithelial cells are notoriously difficult to maintain in culture. This chapter describes the isolation, growth, immortalization, and characterization of the human renal proximal tubule cell. In addition, we describe new paradigms in 3D cell culture which allow the cells to maintain more in vivo-like morphology and function.

  5. In Vivo Chondrogenesis in 3D Bioprinted Human Cell-laden Hydrogel Constructs

    Möller, Thomas; Hägg, Daniel; Brantsing, Camilla; Rotter, Nicole; Apelgren, Peter; Lindahl, Anders; Kölby, Lars; Gatenholm, Paul

    2017-01-01

    Background: The three-dimensional (3D) bioprinting technology allows creation of 3D constructs in a layer-by-layer fashion utilizing biologically relevant materials such as biopolymers and cells. The aim of this study is to investigate the use of 3D bioprinting in a clinically relevant setting to evaluate the potential of this technique for in vivo chondrogenesis. Methods: Thirty-six nude mice (Balb-C, female) received a 5- × 5- × 1-mm piece of bioprinted cell-laden nanofibrillated cellulose/alginate construct in a subcutaneous pocket. Four groups of printed constructs were used: (1) human (male) nasal chondrocytes (hNCs), (2) human (female) bone marrow–derived mesenchymal stem cells (hBMSCs), (3) coculture of hNCs and hBMSCs in a 20/80 ratio, and (4) Cell-free scaffolds (blank). After 14, 30, and 60 days, the scaffolds were harvested for histological, immunohistochemical, and mechanical analysis. Results: The constructs had good mechanical properties and keep their structural integrity after 60 days of implantation. For both the hNC constructs and the cocultured constructs, a gradual increase of glycosaminoglycan production and hNC proliferation was observed. However, the cocultured group showed a more pronounced cell proliferation and enhanced deposition of human collagen II demonstrated by immunohistochemical analysis. Conclusions: In vivo chondrogenesis in a 3D bioprinted human cell-laden hydrogel construct has been demonstrated. The trophic role of the hBMSCs in stimulating hNC proliferation and matrix deposition in the coculture group suggests the potential of 3D bioprinting of human cartilage for future application in reconstructive surgery. PMID:28280669

  6. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models

    Ricci, C.; Mota, C.M.; Moscato, S.; Alessandro, D' D.; Ugel, S.; Sartoris, S.; Bronte, V.; Boggi, U.; Campani, D.; Funel, N.; Moroni, L.; Danti, S.

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol

  7. Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold.

    Ken Ye

    Full Text Available Infrapatellar fat pad adipose stem cells (IPFP-ASCs have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a 'cap' on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

  8. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

    Lee, Wonjae; Park, Jon

    2016-07-01

    Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues.

  9. Electrolytic valving isolation of cell co-culture microenvironment with controlled cell pairing ratios.

    Chen, Yu-Chih; Ingram, Patrick; Yoon, Euisik

    2014-12-21

    Cancer-stromal interaction is a critical process in tumorigenesis. Conventional dish-based co-culture assays simply mix two cell types in the same dish; thus, they are deficient in controlling cell locations and precisely tracking single cell behavior from heterogeneous cell populations. Microfluidic technology can provide a good spatial-temporal control of microenvironments, but the control has been typically realized by using external pumps, making long-term cultures cumbersome and bulky. In this work, we have presented a cell-cell interaction microfluidic platform that can accurately control the co-culture microenvironment by using a novel electrolytic cell isolation scheme without using any valves or pneumatic pumps. The proposed microfluidic platform can also precisely control the number of interacting cells and pairing ratios to emulate cancer niches. More than 80% of the chambers captured the desired number of cells. The duration of cell isolation can be adjusted by electrolytic bubble generation and removal. We have verified that the electrolytic process has a negligible effect on cell viability and proliferation in our platform. To the best of our knowledge, this work is the first attempt to incorporate electrolytic bubble generation as a cell isolation method in microfluidics. For proof of feasibility, we have performed cell-cell interaction assays between prostate cancer (PC3) cells and myoblast (C2C12) cells. The preliminary results demonstrated the potential of using electrolysis for micro-environmental control during cell culture. Also, the ratio controlled cell-cell interaction assays were successfully performed which showed that the cell pairing ratios of PC3 to C2C12 affected the proliferation rate of myoblast cells due to increased secretion of growth factors from prostate cancer cells.

  10. 3D culture increases pluripotent gene expression in mesenchymal stem cells through relaxation of cytoskeleton tension.

    Zhou, Ying; Chen, Haiyan; Li, Hong; Wu, Yaojiong

    2017-03-09

    Three-dimensional (3D) culture has been shown to improve pluripotent gene expression in mesenchymal stem cells (MSCs), but the underlining mechanisms were poorly understood. Here, we found that the relaxation of cytoskeleton tension of MSCs in 3D culture was critically associated with the expressional up-regulation of Nanog. Cultured in spheroids, MSCs showed decreased integrin-based cell-matrix adhesion but increased cadherin-based cell-cell interaction. Different from that in 2D culture, where MSCs exhibited branched and multiple-directed F-actin stress bundles at the cell edge and strengthened stress fibres transversing the cell body, MSCs cultured in spheroids showed compact cell body, relaxed cytoskeleton tension with very thin cortical actin filament outlining the cell, and increased expression of Nanog along with reduced levels of Suv39h1 (H3K9 methyltransferase) and H3K9me3. Notably, pharmaceutical inhibition of actin polymerization with cytochalasin D or silencing Suv39h1 expression with siRNA in 2D-cultured MSCs elevated the expression of Nanog via H3K9 demethylation. Thus, our data suggest that 3D culture increases the expression of Nanog through the relaxation of actin cytoskeleton, which mediates reduced Suv39h1 and H3K9me3 levels.

  11. Rapid Fabrication of Cell-Laden Alginate Hydrogel 3D Structures by Micro Dip-Coating

    Ghanizadeh Tabriz, Atabak; Mills, Christopher G.; Mullins, John J.; Davies, Jamie A.; Shu, Wenmiao

    2017-01-01

    Development of a simple, straightforward 3D fabrication method to culture cells in 3D, without relying on any complex fabrication methods, remains a challenge. In this paper, we describe a new technique that allows fabrication of scalable 3D cell-laden hydrogel structures easily, without complex machinery: the technique can be done using only apparatus already available in a typical cell biology laboratory. The fabrication method involves micro dip-coating of cell-laden hydrogels covering the surface of a metal bar, into the cross-linking reagents calcium chloride or barium chloride to form hollow tubular structures. This method can be used to form single layers with thickness ranging from 126 to 220 µm or multilayered tubular structures. This fabrication method uses alginate hydrogel as the primary biomaterial and a secondary biomaterial can be added depending on the desired application. We demonstrate the feasibility of this method, with survival rate over 75% immediately after fabrication and normal responsiveness of cells within these tubular structures using mouse dermal embryonic fibroblast cells and human embryonic kidney 293 cells containing a tetracycline-responsive, red fluorescent protein (tHEK cells). PMID:28286747

  12. Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

    Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

    2011-01-01

    Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would

  13. Visualization and 3D reconstruction of flame cells of Taenia solium (cestoda.

    Laura E Valverde-Islas

    Full Text Available BACKGROUND: Flame cells are the terminal cells of protonephridial systems, which are part of the excretory systems of invertebrates. Although the knowledge of their biological role is incomplete, there is a consensus that these cells perform excretion/secretion activities. It has been suggested that the flame cells participate in the maintenance of the osmotic environment that the cestodes require to live inside their hosts. In live Platyhelminthes, by light microscopy, the cells appear beating their flames rapidly and, at the ultrastructural, the cells have a large body enclosing a tuft of cilia. Few studies have been performed to define the localization of the cytoskeletal proteins of these cells, and it is unclear how these proteins are involved in cell function. METHODOLOGY/PRINCIPAL FINDINGS: Parasites of two different developmental stages of T. solium were used: cysticerci recovered from naturally infected pigs and intestinal adults obtained from immunosuppressed and experimentally infected golden hamsters. Hamsters were fed viable cysticerci to recover adult parasites after one month of infection. In the present studies focusing on flame cells of cysticerci tissues was performed. Using several methods such as video, confocal and electron microscopy, in addition to computational analysis for reconstruction and modeling, we have provided a 3D visual rendition of the cytoskeletal architecture of Taenia solium flame cells. CONCLUSIONS/SIGNIFICANCE: We consider that visual representations of cells open a new way for understanding the role of these cells in the excretory systems of Platyhelminths. After reconstruction, the observation of high resolution 3D images allowed for virtual observation of the interior composition of cells. A combination of microscopic images, computational reconstructions and 3D modeling of cells appears to be useful for inferring the cellular dynamics of the flame cell cytoskeleton.

  14. Stochastic microstructure modeling and electrochemical simulation of lithium-ion cell anodes in 3D

    Hein, Simon; Feinauer, Julian; Westhoff, Daniel; Manke, Ingo; Schmidt, Volker; Latz, Arnulf

    2016-12-01

    Thermodynamically consistent transport theory is used to compare 3D images of real anode microstructures from lithium-ion batteries to virtual ones created by a parametric stochastic 3D microstructure model. Half-cell simulations in 3D with spatially resolved microstructures at different applied currents show that for low currents the deviations between various electrochemical quantities like current density or overpotential are negligibly small. For larger currents small differences become more pronounced. Qualitative and quantitative differences of these features are discussed with respect to the microstructure and it is shown that the real and virtual structures behave similar during electrochemical simulations. Extensions of the stochastic microstructure model, which overcome small differences in electrochemical behavior, are proposed.

  15. Quantitative data analysis methods for 3D microstructure characterization of Solid Oxide Cells

    Jørgensen, Peter Stanley

    The performance of electrochemical ceramic devices such as solid oxide fuel and electrolyser cells depends on the distribution of constituent phases on the micro or nano scale, also known as the microstructure. The microstructure governs key properties such as ion, electron and gas transport...... task of manually delineating structures within each image slice or the quality of manual and automatic segmentation schemes. To solve this, a framework for the automatic segmentation of 3D image data is developed. The technique is based on a level set method and uses numerical approximations to partial...... differential equations to evolve a 3D surface to capture the phase boundaries. Vector fields derived from the experimentally acquired data are used as the driving forces. The framework performs the segmentation in 3D rather than on a slice by slice basis. It naturally supplies sub-voxel accuracy of segmented...

  16. Fabrication of Dye-Sensitized Solar Cells with a 3D Nanostructured Electrode

    Guo-Yang Chen

    2010-01-01

    Full Text Available A novel Dye-Sensitized Solar Cell (DSSC scheme for better solar conversion efficiency is proposed. The distinctive characteristic of this novel scheme is that the conventional thin film electrode is replaced by a 3D nanostructured indium tin oxide (ITO electrode, which was fabricated using RF magnetron sputtering with an anodic aluminum oxide (AAO template. The template was prepared by immersing the barrier-layer side of an AAO film into a 30 wt% phosphoric acid solution to produce a contrasting surface. RF magnetron sputtering was then used to deposit a 3D nanostructured ITO thin film on the template. The crystallinity and conductivity of the 3D ITO films were further enhanced by annealing. Titanium dioxide nanoparticles were electrophoretically deposited on the 3D ITO film after which the proposed DSSC was formed by filling vacant spaces in the 3D nanostructured ITO electrode with dye. The measured solar conversion efficiency of the device was 0.125%. It presents a 5-fold improvement over that of conventional spin-coated TiO2 film electrode DSSCs.

  17. Stromal cells and integrins: conforming to the needs of the tumor microenvironment.

    Alphonso, Aimee; Alahari, Suresh K

    2009-12-01

    The microenvironment of a tumor is constituted of a heterogenous population of stromal cells, extracellular matrix components, and secreted factors, all of which make the tumor microenvironment distinct from that of normal tissue. Unlike healthy cells, tumor cells require these unique surroundings to metastasize, spread, and form a secondary tumor at a distant site. In this review, we discuss that stromal cells such as fibroblasts and immune cells including macrophages, their secreted factors, such as vascular endothelial growth factor, transforming growth factor beta, and various chemokines, and the integrins that connect the various cell types play a particularly vital role in the survival of a growing tumor mass. Macrophages and fibroblasts are uniquely plastic cells because they are not only able to switch from tumor suppressing to tumor supporting phenotypes but also able to adopt various tumor-supporting functions based on their location within the microenvironment. Integrins serve as the backbone for all of these prometastatic operations because their function as cell-cell and cell-matrix signal transducers are important for the heterogenous components of the microenvironment to communicate.

  18. Investigation of osteoblast cells behavior in polymeric 3D micropatterned scaffolds using digital holographic microscopy.

    Mihailescu, M; Popescu, R C; Matei, A; Acasandrei, A; Paun, I A; Dinescu, M

    2014-08-01

    The effect of micropatterned polymeric scaffolds on the features of the cultured cells at different time intervals after seeding was investigated by digital holographic microscopy. Both parallel and perpendicular walls, with different heights, were fabricated using two-photon lithography on photopolymers. The walls were subsequently coated with polypyrrole-based thin films using the matrix assisted pulsed laser evaporation technique. Osteoblast-like cells, MG-63 line, were cultured on these polymeric 3D micropatterned scaffolds. To analyze these scaffolds with/without cultured cells, an inverted digital holographic microscope, which provides 3D images, was used. Information about the samples' refractive indices and heights was obtained from the phase shift introduced in the optical path. Characteristics of cell adhesion, alignment, orientation, and morphology as a function of the wall heights and time from seeding were highlighted.

  19. Mammary gland development: cell fate specification, stem cells and the microenvironment.

    Inman, Jamie L; Robertson, Claire; Mott, Joni D; Bissell, Mina J

    2015-03-15

    The development of the mammary gland is unique: the final stages of development occur postnatally at puberty under the influence of hormonal cues. Furthermore, during the life of the female, the mammary gland can undergo many rounds of expansion and proliferation. The mammary gland thus provides an excellent model for studying the 'stem/progenitor' cells that allow this repeated expansion and renewal. In this Review, we provide an overview of the different cell types that constitute the mammary gland, and discuss how these cell types arise and differentiate. As cellular differentiation cannot occur without proper signals, we also describe how the tissue microenvironment influences mammary gland development.

  20. Computational Graph Model for 3D Cells Tracking in Zebra Fish Datasets

    Zhang, Lelin; Xiong, Hongkai; Zhao, Yang; Zhang, Kai; Zhou, Xiaobo

    2007-11-01

    This paper leads to a novel technique for tracking and identification of zebra-fish cells in 3D image sequences, extending graph-based multi-objects tracking algorithm to 3D applications. As raised in previous work of 2D graph-based method, separated cells are modeled as vertices that connected by edges. Then the tracking work is simplified to that of vertices matching between graphs generated from consecutive frames. Graph-based tracking is composed of three steps: graph generation, initial source vertices selection and graph saturation. To satisfy demands in this work separated cell records are segmented from original datasets using 3D level-set algorithms. Besides, advancements are achieved in each of the step including graph regulations, multi restrictions on source vertices and enhanced flow quantifications. Those strategies make a good compensation for graph-based multi-objects tracking method in 2D space. Experiments are carried out in 3D datasets sampled from zebra fish, results of which shows that this enhanced method could be potentially applied to tracking of objects with diverse features.

  1. Thermoforming techniques for manufacturing porous scaffolds for application in 3D cell cultivation.

    Borowiec, Justyna; Hampl, Jörg; Gebinoga, Michael; Elsarnagawy, Tarek; Elnakady, Yasser A; Fouad, Hassan; Almajhadi, Fahd; Fernekorn, Uta; Weise, Frank; Singh, Sukhdeep; Elsarnagawy, Dief; Schober, Andreas

    2015-04-01

    Within the scientific community, there is an increasing demand to apply advanced cell cultivation substrates with increased physiological functionalities for studying spatially defined cellular interactions. Porous polymeric scaffolds are utilized for mimicking an organ-like structure or engineering complex tissues and have become a key element for three-dimensional (3D) cell cultivation in the meantime. As a consequence, efficient 3D scaffold fabrication methods play an important role in modern biotechnology. Here, we present a novel thermoforming procedure for manufacturing porous 3D scaffolds from permeable materials. We address the issue of precise thermoforming of porous polymer foils by using multilayer polymer thermoforming technology. This technology offers a new method for structuring porous polymer foils that are otherwise available for non-porous polymers only. We successfully manufactured 3D scaffolds from solvent casted and phase separated polylactic acid (PLA) foils and investigated their biocompatibility and basic cellular performance. The HepG2 cell culture in PLA scaffold has shown enhanced albumin secretion rate in comparison to a previously reported polycarbonate based scaffold with similar geometry.

  2. 3D imaging of Sox2 enhancer clusters in embryonic stem cells.

    Liu, Zhe; Legant, Wesley R; Chen, Bi-Chang; Li, Li; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert

    2014-12-24

    Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.

  3. A 3D in vitro bone organ model using human progenitor cells

    A Papadimitropoulos

    2011-05-01

    Full Text Available Three-dimensional (3D organotypic culture models based on human cells may reduce the use of complex and costly animal models, while gaining clinical relevance. This study aimed at developing a 3D osteoblastic-osteoclastic-endothelial cell co-culture system, as an in vitro model to mimic the process of bone turnover. Osteoprogenitor and endothelial lineage cells were isolated from the stromal vascular fraction (SVF of human adipose tissue, whereas CD14+ osteoclast progenitors were derived from human peripheral blood. Cells were co-cultured within 3D porous ceramic scaffolds using a perfusion-based bioreactor device, in the presence of typical osteoclastogenic factors. After 3 weeks, the scaffolds contained cells with endothelial (2.0 ±0.3%, pre/osteoclastic (14.0 ±1.4% and mesenchymal/osteoblastic (44.0 ±8.4% phenotypes, along with tartrate-resistant acid phosphatase-positive (TRAP+ osteoclastic cells in contact with deposited bone-like matrix. Supernatant analysis demonstrated sustained matrix deposition (by C-terminus procollagen-I propeptides, resorption (by N-terminus collagen-I telopeptides and phosphate levels and osteoclastic activity (by TRAP-5b only when SVF and CD14+ cells were co-cultured. Scanning electron microscopy and magnetic resonance imaging confirmed the pattern of matrix deposition and resorption. The effectiveness of Vitamin D in replacing osteoclastogenic factors indicated a functional osteoblast-osteoclast coupling in the system. The formation of human-origin bone-like tissue, blood vessels and osteoclasts upon ectopic implantation validated the functionality of the developed cell types. The 3D co-culture system and the associated non-invasive analytical tools can be used as an advanced model to capture some aspects of the functional coupling of bone-like matrix deposition and resorption and could be exploited toward the engineering of multi-functional bone substitute implants.

  4. 3D cell culture: a review of current approaches and techniques.

    Haycock, John W

    2011-01-01

    Cell culture in two dimensions has been routinely and diligently undertaken in thousands of laboratories worldwide for the past four decades. However, the culture of cells in two dimensions is arguably primitive and does not reproduce the anatomy or physiology of a tissue for informative or useful study. Creating a third dimension for cell culture is clearly more relevant, but requires a multidisciplinary approach and multidisciplinary expertise. When entering the third dimension, investigators need to consider the design of scaffolds for supporting the organisation of cells or the use of bioreactors for controlling nutrient and waste product exchange. As 3D culture systems become more mature and relevant to human and animal physiology, the ability to design and develop co-cultures becomes possible as does the ability to integrate stem cells. The primary objectives for developing 3D cell culture systems vary widely - and range from engineering tissues for clinical delivery through to the development of models for drug screening. The intention of this review is to provide a general overview of the common approaches and techniques for designing 3D culture models.

  5. Defining an optimal surface chemistry for pluripotent stem cell culture in 2D and 3D

    Zonca, Michael R., Jr.

    Surface chemistry is critical for growing pluripotent stem cells in an undifferentiated state. There is great potential to engineer the surface chemistry at the nanoscale level to regulate stem cell adhesion. However, the challenge is to identify the optimal surface chemistry of the substrata for ES cell attachment and maintenance. Using a high-throughput polymerization and screening platform, a chemically defined, synthetic polymer grafted coating that supports strong attachment and high expansion capacity of pluripotent stem cells has been discovered using mouse embryonic stem (ES) cells as a model system. This optimal substrate, N-[3-(Dimethylamino)propyl] methacrylamide (DMAPMA) that is grafted on 2D synthetic poly(ether sulfone) (PES) membrane, sustains the self-renewal of ES cells (up to 7 passages). DMAPMA supports cell attachment of ES cells through integrin beta1 in a RGD-independent manner and is similar to another recently reported polymer surface. Next, DMAPMA has been able to be transferred to 3D by grafting to synthetic, polymeric, PES fibrous matrices through both photo-induced and plasma-induced polymerization. These 3D modified fibers exhibited higher cell proliferation and greater expression of pluripotency markers of mouse ES cells than 2D PES membranes. Our results indicated that desirable surfaces in 2D can be scaled to 3D and that both surface chemistry and structural dimension strongly influence the growth and differentiation of pluripotent stem cells. Lastly, the feasibility of incorporating DMAPMA into a widely used natural polymer, alginate, has been tested. Novel adhesive alginate hydrogels have been successfully synthesized by either direct polymerization of DMAPMA and methacrylic acid blended with alginate, or photo-induced DMAPMA polymerization on alginate nanofibrous hydrogels. In particular, DMAPMA-coated alginate hydrogels support strong ES cell attachment, exhibiting a concentration dependency of DMAPMA. This research provides a

  6. Regulatory T Cells in the Tumor Microenvironment and Cancer Progression: Role and Therapeutic Targeting

    Chaudhary, Belal; Elkord, Eyad

    2016-01-01

    Recent years have seen significant efforts in understanding and modulating the immune response in cancer. In this context, immunosuppressive cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), have come under intense investigation for their proposed roles in suppressing tumor-specific immune responses and establishing an immunosuppressive tumor microenvironment, thus enabling tumor immune evasion. Additionally, recent evidence indicates that Tregs comprise diverse and heterogeneous subsets; phenotypically and functionally distinct subsets of tumor-infiltrating Tregs could contribute differently to cancer prognosis and clinical outcomes. Understanding Treg biology in the setting of cancer, and specifically the tumor microenvironment, is important for designing effective cancer therapies. In this review, we critically examine the role of Tregs in the tumor microenvironment and in cancer progression focusing on human studies. We also discuss the impact of current therapeutic modalities on Treg biology and the therapeutic opportunities for targeting Tregs to enhance anti-tumor immune responses and clinical benefits. PMID:27509527

  7. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L. James; Lu, Wu

    2015-12-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk

  8. Dynamic Microenvironment Induces Phenotypic Plasticity of Esophageal Cancer Cells Under Flow

    Calibasi Kocal, Gizem; Güven, Sinan; Foygel, Kira; Goldman, Aaron; Chen, Pu; Sengupta, Shiladitya; Paulmurugan, Ramasamy; Baskin, Yasemin; Demirci, Utkan

    2016-12-01

    Cancer microenvironment is a remarkably heterogeneous composition of cellular and non-cellular components, regulated by both external and intrinsic physical and chemical stimuli. Physical alterations driven by increased proliferation of neoplastic cells and angiogenesis in the cancer microenvironment result in the exposure of the cancer cells to elevated levels of flow-based shear stress. We developed a dynamic microfluidic cell culture platform utilizing eshopagael cancer cells as model cells to investigate the phenotypic changes of cancer cells upon exposure to fluid shear stress. We report the epithelial to hybrid epithelial/mesenchymal transition as a result of decreasing E-Cadherin and increasing N-Cadherin and vimentin expressions, higher clonogenicity and ALDH positive expression of cancer cells cultured in a dynamic microfluidic chip under laminar flow compared to the static culture condition. We also sought regulation of chemotherapeutics in cancer microenvironment towards phenotypic control of cancer cells. Such in vitro microfluidic system could potentially be used to monitor how the interstitial fluid dynamics affect cancer microenvironment and plasticity on a simple, highly controllable and inexpensive bioengineered platform.

  9. Rab3D is critical for secretory granule maturation in PC12 cells.

    Tanja Kögel

    Full Text Available Neuropeptide- and hormone-containing secretory granules (SGs are synthesized at the trans-Golgi network (TGN as immature secretory granules (ISGs and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs. Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.

  10. A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.

    Wiley, Luke A; Beebe, David C; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-05-12

    This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.

  11. Glioblastoma-Initiating Cells: Relationship with Neural Stem Cells and the Micro-Environment

    Nicolas Goffart

    2013-08-01

    Full Text Available Glioblastoma multiforme (GBM, WHO grade IV is the most common and lethal subtype of primary brain tumor with a median overall survival of 15 months from the time of diagnosis. The presence in GBM of a cancer population displaying neural stem cell (NSC properties as well as tumor-initiating abilities and resistance to current therapies suggests that these glioblastoma-initiating cells (GICs play a central role in tumor development and are closely related to NSCs. However, it is nowadays still unclear whether GICs derive from NSCs, neural progenitor cells or differentiated cells such as astrocytes or oligodendrocytes. On the other hand, NSCs are located in specific regions of the adult brain called neurogenic niches that have been shown to control critical stem cell properties, to nourish NSCs and to support their self-renewal. This “seed-and-soil” relationship has also been adapted to cancer stem cell research as GICs also require a specific micro-environment to maintain their “stem cell” properties. In this review, we will discuss the controversies surrounding the origin and the identification of GBM stem cells and highlight the micro-environment impact on their biology.

  12. Glioblastoma-Initiating Cells: Relationship with Neural Stem Cells and the Micro-Environment

    Goffart, Nicolas [Laboratory of Developmental Neurobiology, GIGA-Neurosciences Research Center, University of Liège, Liège 4000 (Belgium); Kroonen, Jérôme [Human Genetics, CHU and University of Liège, Liège 4000 (Belgium); The T& P Bohnenn Laboratory for Neuro-Oncology, Department of Neurology and Neurosurgery, UMC Utrecht, Utrecht 3556 (Netherlands); Rogister, Bernard, E-mail: Bernard.Register@ulg.ac.be [Laboratory of Developmental Neurobiology, GIGA-Neurosciences Research Center, University of Liège, Liège 4000 (Belgium); Department of Neurology, CHU and University of Liège, Liège 4000 (Belgium); GIGA-Development, Stem Cells and Regenerative Medicine, University of Liège, Liège 4000 (Belgium)

    2013-08-14

    Glioblastoma multiforme (GBM, WHO grade IV) is the most common and lethal subtype of primary brain tumor with a median overall survival of 15 months from the time of diagnosis. The presence in GBM of a cancer population displaying neural stem cell (NSC) properties as well as tumor-initiating abilities and resistance to current therapies suggests that these glioblastoma-initiating cells (GICs) play a central role in tumor development and are closely related to NSCs. However, it is nowadays still unclear whether GICs derive from NSCs, neural progenitor cells or differentiated cells such as astrocytes or oligodendrocytes. On the other hand, NSCs are located in specific regions of the adult brain called neurogenic niches that have been shown to control critical stem cell properties, to nourish NSCs and to support their self-renewal. This “seed-and-soil” relationship has also been adapted to cancer stem cell research as GICs also require a specific micro-environment to maintain their “stem cell” properties. In this review, we will discuss the controversies surrounding the origin and the identification of GBM stem cells and highlight the micro-environment impact on their biology.

  13. 3D-printed and CNC milled flow-cells for chemiluminescence detection.

    Spilstead, Kara B; Learey, Jessica J; Doeven, Egan H; Barbante, Gregory J; Mohr, Stephan; Barnett, Neil W; Terry, Jessica M; Hall, Robynne M; Francis, Paul S

    2014-08-01

    Herein we explore modern fabrication techniques for the development of chemiluminescence detection flow-cells with features not attainable using the traditional coiled tubing approach. This includes the first 3D-printed chemiluminescence flow-cells, and a milled flow-cell designed to split the analyte stream into two separate detection zones within the same polymer chip. The flow-cells are compared to conventional detection systems using flow injection analysis (FIA) and high performance liquid chromatography (HPLC), with the fast chemiluminescence reactions of an acidic potassium permanganate reagent with morphine and a series of adrenergic phenolic amines.

  14. Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells.

    Karl Gledhill

    Full Text Available The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes.

  15. 3D Printing of Scaffold for Cells Delivery: Advances in Skin Tissue Engineering

    Deepti Singh

    2016-01-01

    Full Text Available Injury or damage to tissue and organs is a major health problem, resulting in about half of the world’s annual healthcare expenditure every year. Advances in the fields of stem cells (SCs and biomaterials processing have provided a tremendous leap for researchers to manipulate the dynamics between these two, and obtain a skin substitute that can completely heal the wounded areas. Although wound healing needs a coordinated interplay between cells, extracellular proteins and growth factors, the most important players in this process are the endogenous SCs, which activate the repair cascade by recruiting cells from different sites. Extra cellular matrix (ECM proteins are activated by these SCs, which in turn aid in cellular migrations and finally secretion of growth factors that can seal and heal the wounds. The interaction between ECM proteins and SCs helps the skin to sustain the rigors of everyday activity, and in an attempt to attain this level of functionality in artificial three-dimensional (3D constructs, tissue engineered biomaterials are fabricated using more advanced techniques such as bioprinting and laser assisted printing of the organs. This review provides a concise summary of the most recent advances that have been made in the area of polymer bio-fabrication using 3D bio printing used for encapsulating stem cells for skin regeneration. The focus of this review is to describe, in detail, the role of 3D architecture and arrangement of cells within this system that can heal wounds and aid in skin regeneration.

  16. Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

    Jagannadh, Veerendra Kalyan; Mackenzie, Mark D; Pal, Parama; Kar, Ajoy K; Gorthi, Sai Siva

    2016-09-19

    Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope.

  17. 3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood.

    Zheng, Siyang; Lin, Henry K; Lu, Bo; Williams, Anthony; Datar, Ram; Cote, Richard J; Tai, Yu-Chong

    2011-02-01

    Detection of circulating tumor cells has emerged as a promising minimally invasive diagnostic and prognostic tool for patients with metastatic cancers. We report a novel three dimensional microfilter device that can enrich viable circulating tumor cells from blood. This device consists of two layers of parylene membrane with pores and gap precisely defined with photolithography. The positions of the pores are shifted between the top and bottom membranes. The bottom membrane supports captured cells and minimize the stress concentration on cell membrane and sustain cell viability during filtration. Viable cell capture on device was investigated with scanning electron microscopy, confocal microscopy, and immunofluorescent staining using model systems of cultured tumor cells spiked in blood or saline. The paper presents and validates this new 3D microfiltration concept for circulation tumor cell enrichment application. The device provides a highly valuable tool for assessing and characterizing viable enriched circulating tumor cells in both research and clinical settings.

  18. Effects of extracellular fiber architecture on cell membrane shear stress in a 3D fibrous matrix.

    Pedersen, John A; Boschetti, Federica; Swartz, Melody A

    2007-01-01

    Interstitial fluid flow has been shown to affect the organization and behavior of cells in 3D environments in vivo and in vitro, yet the forces driving such responses are not clear. Due to the complex architecture of the extracellular matrix (ECM) and the difficulty of measuring fluid flow near cells embedded in it, the levels of shear stress experienced by cells in this environment are typically estimated using bulk-averaged matrix parameters such as hydraulic permeability. While this is useful for estimating average stresses, it cannot yield insight into how local matrix fiber architecture-which is cell-controlled in the immediate pericellular environment-affects the local stresses imposed on the cell surface. To address this, we used computational fluid dynamics to study flow through an idealized mesh constructed of a cubic lattice of fibers simulating a typical in vitro collagen gel. We found that, in such high porosity matrices, the fibers strongly affect the flow fields near the cell, with peak shear stresses up to five times higher than those predicted by the Brinkman equation. We also found that minor remodeling of the fibers near the cell surface had major effects on the shear stress profile on the cell. These findings demonstrate the importance of fiber architecture to the fluid forces on a cell embedded in a 3D matrix, and also show how small modifications in the local ECM can lead to large changes in the mechanical environment of the cell.

  19. The role of the tissue microenvironment in the regulation of cancer cell motility and invasion

    Brábek Jan

    2010-09-01

    Full Text Available Abstract During malignant neoplastic progression the cells undergo genetic and epigenetic cancer-specific alterations that finally lead to a loss of tissue homeostasis and restructuring of the microenvironment. The invasion of cancer cells through connective tissue is a crucial prerequisite for metastasis formation. Although cell invasion is foremost a mechanical process, cancer research has focused largely on gene regulation and signaling that underlie uncontrolled cell growth. More recently, the genes and signals involved in the invasion and transendothelial migration of cancer cells, such as the role of adhesion molecules and matrix degrading enzymes, have become the focus of research. In this review we discuss how the structural and biomechanical properties of extracellular matrix and surrounding cells such as endothelial cells influence cancer cell motility and invasion. We conclude that the microenvironment is a critical determinant of the migration strategy and the efficiency of cancer cell invasion.

  20. The influence of printing parameters on cell survival rate and printability in microextrusion-based 3D cell printing technology.

    Zhao, Yu; Li, Yang; Mao, Shuangshuang; Sun, Wei; Yao, Rui

    2015-11-02

    Three-dimensional (3D) cell printing technology has provided a versatile methodology to fabricate cell-laden tissue-like constructs and in vitro tissue/pathological models for tissue engineering, drug testing and screening applications. However, it still remains a challenge to print bioinks with high viscoelasticity to achieve long-term stable structure and maintain high cell survival rate after printing at the same time. In this study, we systematically investigated the influence of 3D cell printing parameters, i.e. composition and concentration of bioink, holding temperature and holding time, on the printability and cell survival rate in microextrusion-based 3D cell printing technology. Rheological measurements were utilized to characterize the viscoelasticity of gelatin-based bioinks. Results demonstrated that the bioink viscoelasticity was increased when increasing the bioink concentration, increasing holding time and decreasing holding temperature below gelation temperature. The decline of cell survival rate after 3D cell printing process was observed when increasing the viscoelasticity of the gelatin-based bioinks. However, different process parameter combinations would result in the similar rheological characteristics and thus showed similar cell survival rate after 3D bioprinting process. On the other hand, bioink viscoelasticity should also reach a certain point to ensure good printability and shape fidelity. At last, we proposed a protocol for 3D bioprinting of temperature-sensitive gelatin-based hydrogel bioinks with both high cell survival rate and good printability. This research would be useful for biofabrication researchers to adjust the 3D bioprinting process parameters quickly and as a referable template for designing new bioinks.

  1. A complex 3D human tissue culture system based on mammary stromal cells and silk scaffolds for modeling breast morphogenesis and function.

    Wang, Xiuli; Sun, Lin; Maffini, Maricel V; Soto, Ana; Sonnenschein, Carlos; Kaplan, David L

    2010-05-01

    Epithelial-stromal interactions play a crucial role in normal embryonic development and carcinogenesis of the human breast while the underlying mechanisms of these events remain poorly understood. To address this issue, we constructed a physiologically relevant, three-dimensional (3D) culture surrogate of complex human breast tissue that included a tri-culture system made up of human mammary epithelial cells (MCF10A), human fibroblasts and adipocytes, i.e., the two dominant breast stromal cell types, in a Matrigel/collagen mixture on porous silk protein scaffolds. The presence of stromal cells inhibited MCF10A cell proliferation and induced both alveolar and ductal morphogenesis and enhanced casein expression. In contrast to the immature polarity exhibited by co-cultures with either fibroblasts or adipocytes, the alveolar structures formed by the tri-cultures exhibited proper polarity similar to that observed in breast tissue in vivo. Only alveolar structures with reverted polarity were observed in MCF10A monocultures. Consistent with their phenotypic appearance, more functional differentiation of epithelial cells was also observed in the tri-cultures, where casein alpha- and -beta mRNA expression was significantly increased. This in vitro tri-culture breast tissue system sustained on silk scaffold effectively represents a more physiologically relevant 3D microenvironment for mammary epithelial cells and stromal cells than either co-cultures or monocultures. This experimental model provides an important first step for bioengineering an informative human breast tissue system, with which to study normal breast morphogenesis and neoplastic transformation.

  2. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.

  3. Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

    Niu, Chengcheng; Wang, Long; Wang, Zhigang; Xu, Yan; Hu, Yihe; Peng, Qinghai

    2017-03-01

    Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work.

  4. Total 3D imaging of phase objects using defocusing microscopy: application to red blood cells

    Roma, P M S; Amaral, F T; Agero, U; Mesquita, O N

    2014-01-01

    We present Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total 3D imaging of transparent objects. By total 3D imaging we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a new methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic and hypertonic solutions. For each situation the shape of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of RBCs surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine volume, superficial area, sphericity index and RBCs refractive index for each osmotic condition.

  5. Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy

    Park, HyunJoo; Kim, Kyoohyun; Cho, Shin-Hyeong; Lee, Won-Ja; Kim, Youngchan; Lee, SangEun; Park, YongKeun

    2015-01-01

    Babesia microti causes emergency human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability.

  6. Cell pairing ratio controlled micro-environment with valve-less electrolytic isolation

    Chen, Yu-Chih

    2012-01-01

    We present a ratio controlled cell-to-cell interaction chip using valve-less isolation. We incorporated electrolysis in a microfluidic channel. In each microfluidic chamber, we loaded two types of different cells at various pairing ratios. More than 80% of the microchambers were successfully loaded with a specific target pairing ratio. For the proof of concept, we have demonstrated the cell-to-cell interaction between prostate cancer cells and muscle stem cells can be controlled by cell pairing ratios through growth factor secretion. The experimental data shows that sealing of microenvironment by air generated from electrolysis does not affect cell viability and cell interaction assay results. © 2012 IEEE.

  7. The senescent microenvironment promotes the emergence of heterogeneous cancer stem-like cells.

    Castro-Vega, Luis Jaime; Jouravleva, Karina; Ortiz-Montero, Paola; Liu, Win-Yan; Galeano, Jorge Luis; Romero, Martha; Popova, Tatiana; Bacchetti, Silvia; Vernot, Jean Paul; Londoño-Vallejo, Arturo

    2015-10-01

    There is a well-established association between aging and the onset of metastasis. Although the mechanisms through which age impinges upon the malignant phenotype remain uncharacterized, the role of a senescent microenvironment has been emphasized. We reported previously that human epithelial cells that undergo telomere-driven chromosome instability (T-CIN) display global microRNA (miR) deregulation and develop migration and invasion capacities. Here, we show that post-crisis cells are not able to form tumors unless a senescent microenvironment is provided. The characterization of cell lines established from such tumors revealed that these cells have acquired cell autonomous tumorigenicity, giving rise to heterogeneous tumors. Further experiments demonstrate that explanted cells, while displaying differences in cell differentiation markers, are all endowed of enhanced stem cell properties including self-renewal and multilineage differentiation capacity. Treatments of T-CIN+ cells with senescence-conditioned media induce sphere formation exclusively in cells with senescence-associated tumorigenicity, a capacity that depends on miR-145 repression. These results indicate that the senescent microenvironment, while promoting further transdifferentiations in cells with genome instability, is able to propel the progression of premalignant cells towards a malignant, cell stem-like state.

  8. Controlled Positioning of Cells in Biomaterials—Approaches Towards 3D Tissue Printing

    Sandra Hofmann

    2011-08-01

    Full Text Available Current tissue engineering techniques have various drawbacks: they often incorporate uncontrolled and imprecise scaffold geometries, whereas the current conventional cell seeding techniques result mostly in random cell placement rather than uniform cell distribution. For the successful reconstruction of deficient tissue, new material engineering approaches have to be considered to overcome current limitations. An emerging method to produce complex biological products including cells or extracellular matrices in a controlled manner is a process called bioprinting or biofabrication, which effectively uses principles of rapid prototyping combined with cell-loaded biomaterials, typically hydrogels. 3D tissue printing is an approach to manufacture functional tissue layer-by-layer that could be transplanted in vivo after production. This method is especially advantageous for stem cells since a controlled environment can be created to influence cell growth and differentiation. Using printed tissue for biotechnological and pharmacological needs like in vitro drug-testing may lead to a revolution in the pharmaceutical industry since animal models could be partially replaced by biofabricated tissues mimicking human physiology and pathology. This would not only be a major advancement concerning rising ethical issues but would also have a measureable impact on economical aspects in this industry of today, where animal studies are very labor-intensive and therefore costly. In this review, current controlled material and cell positioning techniques are introduced highlighting approaches towards 3D tissue printing.

  9. High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

    Ankam, Soneela; Teo, Benjamin KK; Kukumberg, Marek; Yim, Evelyn KF

    2013-01-01

    Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including ...

  10. High Throughput Studies of Cell Migration in 3D Microtissues Fabricated by a Droplet Microfluidic Chip

    Xiangchen Che

    2016-05-01

    Full Text Available Arrayed three-dimensional (3D micro-sized tissues with encapsulated cells (microtissues have been fabricated by a droplet microfluidic chip. The extracellular matrix (ECM is a polymerized collagen network. One or multiple breast cancer cells were embedded within the microtissues, which were stored in arrayed microchambers on the same chip without ECM droplet shrinkage over 48 h. The migration trajectory of the cells was recorded by optical microscopy. The migration speed was calculated in the range of 3–6 µm/h. Interestingly, cells in devices filled with a continuous collagen network migrated faster than those where only droplets were arrayed in the chambers. This is likely due to differences in the length scales of the ECM network, as cells embedded in thin collagen slabs also migrate slower than those in thick collagen slabs. In addition to migration, this technical platform can be potentially used to study cancer cell-stromal cell interactions and ECM remodeling in 3D tumor-mimicking environments.

  11. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Yeonhee Kim

    Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

  12. Image informatics for studying signal transduction in cells interacting with 3D matrices

    Tzeranis, Dimitrios S.; Guo, Jin; Chen, Chengpin; Yannas, Ioannis V.; Wei, Xunbin; So, Peter T. C.

    2014-03-01

    Cells sense and respond to chemical stimuli on their environment via signal transduction pathways, complex networks of proteins whose interactions transmit chemical information. This work describes an implementation of image informatics, imaging-based methodologies for studying signal transduction networks. The methodology developed focuses on studying signal transduction networks in cells that interact with 3D matrices. It utilizes shRNA-based knock down of network components, 3D high-content imaging of cells inside the matrix by spectral multi-photon microscopy, and single-cell quantification using features that describe both cell morphology and cell-matrix adhesion pattern. The methodology is applied in a pilot study of TGFβ signaling via the SMAD pathway in fibroblasts cultured inside porous collagen-GAG scaffolds, biomaterials similar to the ones used clinically to induce skin regeneration. Preliminary results suggest that knocking down all rSMAD components affects fibroblast response to TGFβ1 and TGFβ3 isoforms in different ways, and suggest a potential role for SMAD1 and SMAD5 in regulating TGFβ isoform response. These preliminary results need to be verified with proteomic results that can provide solid evidence about the particular role of individual components of the SMAD pathway.

  13. Impact of dimensionality and network disruption on microrheology of cancer cells in 3D environments.

    Michael Mak

    2014-11-01

    Full Text Available Dimensionality is a fundamental component that can have profound implications on the characteristics of physical systems. In cell biology, however, the majority of studies on cell physical properties, from rheology to force generation to migration, have been performed on 2D substrates, and it is not clear how a more realistic 3D environment influences cell properties. Here, we develop an integrated approach and demonstrate the combination of mitochondria-tracking microrheology, microfluidics, and Brownian dynamics simulations to explore the impact of dimensionality on intracellular mechanics and on the effects of intracellular disruption. Additionally, we consider both passive thermal and active motor-driven processes within the cell and demonstrate through modeling how active internal fluctuations are modulated via dimensionality. Our results demonstrate that metastatic breast cancer cells (MDA-MB-231 exhibit more solid-like internal motions in 3D compared to 2D, and actin network disruption via Cytochalasin D has a more pronounced effect on internal cell fluctuations in 2D. Our computational results and modeling show that motor-induced active stress fluctuations are enhanced in 2D, leading to increased local intracellular particle fluctuations and apparent fluid-like behavior.

  14. Reconstruction of 3D ion beam micro-tomography data for applications in Cell Biology

    Habchi, C. [Universite de Bordeaux, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France)], E-mail: habchi@cenbg.in2p3.fr; Nguyen, D.T.; Barberet, Ph. [Universite de Bordeaux, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France); Incerti, S. [CNRS/IN2P3, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France); Moretto, Ph. [Universite de Bordeaux, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France); Sakellariou, A. [Research School of Physical Sciences and Engineering, Australian National University, Canberra ACT 0200 (Australia); Seznec, H. [CNRS/IN2P3, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France)

    2009-06-15

    The DISRA (Discrete Image Space Reconstruction Algorithm) reconstruction code, created by A. Sakellariou, was conceived for the ideal case of complete three-dimensional (3D) PIXET (Particle Induced X-ray Emission Tomography) data. This implies two major difficulties for biological samples: first, the long duration of such experiments and second, the subsequent damage that occurs on such fragile specimens. For this reason, the DISRA code was extended at CENBG in order to probe isolated PIXET slices, taking into account the sample structure and mass density provided by 3D STIMT (Scanning Transmission Ion Microscopy Tomography) in the volume of interest. This modified version was tested on a phantom sample and first results on human cancer cells are also presented.

  15. Reconstruction of 3D ion beam micro-tomography data for applications in Cell Biology

    Habchi, C.; Nguyen, D. T.; Barberet, Ph.; Incerti, S.; Moretto, Ph.; Sakellariou, A.; Seznec, H.

    2009-06-01

    The DISRA (Discrete Image Space Reconstruction Algorithm) reconstruction code, created by A. Sakellariou, was conceived for the ideal case of complete three-dimensional (3D) PIXET (Particle Induced X-ray Emission Tomography) data. This implies two major difficulties for biological samples: first, the long duration of such experiments and second, the subsequent damage that occurs on such fragile specimens. For this reason, the DISRA code was extended at CENBG in order to probe isolated PIXET slices, taking into account the sample structure and mass density provided by 3D STIMT (Scanning Transmission Ion Microscopy Tomography) in the volume of interest. This modified version was tested on a phantom sample and first results on human cancer cells are also presented.

  16. Flow-through 3D biofuel cell anode for NAD{sup +}-dependent enzymes

    Rincon, Rosalba A.; Lau, Carolin; Garcia, Kristen E. [Department of Chemical and Nuclear Engineering, Center for Emerging Energy Technologies, University of New Mexico, Albuquerque, NM 87131 (United States); Atanassov, Plamen, E-mail: plamen@unm.ed [Department of Chemical and Nuclear Engineering, Center for Emerging Energy Technologies, University of New Mexico, Albuquerque, NM 87131 (United States)

    2011-02-01

    NAD{sup +}-dependent enzymes require the presence of catalysts for cofactor regeneration in order to be employed in enzymatic biofuel cells. Poly-(methylene green) catalysts have proven to help the oxidation reaction of NADH allowing for the use of such enzymes in electrocatalytic oxidation reactions. In this paper we present the development of 3D anode based on NAD{sup +}-dependent malate dehydrogenase. The 3D material chosen was reticulated vitreous carbon (RVC) which was modified with poly-(MG) for NADH oxidation and it also accommodated the porous immobilization matrix for MDH consisting of MWCNTs embedded in chitosan; allowing for mass transport of the substrate to the electrode. Scanning electron microscopy was used in order to characterize the poly-(MG)-modified RVC, and electrochemical evaluation of the anode was performed.

  17. Heritable Genetic Changes in Cells Recovered From Irradiated 3D Tissue Constructs

    Michael Cornforth

    2012-03-26

    Combining contemporary cytogenetic methods with DNA CGH microarray technology and chromosome flow-sorting increases substantially the ability to resolve exchange breakpoints associated with interstitial deletions and translocations, allowing the consequences of radiation damage to be directly measured at low doses, while also providing valuable insights into molecular mechanisms of misrepair processes that, in turn, identify appropriate biophysical models of risk at low doses. Specific aims apply to cells recovered from 3D tissue constructs of human skin and, for the purpose of comparison, the same cells irradiated in traditional 2D cultures. The project includes research complementary to NASA/HRP space radiation project.

  18. A method for the evaluation of thousands of automated 3D stem cell segmentations.

    Bajcsy, P; Simon, M; Florczyk, S J; Simon, C G; Juba, D; Brady, M C

    2015-12-01

    There is no segmentation method that performs perfectly with any dataset in comparison to human segmentation. Evaluation procedures for segmentation algorithms become critical for their selection. The problems associated with segmentation performance evaluations and visual verification of segmentation results are exaggerated when dealing with thousands of three-dimensional (3D) image volumes because of the amount of computation and manual inputs needed. We address the problem of evaluating 3D segmentation performance when segmentation is applied to thousands of confocal microscopy images (z-stacks). Our approach is to incorporate experimental imaging and geometrical criteria, and map them into computationally efficient segmentation algorithms that can be applied to a very large number of z-stacks. This is an alternative approach to considering existing segmentation methods and evaluating most state-of-the-art algorithms. We designed a methodology for 3D segmentation performance characterization that consists of design, evaluation and verification steps. The characterization integrates manual inputs from projected surrogate 'ground truth' of statistically representative samples and from visual inspection into the evaluation. The novelty of the methodology lies in (1) designing candidate segmentation algorithms by mapping imaging and geometrical criteria into algorithmic steps, and constructing plausible segmentation algorithms with respect to the order of algorithmic steps and their parameters, (2) evaluating segmentation accuracy using samples drawn from probability distribution estimates of candidate segmentations and (3) minimizing human labour needed to create surrogate 'truth' by approximating z-stack segmentations with 2D contours from three orthogonal z-stack projections and by developing visual verification tools. We demonstrate the methodology by applying it to a dataset of 1253 mesenchymal stem cells. The cells reside on 10 different types of biomaterial

  19. Universal lab-on-a-chip platform for complex, perfused 3D cell cultures

    Sonntag, F.; Schmieder, F.; Ströbel, J.; Grünzner, S.; Busek, M.; Günther, K.; Steege, T.; Polk, C.; Klotzbach, U.

    2016-03-01

    The miniaturization, rapid prototyping and automation of lab-on-a-chip technology play nowadays a very important role. Lab-on-a-chip technology is successfully implemented not only for environmental analysis and medical diagnostics, but also as replacement of animals used for the testing of substances in the pharmaceutical and cosmetics industries. For that purpose the Fraunhofer IWS and partners developed a lab-on-a-chip platform for perfused cell-based assays in the last years, which includes different micropumps, valves, channels, reservoirs and customized cell culture modules. This technology is already implemented for the characterization of different human cell cultures and organoids, like skin, liver, endothelium, hair follicle and nephron. The advanced universal lab-on-a-chip platform for complex, perfused 3D cell cultures is divided into a multilayer basic chip with integrated micropump and application-specific 3D printed cell culture modules. Moreover a technology for surface modification of the printed cell culture modules by laser micro structuring and a complex and flexibly programmable controlling device based on an embedded Linux system was developed. A universal lab-on-a-chip platform with an optional oxygenator and a cell culture module for cubic scaffolds as well as first cell culture experiments within the cell culture device will be presented. The module is designed for direct interaction with robotic dispenser systems. This offers the opportunity to combine direct organ printing of cells and scaffolds with the microfluidic cell culture module. The characterization of the developed system was done by means of Micro-Particle Image Velocimetry (μPIV) and an optical oxygen measuring system.

  20. Tracking immune-related cell responses to drug delivery microparticles in 3D dense collagen matrix.

    Obarzanek-Fojt, Magdalena; Curdy, Catherine; Loggia, Nicoletta; Di Lena, Fabio; Grieder, Kathrin; Bitar, Malak; Wick, Peter

    2016-10-01

    Beyond the therapeutic purpose, the impact of drug delivery microparticles on the local tissue and inflammatory responses remains to be further elucidated specifically for reactions mediated by the host immune cells. Such immediate and prolonged reactions may adversely influence the release efficacy and intended therapeutic pathway. The lack of suitable in vitro platforms limits our ability to gain insight into the nature of immune responses at a single cell level. In order to establish an in vitro 3D system mimicking the connective host tissue counterpart, we utilized reproducible, compressed, rat-tail collagen polymerized matrices. THP1 cells (human acute monocytic leukaemia cells) differentiated into macrophage-like cells were chosen as cell model and their functionality was retained in the dense rat-tail collagen matrix. Placebo microparticles were later combined in the immune cell seeded system during collagen polymerization and secreted pro-inflammatory factors: TNFα and IL-8 were used as immune response readout (ELISA). Our data showed an elevated TNFα and IL-8 secretion by macrophage THP1 cells indicating that Placebo microparticles trigger certain immune cell responses under 3D in vivo like conditions. Furthermore, we have shown that the system is sensitive to measure the differences in THP1 macrophage pro-inflammatory responses to Active Pharmaceutical Ingredient (API) microparticles with different API release kinetics. We have successfully developed a tissue-like, advanced, in vitro system enabling selective "readouts" of specific responses of immune-related cells. Such system may provide the basis of an advanced toolbox enabling systemic evaluation and prediction of in vivo microparticle reactions on human immune-related cells.

  1. Fabrication of 3-D Reconstituted Organoid Arrays by DNA-Programmed Assembly of Cells (DPAC).

    Todhunter, Michael E; Weber, Robert J; Farlow, Justin; Jee, Noel Y; Cerchiari, Alec E; Gartner, Zev J

    2016-09-13

    Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) composed into specific three-dimensional (3-D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this article, we describe DNA-programmed assembly of cells (DPAC), a method to fabricate viable, functional arrays of organoid-like tissues within 3-D ECM gels. In DPAC, dissociated cells are chemically functionalized with degradable oligonucleotide "Velcro," allowing rapid, specific, and reversible cell adhesion to a two-dimensional (2-D) template patterned with complementary DNA. An iterative assembly process builds up organoids, layer-by-layer, from this initial 2-D template and into the third dimension. Cleavage of the DNA releases the completed array of tissues that are captured and fully embedded in ECM gels for culture and observation. DPAC controls the size, shape, composition, and spatial heterogeneity of organoids and permits positioning of constituent cells with single-cell resolution even within cultures several centimeters long. © 2016 by John Wiley & Sons, Inc.

  2. Bone Marrow Cells in Acute Lymphoblastic Leukemia Create a Proinflammatory Microenvironment Influencing Normal Hematopoietic Differentiation Fates

    Armando Vilchis-Ordoñez

    2015-01-01

    Full Text Available B-cell acute lymphoblastic leukemia (B-ALL is a serious public health problem in the pediatric population worldwide, contributing to 85% of deaths from childhood cancers. Understanding the biology of the disease is crucial for its clinical management and the development of therapeutic strategies. In line with that observed in other malignancies, chronic inflammation may contribute to a tumor microenvironment resulting in the damage of normal processes, concomitant to development and maintenance of neoplastic cells. We report here that hematopoietic cells from bone marrow B-ALL have the ability to produce proinflammatory and growth factors, including TNFα, IL-1β, IL-12, and GM-CSF that stimulate proliferation and differentiation of normal stem and progenitor cells. Our findings suggest an apparently distinct CD13+CD33+ population of leukemic cells contributing to a proinflammatory microenvironment that may be detrimental to long-term normal hematopoiesis within B-ALL bone marrow.

  3. Current automated 3D cell detection methods are not a suitable replacement for manual stereologic cell counting

    Christoph eSchmitz

    2014-05-01

    Full Text Available Stereologic cell counting has had a major impact on the field of neuroscience. A major bottleneck in stereologic cell counting is that the user must manually decide whether or not each cell is counted according to three-dimensional (3D stereologic counting rules by visual inspection within hundreds of microscopic fields-of-view per investigated brain or brain region. Reliance on visual inspection forces stereologic cell counting to be very labor-intensive and time-consuming, and is the main reason why biased, non-stereologic two-dimensional (2D cell counting approaches have remained in widespread use. We present an evaluation of the performance of modern automated cell detection and segmentation algorithms as a potential alternative to the manual approach in stereologic cell counting. The image data used in this study were 3D microscopic images of thick brain tissue sections prepared with a variety of commonly used nuclear and cytoplasmic stains. The evaluation compared the numbers and locations of cells identified unambiguously and counted exhaustively by an expert observer with those found by three automated 3D cell detection algorithms: nuclei segmentation from the FARSIGHT toolkit, nuclei segmentation by 3D multiple level set methods, and the 3D object counter plug-in for ImageJ. Of these methods, FARSIGHT performed best, with true-positive detection rates between 38–99% and false-positive rates from 3.6–82%. The results demonstrate that the current automated methods suffer from lower detection rates and higher false-positive rates than are acceptable for obtaining valid estimates of cell numbers. Thus, at present, stereologic cell counting with manual decision for object inclusion according to unbiased stereologic counting rules remains the only adequate method for unbiased cell quantification in histologic tissue sections.

  4. Oxygen microenvironment affects the uptake of nanoparticles in head and neck tumor cells

    Chen, Eunice Y.; Hodge, Sasson; Tai, Katherine; Hou, Huagang; Khan, Nadeem; Hoopes, P. Jack; Samkoe, Kimberley S.

    2013-02-01

    Survival of head and neck cancer patients has not improved in several decades despite advances in diagnostic and therapeutic techniques. Tumor hypoxia in head and neck cancers is a critical factor that leads to poor prognosis, resistance to radiation and chemotherapies, and increased metastatic potential. Magnetic nanoparticle hyperthermia (mNPHT) is a promising therapy for hypoxic tumors because nanoparticles (NP) can be directly injected into, or targeted to, hypoxic tumor cells and exposed to alternating magnetic fields (AMF) to induce hyperthermia. Magnetic NPHT can improve therapeutic effectiveness by two modes of action: 1) direct killing of hypoxic tumor cells; and 2) increase in tumor oxygenation, which has the potential to make the tumor more susceptible to adjuvant therapies such as radiation and chemotherapy. Prior studies in breast cancer cells demonstrated that a hypoxic microenvironment diminished NP uptake in vitro; however, mNPHT with intratumoral NP injection in hypoxic tumors increased tumor oxygenation and delayed tumor growth. In this study, head and neck squamous cell carcinoma (HNSCC) cell lines were incubated in normoxic, hypoxic, and hyperoxic conditions with iron oxide NP for 4-72 hours. After incubation, the cells were analyzed for iron uptake by mass spectrometry, Prussian blue staining, and electron microscopy. In contrast to breast cancer cells, uptake of NPs was increased in hypoxic microenvironments as compared to normoxic conditions in HNSCC cells. In future studies, we will confirm the effect of the oxygen microenvironment on NP uptake and efficacy of mNPHT both in vitro and in vivo.

  5. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    Park, Jun Yong; Oser, Dorian; Iapozzuto, Peter; Norbury, Sean; Mahajan, Supriya; Khmaladze, Alexander; Sharikova, Anna

    2016-03-01

    This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug "Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

  6. Optimum 3D Matrix Stiffness for Maintenance of Cancer Stem Cells Is Dependent on Tissue Origin of Cancer Cells.

    Esmaiel Jabbari

    Full Text Available The growth and expression of cancer stem cells (CSCs depend on many factors in the tumor microenvironment. The objective of this work was to investigate the effect of cancer cells' tissue origin on the optimum matrix stiffness for CSC growth and marker expression in a model polyethylene glycol diacrylate (PEGDA hydrogel without the interference of other factors in the microenvironment.Human MCF7 and MDA-MB-231 breast carcinoma, HCT116 colorectal and AGS gastric carcinoma, and U2OS osteosarcoma cells were used. The cells were encapsulated in PEGDA gels with compressive moduli in the 2-70 kPa range and optimized cell seeding density of 0.6x106 cells/mL. Micropatterning was used to optimize the growth of encapsulated cells with respect to average tumorsphere size. The CSC sub-population of the encapsulated cells was characterized by cell number, tumorsphere size and number density, and mRNA expression of CSC markers.The optimum matrix stiffness for growth and marker expression of CSC sub-population of cancer cells was 5 kPa for breast MCF7 and MDA231, 25 kPa for colorectal HCT116 and gastric AGS, and 50 kPa for bone U2OS cells. Conjugation of a CD44 binding peptide to the gel stopped tumorsphere formation by cancer cells from different tissue origin. The expression of YAP/TAZ transcription factors by the encapsulated cancer cells was highest at the optimum stiffness indicating a link between the Hippo transducers and CSC growth. The optimum average tumorsphere size for CSC growth and marker expression was 50 μm.The marker expression results suggest that the CSC sub-population of cancer cells resides within a niche with optimum stiffness which depends on the cancer cells' tissue origin.

  7. Crosstalk between Innate Lymphoid Cells and Other Immune Cells in the Tumor Microenvironment

    Irshad, Sheeba; Gordon, Peter; Wong, Felix; Sheriff, Ibrahim; Tutt, Andrew; Ng, Tony

    2016-01-01

    Our knowledge and understanding of the tumor microenvironment (TME) have been recently expanded with the recognition of the important role of innate lymphoid cells (ILC). Three different groups of ILC have been described based on their ability to produce cytokines that mediate the interactions between innate and adaptive immune cells in a variety of immune responses in infection, allergy, and autoimmunity. However, recent evidence from experimental models and clinical studies has demonstrated that ILC contribute to the mechanisms that generate suppressive or tolerant environments that allow tumor regression or progression. Defining the complex network of interactions and crosstalk of ILC with other immune cells and understanding the specific contributions of each type of ILC leading to tumor development will allow the manipulation of their function and will be important to develop new interventions and therapeutic strategies. PMID:27882334

  8. Crosstalk between Innate Lymphoid Cells and Other Immune Cells in the Tumor Microenvironment

    Fabian Flores-Borja

    2016-01-01

    Full Text Available Our knowledge and understanding of the tumor microenvironment (TME have been recently expanded with the recognition of the important role of innate lymphoid cells (ILC. Three different groups of ILC have been described based on their ability to produce cytokines that mediate the interactions between innate and adaptive immune cells in a variety of immune responses in infection, allergy, and autoimmunity. However, recent evidence from experimental models and clinical studies has demonstrated that ILC contribute to the mechanisms that generate suppressive or tolerant environments that allow tumor regression or progression. Defining the complex network of interactions and crosstalk of ILC with other immune cells and understanding the specific contributions of each type of ILC leading to tumor development will allow the manipulation of their function and will be important to develop new interventions and therapeutic strategies.

  9. Fabrication of solution processed 3D nanostructured CuInGaS₂ thin film solar cells.

    Chu, Van Ben; Cho, Jin Woo; Park, Se Jin; Hwang, Yun Jeong; Park, Hoo Keun; Do, Young Rag; Min, Byoung Koun

    2014-03-28

    In this study we demonstrate the fabrication of CuInGaS₂ (CIGS) thin film solar cells with a three-dimensional (3D) nanostructure based on indium tin oxide (ITO) nanorod films and precursor solutions (Cu, In and Ga nitrates in alcohol). To obtain solution processed 3D nanostructured CIGS thin film solar cells, two different precursor solutions were applied to complete gap filling in ITO nanorods and achieve the desirable absorber film thickness. Specifically, a coating of precursor solution without polymer binder material was first applied to fill the gap between ITO nanorods followed by deposition of the second precursor solution in the presence of a binder to generate an absorber film thickness of ∼1.3 μm. A solar cell device with a (Al, Ni)/AZO/i-ZnO/CdS/CIGS/ITO nanorod/glass structure was constructed using the CIGS film, and the highest power conversion efficiency was measured to be ∼6.3% at standard irradiation conditions, which was 22.5% higher than the planar type of CIGS solar cell on ITO substrate fabricated using the same precursor solutions.

  10. 3D cut-cell modelling for high-resolution atmospheric simulations

    Yamazaki, H; Nikiforakis, N

    2015-01-01

    With the recent, rapid development of computer technology, the resolution of atmospheric numerical models has increased substantially. As a result, steep gradients in mountainous terrain are now being resolved in high-resolution models. This results in large truncation errors in those models using terrain-following coordinates. In this study, a new 3D Cartesian coordinate non-hydrostatic atmospheric model is developed. A cut-cell representation of topography based on finite-volume discretization is combined with a cell-merging approach, in which small cut-cells are merged with neighboring cells either vertically or horizontally. In addition, a block-structured mesh-refinement technique achieves a variable resolution on the model grid with the finest resolution occurring close to the terrain surface. The model successfully reproduces a flow over a 3D bell-shaped hill that shows a good agreement with the flow predicted by the linear theory. The ability of the model to simulate flows over steep terrain is demons...

  11. Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

    Rimann, Markus; Bono, Epifania; Annaheim, Helene; Bleisch, Matthias; Graf-Hausner, Ursula

    2016-08-01

    Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.

  12. 3D bioprinting of biomimetic aortic vascular constructs with self-supporting cells.

    Kucukgul, Can; Ozler, S Burce; Inci, Ilyas; Karakas, Ezgi; Irmak, Ster; Gozuacik, Devrim; Taralp, Alpay; Koc, Bahattin

    2015-04-01

    Cardiovascular diseases are the leading cause of deaths throughout the world. Vascular diseases are mostly treated with autografts and blood vessel transplantations. However, traditional grafting methods have several problems including lack of suitable harvest sites, additional surgical costs for harvesting procedure, pain, infection, lack of donors, and even no substitutes at all. Recently, tissue engineering and regenerative medicine approaches are used to regenerate damaged or diseased tissues. Most of the tissue engineering investigations have been based on the cell seeding into scaffolds by providing a suitable environment for cell attachment, proliferation, and differentiation. Because of the challenges such as difficulties in seeding cells spatially, rejection, and inflammation of biomaterials used, the recent tissue engineering studies focus on scaffold-free techniques. In this paper, the development of novel computer aided algorithms and methods are developed for 3D bioprinting of scaffold-free biomimetic macrovascular structures. Computer model mimicking a real human aorta is generated using imaging techniques and the proposed computational algorithms. An optimized three-dimensional bioprinting path planning are developed with the proposed self-supported model. Mouse embryonic fibroblast (MEF) cell aggregates and support structures (hydrogels) are 3D bioprinted layer-by-layer according to the proposed self-supported method to form an aortic tissue construct.

  13. An automated tool for 3D tracking of single molecules in living cells

    Gardini, L.; Capitanio, M.; Pavone, F. S.

    2015-07-01

    Recently, tremendous improvements have been achieved in the precision of localization of single fluorescent molecules, allowing localization and tracking of biomolecules at the nm level. Since the behaviour of proteins and biological molecules is tightly influenced by the cell's environment, a growing number of microscopy techniques are moving from in vitro to live cell experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). To satisfy these requirements we developed an automated routine that allow 3D tracking of single fluorescent molecules in living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.

  14. Construction of 3D micropatterned surfaces with wormlike and superhydrophilic PEG brushes to detect dysfunctional cells.

    Hou, Jianwen; Shi, Qiang; Ye, Wei; Fan, Qunfu; Shi, Hengchong; Wong, Shing-Chung; Xu, Xiaodong; Yin, Jinghua

    2014-12-10

    Detection of dysfunctional and apoptotic cells plays an important role in clinical diagnosis and therapy. To develop a portable and user-friendly platform for dysfunctional and aging cell detection, we present a facile method to construct 3D patterns on the surface of styrene-b-(ethylene-co-butylene)-b-styrene elastomer (SEBS) with poly(ethylene glycol) brushes. Normal red blood cells (RBCs) and lysed RBCs (dysfunctional cells) are used as model cells. The strategy is based on the fact that poly(ethylene glycol) brushes tend to interact with phosphatidylserine, which is in the inner leaflet of normal cell membranes but becomes exposed in abnormal or apoptotic cell membranes. We demonstrate that varied patterned surfaces can be obtained by selectively patterning atom transfer radical polymerization (ATRP) initiators on the SEBS surface via an aqueous-based method and growing PEG brushes through surface-initiated atom transfer radical polymerization. The relatively high initiator density and polymerization temperature facilitate formation of PEG brushes in high density, which gives brushes worm-like morphology and superhydrophilic property; the tendency of dysfunctional cells adhered on the patterned surfaces is completely different from well-defined arrays of normal cells on the patterned surfaces, providing a facile method to detect dysfunctional cells effectively. The PEG-patterned surfaces are also applicable to detect apoptotic HeLa cells. The simplicity and easy handling of the described technique shows the potential application in microdiagnostic devices.

  15. Stem Cell Niche, the Microenvironment and Immunological Crosstalk

    Law Sujata; S. Chaudhuri

    2008-01-01

    The concept of stem cells, their physiological existence, the intricate anatomical localization, the known and the unknown functions, and their exclusive utility for the purpose of regenerative medicine, are all now encompassed within an emergent question, 'how compatible these cells are immunologically?'Indeed, the medical aspects of stem cells are dependent on a large number of queries based on the basic properties of the cells. It has greatly been emphasized to probe into the basic research on stem cells before any successful therapeutic attempts are made. One of the intricate aspects of the adult stem cells is its immunological behavior in relation to the microenvironmental associates, the stromal ceils in the presence of a suitable target.

  16. Enabling Flexible Polymer Tandem Solar Cells by 3D Ptychographic Imaging

    Dam, Henrik Friis; Andersen, Thomas Rieks; Pedersen, Emil Bøje Lind

    2015-01-01

    one after the other by wet processing leaves plenty of room for error and the process development calls for an analytical technique that enables 3D reconstruction of the layer stack with the possibility to probe thickness, density, and chemistry of the individual layers in the stack. The use......The realization of a complete tandem polymer solar cell under ambient conditions using only printing and coating methods on a flexible substrate results in a fully scalable process but also requires accurate control during layer formation to succeed. The serial process where the layers are added...

  17. Brownian nanoimaging of interface dynamics and ligand-receptor binding at cell surfaces in 3-D.

    Kuznetsov, Igor R; Evans, Evan A

    2013-04-01

    We describe a method for nanoimaging interfacial dynamics and ligand-receptor binding at surfaces of live cells in 3-D. The imaging probe is a 1-μm diameter glass bead confined by a soft laser trap to create a "cloud" of fluctuating states. Using a facile on-line method of video image analysis, the probe displacements are reported at ~10 ms intervals with bare precisions (±SD) of 4-6 nm along the optical axis (elevation) and 2 nm in the transverse directions. We demonstrate how the Brownian distributions are analyzed to characterize the free energy potential of each small probe in 3-D taking into account the blur effect of its motions during CCD image capture. Then, using the approach to image interactions of a labeled probe with lamellae of leukocytic cells spreading on cover-glass substrates, we show that deformations of the soft distribution in probe elevations provide both a sensitive long-range sensor for defining the steric topography of a cell lamella and a fast telemetry for reporting rare events of probe binding with its surface receptors. Invoking established principles of Brownian physics and statistical thermodynamics, we describe an off-line method of super resolution that improves precision of probe separations from a non-reactive steric boundary to ~1 nm.

  18. Tuning 3D Collagen Matrix Stiffness Independently of Collagen Concentration Modulates Endothelial Cell Behavior

    Mason, Brooke N.; Starchenko, Alina; Williams, Rebecca M.; Bonassar, Lawrence J.; Reinhart-King, Cynthia A.

    2012-01-01

    Numerous studies have described the effects of matrix stiffening on cell behavior using two dimensional (2D) synthetic surfaces; however less is known about the effects of matrix stiffening on cells embedded in three dimensional (3D) in vivo-like matrices. A primary limitation in investigating the effects of matrix stiffness in 3D is the lack of materials that can be tuned to control stiffness independently of matrix density. Here, we use collagen-based scaffolds where the mechanical properties are tuned using non-enzymatic glycation of the collagen in solution, prior to polymerization. Collagen solutions glycated prior to polymerization result in collagen gels with a 3-fold increase in compressive modulus without significant changes to the collagen architecture. Using these scaffolds, we show that endothelial cell spreading increases with matrix stiffness, as does the number and length of angiogenic sprouts and the overall spheroid outgrowth. Differences in sprout length are maintained even when the receptor for advanced glycation endproducts is inhibited. Our results demonstrate the ability to de-couple matrix stiffness from matrix density and structure in collagen gels, and that increased matrix stiffness results in increased sprouting and outgrowth. PMID:22902816

  19. Scaffolds fabricated by 3D two-photon photopolymerization for live cell studies

    Teplicky, T.; Cunderlikova, B.; Mateasik, A.; Vincze, A.; Chorvat, D.; Marcek Chorvatova, A.

    2016-12-01

    Design and fabrication of appropriate biocompatible microstructures that ensure fixation and control of experimental conditions for live cell and bacteria observations is an important prerequisite for number of real time experiments. Our approach is to design engineered microfabricated 3D structures for growth of cells in culture without significant modification of their metabolic state. Presented approach is aimed at evaluation of the potential applicability of biocompatible constructs in the biomedical field and thus live cell monitoring in controlled conditions. Design and evaluation of properties of materials and structures with mesoscopic arrangement and their interaction with biological objects is a prerequisite for establishment of physiologically relevant in vitro models of pathologies as well as for development of a new generation of nano / micro / bio-sensors.

  20. AC electric field induced dipole-based on-chip 3D cell rotation.

    Benhal, Prateek; Chase, J Geoffrey; Gaynor, Paul; Oback, Björn; Wang, Wenhui

    2014-08-01

    The precise rotation of suspended cells is one of the many fundamental manipulations used in a wide range of biotechnological applications such as cell injection and enucleation in nuclear transfer (NT) cloning. Noticeably scarce among the existing rotation techniques is the three-dimensional (3D) rotation of cells on a single chip. Here we present an alternating current (ac) induced electric field-based biochip platform, which has an open-top sub-mm square chamber enclosed by four sidewall electrodes and two bottom electrodes, to achieve rotation about the two axes, thus 3D cell rotation. By applying an ac potential to the four sidewall electrodes, an in-plane (yaw) rotating electric field is generated and in-plane rotation is achieved. Similarly, by applying an ac potential to two opposite sidewall electrodes and the two bottom electrodes, an out-of-plane (pitch) rotating electric field is generated and rolling rotation is achieved. As a prompt proof-of-concept, bottom electrodes were constructed with transparent indium tin oxide (ITO) using the standard lift-off process and the sidewall electrodes were constructed using a low-cost micro-milling process and then assembled to form the chip. Through experiments, we demonstrate rotation of bovine oocytes of ~120 μm diameter about two axes, with the capability of controlling the rotation direction and the rate for each axis through control of the ac potential amplitude, frequency, and phase shift, and cell medium conductivity. The maximum observed rotation rate reached nearly 140° s⁻¹, while a consistent rotation rate reached up to 40° s⁻¹. Rotation rate spectra for zona pellucida-intact and zona pellucida-free oocytes were further compared and found to have no effective difference. This simple, transparent, cheap-to-manufacture, and open-top platform allows additional functional modules to be integrated to become a more powerful cell manipulation system.

  1. One-step fabrication of 3D silver paste electrodes into microfluidic devices for enhanced droplet-based cell sorting

    Lang Rao

    2015-05-01

    Full Text Available 3D microelectrodes are one-step fabricated into a microfluidic droplet separator by filling conductive silver paste into PDMS microchambers. The advantages of 3D silver paste electrodes in promoting droplet sorting accuracy are systematically demonstrated by theoretical calculation, numerical simulation and experimental validation. The employment of 3D electrodes also helps to decrease the droplet sorting voltage, guaranteeing that cells encapsulated in droplets undergo chip-based sorting processes are at better metabolic status for further potential cellular assays. At last, target droplet containing single cell are selectively sorted out from others by an appropriate electric pulse. This method provides a simple and inexpensive alternative to fabricate 3D electrodes, and it is expected our 3D electrode-integrated microfluidic droplet separator platform can be widely used in single cell operation and analysis.

  2. Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer.

    Augustine, Tanya N; Dix-Peek, Thérèse; Duarte, Raquel; Candy, Geoffrey P

    2015-11-01

    Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell-cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines.

  3. Accessible bioprinting: adaptation of a low-cost 3D-printer for precise cell placement and stem cell differentiation.

    Reid, John A; Mollica, Peter A; Johnson, Garett D; Ogle, Roy C; Bruno, Robert D; Sachs, Patrick C

    2016-06-07

    The precision and repeatability offered by computer-aided design and computer-numerically controlled techniques in biofabrication processes is quickly becoming an industry standard. However, many hurdles still exist before these techniques can be used in research laboratories for cellular and molecular biology applications. Extrusion-based bioprinting systems have been characterized by high development costs, injector clogging, difficulty achieving small cell number deposits, decreased cell viability, and altered cell function post-printing. To circumvent the high-price barrier to entry of conventional bioprinters, we designed and 3D printed components for the adaptation of an inexpensive 'off-the-shelf' commercially available 3D printer. We also demonstrate via goal based computer simulations that the needle geometries of conventional commercially standardized, 'luer-lock' syringe-needle systems cause many of the issues plaguing conventional bioprinters. To address these performance limitations we optimized flow within several microneedle geometries, which revealed a short tapered injector design with minimal cylindrical needle length was ideal to minimize cell strain and accretion. We then experimentally quantified these geometries using pulled glass microcapillary pipettes and our modified, low-cost 3D printer. This systems performance validated our models exhibiting: reduced clogging, single cell print resolution, and maintenance of cell viability without the use of a sacrificial vehicle. Using this system we show the successful printing of human induced pluripotent stem cells (hiPSCs) into Geltrex and note their retention of a pluripotent state 7 d post printing. We also show embryoid body differentiation of hiPSC by injection into differentiation conducive environments, wherein we observed continuous growth, emergence of various evaginations, and post-printing gene expression indicative of the presence of all three germ layers. These data demonstrate an

  4. Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

    Niu, Chengcheng; Wang, Long; Wang, Zhigang; Xu, Yan; Hu, Yihe; Peng, Qinghai

    2017-01-01

    Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work. PMID:28262671

  5. Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

    Esch, Mandy B; Prot, Jean-Matthieu; Wang, Ying I; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A; Applegate, Dawn R; Shuler, Michael L

    2015-05-21

    We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation

  6. High power density microbial fuel cell with flexible 3D graphene-nickel foam as anode

    Wang, Hanyu; Wang, Gongming; Ling, Yichuan; Qian, Fang; Song, Yang; Lu, Xihong; Chen, Shaowei; Tong, Yexiang; Li, Yat

    2013-10-01

    The structure and electrical conductivity of anode play a significant role in the power generation of microbial fuel cells (MFCs). In this study, we developed a three-dimensional (3D) reduced graphene oxide-nickel (denoted as rGO-Ni) foam as an anode for MFC through controlled deposition of rGO sheets onto the nickel foam substrate. The loading amount of rGO sheets and electrode surface area can be controlled by the number of rGO loading cycles. 3D rGO-Ni foam anode provides not only a large accessible surface area for microbial colonization and electron mediators, but also a uniform macro-porous scaffold for effective mass diffusion of the culture medium. Significantly, at a steady state of the power generation, the MFC device with flexible rGO-Ni electrodes produced an optimal volumetric power density of 661 W m-3 calculated based on the volume of anode material, or 27 W m-3 based on the volume of the anode chamber. These values are substantially higher than that of plain nickel foam, and other conventional carbon based electrodes (e.g., carbon cloth, carbon felt, and carbon paper) measured in the same conditions. To our knowledge, this is the highest volumetric power density reported for mL-scale MFC device with a pure strain of Shewanella oneidensis MR-1. We also demonstrated that the MFC device can be operated effectively in a batch-mode at least for a week. These new 3D rGO-Ni electrodes show great promise for improving the power generation of MFC devices.The structure and electrical conductivity of anode play a significant role in the power generation of microbial fuel cells (MFCs). In this study, we developed a three-dimensional (3D) reduced graphene oxide-nickel (denoted as rGO-Ni) foam as an anode for MFC through controlled deposition of rGO sheets onto the nickel foam substrate. The loading amount of rGO sheets and electrode surface area can be controlled by the number of rGO loading cycles. 3D rGO-Ni foam anode provides not only a large accessible

  7. Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity.

    Blaeser, Andreas; Duarte Campos, Daniela Filipa; Puster, Uta; Richtering, Walter; Stevens, Molly M; Fischer, Horst

    2016-02-04

    A microvalve-based bioprinting system for the manufacturing of high-resolution, multimaterial 3D-structures is reported. Applying a straightforward fluid-dynamics model, the shear stress at the nozzle site can precisely be controlled. Using this system, a broad study on how cell viability and proliferation potential are affected by different levels of shear stress is conducted. Complex, multimaterial 3D structures are printed with high resolution. This work pioneers the investigation of shear stress-induced cell damage in 3D bioprinting and might help to comprehend and improve the outcome of cell-printing studies in the future.

  8. Crosstalk between tumor cells and microenvironment via Wnt pathway in colorectal cancer dissemination

    Dan Huang; Xiang Du

    2008-01-01

    Invasion and metastasis are the deadly face of malignant tumors. Considering the high rate of incidence and mortality of colorectal cancer, it is critical to determine the mechanisms of its dissemination. In the parallel investigation of the invasive front and tumor center area of colorectal cancer (CRC), observation of heterogeneous p-catenin distribution and epithelial-mesenchymal transition (EMT) at the invasive front suggested that there might be a crosstalk between tumor cells and the tumor microenvironment. Wnt signaling pathway is also involved in the cancer progression due to its key role in CRC tumorigenesis. Moreover, in recent years, there is increasing evidence that the regulators of microenvironment, including extracellular matrix, growth factors and inflammatory factors, are associated with the activation of Wnt pathway and the mobility of tumor cells. In this review, we will try to explain how these molecules trigger metastasis via the Wnt pathway.

  9. Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

    Neves, Sara C; Mota, Carlos; Longoni, Alessia; Barrias, Cristina C; Granja, Pedro L; Moroni, Lorenzo

    2016-05-24

    Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 μm to 1.950 ± 0.553 μm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 μm and 396.1 ± 32.3 μm, in the z-axis between 36.5 ± 5.3 μm and 70.7 ± 8.8 μm, average fiber diameters between 69.4 ± 6.1 μm and 99.0 ± 9.4 μm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 μm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 μm).

  10. Dynamic 3D cell rearrangements guided by a fibronectin matrix underlie somitogenesis.

    Gabriel G Martins

    Full Text Available Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.

  11. Characterization of Porcine Ventral Mesencephalic Precursor Cells following Long-Term Propagation in 3D Culture

    Pia S. Jensen

    2012-01-01

    Full Text Available The potential use of predifferentiated neural precursor cells for treatment of a neurological disorder like Parkinson’s disease combines stem cell research with previous experimental and clinical transplantation of developing dopaminergic neurons. One current obstacle is, however, the lack of ability to generate dopaminergic neurons after long-term in vitro propagation of the cells. The domestic pig is considered a useful nonprimate large animal model in neuroscience, because of a better resemblance of the larger gyrencephalic pig brain to the human brain than the commonly used brains of smaller rodents. In the present study, porcine embryonic (28–30 days, ventral mesencephalic precursor cells were isolated and propagated as free-floating neural tissue spheres in medium containing epidermal growth factor and fibroblast growth factor 2. For passaging, the tissue spheres were cut into quarters, avoiding mechanical or enzymatic dissociation in order to minimize cellular trauma and preserve intercellular contacts. Spheres were propagated for up to 237 days with analysis of cellular content and differentiation at various time points. Our study provides the first demonstration that porcine ventral mesencephalic precursor cells can be long-term propagated as neural tissue spheres, thereby providing an experimental 3D in vitro model for studies of neural precursor cells, their niche, and differentiation capacity.

  12. Bone Marrow Cells in Acute Lymphoblastic Leukemia Create a Proinflammatory Microenvironment Influencing Normal Hematopoietic Differentiation Fates

    2015-01-01

    B-cell acute lymphoblastic leukemia (B-ALL) is a serious public health problem in the pediatric population worldwide, contributing to 85% of deaths from childhood cancers. Understanding the biology of the disease is crucial for its clinical management and the development of therapeutic strategies. In line with that observed in other malignancies, chronic inflammation may contribute to a tumor microenvironment resulting in the damage of normal processes, concomitant to development and maintena...

  13. Concise Review: Stem Cell Microenvironment on a Chip: Current Technologies for Tissue Engineering and Stem Cell Biology

    Park, DoYeun; Lim, Jaeho; Park, Joong Yull

    2015-01-01

    Stem cells have huge potential in many therapeutic areas. With conventional cell culture methods, however, it is difficult to achieve in vivo-like microenvironments in which a number of well-controlled stimuli are provided for growing highly sensitive stem cells. In contrast, microtechnology-based platforms offer advantages of high precision, controllability, scalability, and reproducibility, enabling imitation of the complex physiological context of in vivo. This capability may fill the gap between the present knowledge about stem cells and that required for clinical stem cell-based therapies. We reviewed the various types of microplatforms on which stem cell microenvironments are mimicked. We have assigned the various microplatforms to four categories based on their practical uses to assist stem cell biologists in using them for research. In particular, many examples are given of microplatforms used for the production of embryoid bodies and aggregates of stem cells in vitro. We also categorized microplatforms based on the types of factors controlling the behaviors of stem cells. Finally, we outline possible future directions for microplatform-based stem cell research, such as research leading to the production of well-defined environments for stem cells to be used in scaled-up systems or organs-on-a-chip, the regulation of induced pluripotent stem cells, and the study of the genetic states of stem cells on microplatforms. Significance Stem cells are highly sensitive to a variety of physicochemical cues, and their fate can be easily altered by a slight change of environment; therefore, systematic analysis and discrimination of the extracellular signals and intracellular pathways controlling the fate of cells and experimental realization of sensitive and controllable niche environments are critical. This review introduces diverse microplatforms to provide in vitro stem cell niches. Microplatforms could control microenvironments around cells and have recently

  14. Redirection of Human Cancer Cells upon the Interaction with the Regenerating Mouse Mammary Gland Microenvironment

    Sonia M. Rosenfield

    2013-01-01

    Full Text Available Tumorigenesis is often described as a result of accumulated mutations that lead to growth advantage and clonal expansion of mutated cells. There is evidence in the literature that cancer cells are influenced by the microenvironment. Our previous studies demonstrated that the mouse mammary gland is capable of redirecting mouse cells of non-mammary origins as well as Mouse Mammary Tumor Virus (MMTV-neu transformed cells toward normal mammary epithelial cell fate during gland regeneration. Interestingly, the malignant phenotype of MMTV-neu transformed cells was suppressed during serial transplantation experiments. Here, we discuss our studies that demonstrated the potential of the regenerating mouse mammary gland to redirect cancer cells of different species into a functional tumor-free mammary epithelial cell progeny. Immunochemistry for human specific CD133, mitochondria, cytokeratins as well as milk proteins and FISH for human specific probe identified human epithelial cell progeny in ducts, lobules, and secretory acini. Fluorescent In Situ Hybridization (FISH for human centromeric DNA and FACS analysis of propidium iodine staining excluded the possibility of mouse-human cell fusion. To our knowledge this is the first evidence that human cancer cells of embryonic or somatic origins respond to developmental signals generated by the mouse mammary gland microenvironment during gland regeneration in vivo.

  15. How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication?

    Dostert, Gabriel; Mesure, Benjamin; Menu, Patrick; Velot, Émilie

    2017-01-01

    Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal, their multipotency, and their immunomodulatory properties that make them an attractive tool for regenerative medicine. A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types. Extracellular vesicles (EVs) belong to cellular secretions. They are produced by cells continuously or after stimulation (e.g., calcium flux, cellular stress) and act in tissue homeostasis and intercellular communication. The understanding of the role of EVs is growing, more particularly their impact on cell migration, differentiation, or immunomodulation. EVs derived from MSCs show these interesting properties that may be considered in therapeutics, although they can have adverse effects by facilitating cancer propagation. Moreover, MSC behavior may also be influenced (proliferation, differentiation) by EVs derived from other donor cells. The aim of this mini review is to summarize the two-way communication between MSCs and other cell types, and how they can affect each other with their microenvironment through EVs. On the one hand, the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand, the effects of EVs derived from various donor cells on MSCs. The discrepancies between cancer cells and MSCs communication according to the sources of MSCs but also the tumor origins are also mentioned. PMID:28224125

  16. A MULTISCALE APPROACH TO THE REPRESENTATION OF 3D IMAGES, WITH APPLICATION TO POLYMER SOLAR CELLS

    Ralf Thiedmann

    2011-03-01

    Full Text Available A multiscale approach to the description of geometrically complex 3D image data is proposed which distinguishes between morphological features on a ‘macro-scale’ and a ‘micro-scale’. Since our method is mainly tailored to nanostructures observed in composite materials consisting of two different phases, an appropriate binarization of grayscale images is required first. Then, a morphological smoothing is applied to extract the structural information from binarized image data on the ‘macro-scale’. A stochastic algorithm is developed for the morphologically smoothed images whose goal is to find a suitable representation of the macro-scale structure by unions of overlapping spheres. Such representations can be interpreted as marked point patterns. They lead to an enormous reduction of data and allow the application of well-known tools from point-process theory for their analysis and structural modeling. All those voxels which have been ‘misspecified’ by the morphological smoothing and subsequent representation by unions of overlapping spheres are interpreted as ‘micro-scale’ structure. The exemplary data sets considered in this paper are 3D grayscale images of photoactive layers in hybrid solar cells gained by electron tomography. These composite materials consist of two phases: a polymer phase and a zinc oxide phase. The macro-scale structure of the latter is represented by unions of overlapping spheres.

  17. A miniature microbial fuel cell with conducting nanofibers-based 3D porous biofilm

    Jiang, Huawei; Halverson, Larry J.; Dong, Liang

    2015-12-01

    Miniature microbial fuel cell (MFC) technology has received growing interest due to its potential applications in high-throughput screening of bacteria and mutants to elucidate mechanisms of electricity generation. This paper reports a novel miniature MFC with an improved output power density and short startup time, utilizing electrospun conducting poly(3,4-ethylenedioxythiophene) (PEDOT) nanofibers as a 3D porous anode within a 12 μl anolyte chamber. This device results in 423 μW cm-3 power density based on the volume of the anolyte chamber, using Shewanella oneidensis MR-1 as a model biocatalyst without any optimization of bacterial culture. The device also excels in a startup time of only 1hr. The high conductivity of the electrospun nanofibers makes them suitable for efficient electron transfer. The mean pore size of the conducting nanofibers is several micrometers, which is favorable for bacterial penetration and colonization of surfaces of the nanofibers. We demonstrate that S. oneidensis can fully colonize the interior region of this nanofibers-based porous anode. This work represents a new attempt to explore the use of electrospun PEDOT nanofibers as a 3D anode material for MFCs. The presented miniature MFC potentially will provide a high-sensitivity, high-throughput tool to screen suitable bacterial species and mutant strains for use in large-size MFCs.

  18. Pico-projector-based optical sectioning microscopy for 3D chlorophyll fluorescence imaging of mesophyll cells

    Chen, Szu-Yu; Hsu, Yu John; Yeh, Chia-Hua; Chen, S.-Wei; Chung, Chien-Han

    2015-03-01

    A pico-projector-based optical sectioning microscope (POSM) was constructed using a pico-projector to generate structured illumination patterns. A net rate of 5.8 × 106 pixel/s and sub-micron spatial resolution in three-dimensions (3D) were achieved. Based on the pico-projector’s flexibility in pattern generation, the characteristics of POSM with different modulation periods and at different imaging depths were measured and discussed. With the application of different modulation periods, 3D chlorophyll fluorescence imaging of mesophyll cells was carried out in freshly plucked leaves of four species without sectioning or staining. For each leaf, an average penetration depth of 120 μm was achieved. Increasing the modulation period along with the increment of imaging depth, optical sectioning images can be obtained with a compromise between the axial resolution and signal-to-noise ratio. After ∼30 min imaging on the same area, photodamage was hardly observed. Taking the advantages of high speed and low damages of POSM, the investigation of the dynamic fluorescence responses to temperature changes was performed under three different treatment temperatures. The three embedded blue, green and red light-emitting diode light sources were applied to observe the responses of the leaves with different wavelength excitation.

  19. 2D/3D perovskite hybrids as moisture-tolerant and efficient light absorbers for solar cells.

    Ma, Chaoyan; Leng, Chongqian; Ji, Yixiong; Wei, Xingzhan; Sun, Kuan; Tang, Linlong; Yang, Jun; Luo, Wei; Li, Chaolong; Deng, Yunsheng; Feng, Shuanglong; Shen, Jun; Lu, Shirong; Du, Chunlei; Shi, Haofei

    2016-11-03

    The lifetime and power conversion efficiency are the key issues for the commercialization of perovskite solar cells (PSCs). In this paper, the development of 2D/3D perovskite hybrids (CA2PbI4/MAPbIxCl3-x) was firstly demonstrated to be a reliable method to combine their advantages, and provided a new concept for achieving both stable and efficient PSCs through the hybridization of perovskites. 2D/3D perovskite hybrids afforded significantly-improved moisture stability of films and devices without encapsulation in a high humidity of 63 ± 5%, as compared with the 3D perovskite (MAPbIxCl3-x). The 2D/3D perovskite-hybrid film did not undergo any degradation after 40 days, while the 3D perovskite decomposed completely under the same conditions after 8 days. The 2D/3D perovskite-hybrid device maintained 54% of the original efficiency after 220 hours, whereas the 3D perovskite device lost all the efficiency within only 50 hours. Moreover, the 2D/3D perovskite hybrid achieved comparable device performances (PCE: 13.86%) to the 3D perovskite (PCE: 13.12%) after the optimization of device fabrication conditions.

  20. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity.

    Chih-Hao Chang

    Full Text Available Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP techniques. A self-developed 3D printer with laser-aided gelling (LAG process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w. Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity.

  1. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity.

    Chang, Chih-Hao; Lin, Chih-Yang; Liu, Fwu-Hsing; Chen, Mark Hung-Chih; Lin, Chun-Pin; Ho, Hong-Nerng; Liao, Yunn-Shiuan

    2015-01-01

    Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP) techniques. A self-developed 3D printer with laser-aided gelling (LAG) process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w). Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity.

  2. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity

    Chang, Chih-Hao; Lin, Chih-Yang; Liu, Fwu-Hsing; Chen, Mark Hung-Chih; Lin, Chun-Pin; Ho, Hong-Nerng; Liao, Yunn-Shiuan

    2015-01-01

    Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP) techniques. A self-developed 3D printer with laser-aided gelling (LAG) process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w). Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity. PMID:26618362

  3. Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis.

    Barott, Katie L; Venn, Alexander A; Perez, Sidney O; Tambutté, Sylvie; Tresguerres, Martin

    2015-01-13

    Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

  4. Label-free optical detection of cells grown in 3D silicon microstructures.

    Merlo, Sabina; Carpignano, Francesca; Silva, Gloria; Aredia, Francesca; Scovassi, A Ivana; Mazzini, Giuliano; Surdo, Salvatore; Barillaro, Giuseppe

    2013-08-21

    We demonstrate high aspect-ratio photonic crystals that could serve as three-dimensional (3D) microincubators for cell culture and also provide label-free optical detection of the cells. The investigated microstructures, fabricated by electrochemical micromachining of standard silicon wafers, consist of periodic arrays of silicon walls separated by narrow deeply etched air-gaps (50 μm high and 5 μm wide) and feature the typical spectral properties of photonic crystals in the wavelength range 1.0-1.7 μm: their spectral reflectivity is characterized by wavelength regions where reflectivity is high (photonic bandgaps), separated by narrow wavelength regions where reflectivity is very low. In this work, we show that the presence of cells, grown inside the gaps, strongly affects light propagation across the photonic crystal and, therefore, its spectral reflectivity. Exploiting a label-free optical detection method, based on a fiberoptic setup, we are able to probe the extension of cells adherent to the vertical silicon walls with a non-invasive direct testing. In particular, the intensity ratio at two wavelengths is the experimental parameter that can be well correlated to the cell spreading on the silicon wall inside the gaps.

  5. Human pluripotent stem cells on artificial microenvironments: a high content perspective.

    Priyalakshmi eViswanathan

    2014-07-01

    Full Text Available Self-renewing stem cell populations are increasingly considered as resources for cell therapy and tools for drug discovery. Human pluripotent stem (hPS cells in particular offer a virtually unlimited reservoir of homogeneous cells and can be differentiated towards diverse lineages. Many diseases show impairment in self-renewal or differentiation, abnormal lineage choice or other aberrant cell behavior in response to chemical or physical cues. To investigate these responses, there is a growing interest in the development of specific assays using hPS cells artificial microenvironments and high content analysis. Several hurdles need to be overcome that can be grouped in: (i availability of robust, homogeneous and consistent cell populations as a starting point; (ii appropriate understanding and use of chemical and physical microenvironments; (iii development of assays that dissect the complexity of cell populations in tissues while mirroring specific aspects of their behavior. Here we review recent progress in the culture of hPS cells and we detail the importance of the environment surrounding the cells with a focus on synthetic material and suitable high content analysis approaches. The technologies described if properly combined have the potential to create a paradigm shift in the way diseases are modelled and drug discovery is performed.

  6. Altering the Microenvironment to Promote Dormancy of Metastatic Breast Cancer Cell in a 3D Bone Culture System

    2015-12-01

    1138-1148. Carlsten, H. 2005. Immune responses and bone loss: the estrogen connection. Immunological reviews . 208:194- 206. 28 Gehler, S., S.M...factors to be considered in cancer recurrence ( reviewed by [6]). Das Roy et al. found an increase in lung and bone marrow metastasis using an...activation of COX-2, plays an important role in normal bone physiology as well as in cancer and bone metastasis ( review [43]). In the normal bone, PGE2 is the

  7. Identifying same-cell contours in image stacks: a key step in making 3D reconstructions.

    Leung, Tony Kin Shun; Veldhuis, Jim H; Krens, S F Gabby; Heisenberg, C P; Brodland, G Wayne

    2011-02-01

    Identification of contours belonging to the same cell is a crucial step in the analysis of confocal stacks and other image sets in which cell outlines are visible, and it is central to the making of 3D cell reconstructions. When the cells are close packed, the contour grouping problem is more complex than that found in medical imaging, for example, because there are multiple regions of interest, the regions are not separable from each other by an identifiable background and regions cannot be distinguished by intensity differences. Here, we present an algorithm that uses three primary metrics-overlap of contour areas in adjacent images, co-linearity of the centroids of these areas across three images in a stack, and cell taper-to assign cells to groups. Decreasing thresholds are used to successively assign contours whose membership is less obvious. In a final step, remaining contours are assigned to existing groups by setting all thresholds to zero and groups having strong hour-glass shapes are partitioned. When applied to synthetic data from isotropic model aggregates, a curved model epithelium in which the long axes of the cells lie at all possible angles to the transection plane, and a confocal image stack, algorithm assignments were between 97 and 100% accurate in sets having at least four contours per cell. The algorithm is not particularly sensitive to the thresholds used, and a single set of parameters was used for all of the tests. The algorithm, which could be extended to time-lapse data, solves a key problem in the translation of image data into cell information.

  8. 3D differentiation of neural stem cells in macroporous photopolymerizable hydrogel scaffolds.

    Hang Li

    Full Text Available Neural stem/progenitor cells (NSPCs are the stem cell of the adult central nervous system (CNS. These cells are able to differentiate into the major cell types found in the CNS (neurons, oligodendrocytes, astrocytes, thus NSPCs are the mechanism by which the adult CNS could potentially regenerate after injury or disorder. Microenviromental factors are critical for guiding NSPC differentiation and are thus important for neural tissue engineering. In this study, D-mannitol crystals were mixed with photocrosslinkable methacrylamide chitosan (MAC as a porogen to enhance pore size during hydrogel formation. D-mannitol was admixed to MAC at 5, 10 and 20 wt% D-mannitol per total initial hydrogel weight. D-mannitol crystals were observed to dissolve and leave the scaffold within 1 hr. Quantification of resulting average pore sizes showed that D-mannitol addition resulted in larger average pore size (5 wt%, 4060±160 µm(2, 10 wt%, 6330±1160 µm(2, 20 wt%, 7600±1550 µm(2 compared with controls (0 wt%, 3150±220 µm(2. Oxygen diffusion studies demonstrated that larger average pore area resulted in enhanced oxygen diffusion through scaffolds. Finally, the differentiation responses of NSPCs to phenotypic differentiation conditions were studied for neurons, astrocytes and oligodendrocytes in hydrogels of varied porosity over 14 d. Quantification of total cell numbers at day 7 and 14, showed that cell numbers decreased with increased porosity and over the length of the culture. At day 14 immunohistochemistry quantification for primary cell types demonstrated significant differentiation to the desired cells types, and that total percentages of each cell type was greatest when scaffolds were more porous. These results suggest that larger pore sizes in MAC hydrogels effectively promote NSPC 3D differentiation.

  9. SURVIVAL OF LIVER CELLS, IMMOBILIZED ON 3D-MATRIXES, IN LIVER FAILURE MODEL

    M. Y. Shagidulin

    2011-01-01

    Full Text Available It was examined a new method for correction of hepatic failure by transplantation of liver support biounit (liver cells, immobilized on biocompatible and biodegradable 3D-matrixes ElastoPOB® into small intestine mesentery. It was determined that after modeling of acute hepatic failure on dogs by 65–70% liver resection and transplantation liver support biounit the restoration of disturbed biochemical indecies (such as total protein, lactate, cytolytic ensymes-ALT, AST, ALP, LDH, fibrinogen, protrombine index and others took place more rapidly on 9–14th day instead of 18th day in control. It was made a preposition about efficiency of the suggested method for correction both acute hepatic failure because even 90 days after transplantation of liver support biounit alive hepatocytes and neogenic plethoric vessels, growing through matrix were revealed. 

  10. Immersed Boundary Models for Quantifying Flow-Induced Mechanical Stimuli on Stem Cells Seeded on 3D Scaffolds in Perfusion Bioreactors

    Smeets, Bart; Odenthal, Tim; Luyten, Frank P.; Ramon, Herman; Papantoniou, Ioannis; Geris, Liesbet

    2016-01-01

    Perfusion bioreactors regulate flow conditions in order to provide cells with oxygen, nutrients and flow-associated mechanical stimuli. Locally, these flow conditions can vary depending on the scaffold geometry, cellular confluency and amount of extra cellular matrix deposition. In this study, a novel application of the immersed boundary method was introduced in order to represent a detailed deformable cell attached to a 3D scaffold inside a perfusion bioreactor and exposed to microscopic flow. The immersed boundary model permits the prediction of mechanical effects of the local flow conditions on the cell. Incorporating stiffness values measured with atomic force microscopy and micro-flow boundary conditions obtained from computational fluid dynamics simulations on the entire scaffold, we compared cell deformation, cortical tension, normal and shear pressure between different cell shapes and locations. We observed a large effect of the precise cell location on the local shear stress and we predicted flow-induced cortical tensions in the order of 5 pN/μm, at the lower end of the range reported in literature. The proposed method provides an interesting tool to study perfusion bioreactors processes down to the level of the individual cell’s micro-environment, which can further aid in the achievement of robust bioprocess control for regenerative medicine applications. PMID:27658116

  11. Self-Organization of Polarized Cerebellar Tissue in 3D Culture of Human Pluripotent Stem Cells

    Keiko Muguruma

    2015-02-01

    Full Text Available During cerebellar development, the main portion of the cerebellar plate neuroepithelium gives birth to Purkinje cells and interneurons, whereas the rhombic lip, the germinal zone at its dorsal edge, generates granule cells and cerebellar nuclei neurons. However, it remains elusive how these components cooperate to form the intricate cerebellar structure. Here, we found that a polarized cerebellar structure self-organizes in 3D human embryonic stem cell (ESC culture. The self-organized neuroepithelium differentiates into electrophysiologically functional Purkinje cells. The addition of fibroblast growth factor 19 (FGF19 promotes spontaneous generation of dorsoventrally polarized neural-tube-like structures at the level of the cerebellum. Furthermore, addition of SDF1 and FGF19 promotes the generation of a continuous cerebellar plate neuroepithelium with rhombic-lip-like structure at one end and a three-layer cytoarchitecture similar to the embryonic cerebellum. Thus, human-ESC-derived cerebellar progenitors exhibit substantial self-organizing potential for generating a polarized structure reminiscent of the early human cerebellum at the first trimester.

  12. Using Polymer Confinement for Stem Cell Differentiation: 3D Printed vs Molded Scaffolds

    Rafailovich, Miriam

    Additive manufacturing technologies are increasingly being used to replace standard extrusion or molding methods in engineering polymeric biomedical implants, which can be further seeded with cells for tissue regeneration. The principal advantage of this new technology is the ability to print directly from a scan and hence produce parts which are an ideal fit for an individual, eliminating much of the sizing and fitting associated with standard manufacturing methods. The question though arises whether devices which may be macroscopically similar, serve identical functions and are produced from the same material, interact in the same manner with cells and living tissue. Here we show that fundamental differences can exist between 3-D printed and extruded scaffolds which can impact stem cell differentiation and lineage selection. We will show how polymer confinement inherent in these methods affect the printed features on multiple length scales. We will also and how the differentiation of stem cells is affected by substrate heterogeneity in both morphological and mechanical features. NSF-Inspire award # 1344267.

  13. A 3D model for PEM fuel cells operated on reformate

    Zhou, Tianhong; Liu, Hongtan

    A three-dimensional mathematical model for PEM fuel cells operated on reformate is developed based on our previous established fuel cell model [Int. J. Transport Phenomena 3 (2001) 177], by incorporating the adsorption and oxidation kinetics of CO on platinum surface proposed by Springer et al. [Proceedings of the Electrochemical Society, Montreal, Canada, 1997; J. Electrochem. Soc. 148 (2001) A11]. This model is capable of studying the effect of CO poisoning as well as the hydrogen dilution effect by inert gases. The adsorption and oxidation kinetics of CO on a platinum surface are incorporated in the source terms of the species equations; thus, the basic form of the mathematical equations are the same as those used for PEM fuel cells operated on pure hydrogen. With this model, we can obtain detailed information on the CO poisoning and variation of CO and hydrogen concentrations inside the anode. The results from this 3D model reveal many new phenomena that cannot be obtained from previous 1D or 2D models. Results of the effects of various operating and design parameters, such as anode flow rate, gas diffuser porosity, gas diffuser thickness, and the width of the collector plate shoulder, are also presented. The modeling results demonstrate the value of this model as a design and optimization tool for the anode of PEM fuel cells operating on reformate.

  14. Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells

    Nair MS

    2015-05-01

    Full Text Available Maya S Nair,1 Ullas Mony,1 Deepthy Menon,1 Manzoor Koyakutty,1 Neeraj Sidharthan,2 Keechilat Pavithran,2 Shantikumar V Nair,1 Krishnakumar N Menon11Amrita Centre for Nanosciences and Molecular Medicine, 2Department of Oncology, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kerala, IndiaAbstract: Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU/poly-L-lactic acid (PLLA in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34+/CD38-/CD33- phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27Kip1leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27

  15. Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells

    Alain Carpentier

    2012-06-01

    Full Text Available Electrostimulation (ES can be defined as a safe physical method to induce stem cell differentiation. The aim of this study is to evaluate the effectiveness of ES on bone marrow mesenchymal stem cells (BMSCs seeded in collagen scaffolds in terms of proliferation and differentiation into cardiomyocytes. BMSCs were isolated from Wistar rats and seeded into 3D collagen type 1 templates measuring 25 x 25 x 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical impedance and cell proliferation were measured. Expression of cardiac markers was assessed by immunocytochemistry. Viscoelasticity of collagen matrix was evaluated. Electrical impedance assessments showed a low resistance of 234±41 Ohms which indicates good electrical conductivity of collagen matrix. Cell proliferation at 570 nm as significantly increased in ES groups after seven day (ES 0.129±0.03 vs non-stimulated control matrix 0.06±0.01, P=0.002 and after 21 days, (ES 0.22±0.04 vs control 0.13±0.01, P=0.01. Immunocytochemistry of BMSCs after 21 days ES showed positive staining of cardiac markers, troponin I, connexin 43, sarcomeric alpha-actinin, slow myosin, fast myosin and desmin. Staining for BMSCs marker CD29 after 21 days was negative. Electrostimulation of cell-seeded collagen matrix changed stem cell morphology and bio- chemical characteristics, increasing the expression of cardiac markers. Thus, MSC-derived differentiated cells by electrostimulation grafted in biological scaffolds might result in a convenient tissue engineering source for myocardial diseases.

  16. Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells.

    Haneef, Kanwal; Lila, Nermine; Benadda, Samira; Legrand, Fabien; Carpentier, Alain; Chachques, Juan C

    2012-06-01

    Electrostimulation (ES) can be defined as a safe physical method to induce stem cell differentiation. The aim of this study is to evaluate the effectiveness of ES on bone marrow mesenchymal stem cells (BMSCs) seeded in collagen scaffolds in terms of proliferation and differentiation into cardiomyocytes. BMSCs were isolated from Wistar rats and seeded into 3D collagen type 1 templates measuring 25 × 25 × 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical impedance and cell proliferation were measured. Expression of cardiac markers was assessed by immunocytochemistry. Viscoelasticity of collagen matrix was evaluated. Electrical impedance assessments showed a low resistance of 234±41 Ohms which indicates good electrical conductivity of collagen matrix. Cell proliferation at 570 nm as significantly increased in ES groups after seven day (ES 0.129±0.03 vs non-stimulated control matrix 0.06±0.01, P=0.002) and after 21 days, (ES 0.22±0.04 vs control 0.13±0.01, P=0.01). Immunocytoche mistry of BMSCs after 21 days ES showed positive staining of cardiac markers, troponin I, connexin 43, sarcomeric alpha-actinin, slow myosin, fast myosin and desmin. Staining for BMSCs marker CD29 after 21 days was negative. Electrostimulation of cell-seeded collagen matrix changed stem cell morphology and biochemical characteristics, increasing the expression of cardiac markers. Thus, MSC-derived differentiated cells by electrostimulation grafted in biological scaffolds might result in a convenient tissue engineering source for myocardial diseases.

  17. Human Pluripotent Stem Cell Mechanobiology: Manipulating the Biophysical Microenvironment for Regenerative Medicine and Tissue Engineering Applications.

    Ireland, Ronald G; Simmons, Craig A

    2015-11-01

    A stem cell in its microenvironment is subjected to a myriad of soluble chemical cues and mechanical forces that act in concert to orchestrate cell fate. Intuitively, many of these soluble and biophysical factors have been the focus of intense study to successfully influence and direct cell differentiation in vitro. Human pluripotent stem cells (hPSCs) have been of considerable interest in these studies due to their great promise for regenerative medicine. Culturing and directing differentiation of hPSCs, however, is currently extremely labor-intensive and lacks the efficiency required to generate large populations of clinical-grade cells. Improved efficiency may come from efforts to understand how the cell biophysical signals can complement biochemical signals to regulate cell pluripotency and direct differentiation. In this concise review, we explore hPSC mechanobiology and how the hPSC biophysical microenvironment can be manipulated to maintain and differentiate hPSCs into functional cell types for regenerative medicine and tissue engineering applications.

  18. Cancer Microenvironment: What Can We Learn from the Stem Cell Niche.

    Lacina, Lukas; Plzak, Jan; Kodet, Ondrej; Szabo, Pavol; Chovanec, Martin; Dvorankova, Barbora; Smetana, Karel

    2015-10-12

    Epidermal stem cells (ESCs) are crucial for maintenance and self- renewal of skin epithelium and also for regular hair cycling. Their role in wound healing is also indispensable. ESCs reside in a defined outer root sheath portion of hair follicle-also known as the bulge region. ECS are also found between basal cells of the interfollicular epidermis or mucous membranes. The non-epithelial elements such as mesenchymal stem cell-like elements of dermis or surrounding adipose tissue can also contribute to this niche formation. Cancer stem cells (CSCs) participate in formation of common epithelial malignant diseases such as basal cell or squamous cell carcinoma. In this review article, we focus on the role of cancer microenvironment with emphasis on the effect of cancer-associated fibroblasts (CAFs). This model reflects various biological aspects of interaction between cancer cell and CAFs with multiple parallels to interaction of normal epidermal stem cells and their niche. The complexity of intercellular interactions within tumor stroma is depicted on example of malignant melanoma, where keratinocytes also contribute the microenvironmental landscape during early phase of tumor progression. Interactions seen in normal bulge region can therefore be an important source of information for proper understanding to melanoma. The therapeutic consequences of targeting of microenvironment in anticancer therapy and for improved wound healing are included to article.

  19. Cancer Microenvironment: What Can We Learn from the Stem Cell Niche

    Lukas Lacina

    2015-10-01

    Full Text Available Epidermal stem cells (ESCs are crucial for maintenance and self- renewal of skin epithelium and also for regular hair cycling. Their role in wound healing is also indispensable. ESCs reside in a defined outer root sheath portion of hair follicle—also known as the bulge region. ECS are also found between basal cells of the interfollicular epidermis or mucous membranes. The non-epithelial elements such as mesenchymal stem cell-like elements of dermis or surrounding adipose tissue can also contribute to this niche formation. Cancer stem cells (CSCs participate in formation of common epithelial malignant diseases such as basal cell or squamous cell carcinoma. In this review article, we focus on the role of cancer microenvironment with emphasis on the effect of cancer-associated fibroblasts (CAFs. This model reflects various biological aspects of interaction between cancer cell and CAFs with multiple parallels to interaction of normal epidermal stem cells and their niche. The complexity of intercellular interactions within tumor stroma is depicted on example of malignant melanoma, where keratinocytes also contribute the microenvironmental landscape during early phase of tumor progression. Interactions seen in normal bulge region can therefore be an important source of information for proper understanding to melanoma. The therapeutic consequences of targeting of microenvironment in anticancer therapy and for improved wound healing are included to article.

  20. Improved Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation.

    Zhou, Xuan; Castro, Nathan J; Zhu, Wei; Cui, Haitao; Aliabouzar, Mitra; Sarkar, Kausik; Zhang, Lijie Grace

    2016-09-06

    3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm(2) intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

  1. Improved Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation

    Zhou, Xuan; Castro, Nathan J.; Zhu, Wei; Cui, Haitao; Aliabouzar, Mitra; Sarkar, Kausik; Zhang, Lijie Grace

    2016-01-01

    3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application. PMID:27597635

  2. Generation of Multilayered 3D Structures of HepG2 Cells Using a Bio-printing Technique

    Jeon, Hyeryeon; Kang, Kyojin; Park, Su A; Kim, Wan Doo; Paik, Seung Sam; Lee, Sang-Hun; Jeong, Jaemin; Choi, Dongho

    2017-01-01

    Background/Aims Chronic liver disease is a major widespread cause of death, and whole liver transplantation is the only definitive treatment for patients with end-stage liver diseases. However, many problems, including donor shortage, surgical complications and cost, hinder their usage. Recently, tissue-engineering technology provided a potential breakthrough for solving these problems. Three-dimensional (3D) printing technology has been used to mimic tissues and organs suitable for transplantation, but applications for the liver have been rare. Methods A 3D bioprinting system was used to construct 3D printed hepatic structures using alginate. HepG2 cells were cultured on these 3D structures for 3 weeks and examined by fluorescence microscopy, histology and immunohistochemistry. The expression of liver-specific markers was quantified on days 1, 7, 14, and 21. Results The cells grew well on the alginate scaffold, and liver-specific gene expression increased. The cells grew more extensively in 3D culture than two-dimensional culture and exhibited better structural aspects of the liver, indicating that the 3D bioprinting method recapitulates the liver architecture. Conclusions The 3D bioprinting of hepatic structures appears feasible. This technology may become a major tool and provide a bridge between basic science and the clinical challenges for regenerative medicine of the liver. PMID:27559001

  3. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models.

    Ricci, Claudio; Mota, Carlos; Moscato, Stefania; D'Alessandro, Delfo; Ugel, Stefano; Sartoris, Silvia; Bronte, Vincenzo; Boggi, Ugo; Campani, Daniela; Funel, Niccola; Moroni, Lorenzo; Danti, Serena

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.

  4. Optimizing fuel cell parts by using 3D screen printed metals

    Studnitzky, Thomas [Fraunhofer-Institue for Manufacturing and Advanced Materials, Dresden (Germany). Dept. of Powder Metallurgy and Composite Materials; Strauss, Alexander [Centre for Fuel Cell Technology, Duisburg (Germany). Dept. of Microsystems

    2010-07-01

    Miniature fuel cells have the potential to extend the runtime of various portable applications. In this context, sufficient energy densities have to be achieved within a stack in order to build fuel cell systems competitive to established battery technologies. Metallic bipolar plates composed of stainless steels permit the construction of thin and mechanically robust cells. Moreover the utilisation of the presented manufacturing method enables a new freedom in design of Bipolar Plates and their integrated flow field structures for future improvements of cell efficiency. In this study different miniature PEM fuel cells have been designed and tested. Bases for their construction are micro structured plates composed of 316L stainless steel. This design can include very fine walls down to 60 {mu}m as well as undercuts, which is impossible with other manufacturing methods. These designs were adapted by Fraunhofer IFAM for the screen printing process. As a first result a proof of concept has been established for 3D screen printing as a method for the manufacturing of fuel cell bipolar elements. To this end, a modified screen printing process is used to manufacture 3-dimensional parts layer-on-layer by depositing a suitable metallic powder which is mixed with a binder. The resulting green parts might include closed channels and channel wall thicknesses may reach 80 {mu}m or less. The green parts are debindered and sintered in order to obtain purely metallic structures. The manufactured bipolar plates show promising electric behaviour. In the current state of the project, bipolar plates with undercuts and new materials combinations are in the centre of the investigations. (orig.)

  5. 3D optical simulation formalism OPTOS for textured silicon solar cells.

    Tucher, Nico; Eisenlohr, Johannes; Kiefel, Peter; Höhn, Oliver; Hauser, Hubert; Peters, Marius; Müller, Claas; Goldschmidt, Jan Christoph; Bläsi, Benedikt

    2015-11-30

    In this paper we introduce the three-dimensional formulation of the OPTOS formalism, a matrix-based method that allows for the efficient simulation of non-coherent light propagation and absorption in thick textured sheets. As application examples, we calculate the absorptance of solar cells featuring textures on front and rear side with different feature sizes operating in different optical regimes. A discretization of polar and azimuth angle enables a three-dimensional description of systems with arbitrary surface textures. We present redistribution matrices for 3D surface textures, including pyramidal textures, binary crossed gratings and a Lambertian scatterer. The results of the OPTOS simulations for silicon sheets with different combinations of these surfaces are in accordance with both optical measurements and results based on established simulation methods like ray tracing. Using OPTOS, we show that the integration of a diffractive grating at the rear side of a silicon solar cell featuring a pyramidal front side results in absorption close to the Yablonovitch Limit enhancing the photocurrent density by 0.6 mA/cm2 for a 200 µm thick cell.

  6. A Novel Flow-Perfusion Bioreactor Supports 3D Dynamic Cell Culture

    Alexander M. Sailon

    2009-01-01

    Full Text Available Background. Bone engineering requires thicker three-dimensional constructs than the maximum thickness supported by standard cell-culture techniques (2 mm. A flow-perfusion bioreactor was developed to provide chemotransportation to thick (6 mm scaffolds. Methods. Polyurethane scaffolds, seeded with murine preosteoblasts, were loaded into a novel bioreactor. Control scaffolds remained in static culture. Samples were harvested at days 2, 4, 6, and 8 and analyzed for cellular distribution, viability, metabolic activity, and density at the periphery and core. Results. By day 8, static scaffolds had a periphery cell density of 67%±5.0%, while in the core it was 0.3%±0.3%. Flow-perfused scaffolds demonstrated peripheral cell density of 94%±8.3% and core density of 76%±3.1% at day 8. Conclusions. Flow perfusion provides chemotransportation to thick scaffolds. This system may permit high throughput study of 3D tissues in vitro and enable prefabrication of biological constructs large enough to solve clinical problems.

  7. Cold Atmospheric Plasma Induces a Predominantly Necrotic Cell Death via the Microenvironment.

    François Virard

    Full Text Available Cold plasma is a partially ionized gas generated by an electric field at atmospheric pressure that was initially used in medicine for decontamination and sterilization of inert surfaces. There is currently growing interest in using cold plasma for more direct medical applications, mainly due to the possibility of tuning it to obtain selective biological effects in absence of toxicity for surrounding normal tissues,. While the therapeutic potential of cold plasma in chronic wound, blood coagulation, and cancer treatment is beginning to be documented, information on plasma/cell interaction is so far limited and controversial.Using normal primary human fibroblast cultures isolated from oral tissue, we sought to decipher the effects on cell behavior of a proprietary cold plasma device generating guided ionization waves carried by helium. In this model, cold plasma treatment induces a predominantly necrotic cell death. Interestingly, death is not triggered by a direct interaction of the cold plasma with cells, but rather via a transient modification in the microenvironment. We show that modification of the microenvironment redox status suppresses treatment toxicity and protects cells from death. Moreover, necrosis is not accidental and seems to be an active response to an environmental cue, as its execution can be inhibited to rescue cells.These observations will need to be taken into account when studying in vitro plasma/cell interaction and may have implications for the design and future evaluation of the efficacy and safety of this new treatment strategy.

  8. Lactate Contribution to the Tumor Microenvironment: Mechanisms, Effects on Immune Cells and Therapeutic Relevance

    Romero-Garcia, Susana; Moreno-Altamirano, María Maximina B.; Prado-Garcia, Heriberto; Sánchez-García, Francisco Javier

    2016-01-01

    Malignant transformation of cells leads to enhanced glucose uptake and the conversion of a larger fraction of pyruvate into lactate, even under normoxic conditions; this phenomenon of aerobic glycolysis is largely known as the Warburg effect. This metabolic reprograming serves to generate biosynthetic precursors, thus facilitating the survival of rapidly proliferating malignant cells. Extracellular lactate directs the metabolic reprograming of tumor cells, thereby serving as an additional selective pressure. Besides tumor cells, stromal cells are another source of lactate production in the tumor microenvironment, whose role in both tumor growth and the antitumor immune response is the subject of intense research. In this review, we provide an integral perspective of the relationship between lactate and the overall tumor microenvironment, from lactate structure to metabolic pathways for its synthesis, receptors, signaling pathways, lactate-producing cells, lactate-responding cells, and how all contribute to the tumor outcome. We discuss the role of lactate as an immunosuppressor molecule that contributes to tumor evasion and we explore the possibility of targeting lactate metabolism for cancer treatment, as well as of using lactate as a prognostic biomarker. PMID:26909082

  9. Lactate contribution to the tumor microenvironment: mechanisms, effects on immune cells and therapeutic relevance

    Susana eRomero-Garcia

    2016-02-01

    Full Text Available Malignant transformation of cells leads to enhanced glucose uptake and the conversion of a larger fraction of pyruvate into lactate, even under normoxic conditions; this phenomenon of aerobic glycolysis is largely known as the Warburg effect. This metabolic reprogramming serves to generate biosynthetic precursors, thus facilitating the survival of rapidly proliferating malignant cells. Extracellular lactate directs the metabolic reprogramming of tumor cells, thereby serving as an additional selective pressure. Besides tumor cells, stromal cells are another source of lactate production in the tumor microenvironment, whose role in both tumor growth and the anti-tumor immune response is the subject of intense research. In this review, we provide an integral perspective of the relationship between lactate and the overall tumor microenvironment, from lactate structure to metabolic pathways for its synthesis, receptors, signaling pathways, lactate-producing cells, lactate-responding cells, and how all contribute to the tumor outcome. We discuss the role of lactate as a immunosuppressor molecule that contributes to tumor evasion and explore the possibility of targeting lactate metabolism for cancer treatment, as well as of using lactate as a prognostic biomarker.

  10. Hypoxia-induced reactive oxygen species cause chromosomal abnormalities in endothelial cells in the tumor microenvironment.

    Miyako Kondoh

    Full Text Available There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.

  11. Challenges and limitations of targeting cancer stem cells and/or the tumour microenvironment

    Juan Sebastian Yakisich

    2012-05-01

    Full Text Available The existence of cancer cells with stem cell properties (Cancer Stem Cells, CSCs and their association with tumor resistance and relapse has led to the search for active compounds to eliminate these cells or modulate their stemness in the hope of curing cancer. So far, three classes of drugs that target cancer stemness (Stemness Modulator Drugs have been identified: i drugs that selectively eliminate CSCs (stem cell targeting drugs; ii drugs that decrease stemness (stemness inhibitor drugs; and iii drugs that promote stemness (stemness promoting drugs. In addition, microenvironment modulating drugs aimed at selectively targeting the stem cell niche are being investigated and may represent an important class of drug for cancer therapy. This article will briefly review the current use of these substances and discuss the potential outcomes, challenges and limitations of treatment modalities using these classes of drugs for cancer treatment. Finally, a modular tumor model will be proposed as a guide to integrate our knowledge on the biology of cancer stem cell with that of the tumor microenvironment to promote a more rational development of anticancer therapy.

  12. Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

    Hida, Yasuhiro; Maishi, Nako; Towfik, Alam Mohammad; Inoue, Nobuo; Shindoh, Masanobu; Hida, Kyoko

    2013-01-01

    There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment. PMID:24260373

  13. Microenvironment is involved in cellular response to hydrostatic pressures during chondrogenesis of mesenchymal stem cells.

    Ye, Rui; Hao, Jin; Song, Jinlin; Zhao, Zhihe; Fang, Shanbao; Wang, Yating; Li, Juan

    2014-06-01

    Chondrocytes integrate numerous microenvironmental cues to mount physiologically relevant differentiation responses, and the regulation of mechanical signaling in chondrogenic differentiation is now coming into intensive focus. To facilitate tissue-engineered chondrogenesis by mechanical strategy, a thorough understanding about the interactional roles of chemical factors under mechanical stimuli in regulating chondrogenesis is in great need. Therefore, this study attempts to investigate the interaction of rat MSCs with their microenvironment by imposing dynamic and static hydrostatic pressure through modulating gaseous tension above the culture medium. Under dynamic pressure, chemical parameters (pH, pO2, and pCO2) were kept in homeostasis. In contrast, pH was remarkably reduced due to increased pCO2 under static pressure. MSCs under the dynamically pressured microenvironment exhibited a strong accumulation of GAG within and outside the alginate beads, while cells under the statically pressured environment lost newly synthesized GAG into the medium with a speed higher than its production. In addition, the synergic influence on expression of chondrogenic genes was more persistent under dynamic pressure than that under static pressure. This temporal contrast was similar to that of activation of endogenous TGF-β1. Taken altogether, it indicates that a loading strategy which can keep a homeostatic chemical microenvironment is preferred, since it might sustain the stimulatory effects of mechanical stimuli on chondrogenesis via activation of endogenous TGF-β1.

  14. Related pituitary cell lineages develop into interdigitated 3D cell networks.

    Budry, Lionel; Lafont, Chrystel; El Yandouzi, Taoufik; Chauvet, Norbert; Conéjero, Geneviève; Drouin, Jacques; Mollard, Patrice

    2011-07-26

    The pituitary gland has long been considered to be a random patchwork of hormone-producing cells. By using pituitary-scale tridimensional imaging for two of the least abundant cell lineages, the corticotropes and gonadotropes, we have now uncovered highly organized and interdigitated cell networks that reflect homotypic and heterotypic interactions between cells. Although newly differentiated corticotrope cells appear on the ventral surface of the gland, they rapidly form homotypic strands of cells that extend from the lateral tips of the anterior pituitary along its ventral surface and into the medial gland. As the corticotrope network is established away from the microvasculature, cell morphology changes from rounded, to polygonal, and finally to cells with long cytoplasmic processes or cytonemes that connect corticotropes to the perivascular space. Gonadotropes differentiate later and are positioned in close proximity to corticotropes and capillaries. Blockade of corticotrope terminal differentiation produced by knockout of the gene encoding the transcription factor Tpit results in smaller gonadotropes within an expanded cell network, particularly in the lateral gland. Thus, pituitary-scale tridimensional imaging reveals highly structured cell networks of unique topology for each pituitary lineage. The sequential development of interdigitated cell networks during organogenesis indicate that extensive cell:cell interactions lead to a highly ordered cell positioning rather than random patchwork.

  15. DDR-mediated crosstalk between DNA-damaged cells and their microenvironment.

    Malaquin, Nicolas; Carrier-Leclerc, Audrey; Dessureault, Mireille; Rodier, Francis

    2015-01-01

    The DNA damage response (DDR) is an evolutionarily conserved signaling cascade that senses and responds to double-strand DNA breaks by organizing downstream cellular events, ranging from appropriate DNA repair to cell cycle checkpoints. In higher organisms, the DDR prevents neoplastic transformation by directly protecting the information contained in the genome and by regulating cell fate decisions, like apoptosis and senescence, to ensure the removal of severely damaged cells. In addition to these well-studied cell-autonomous effects, emerging evidence now shows that the DDR signaling cascade can also function in a paracrine manner, thus influencing the biology of the surrounding cellular microenvironment. In this context, the DDR plays an emerging role in shaping the damaged tumor microenvironment through the regulation of tissue repair and local immune responses, thereby providing a promising avenue for novel therapeutic interventions. Additionally, while DDR-mediated extracellular signals can convey information to surrounding, undamaged cells, they can also feedback onto DNA-damaged cells to reinforce selected signaling pathways. Overall, these extracellular DDR signals can be subdivided into two time-specific waves: a rapid bystander effect occurring within a few hours of DNA damage; and a late, delayed, senescence-associated secretory phenotype generally requiring multiple days to establish. Here, we highlight and discuss examples of rapid and late DDR-mediated extracellular alarm signals.

  16. Mesenchymal Stromal Cells Can Regulate the Immune Response in the Tumor Microenvironment

    Alessandro Poggi

    2016-11-01

    Full Text Available The tumor microenvironment is a good target for therapy in solid tumors and hematological malignancies. Indeed, solid tumor cells’ growth and expansion can influence neighboring cells’ behavior, leading to a modulation of mesenchymal stromal cell (MSC activities and remodeling of extracellular matrix components. This leads to an altered microenvironment, where reparative mechanisms, in the presence of sub-acute inflammation, are not able to reconstitute healthy tissue. Carcinoma cells can undergo epithelial mesenchymal transition (EMT, a key step to generate metastasis; these mesenchymal-like cells display the functional behavior of MSC. Furthermore, MSC can support the survival and growth of leukemic cells within bone marrow participating in the leukemic cell niche. Notably, MSC can inhibit the anti-tumor immune response through either carcinoma-associated fibroblasts or bone marrow stromal cells. Experimental data have indicated their relevance in regulating cytolytic effector lymphocytes of the innate and adaptive arms of the immune system. Herein, we will discuss some of the evidence in hematological malignancies and solid tumors. In particular, we will focus our attention on the means by which it is conceivable to inhibit MSC-mediated immune suppression and trigger anti-tumor innate immunity.

  17. Metabolic Hallmarks of Tumor and Immune Cells in the Tumor Microenvironment

    Renner, Kathrin; Singer, Katrin; Koehl, Gudrun E.; Geissler, Edward K.; Peter, Katrin; Siska, Peter J.; Kreutz, Marina

    2017-01-01

    Cytotoxic T lymphocytes and NK cells play an important role in eliminating malignant tumor cells and the number and activity of tumor-infiltrating T cells represent a good marker for tumor prognosis. Based on these findings, immunotherapy, e.g., checkpoint blockade, has received considerable attention during the last couple of years. However, for the majority of patients, immune control of their tumors is gray theory as malignant cells use effective mechanisms to outsmart the immune system. Increasing evidence suggests that changes in tumor metabolism not only ensure an effective energy supply and generation of building blocks for tumor growth but also contribute to inhibition of the antitumor response. Immunosuppression in the tumor microenvironment is often based on the mutual metabolic requirements of immune cells and tumor cells. Cytotoxic T and NK cell activation leads to an increased demand for glucose and amino acids, a well-known feature shown by tumor cells. These close metabolic interdependencies result in metabolic competition, limiting the proliferation, and effector functions of tumor-specific immune cells. Moreover, not only nutrient restriction but also tumor-driven shifts in metabolite abundance and accumulation of metabolic waste products (e.g., lactate) lead to local immunosuppression, thereby facilitating tumor progression and metastasis. In this review, we describe the metabolic interplay between immune cells and tumor cells and discuss tumor cell metabolism as a target structure for cancer therapy. Metabolic (re)education of tumor cells is not only an approach to kill tumor cells directly but could overcome metabolic immunosuppression in the tumor microenvironment and thereby facilitate immunotherapy. PMID:28337200

  18. The role of the cytoskeleton in cellular force generation in 2D and 3D environments

    Kraning-Rush, Casey M.; Carey, Shawn P.; Califano, Joseph P.; Smith, Brooke N.; Reinhart-King, Cynthia A.

    2011-02-01

    To adhere and migrate, cells generate forces through the cytoskeleton that are transmitted to the surrounding matrix. While cellular force generation has been studied on 2D substrates, less is known about cytoskeletal-mediated traction forces of cells embedded in more in vivo-like 3D matrices. Recent studies have revealed important differences between the cytoskeletal structure, adhesion, and migration of cells in 2D and 3D. Because the cytoskeleton mediates force, we sought to directly compare the role of the cytoskeleton in modulating cell force in 2D and 3D. MDA-MB-231 cells were treated with agents that perturbed actin, microtubules, or myosin, and analyzed for changes in cytoskeletal organization and force generation in both 2D and 3D. To quantify traction stresses in 2D, traction force microscopy was used; in 3D, force was assessed based on single cell-mediated collagen fibril reorganization imaged using confocal reflectance microscopy. Interestingly, even though previous studies have observed differences in cell behaviors like migration in 2D and 3D, our data indicate that forces generated on 2D substrates correlate with forces within 3D matrices. Disruption of actin, myosin or microtubules in either 2D or 3D microenvironments disrupts cell-generated force. These data suggest that despite differences in cytoskeletal organization in 2D and 3D, actin, microtubules and myosin contribute to contractility and matrix reorganization similarly in both microenvironments.

  19. Scaffolds for 3D in vitro culture of neural lineage cells.

    Murphy, Ashley R; Laslett, Andrew; O'Brien, Carmel M; Cameron, Neil R

    2017-03-01

    Understanding how neurodegenerative disorders develop is not only a key challenge for researchers but also for the wider society, given the rapidly aging populations in developed countries. Advances in this field require new tools with which to recreate neural tissue in vitro and produce realistic disease models. This in turn requires robust and reliable systems for performing 3D in vitro culture of neural lineage cells. This review provides a state of the art update on three-dimensional culture systems for in vitro development of neural tissue, employing a wide range of scaffold types including hydrogels, solid porous polymers, fibrous materials and decellularised tissues as well as microfluidic devices and lab-on-a-chip systems. To provide some context with in vivo development of the central nervous system (CNS), we also provide a brief overview of the neural stem cell niche, neural development and neural differentiation in vitro. We conclude with a discussion of future directions for this exciting and important field of biomaterials research.

  20. Heritable Genetic Changes in Cells Recovered From Irradiated 3D Tissue Contracts. Final report

    Cornforth, Michael N. [The University of Texas Medical Branch at Galveston, TX (United States)

    2013-05-03

    Combining contemporary cytogenetic methods with DNA CGH microarray technology and chromosome flow-sorting increases substantially the ability to resolve exchange breakpoints associated with interstitial deletions and translocations, allowing the consequences of radiation damage to be directly measured at low doses, while also providing valuable insights into molecular mechanisms of misrepair processes that, in turn, identify appropriate biophysical models of risk at low doses. The aims of this work apply to cells recovered from 3D tissue constructs of human skin and, for the purpose of comparison, the same cells irradiated in traditional 2D cultures. These aims are: to analyze by multi-flour fluorescence in situ hybridization (mFISH) the chromosomes in clonal descendents of individual human fibroblasts that were previously irradiated; to examine irradiated clones from Aim 1 for submicroscopic deletions by subjecting their DNA to comparative genomic hybridization (CGH) microarray analysis; and to flow-sort aberrant chromosomes from clones containing stable radiation-induced translocations and map the breakpoints to within an average resolution of 100 kb using the technique of 'array painting'.

  1. The Interactorium: visualising proteins, complexes and interaction networks in a virtual 3-D cell.

    Widjaja, Yose Y; Pang, Chi Nam Ignatius; Li, Simone S; Wilkins, Marc R; Lambert, Tim D

    2009-12-01

    Here, we describe the Interactorium, a tool in which a Virtual Cell is used as the context for the seamless visualisation of the yeast protein interaction network, protein complexes and protein 3-D structures. The tool has been designed to display very complex networks of up to 40 000 proteins or 6000 multiprotein complexes and has a series of toolboxes and menus to allow real-time data manipulation and control the manner in which data are displayed. It incorporates new algorithms that reduce the complexity of the visualisation by the generation of putative new complexes from existing data and by the reduction of edges through the use of protein "twins" when they occur in multiple locations. Since the Interactorium permits multi-level viewing of the molecular biology of the cell, it is a considerable advance over existing approaches. We illustrate its use for Saccharomyces cerevisiae but note that it will also be useful for the analysis of data from simpler prokaryotes and higher eukaryotes, including humans. The Interactorium is available for download at http://www.interactorium.net.

  2. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated doma

  3. The performance of 3-D graphite doped anodes in microbial electrolysis cells

    Yasri, Nael G.; Nakhla, George

    2017-02-01

    This study investigated the use of granular activated carbon (GAC) as high surface area 3-dimensional (3-D) anode in MECs systems. The interfacial anodes' charge transfer resistance of the doped GAC did not impact the overall performance of MECs. Based on our finding, the 3-D anode packed with GAC-doped with nonconductive calcium sulfide (CaS) outperformed the more conductive iron (II) sulfide (FeS), magnetite (Fe3O4), or GAC without doping. The results showed higher current densities for 3-D CaS (40.1 A/m3), as compared with 3-D FeS (34.4 A/m3), 3-D Fe3O4 (29.8 A/m3), and 3-D GAC (23.1 A/m3). The higher current density in the 3-D CaS translated to higher coulombic efficiency (96.7%), hydrogen yield (3.6 mol H2/mol acetate), and attached biomass per anode mass (54.01 mg COD biomass/g GAC). Although the 3-D MEC achieved similar hydrogen yield, hydrogen recovery efficiency, and COD removal rate to a conventional sandwich type MEC, the current density, coulombic efficiency, and overall energy efficiency were higher.

  4. A tetraphenylethylene core-based 3D structure small molecular acceptor enabling efficient non-fullerene organic solar cells.

    Liu, Yuhang; Mu, Cheng; Jiang, Kui; Zhao, Jingbo; Li, Yunke; Zhang, Lu; Li, Zhengke; Lai, Joshua Yuk Lin; Hu, Huawei; Ma, Tingxuan; Hu, Rongrong; Yu, Demei; Huang, Xuhui; Tang, Ben Zhong; Yan, He

    2015-02-01

    A tetraphenylethylene core-based small molecular acceptor with a unique 3D molecular structure is developed. Bulk-heterojunction blend films with a small feature size (≈20 nm) are obtained, which lead to non-fullerene organic solar cells (OSCs) with 5.5% power conversion efficiency. The work provides a new molecular design approach to efficient non-fullerene OSCs based on 3D-structured small-molecule acceptors.

  5. Cells in 3D matrices under interstitial flow: effects of extracellular matrix alignment on cell shear stress and drag forces.

    Pedersen, John A; Lichter, Seth; Swartz, Melody A

    2010-03-22

    Interstitial flow is an important regulator of various cell behaviors both in vitro and in vivo, yet the forces that fluid flow imposes on cells embedded in a 3D extracellular matrix (ECM), and the effects of matrix architecture on those forces, are not well understood. Here, we demonstrate how fiber alignment can affect the shear and pressure forces on the cell and ECM. Using computational fluid dynamics simulations, we show that while the solutions of the Brinkman equation accurately estimate the average fluid shear stress and the drag forces on a cell within a 3D fibrous medium, the distribution of shear stress on the cellular surface as well as the peak shear stresses remain intimately related to the pericellular fiber architecture and cannot be estimated using bulk-averaged properties. We demonstrate that perpendicular fiber alignment of the ECM yields lower shear stress and pressure forces on the cells and higher stresses on the ECM, leading to decreased permeability, while parallel fiber alignment leads to higher stresses on cells and increased permeability, as compared to a cubic lattice arrangement. The Spielman-Goren permeability relationships for fibrous media agreed well with CFD simulations of flow with explicitly considered fibers. These results suggest that the experimentally observed active remodeling of ECM fibers by fibroblasts under interstitial flow to a perpendicular alignment could serve to decrease the shear and drag forces on the cell.

  6. Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture.

    Michael S Weiss

    Full Text Available BACKGROUND: Extracellular activation of signal transduction pathways and their downstream target transcription factors (TFs are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture. METHODOLOGY/PRINCIPAL FINDINGS: TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A. CONCLUSIONS/SIGNIFICANCE: This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a

  7. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration

    Jyuhn-Huarng Juang

    2015-02-01

    Full Text Available The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

  8. Melanoma cells undergo aggressive coalescence in a 3D Matrigel model that is repressed by anti-CD44

    Voss, Edward; Kuhl, Spencer; Buchele, Emma C.; Klemme, Michael R.; Russell, Kanoe B.; Ambrose, Joseph; Soll, Benjamin A.; Bossler, Aaron; Milhem, Mohammed; Goldman, Charles

    2017-01-01

    Using unique computer-assisted 3D reconstruction software, it was previously demonstrated that tumorigenic cell lines derived from breast tumors, when seeded in a 3D Matrigel model, grew as clonal aggregates which, after approximately 100 hours, underwent coalescence mediated by specialized cells, eventually forming a highly structured large spheroid. Non-tumorigenic cells did not undergo coalescence. Because histological sections of melanomas forming in patients suggest that melanoma cells migrate and coalesce to form tumors, we tested whether they also underwent coalescence in a 3D Matrigel model. Melanoma cells exiting fragments of three independent melanomas or from secondary cultures derived from them, and cells from the melanoma line HTB-66, all underwent coalescence mediated by specialized cells in the 3D model. Normal melanocytes did not. However, coalescence of melanoma cells differed from that of breast-derived tumorigenic cell lines in that they 1) coalesced immediately, 2) underwent coalescence as individual cells as well as aggregates, 3) underwent coalescence far faster and 4) ultimately formed long, flat, fenestrated aggregates that were extremely dynamic. A screen of 51 purified monoclonal antibodies (mAbs) targeting cell surface-associated molecules revealed that two mAbs, anti-beta 1 integrin/(CD29) and anti-CD44, blocked melanoma cell coalescence. They also blocked coalescence of tumorigenic cells derived from a breast tumor. These results add weight to the commonality of coalescence as a characteristic of tumorigenic cells, as well as the usefulness of the 3D Matrigel model and software for both investigating the mechanisms regulating tumorigenesis and screening for potential anti-tumorigenesis mAbs. PMID:28264026

  9. Trehalose effectiveness as a cryoprotectant in 2D and 3D cell cultures of human embryonic kidney cells.

    Hara, Jared; Tottori, Jordan; Anders, Megan; Dadhwal, Smritee; Asuri, Prashanth; Mobed-Miremadi, Maryam

    2017-05-01

    Post cryopreservation viability of human embryonic kidney (HEK) cells under two-dimensional (2D) and three-dimensional (3D) culture conditions was studied using trehalose as the sole cryoprotective agent. An L9 (3(4)) Taguchi design was used to optimize the cryoprotection cocktail seeding process prior to slow-freezing with the specific aim of maximizing cell viability measured 7 days post thaw, using the combinatorial cell viability and in-vitro cytotoxicity WST assay. At low (200 mM) and medium (800 mM) levels of trehalose concentration, encapsulation in alginate offered a greater protection to cryopreservation. However, at the highest trehalose concentration (1200 mM) and in the absence of the pre-incubation step, there was no statistical difference at the 95% CI (p = 0.0212) between the viability of the HEK cells under 2D and 3D culture conditions estimated to be 17.9 ± 4.6% and 14.0 ± 3.6%, respectively. A parallel comparison between cryoprotective agents conducted at the optimal levels of the L9 study, using trehalose, dimethylsulfoxide and glycerol in alginate microcapsules yielded a viability of 36.0 ± 7.4% for trehalose, in average 75% higher than the results associated with the other two cell membrane-permeating compounds. In summary, the effectiveness of trehalose has been demonstrated by the fact that 3D cell cultures can readily be equilibrated with trehalose before cryopreservation, thus mitigating the cytotoxic effects of glycerol and dimethylsulfoxide.

  10. Tumor microenvironment:bidirectional interactions between cancer cells and normal cells

    Lu-Yuan Li

    2010-01-01

    @@ "The road to metastasis is paved with tumor-microenvironment interactions",claimed Dr.Isaac Witz from Tel Aviv University,Israel,in his keynote speech at the first Tianjin Forum on Tumor Microenvironment(http://TFTM.nankai.edu.cn),an international conference held at Nankai University in Tianjin,China,on July 2-4,2010.About 300 cancer researchers and students attended the conference.

  11. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix.

    Ma, Yufei; Ji, Yuan; Huang, Guoyou; Ling, Kai; Zhang, Xiaohui; Xu, Feng

    2015-12-22

    Periodontitis is an inflammatory disease negatively affecting up to 15% of adults worldwide. Periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, where it is necessary to find proper extracellular matrix (ECM) materials (e.g., composition, concentration). In this study, we proposed a bioprinting-based approach to generate nano-liter sized three-dimensional (3D) cell-laden hydrogel array with gradient of ECM components, through controlling the volume ratio of two hydrogels, such as gelatin methacrylate (GelMA) and poly(ethylene glycol) (PEG) dimethacrylate. The resulting cell-laden array with a gradient of GelMA/PEG composition was used to screen human PDLSC response to ECM. The behavior (e.g., cell viability, spreading) of human PDLSCs in GelMA/PEG array were found to be depended on the volume ratios of GelMA/PEG, with cell viability and spreading area decreased along with increasing the ratio of PEG. The developed approach would be useful for screening cell-biomaterial interaction in 3D and promoting regeneration of functional tissue.

  12. 3D Biomaterial Microarrays for Regenerative Medicine

    Gaharwar, Akhilesh K.; Arpanaei, Ayyoob; Andresen, Thomas Lars;

    2015-01-01

    Three dimensional (3D) biomaterial microarrays hold enormous promise for regenerative medicine because of their ability to accelerate the design and fabrication of biomimetic materials. Such tissue-like biomaterials can provide an appropriate microenvironment for stimulating and controlling stem...

  13. 3D In Vitro Model for Breast Cancer Research Using Magnetic Levitation and Bioprinting Method.

    Leonard, Fransisca; Godin, Biana

    2016-01-01

    Tumor microenvironment composition and architecture are known as a major factor in orchestrating the tumor growth and its response to various therapies. In this context, in vivo studies are necessary to evaluate the responses. However, while tumor cells can be of human origin, tumor microenvironment in the in vivo models is host-based. On the other hand, in vitro studies in a flat monoculture of tumor cells (the most frequently used in vitro tumor model) are unable to recapitulate the complexity of tumor microenvironment. Three-dimensional (3D) in vitro cell cultures of tumor cells have been proven to be an important experimental tool in understanding mechanisms of tumor growth, response to therapeutics, and transport of nutrients/drugs. We have recently described a novel tool to create 3D co-cultures of tumor cells and cells in the tumor microenvironment. Our method utilizes magnetic manipulation/levitation of the specific ratios of tumor cells and cells in the tumor microenvironment (from human or animal origin) aiding in the formation of tumor spheres with defined cellular composition and density, as quickly as within 24 h. This chapter describes the experimental protocols developed to model the 3D structure of the cancer environment using the above method.

  14. A Bio-Acoustic Levitational (BAL) Assembly Method for Engineering of Multilayered, 3D Brain-Like Constructs, Using Human Embryonic Stem Cell Derived Neuro-Progenitors.

    Bouyer, Charlène; Chen, Pu; Güven, Sinan; Demirtaş, Tuğrul Tolga; Nieland, Thomas J F; Padilla, Frédéric; Demirci, Utkan

    2016-01-06

    A bio-acoustic levitational assembly method for engineering of multilayered, 3D brainlike constructs is presented. Acoustic radiation forces are used to levitate neuroprogenitors derived from human embryonic stem cells in 3D multilayered fibrin tissue constructs. The neuro-progenitor cells are subsequently differentiated in neural cells, resulting in a 3D neuronal construct with inter and intralayer neurite elongations.

  15. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.

    Moreno, Edinson Lucumi; Hachi, Siham; Hemmer, Kathrin; Trietsch, Sebastiaan J; Baumuratov, Aidos S; Hankemeier, Thomas; Vulto, Paul; Schwamborn, Jens C; Fleming, Ronan M T

    2015-06-07

    A hallmark of Parkinson's disease is the progressive loss of nigrostriatal dopaminergic neurons. We derived human neuroepithelial cells from induced pluripotent stem cells and successfully differentiated them into dopaminergic neurons within phase-guided, three-dimensional microfluidic cell culture bioreactors. After 30 days of differentiation within the microfluidic bioreactors, in situ morphological, immunocytochemical and calcium imaging confirmed the presence of dopaminergic neurons that were spontaneously electrophysiologically active, a characteristic feature of nigrostriatal dopaminergic neurons in vivo. Differentiation was as efficient as in macroscopic culture, with up to 19% of differentiated neurons immunoreactive for tyrosine hydroxylase, the penultimate enzyme in the synthesis of dopamine. This new microfluidic cell culture model integrates the latest innovations in developmental biology and microfluidic cell culture to generate a biologically realistic and economically efficient route to personalised drug discovery for Parkinson's disease.

  16. Occurrence of thymosin ß4 in human breast cancer cells and in other cell types of the tumor microenvironment

    Larsson, Lars-Inge; Holck, Susanne

    2007-01-01

    that there is a considerable heterogeneity in the cellular distribution of thymosin ß4 in breast cancer. In most tumors examined, cancer cells showed low or intermediate reactivity for thymosin ß4, whereas leukocytes and macrophages showed intense reactivity. In addition, endothelial cells showed variable reactivity...... microenvironment produce thymosin ß4 and that such expression varies from tumor to tumor. Such heterogeneity of expression should be taken into account when the role of thymosin ß4 in tumor biology is assessed....

  17. Occurrence of thymosin beta4 in human breast cancer cells and in other cell types of the tumor microenvironment

    Larsson, L.-I.; Holck, Susanne

    2007-01-01

    that there is a considerable heterogeneity in the cellular distribution of thymosin beta4 in breast cancer. In most tumors examined, cancer cells showed low or intermediate reactivity for thymosin beta4, whereas leukocytes and macrophages showed intense reactivity. In addition, endothelial cells showed variable reactivity...... the tumor microenvironment produce thymosin beta4 and that such expression varies from tumor to tumor. Such heterogeneity of expression should be taken into account when the role of thymosin beta4 in tumor biology is assessed....

  18. Tumour tissue microenvironment can inhibit dendritic cell maturation in colorectal cancer.

    Michielsen, Adriana J

    2011-01-01

    Inflammatory mediators in the tumour microenvironment promote tumour growth, vascular development and enable evasion of anti-tumour immune responses, by disabling infiltrating dendritic cells. However, the constituents of the tumour microenvironment that directly influence dendritic cell maturation and function are not well characterised. Our aim was to identify tumour-associated inflammatory mediators which influence the function of dendritic cells. Tumour conditioned media obtained from cultured colorectal tumour explant tissue contained high levels of the chemokines CCL2, CXCL1, CXCL5 in addition to VEGF. Pre-treatment of monocyte derived dendritic cells with this tumour conditioned media inhibited the up-regulation of CD86, CD83, CD54 and HLA-DR in response to LPS, enhancing IL-10 while reducing IL-12p70 secretion. We examined if specific individual components of the tumour conditioned media (CCL2, CXCL1, CXCL5) could modulate dendritic cell maturation or cytokine secretion in response to LPS. VEGF was also assessed as it has a suppressive effect on dendritic cell maturation. Pre-treatment of immature dendritic cells with VEGF inhibited LPS induced upregulation of CD80 and CD54, while CXCL1 inhibited HLA-DR. Interestingly, treatment of dendritic cells with CCL2, CXCL1, CXCL5 or VEGF significantly suppressed their ability to secrete IL-12p70 in response to LPS. In addition, dendritic cells treated with a combination of CXCL1 and VEGF secreted less IL-12p70 in response to LPS compared to pre-treatment with either cytokine alone. In conclusion, tumour conditioned media strongly influences dendritic cell maturation and function.

  19. Highly ordered large-scale neuronal networks of individual cells - toward single cell to 3D nanowire intracellular interfaces.

    Kwiat, Moria; Elnathan, Roey; Pevzner, Alexander; Peretz, Asher; Barak, Boaz; Peretz, Hagit; Ducobni, Tamir; Stein, Daniel; Mittelman, Leonid; Ashery, Uri; Patolsky, Fernando

    2012-07-25

    The use of artificial, prepatterned neuronal networks in vitro is a promising approach for studying the development and dynamics of small neural systems in order to understand the basic functionality of neurons and later on of the brain. The present work presents a high fidelity and robust procedure for controlling neuronal growth on substrates such as silicon wafers and glass, enabling us to obtain mature and durable neural networks of individual cells at designed geometries. It offers several advantages compared to other related techniques that have been reported in recent years mainly because of its high yield and reproducibility. The procedure is based on surface chemistry that allows the formation of functional, tailormade neural architectures with a micrometer high-resolution partition, that has the ability to promote or repel cells attachment. The main achievements of this work are deemed to be the creation of a large scale neuronal network at low density down to individual cells, that develop intact typical neurites and synapses without any glia-supportive cells straight from the plating stage and with a relatively long term survival rate, up to 4 weeks. An important application of this method is its use on 3D nanopillars and 3D nanowire-device arrays, enabling not only the cell bodies, but also their neurites to be positioned directly on electrical devices and grow with registration to the recording elements underneath.

  20. Recent advance in fabricating monolithic 3D porous graphene and their applications in biosensing and biofuel cells.

    Qiu, Hua-Jun; Guan, Yongxin; Luo, Pan; Wang, Yu

    2017-03-15

    Graphene shows great potential in biosensing and bioelectronics. To facilitate graphene's applications and enhance its performance, recently, three-dimensional (3D) graphene-based materials especially free-standing porous graphene with tunable pore size and void space, have attracted increasing attention for bio-related applications owing to their special features. 3D graphene usually shows the following merits such as an interconnected porous network, a high electronic conductivity, a large active surface area, good chemical/thermal stability and can be more easily handled compared with dispersed graphene sheets. With modified surface properties, graphene can also be bio-friendly. These properties make 3D graphene a perfect candidate as high-performance electrode materials in bioelectronics devices. In this review, we discuss recent advance in fabricating monolithic 3D graphene and their applications in biosensing and biofuel cells.

  1. Prospective use of the 3D printing technology for the microstructural engineering of Solid Oxide Fuel Cell components

    Hernandez-Rodriguez, E. M.; Acosta-Mora, P.; Mendez-Ramos, J.; Borges Chinea, E.; Esparza Ferrera, P.; Canales-Vazquez, J.; Nunez, P.; Ruiz-Morales, J.

    2014-07-01

    A cost-effective micro-manufacturing process to accurately build 3D microstructures for their prospective use in the fabrication of Solid Oxide Fuel Cells components has been tested. The 3D printing method, based on the stereo lithography, allows solidifying layer by layer a dispersion of ceramic material in a liquid photosensitive organic monomer. A simple projector, a computer-controlled z-stage and a few PowerPoint slides may be used for the fabrication of a wide range of complex 3D microstructures in few minutes. In this work, 3D ceramic microstructures based on the yttria-stabilized zirconia (YSZ) were successfully fabricated. The micro structured ceramic components produced were stable after sintering at 1400 degree centigrade for 4 h. Impedance measurements show that the fabrication process does not have any detrimental effect on the electrical properties of the structured material. (Author)

  2. An impedance method for spatial sensing of 3D cell constructs – towards applications in tissue engineering

    Canali, Chiara; Mazzoni, Chiara; Larsen, Layla Bashir

    2015-01-01

    ) cells were encapsulated in gelatin to form artificial 3D cell constructs and detected when placed in different positions inside large gelatin scaffolds. Taken together, these results open new perspectives for impedance-based sensing technologies for non-invasive monitoring in tissue engineering...

  3. A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

    Robert J Polackwich

    Full Text Available New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1 increased the strength of cell-induced forces on the ECM, 2 did not significantly change migration speed, and 3 increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity.

  4. Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

    Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

    2017-01-27

    Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

  5. Cell and Signal Components of the Microenvironment of Bone Metastasis Are Affected by Hypoxia

    Bendinelli, Paola; Maroni, Paola; Matteucci, Emanuela; Desiderio, Maria Alfonsina

    2016-01-01

    Bone metastatic cells release bone microenvironment proteins, such as the matricellular protein SPARC (secreted protein acidic and rich in cysteine), and share a cell signaling typical of the bone metabolism controlled by Runx2. The megakaryocytes in the bone marrow engrafted by the metastases seem to be one of the principal microenvironment sources of the biological stimuli, implicated in the formation of an osteoblastic niche, and affecting metastasis phenotype and colonization. Educated platelets in the circulation might derive from megakaryocytes in bone metastasis. The evaluation of predictive markers in the circulating platelets might be useful for the stratification of patients for therapeutic purposes. The hypoxic environment in bone metastasis is one of the key regulators of the network of the biological soluble and structural components of the matrix. In bone metastatic cells under hypoxia, similar patterns of Runx2 and SPARC are observed, both showing downregulation. Conversely, hypoxia induces Endothelin 1, which upregulates SPARC, and these biological stimuli may be considered prognostic markers of bone metastasis in breast carcinoma patients. PMID:27187355

  6. Cell and Signal Components of the Microenvironment of Bone Metastasis Are Affected by Hypoxia

    Paola Bendinelli

    2016-05-01

    Full Text Available Bone metastatic cells release bone microenvironment proteins, such as the matricellular protein SPARC (secreted protein acidic and rich in cysteine, and share a cell signaling typical of the bone metabolism controlled by Runx2. The megakaryocytes in the bone marrow engrafted by the metastases seem to be one of the principal microenvironment sources of the biological stimuli, implicated in the formation of an osteoblastic niche, and affecting metastasis phenotype and colonization. Educated platelets in the circulation might derive from megakaryocytes in bone metastasis. The evaluation of predictive markers in the circulating platelets might be useful for the stratification of patients for therapeutic purposes. The hypoxic environment in bone metastasis is one of the key regulators of the network of the biological soluble and structural components of the matrix. In bone metastatic cells under hypoxia, similar patterns of Runx2 and SPARC are observed, both showing downregulation. Conversely, hypoxia induces Endothelin 1, which upregulates SPARC, and these biological stimuli may be considered prognostic markers of bone metastasis in breast carcinoma patients.

  7. Metastatic breast cancer cells in the bone marrow microenvironment: novel insights into oncoprotection

    Shyam A. Patel

    2011-06-01

    Full Text Available Among all cancers, malignancies of the breast are the second leading cause of cancer death in the United States after carcinoma of the lung. One of the major factors considered when assessing the prognosis of breast cancer patients is whether the tumor has metastasized to distant organs. Although the exact phenotype of the malignant cells responsible for metastasis and dormancy is still unknown, growing evidence has revealed that they may have stem cell-like properties that may account for resistance to chemotherapy and radiation. One process that has been attributed to primary tumor metastasis is the epithelial- to-mesenchymal transition. In this review, we specifically discuss breast cancer dissemination to the bone marrow and factors that ultimately serve to shelter and promote tumor growth, including the complex relationship between mesenchymal stem cells (MSCs and various aspects of the immune system, carcinoma-associated fibroblasts, and the diverse components of the tumor microenvironment. A better understanding of the journey from the primary tumor site to the bone marrow and subsequently the oncoprotective role of MSCs and other factors within that microenvironment can potentially lead to development of novel therapeutic targets.

  8. Effect of the Premalignant and Tumor Microenvironment on Immune Cell Cytokine Production in Head and Neck Cancer

    Johnson, Sara D. [Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425 (United States); De Costa, Anna-Maria A. [Department of Otolaryngology, Head and Neck Surgery, Medical University of South Carolina, 135 Rutledge Avenue, Charleston, SC 29425 (United States); Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425 (United States); Young, M. Rita I., E-mail: rita.young@va.gov [Department of Otolaryngology, Head and Neck Surgery, Medical University of South Carolina, 135 Rutledge Avenue, Charleston, SC 29425 (United States); Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425 (United States); Medical Research Service (151), Ralph H. Johnson Veterans Affairs Medical Center, 109 Bee Street, Charleston, SC 29401 (United States)

    2014-04-02

    Head and neck squamous cell carcinoma (HNSCC) is marked by immunosuppression, a state in which the established tumor escapes immune attack. However, the impact of the premalignant and tumor microenvironments on immune reactivity has yet to be elucidated. The purpose of this study was to determine how soluble mediators from cells established from carcinogen-induced oral premalignant lesions and HNSCC modulate immune cell cytokine production. It was found that premalignant cells secrete significantly increased levels of G-CSF, RANTES, MCP-1, and PGE{sub 2} compared to HNSCC cells. Splenocytes incubated with premalignant supernatant secreted significantly increased levels of Th1-, Th2-, and Th17-associated cytokines compared to splenocytes incubated with HNSCC supernatant. These studies demonstrate that whereas the premalignant microenvironment elicits proinflammatory cytokine production, the tumor microenvironment is significantly less immune stimulatory and may contribute to immunosuppression in established HNSCC.

  9. A multimaterial bioink method for 3D printing tunable, cell-compatible hydrogels.

    Rutz, Alexandra L; Hyland, Kelly E; Jakus, Adam E; Burghardt, Wesley R; Shah, Ramille N

    2015-03-04

    A multimaterial bio-ink method using polyethylene glycol crosslinking is presented for expanding the biomaterial palette required for 3D bioprinting of more mimetic and customizable tissue and organ constructs. Lightly crosslinked, soft hydrogels are produced from precursor solutions of various materials and 3D printed. Rheological and biological characterizations are presented, and the promise of this new bio-ink synthesis strategy is discussed.

  10. Osteogenic potential of human adipose-tissue-derived mesenchymal stromal cells cultured on 3D-printed porous structured titanium.

    Lewallen, Eric A; Jones, Dakota L; Dudakovic, Amel; Thaler, Roman; Paradise, Christopher R; Kremers, Hilal M; Abdel, Matthew P; Kakar, Sanjeev; Dietz, Allan B; Cohen, Robert C; Lewallen, David G; van Wijnen, Andre J

    2016-05-01

    Integration of porous metal prosthetics, which restore form and function of irreversibly damaged joints, into remaining healthy bone is critical for implant success. We investigated the biological properties of adipose-tissue-derived mesenchymal stromal/stem cells (AMSCs) and addressed their potential to alter the in vitro microenvironment of implants. We employed human AMSCs as a practical source for musculoskeletal applications because these cells can be obtained in large quantities, are multipotent, and have trophic paracrine functions. AMSCs were cultured on surgical-grade porous titanium disks as a model for orthopedic implants. We monitored cell/substrate attachment, cell proliferation, multipotency, and differentiation phenotypes of AMSCs upon osteogenic induction. High-resolution scanning electron microscopy and histology revealed that AMSCs adhere to the porous metallic surface. Compared to standard tissue culture plastic, AMSCs grown in the porous titanium microenvironment showed differences in temporal expression for genes involved in cell cycle progression (CCNB2, HIST2H4), extracellular matrix production (COL1A1, COL3A1), mesenchymal lineage identity (ACTA2, CD248, CD44), osteoblastic transcription factors (DLX3, DLX5, ID3), and epigenetic regulators (EZH1, EZH2). We conclude that metal orthopedic implants can be effectively seeded with clinical-grade stem/stromal cells to create a pre-conditioned implant.

  11. Pulmonary surfactant expression analysis--role of cell-cell interactions and 3-D tissue-like architecture.

    Nandkumar, Maya A; Ashna, U; Thomas, Lynda V; Nair, Prabha D

    2015-03-01

    Surfactant production is important in maintaining alveolar function both in vivo and in vitro, but surfactant expression is the primary property lost by alveolar Type II Pneumocytes in culture and its maintenance is a functional requirement. To develop a functional tissue-like model, the in vivo cell-cell interactions and three dimensional architecture has to be reproduced. To this end, 3D button-shaped synthetic gelatin vinyl acetate (GeVAc) co-polymer scaffold was seeded with different types of lung cells. Functionality of the construct was studied under both static and dynamic conditions. The construct was characterized by Environmental Scanning Electron and fluorescent microscopy, and functionality of the system was analyzed by studying mRNA modulations of all four surfactant genes A, B, C, and D by real time-PCR and varying culture conditions. The scaffold supports alveolar cell adhesion and maintenance of cuboidal morphology, and the alveolar-specific property of surfactant synthesis, which would otherwise be rapidly lost in culture. This is a novel 3D system that expresses all 4 surfactants for a culture duration of 3 weeks.

  12. The Brain Microenvironment Preferentially Enhances the Radioresistance of CD133+ Glioblastoma Stem-like Cells

    Muhammad Jamal

    2012-02-01

    Full Text Available Brain tumor xenografts initiated from glioblastoma (GBM CD133+ tumor stem-like cells (TSCs are composed of TSC and non-TSC subpopulations, simulating the phenotypic heterogeneity of GBMs in situ. Given that the discrepancies between the radiosensitivity of GBM cells in vitro and the treatment response of patients suggest a role for the microenvironment in GBM radioresistance, we compared the response of TSCs and non-TSCs irradiated under in vitro and orthotopic conditions. As a measure of radioresponse determined at the individual cell level, γH2AX and 53BP1 foci were quantified in CD133+ cells and their differentiated (CD133- progeny. Under in vitro conditions, no difference was detected between CD133+ and CD133- cells in foci induction or dispersal after irradiation. However, irradiation of orthotopic xenografts initiated from TSCs resulted in the induction of fewer γH2AX and 53BP1 foci in CD133+ cells compared to their CD133- counterparts within the same tumor. Xenograft irradiation resulted in a tumor growth delay of approximately 7 days with a corresponding increase in the percentage of CD133+ cells at 7 days after radiation, which persisted to the onset of neurologic symptoms. These results suggest that, although the radioresponse of TSCs and non-TSCs does not differ under in vitro growth conditions, CD133+ cells are relatively radioresistant under intracerebral growth conditions. Whereas these findings are consistent with the suspected role for TSCs as a determinant of GBM radioresistance, these data also illustrate the dependence of the cellular radioresistance on the brain microenvironment.

  13. Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation

    Hiruma, Yuko; Honjo, Tadashi; Jelinek, Diane F.; Windle, Jolene J.; Shin, Jaekyoon; Roodman, G. David

    2009-01-01

    Adhesive interactions between multiple myeloma (MM) cells and marrow stromal cells activate multiple signaling pathways including nuclear factor κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and Jun N-terminal kinase (JNK) in stromal cells, which promote tumor growth and bone destruction. Sequestosome-1 (p62), an adapter protein that has no intrinsic enzymatic activity, serves as a platform to facilitate formation of signaling complexes for these pathways. Therefore, we determined if targeting only p62 would inhibit multiple signaling pathways activated in the MM microenvironment and thereby decrease MM cell growth and osteoclast formation. Signaling through NF-κB and p38 MAPK was increased in primary stromal cells from MM patients. Increased interleukin-6 (IL-6) production by MM stromal cells was p38 MAPK-dependent while increased vascular cell adhesion molecule-1 (VCAM-1) expression was NF-κB–dependent. Knocking-down p62 in patient-derived stromal cells significantly decreased protein kinase Cζ (PKCζ), VCAM-1, and IL-6 levels as well as decreased stromal cell support of MM cell growth. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, tumor necrosis factor-α (TNF-α), and receptor activator of NF-κB ligand (RANKL) and supported MM cell growth and osteoclast formation to a much lower extent than normal cells. Thus, p62 is an attractive therapeutic target for MM. PMID:19282458

  14. Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE

    Carmona, Guillaume; Perera, Upamali; Gillett, Cheryl; Naba, Alexandra; Law, Ah-Lai; Sharma, Ved P.; Wang, Jian; Wyckoff, Jeffrey; Balsamo, Michele; Mosis, Fuad; De Piano, Mario; Monypenny, James; Woodman, Natalie; McConnell, Russell E.; Mouneimne, Ghassan; Van Hemelrijck, Mieke; Cao, Yihai; Condeelis, John; Hynes, Richard O.; Gertler, Frank B.; Krause, Matthias

    2016-01-01

    Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlates with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation, and matrix degradation were impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not Ena/VASP is required for random 2D cell migration. We identify a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, while Src-dependent phosphorylation enhances binding to Scar/WAVE but not Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of EGF gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis. PMID:26996666

  15. Glioblastoma Stem Cells Microenvironment: The Paracrine Roles of the Niche in Drug and Radioresistance

    Alessia Fidoamore

    2016-01-01

    Full Text Available Among all solid tumors, the high-grade glioma appears to be the most vascularized one. In fact, “microvascular hyperplasia” is a hallmark of GBM. An altered vascular network determines irregular blood flow, so that tumor cells spread rapidly beyond the diffusion distance of oxygen in the tissue, with the consequent formation of hypoxic or anoxic areas, where the bulk of glioblastoma stem cells (GSCs reside. The response to this event is the induction of angiogenesis, a process mediated by hypoxia inducible factors. However, this new capillary network is not efficient in maintaining a proper oxygen supply to the tumor mass, thereby causing an oxygen gradient within the neoplastic zone. This microenvironment helps GSCs to remain in a “quiescent” state preserving their potential to proliferate and differentiate, thus protecting them by the effects of chemo- and radiotherapy. Recent evidences suggest that responses of glioblastoma to standard therapies are determined by the microenvironment of the niche, where the GSCs reside, allowing a variety of mechanisms that contribute to the chemo- and radioresistance, by preserving GSCs. It is, therefore, crucial to investigate the components/factors of the niche in order to formulate new adjuvant therapies rendering more efficiently the gold standard therapies for this neoplasm.

  16. Development of 3D in vitro platform technology to engineer mesenchymal stem cells

    Hosseinkhani H

    2012-06-01

    Full Text Available Hossein Hosseinkhani,1 Po-Da Hong,1 Dah-Shyong Yu,2 Yi-Ru Chen,3 Diana Ickowicz,4 Ira-Yudovin Farber,4 Abraham J Domb41Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology (TAIWANTECH, 2Nanomedicine Research Center, National Defense Medical Center, Taipei, Taiwan, 3Department of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan, 4Institute of Drug Research, The Center for Nanoscience and Nanotechnology, School of Pharmacy-Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, IsraelAbstract: This study aims to develop a three-dimensional in vitro culture system to genetically engineer mesenchymal stem cells (MSC to express bone morphogenic protein-2. We employed nanofabrication technologies borrowed from the spinning industry, such as electrospinning, to mass-produce identical building blocks in a variety of shapes and sizes to fabricate electrospun nanofiber sheets comprised of composites of poly (glycolic acid and collagen. Homogenous nanoparticles of cationic biodegradable natural polymer were formed by simple mixing of an aqueous solution of plasmid DNA encoded bone morphogenic protein-2 with the same volume of cationic polysaccharide, dextran-spermine. Rat bone marrow MSC were cultured on electrospun nanofiber sheets comprised of composites of poly (glycolic acid and collagen prior to the incorporation of the nanoparticles into the nanofiber sheets. Bone morphogenic protein-2 was significantly detected in MSC cultured on nanofiber sheets incorporated with nanoparticles after 2 days compared with MSC cultured on nanofiber sheets incorporated with naked plasmid DNA. We conclude that the incorporation of nanoparticles into nanofiber sheets is a very promising strategy to genetically engineer MSC and can be used for further applications in regenerative medicine therapy.Keywords: 3D culture, nanoparticles, nanofibers, polycations, tissue engineering

  17. A 3D Poly(ethylene glycol)-based Tumor Angiogenesis Model to Study the Influence of Vascular Cells on Lung Tumor Cell Behavior

    Laila C. Roudsari; Jeffs, Sydney E.; Witt, Amber S.; Gill, Bartley J.; West, Jennifer L.

    2016-01-01

    Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. In vitro models that mimic in vivo tumor neovascularization facilitate exploration of this role. Here we investigated lung adenocarcinoma cancer cells (344SQ) and endothelial and pericyte vascular cells encapsulated in cell-adhesive, proteolytically-degradable poly(ethylene) glycol-based hydrogels. 344SQ in hydrogels formed spheroids and secreted...

  18. FRET imaging of calcium signaling in live cells in the microenvironment.

    Qian, Tongcheng; Lu, Shaoying; Ma, Hongwei; Fang, Jing; Zhong, Wenxuan; Wang, Yingxiao

    2013-02-01

    The microenvironment has been shown to regulate cellular functions including cell growth, differentiation, proliferation, migration, cancer development and metastasis. However, the underlying molecular mechanism remains largely unclear. We have integrated micro-pattern technology and molecular biosensors based on fluorescence resonance energy transfer (FRET) to visualize calcium responses in cells constrained to grow on a micro-patterned surface. Upon ATP stimulation, human umbilical vein endothelial cells (HUVECs) cultured on different surface micro-patterns had a shorter decay time and a reduced peak of a transient intracellular calcium rise compared to control cells without constraints. The decay time is regulated by the plasma membrane and the membrane calcium channels, while the peak by endoplasmic reticulum (ER) calcium release. Further results revealed that voltage operated channels (VOCs), coupling the plasma membrane and ER, can affect both the decay time and the peak of calcium response. The inhibition of VOCs can eliminate the effect of different micro-patterns on calcium signals. When two connected HUVECs were constrained to grow on a micro-pattern, drastically distinct calcium responses upon ATP stimulation can be observed, in contrast to the similar responses of two connected cells cultured without patterns. Interestingly, the inhibition of VOCs also blocked this difference of calcium responses between two connected cells on micro-patterns. These results indicate that a micro-patterned surface can have a profound effect on the calcium responses of HUVECs under ATP stimulation, largely mediated by VOCs. Therefore, our results shed new light on the molecular mechanism by which HUVECs perceive the microenvironment and regulate intracellular calcium signals.

  19. Quantifying transient 3D dynamical phenomena of single mRNA particles in live yeast cell measurements.

    Calderon, Christopher P; Thompson, Michael A; Casolari, Jason M; Paffenroth, Randy C; Moerner, W E

    2013-12-12

    Single-particle tracking (SPT) has been extensively used to obtain information about diffusion and directed motion in a wide range of biological applications. Recently, new methods have appeared for obtaining precise (10s of nm) spatial information in three dimensions (3D) with high temporal resolution (measurements obtained every 4 ms), which promise to more accurately sense the true dynamical behavior in the natural 3D cellular environment. Despite the quantitative 3D tracking information, the range of mathematical methods for extracting information about the underlying system has been limited mostly to mean-squared displacement analysis and other techniques not accounting for complex 3D kinetic interactions. There is a great need for new analysis tools aiming to more fully extract the biological information content from in vivo SPT measurements. High-resolution SPT experimental data has enormous potential to objectively scrutinize various proposed mechanistic schemes arising from theoretical biophysics and cell biology. At the same time, methods for rigorously checking the statistical consistency of both model assumptions and estimated parameters against observed experimental data (i.e., goodness-of-fit tests) have not received great attention. We demonstrate methods enabling (1) estimation of the parameters of 3D stochastic differential equation (SDE) models of the underlying dynamics given only one trajectory; and (2) construction of hypothesis tests checking the consistency of the fitted model with the observed trajectory so that extracted parameters are not overinterpreted (the tools are applicable to linear or nonlinear SDEs calibrated from nonstationary time series data). The approach is demonstrated on high-resolution 3D trajectories of single ARG3 mRNA particles in yeast cells in order to show the power of the methods in detecting signatures of transient directed transport. The methods presented are generally relevant to a wide variety of 2D and 3D SPT

  20. Sustained PDGF-BB release from PHBHHx loaded nanoparticles in 3D hydrogel/stem cell model.

    Dong, Cui-Ling; Webb, William R; Peng, Qiang; Tang, James Z; Forsyth, Nicholas R; Chen, Guo-Qiang; El Haj, Alicia J

    2015-01-01

    This study aimed to design a growth factor loaded copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) nanoparticles containing 3D collagen matrix to achieve growth factor sustained release for long-term stimulation of human mesenchymal stem cells (hMSCs) proliferation/differentiation for tissue engineer application. Platelet-derived growth factor-BB (PDGF-BB), which is known to enhance hMSCs proliferation in human serum, was selected as a model growth factor, and biodegradable copolyester of PHBHHx was chosen to be the sustained release vehicle. PDGF-BB phospholipid complex encapsulated PHBHHx nanoparticles were fabricated, and their effect on hMSCs proliferation was investigated via assays of CCK-8 and live-dead staining to cells inoculated in 2D tissue culture plates and 3D collagen gel scaffolds, respectively. The resulting spherical PHBHHx nanoparticles were stable in terms of their mean particle size, polydispersity index and zeta potential before and after lyophilization. In vitro study revealed a sustained release of PDGF-BB with a low burst release. Furthermore, sustained released PDGF-BB was revealed to significantly promote hMSCs proliferation in both cell monolayer and cell seeded 3D collagen scaffolds inoculated in serum-free media. Therefore, the 3D collagen matrices with locally sustained release growth factor nanoparticles hold promise to be used for stem cell tissue engineering.

  1. The effect of porosity on cell ingrowth into accurately defined, laser-made, polylactide-based 3D scaffolds

    Danilevicius, Paulius; Georgiadi, Leoni [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Pateman, Christopher J.; Claeyssens, Frederik [Kroto Research Institute, Department of Materials Science and Engineering, University of Sheffield, Broad Lane, Sheffield S3 7HQ (United Kingdom); Chatzinikolaidou, Maria, E-mail: mchatzin@materials.uoc.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Department of Materials Science and Technology, University of Crete, PO Box 2208, 71303 Heraklion (Greece); Farsari, Maria, E-mail: mfarsari@iesl.forth.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece)

    2015-05-01

    Highlights: • We studied the porosity of laser-made 3D scaffolds on MC3T3-E1 pre-osteoblastic cells. • We made polylactide 3D scaffolds with pores 25–110 μm. - Abstract: The aim of this study is to demonstrate the accuracy required for the investigation of the role of solid scaffolds’ porosity in cell proliferation. We therefore present a qualitative investigation into the effect of porosity on MC3T3-E1 pre-osteoblastic cell ingrowth of three-dimensional (3D) scaffolds fabricated by direct femtosecond laser writing. The material we used is a purpose made photosensitive pre-polymer based on polylactide. We designed and fabricated complex, geometry-controlled 3D scaffolds with pore sizes ranging from 25 to 110 μm, representing porosities 70%, 82%, 86%, and 90%. The 70% porosity scaffolds did not support cell growth initially and in the long term. For the other porosities, we found a strong adhesion of the pre-osteoblastic cells from the first hours after seeding and a remarkable proliferation increase after 3 weeks and up to 8 weeks. The 86% porosity scaffolds exhibited a higher efficiency compared to 82% and 90%. In addition, bulk material degradation studies showed that the employed, highly-acrylated polylactide is degradable. These findings support the potential use of the proposed material and the scaffold fabrication technique in bone tissue engineering.

  2. Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

    Novikov, I A; Vakhrushev, I V; Antonov, E N; Yarygin, K N; Subbot, A M

    2017-02-01

    Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

  3. Preparation of 3D electrode microarrays of multi-walled carbon nanotubes/nafion nanocomposites for microfluidic biofuel cells.

    Choi, Jin Ho; Kim, Young Ho; Choi, Sung Deuk; Kim, Gyu Man

    2014-12-01

    Three-dimensional (3D) electrode microarrays with multi-walled carbon nanotubes (MWCNTs) reinforced Nafion nanocomposites were prepared for microfluidic biofuel cells. The oxidized MWCNTs (ox-MWCNTs) were prepared using chemical reactions with 60% nitric acid solution with pristine MWCNTs at 120 degrees C for 12 hrs with a nitrogen gas flow environment. Ox-MWCNTs in the range of 1 to 20 wt.% based on the Nafion polymer weight were reinforced to Nafion nanocomposites by solution casting. The micro-porous structure of the ox-MWCNTs reinforced Nafion nanocomposites was prepared by plasma etching for 5 to 20 min. The 10 wt.% ox-MWCNTs reinforced Nafion nanocomposite produced stable micro-porous structures of 3D electrodes by 10 min plasma etching. Micro-scale 3D structures of MWCNTs reinforced Nafion nanocomposites in a diameter range of 47 to 300 μm were prepared by the micro-stencil assisted casting. To characterize the 3D electrode microarrays, the physical geometry and the reinforced MWCNT dispersion in the nanocomposite structure were examined using a scanning electron microscope (SEM) and an optical microscope. Thermal property measurements of the ox-MWCNTs reinforced Nafion nanocomposites with 10 min of plasma etching, and without plasma etching were made. Both showed stable thermal properties over 300 degrees C. The proposed 3D electrode microarray of MWCNT/Nafion nanocomposites with micro-porous structures can be applied to miniaturized fuel cell devices.

  4. Tumor Irradiation Increases the Recruitment of Circulating Mesenchymal Stem Cells into the Tumor Microenvironment

    Klopp, Ann H.; Spaeth, Erika L.; Dembinski, Jennifer L.; Woodward, Wendy A.; Munshi, Anupama; Meyn, Raymond E.; Cox, James D.; Andreeff, Michael; Marini, Frank C.

    2011-01-01

    Mesenchymal stem cells (MSC) migrate to and proliferate within sites of inflammation and tumors as part of the tissue remodeling process. Radiation increases the expression of inflammatory mediators that could enhance the recruitment of MSC into the tumor microenvironment. To investigate this, bilateral murine 4T1 breast carcinomas (expressing renilla luciferase) were irradiated unilaterally (1 or 2 Gy). Twenty-four hours later, 2 × 105 MSC-expressing firefly luciferase were injected i.v. Mice were then monitored with bioluminescent imaging for expression of both renilla (tumor) and firefly (MSC) luciferase. Forty-eight hours postirradiation, levels of MSC engraftment were 34% higher in tumors receiving 2 Gy (P = 0.004) than in the contralateral unirradiated limb. Immunohistochemical staining of tumor sections from mice treated unilaterally with 2 Gy revealed higher levels of MSC in the parenchyma of radiated tumors, whereas a higher proportion of MSC remained vasculature-associated in unirradiated tumors. To discern the potential mediators involved in MSC attraction, in vitro migration assays showed a 50% to 80% increase in MSC migration towards conditioned media from 1 to 5 Gy-irradiated 4T1 cells compared with unirradiated 4T1 cells. Irradiated 4T1 cells had increased expression of the cytokines, transforming growth factor-β1, vascular endothelial growth factor, and platelet-derived growth factor-BB, and this up-regulation was confirmed by immunohistochemistry in tumors irradiated in vivo. Interestingly, the chemokine receptor CCR2 was found to be up-regulated in MSC exposed to irradiated tumor cells and inhibition of CCR2 led to a marked decrease of MSC migration in vitro. In conclusion, clinically relevant low doses of irradiation increase the tropism for and engraftment of MSC in the tumor microenvironment. PMID:18089798

  5. Cytokines from the tumor microenvironment modulate sirtinol cytotoxicity in A549 lung carcinoma cells.

    Pal, Shyama; Shankar, Bhavani S; Sainis, Krishna B

    2013-10-01

    Cytokines in tumor microenvironment play an important role in the success or failure of molecular targeted therapies. We have chosen tumor necrosis factor α (TNF-α), TNF related apoptosis inducing ligand (TRAIL), insulin-like growth factor 1 (IGF-1) and transforming growth factor β (TGF-β) as representative pro-inflammatory, pro-apoptotic, anti-apoptotic and anti-inflammatory tumor derived cytokines. Analysis of Oncomine database revealed the differential expression of these cytokines in a subset of cancer patients. The effects of these cytokines on cytotoxicity of FDA approved drugs - cisplatin and taxol and inhibitors of epidermal growth factor receptor - AG658, Janus kinase - AG490 and SIRT1 - sirtinol were assessed in A549 lung cancer cells. TRAIL augmented cytotoxicity of sirtinol and IGF-1 had a sparing effect. Since TRAIL and IGF-1 differentially modulated sirtinol cytotoxicity, further studies were carried out to identify the mechanisms. Sirtinol or knockdown of SIRT1 increased the expression of death receptors DR4 and DR5 and sensitized A549 cells to TRAIL. Increased cell death in presence of TRAIL and sirtinol was caspase independent and demonstrated classical features of necroptosis. Inhibition of iNOS increased caspase activity and switched the mode of cell death to caspase mediated apoptosis. Interestingly, sirtinol or SIRT1 knockdown did not increase IGF-1R expression. Instead, it abrogated ligand induced downregulation of IGF-1R and increased cell survival through PI3K-AKT pathway. In conclusion, these findings reveal that the tumor microenvironment contributes to modulation of cytotoxicity of drugs and that combination therapy, with agents that increase TRAIL signaling and suppress IGF-1 pathway may potentiate anticancer effect.

  6. Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments

    LaBarge, Mark A; Nelson, Celeste M; Villadsen, Rene; Fridriksdottir, Agla; Ruth, Jason R; Stampfer, Martha R; Petersen, Ole W; Bissell, Mina J

    2008-09-19

    In adult tissues, multi-potent progenitor cells are some of the most primitive members of the developmental hierarchies that maintain homeostasis. That progenitors and their more mature progeny share identical genomes, suggests that fate decisions are directed by interactions with extrinsic soluble factors, ECM, and other cells, as well as physical properties of the ECM. To understand regulation of fate decisions, therefore, would require a means of understanding carefully choreographed combinatorial interactions. Here we used microenvironment protein microarrays to functionally identify combinations of cell-extrinsic mammary gland proteins and ECM molecules that imposed specific cell fates on bipotent human mammary progenitor cells. Micropatterned cell culture surfaces were fabricated to distinguish between the instructive effects of cell-cell versus cell-ECM interactions, as well as constellations of signaling molecules; and these were used in conjunction with physiologically relevant 3 dimensional human breast cultures. Both immortalized and primary human breast progenitors were analyzed. We report on the functional ability of those proteins of the mammary gland that maintain quiescence, maintain the progenitor state, and guide progenitor differentiation towards myoepithelial and luminal lineages.

  7. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions

    Williams, David A.; Rios, Maribel; Stephens, Carmella; Patel, Vikram P.

    1991-08-01

    THE self-renewal and differentiation of haematopoietic stem cells occurs in vivo and in vitro in direct contact with cells making up the haematopoietic microenvironment1-4. In this study we used adhesive ligands and blocking antibodies to identify stromal cell-derived extracellular matrix proteins involved in promoting attachment of murine haematopoietic stem cells. Here we report that day-12 colony-forming-unit spleen (CFU-S12)5 cells and reconstituting haematopoietic stem cells attach to the C-terminal, heparin-binding fragment of fibronectin by recognizing the CS-1 peptide of the alternatively spliced non-type III connecting segment (IIICS) of human plasma fibronectin. Furthermore, CFU-S12 stem cells express the α4 subunit of the VLA-4 integrin receptor, which is known to be a receptor for the CS-1 sequence, and monoclonal antibodies against the integrin α4 subunit of VLA-4 block adhesion of CFU-S12 stem cells to plates coated with the C-terminal fibronectin fragment. Finally, polyclonal antibodies against the integrin β1 subunit of VLA-4 inhibit the formation of CFU-S12-derived spleen colonies and medullary haematopoiesis in vivo following intravenous infusion of antibody-treated bone marrow cells.

  8. Periodontal Ligament Stem Cells in the Periodontitis Microenvironment Are Sensitive to Static Mechanical Strain

    Jia Liu

    2017-01-01

    Full Text Available During orthodontic treatment, periodontium remodeling of periodontitis patients under mechanical force was abnormal. We have previously confirmed the function impairment of periodontal ligament stem cells (PDLSCs in the periodontitis microenvironment which might be involved in this pathological process. However, the response of PDLSCs in periodontitis microenvironment to mechanical force remains unclear. Therefore, in the present study, we introduced a Flexcell tension apparatus and investigated the response of PDLSCs obtained from periodontal tissues of periodontitis patients (PPDLSCs and of those obtained from healthy periodontal tissues (HPDLSCs to different magnitudes of static mechanical strain (SMS. PPDLSCs showed increased proliferation, decreased osteogenic activity, activated osteoclastogenesis, and greater secretion of inflammatory cytokines. Different magnitudes of SMS exerted distinct effects on HPDLSCs and PPDLSCs. An SMS of 12% induced optimal effects in HPDLSCs, including the highest proliferation, the best osteogenic ability, the lowest osteoclastogenesis, and the lowest secretion of inflammatory cytokines, while the optimal SMS for PPDLSCs was 8%. Excessive SMS damaged PPDLSCs function, including decreased proliferation, an imbalance between osteogenesis and osteoclastogenesis, and an activated inflammatory response. Our data suggest that PPDLSCs are more sensitive and less tolerant to SMS, and this may explain why mechanical force results in undesirable effects in periodontitis patients.

  9. Periodontal Ligament Stem Cells in the Periodontitis Microenvironment Are Sensitive to Static Mechanical Strain

    Liu, Jia; Liu, Shiyu; Gao, Jie; Qin, Wen; Song, Yang

    2017-01-01

    During orthodontic treatment, periodontium remodeling of periodontitis patients under mechanical force was abnormal. We have previously confirmed the function impairment of periodontal ligament stem cells (PDLSCs) in the periodontitis microenvironment which might be involved in this pathological process. However, the response of PDLSCs in periodontitis microenvironment to mechanical force remains unclear. Therefore, in the present study, we introduced a Flexcell tension apparatus and investigated the response of PDLSCs obtained from periodontal tissues of periodontitis patients (PPDLSCs) and of those obtained from healthy periodontal tissues (HPDLSCs) to different magnitudes of static mechanical strain (SMS). PPDLSCs showed increased proliferation, decreased osteogenic activity, activated osteoclastogenesis, and greater secretion of inflammatory cytokines. Different magnitudes of SMS exerted distinct effects on HPDLSCs and PPDLSCs. An SMS of 12% induced optimal effects in HPDLSCs, including the highest proliferation, the best osteogenic ability, the lowest osteoclastogenesis, and the lowest secretion of inflammatory cytokines, while the optimal SMS for PPDLSCs was 8%. Excessive SMS damaged PPDLSCs function, including decreased proliferation, an imbalance between osteogenesis and osteoclastogenesis, and an activated inflammatory response. Our data suggest that PPDLSCs are more sensitive and less tolerant to SMS, and this may explain why mechanical force results in undesirable effects in periodontitis patients. PMID:28316629

  10. Recreating the Cardiac Microenvironment in Pluripotent Stem Cell Models of Human Physiology and Disease.

    Atmanli, Ayhan; Domian, Ibrahim John

    2016-12-19

    The advent of human pluripotent stem cell (hPSC) biology has opened unprecedented opportunities for the use of tissue engineering to generate human cardiac tissue for in vitro study. Engineering cardiac constructs that recapitulate human development and disease requires faithful recreation of the cardiac niche in vitro. Here we discuss recent progress in translating the in vivo cardiac microenvironment into PSC models of the human heart. We review three key physiologic features required to recreate the cardiac niche and facilitate normal cardiac differentiation and maturation: the biochemical, biophysical, and bioelectrical signaling cues. Finally, we discuss key barriers that must be overcome to fulfill the promise of stem cell biology in preclinical applications and ultimately in clinical practice.

  11. Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line

    Fey, S. J.; Wrzesinski, Krzysztof

    2012-01-01

    , diclofenac, metformin, phenformin, and valproic acid) to LD50 data (mg compound/mg cellular protein) showed that the variation in LD50 values was generally less than that suggested by the original LC50 data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented...

  12. Electric field-controlled directed migration of neural progenitor cells in 2D and 3D environments.

    Meng, Xiaoting; Li, Wenfei; Young, Fraser; Gao, Runchi; Chalmers, Laura; Zhao, Min; Song, Bing

    2012-02-16

    Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system(1,2). These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans(3,4). In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types(5,6), including neural progenitor cells (NPCs)(7,8). Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously(5,11). Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures(9,10). Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

  13. Enabling Lorentz boosted frame particle-in-cell simulations of laser wakefield acceleration in quasi-3D geometry

    Yu, Peicheng; Xu, Xinlu; Davidson, Asher; Tableman, Adam; Dalichaouch, Thamine; Li, Fei; Meyers, Michael D.; An, Weiming; Tsung, Frank S.; Decyk, Viktor K.; Fiuza, Frederico; Vieira, Jorge; Fonseca, Ricardo A.; Lu, Wei; Silva, Luis O.; Mori, Warren B.

    2016-07-01

    When modeling laser wakefield acceleration (LWFA) using the particle-in-cell (PIC) algorithm in a Lorentz boosted frame, the plasma is drifting relativistically at βb c towards the laser, which can lead to a computational speedup of ∼ γb2 = (1 - βb2)-1. Meanwhile, when LWFA is modeled in the quasi-3D geometry in which the electromagnetic fields and current are decomposed into a limited number of azimuthal harmonics, speedups are achieved by modeling three dimensional (3D) problems with the computational loads on the order of two dimensional r - z simulations. Here, we describe a method to combine the speedups from the Lorentz boosted frame and quasi-3D algorithms. The key to the combination is the use of a hybrid Yee-FFT solver in the quasi-3D geometry that significantly mitigates the Numerical Cerenkov Instability (NCI) which inevitably arises in a Lorentz boosted frame due to the unphysical coupling of Langmuir modes and EM modes of the relativistically drifting plasma in these simulations. In addition, based on the space-time distribution of the LWFA data in the lab and boosted frame, we propose to use a moving window to follow the drifting plasma, instead of following the laser driver as is done in the LWFA lab frame simulations, in order to further reduce the computational loads. We describe the details of how the NCI is mitigated for the quasi-3D geometry, the setups for simulations which combine the Lorentz boosted frame, quasi-3D geometry, and the use of a moving window, and compare the results from these simulations against their corresponding lab frame cases. Good agreement is obtained among these sample simulations, particularly when there is no self-trapping, which demonstrates it is possible to combine the Lorentz boosted frame and the quasi-3D algorithms when modeling LWFA. We also discuss the preliminary speedups achieved in these sample simulations.

  14. Advanced 3D Poisson solvers and particle-in-cell methods for accelerator modeling

    Serafini, David B; McCorquodale, Peter; Colella, Phillip [Lawrence Berkeley National Lab, Applied Numerical Algorithms Group, SciDAC Applied Differential Equations Center (United States)

    2005-01-01

    We seek to improve on the conventional FFT-based algorithms for solving the Poisson equation with infinite-domain (open) boundary conditions for large problems in accelerator modeling and related areas. In particular, improvements in both accuracy and performance are possible by combining several technologies: the method of local corrections (MLC); the James algorithm; and adaptive mesh refinement (AMR). The MLC enables the parallelization (by domain decomposition) of problems with large domains and many grid points. This improves on the FFT-based Poisson solvers typically used as it doesn't require the all-to-all communication pattern that parallel 3d FFT algorithms require, which tends to be a performance bottleneck on current (and foreseeable) parallel computers. In initial tests, good scalability up to 1000 processors has been demonstrated for our new MLC solver. An essential component of our approach is a new version of the James algorithm for infinite-domain boundary conditions for the case of three dimensions. By using a simplified version of the fast multipole method in the boundary-to-boundary potential calculation, we improve on the performance of the Hockney algorithm typically used by reducing the number of grid points by a factor of 8, and the CPU costs by a factor of 3. This is particularly important for large problems where computer memory limits are a consideration. The MLC allows for the use of adaptive mesh refinement, which reduces the number of grid points and increases the accuracy in the Poisson solution. This improves on the uniform grid methods typically used in PIC codes, particularly in beam problems where the halo is large. Also, the number of particles per cell can be controlled more closely with adaptivity than with a uniform grid. To use AMR with particles is more complicated than using uniform grids. It affects depositing particles on the non-uniform grid, reassigning particles when the adaptive grid changes and maintaining the

  15. Hematopoietic stem cell-derived adipocytes and fibroblastsin the tumor microenvironment

    Ying Xiong; Lindsay T McDonald; Dayvia L Russell; Ryan R Kelly; Katie R Wilson; Meenal Mehrotra; Adam C Soloff; Amanda C LaRue

    2015-01-01

    The tumor microenvironment (TME) is complex andconstantly evolving. This is due, in part, to the crosstalkbetween tumor cells and the multiple cell types thatcomprise the TME, which results in a heterogeneouspopulation of tumor cells and TME cells. This reviewwill focus on two stromal cell types, the cancerassociatedadipocyte (CAA) and the cancer-associatedfibroblast (CAF). In the clinic, the presence of CAAsand CAFs in the TME translates to poor prognosis inmultiple tumor types. CAAs and CAFs have an activatedphenotype and produce growth factors, inflammatoryfactors, cytokines, chemokines, extracellular matrixcomponents, and proteases in an accelerated andaberrant fashion. Through this activated state, CAAs andCAFs remodel the TME, thereby driving all aspects oftumor progression, including tumor growth and survival,chemoresistance, tumor vascularization, tumor invasion,and tumor cell metastasis. Similarities in the tumorpromotingfunctions of CAAs and CAFs suggest that amultipronged therapeutic approach may be necessaryto achieve maximal impact on disease. While CAAsand CAFs are thought to arise from tissues adjacentto the tumor, multiple alternative origins for CAAs andCAFs have recently been identified. Recent studiesfrom our lab and others suggest that the hematopoieticstem cell, through the myeloid lineage, may serve asa progenitor for CAAs and CAFs. We hypothesize thatthe multiple origins of CAAs and CAFs may contributeto the heterogeneity seen in the TME. Thus, a betterunderstanding of the origin of CAAs and CAFs, howthis origin impacts their functions in the TME, and the temporal participation of uniquely originating TME cells may lead to novel or improved anti-tumor therapeutics.

  16. Modulating the physical microenvironment to study regenerative processes in vitro using cells from mouse phalangeal elements.

    Lynch, Kristen M; Ahsan, Tabassum

    2013-06-01

    Epimorphic regeneration in humans of complex multitissue structures is primarily limited to the digit tip. In a comparable mouse model, the response is level-specific in that regeneration occurs after amputation at the distal end of the terminal phalanx, but not more proximally. Recent isolation of stromal cells from CD1 murine phalangeal elements two and three (P2 and P3) allow for comparative studies of cells prevalent at the amputation plane of a more proximal region (considered nonregenerative) and a more distal region (considered regenerative), respectively. This study used adherent, suspension, and collagen gel cultures to investigate cellular processes relevant to the initial response to injury. Overall, P2 cells were both more migratory and able to compact collagen gels to a greater extent compared to P3 cells. This observed increased capacity of P2 cells to generate traction forces was likely related to the higher expression of key cytoskeletal proteins (e.g., microfilament, nonkeratin intermediate filaments, and microtubules) compared to P3 cells. In contrast, P3 cells were found to be more proliferative than P2 cells under all three culture conditions and to have higher expression of keratin proteins. In addition, when cultured in suspension rather than on adherent surfaces, P3 cells were both more proliferative and had greater gene expression for matrix proteins. Together these results add to the known inherent differences in these stromal cells by characterizing responses to the physical microenvironment. Further, while compaction by P2 cells confirm that collagen gels is a useful model to study wound healing, the response of P3 cells indicate that suspension culture, in which cell-cell interactions dominate like in the blastema, may be better suited to study regeneration. Therefore, this study can help develop clinical strategies for promoting regeneration through increased understanding in the properties of cells involved in endogenous repair as well

  17. 3D bioprinting of neural stem cell-laden thermoresponsive biodegradable polyurethane hydrogel and potential in central nervous system repair.

    Hsieh, Fu-Yu; Lin, Hsin-Hua; Hsu, Shan-Hui

    2015-12-01

    The 3D bioprinting technology serves as a powerful tool for building tissue in the field of tissue engineering. Traditional 3D printing methods involve the use of heat, toxic organic solvents, or toxic photoinitiators for fabrication of synthetic scaffolds. In this study, two thermoresponsive water-based biodegradable polyurethane dispersions (PU1 and PU2) were synthesized which may form gel near 37 °C without any crosslinker. The stiffness of the hydrogel could be easily fine-tuned by the solid content of the dispersion. Neural stem cells (NSCs) were embedded into the polyurethane dispersions before gelation. The dispersions containing NSCs were subsequently printed and maintained at 37 °C. The NSCs in 25-30% PU2 hydrogels (∼680-2400 Pa) had excellent proliferation and differentiation but not in 25-30% PU1 hydrogels. Moreover, NSC-laden 25-30% PU2 hydrogels injected into the zebrafish embryo neural injury model could rescue the function of impaired nervous system. However, NSC-laden 25-30% PU1 hydrogels only showed a minor repair effect in the zebrafish model. In addition, the function of adult zebrafish with traumatic brain injury was rescued after implantation of the 3D-printed NSC-laden 25% PU2 constructs. Therefore, the newly developed 3D bioprinting technique involving NSCs embedded in the thermoresponsive biodegradable polyurethane ink offers new possibilities for future applications of 3D bioprinting in neural tissue engineering.

  18. The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability.

    Billiet, Thomas; Gevaert, Elien; De Schryver, Thomas; Cornelissen, Maria; Dubruel, Peter

    2014-01-01

    In the present study, we report on the combined efforts of material chemistry, engineering and biology as a systemic approach for the fabrication of high viability 3D printed macroporous gelatin methacrylamide constructs. First, we propose the use and optimization of VA-086 as a photo-initiator with enhanced biocompatibility compared to the conventional Irgacure 2959. Second, a parametric study on the printing of gelatins was performed in order to characterize and compare construct architectures. Hereby, the influence of the hydrogel building block concentration, the printing temperature, the printing pressure, the printing speed, and the cell density were analyzed in depth. As a result, scaffolds could be designed having a 100% interconnected pore network in the gelatin concentration range of 10-20 w/v%. In the last part, the fabrication of cell-laden scaffolds was studied, whereby the application for tissue engineering was tested by encapsulation of the hepatocarcinoma cell line (HepG2). Printing pressure and needle shape was revealed to impact the overall cell viability. Mechanically stable cell-laden gelatin methacrylamide scaffolds with high cell viability (>97%) could be printed.

  19. A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.

    Campbell, Jonathan J; Davidenko, Natalia; Caffarel, Maria M; Cameron, Ruth E; Watson, Christine J

    2011-01-01

    Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

  20. A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.

    Jonathan J Campbell

    Full Text Available Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM in three dimensional (3D space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

  1. Cell membrane-anchored biosensors for real-time monitoring of the cellular microenvironment.

    Qiu, Liping; Zhang, Tao; Jiang, Jianhui; Wu, Cuichen; Zhu, Guizhi; You, Mingxu; Chen, Xigao; Zhang, Liqin; Cui, Cheng; Yu, Ruqin; Tan, Weihong

    2014-09-24

    Cell membrane-anchored biochemical sensors that allow real-time monitoring of the interactions of cells with their microenvironment would be powerful tools for studying the mechanisms underlying various biological processes, such as cell metabolism and signaling. Despite the significance of these techniques, unfortunately, their development has lagged far behind due to the lack of a desirable membrane engineering method. Here, we propose a simple, efficient, biocompatible, and universal strategy for one-step self-construction of cell-surface sensors using diacyllipid-DNA conjugates as the building and sensing elements. The sensors exploit the high membrane-insertion capacity of a diacyllipid tail and good sensing performance of the DNA probes. Based on this strategy, we have engineered specific DNAzymes on the cell membrane for metal ion assay in the extracellular microspace. The immobilized DNAzyme showed excellent performance for reporting and semiquantifying both exogenous and cell-extruded target metal ions in real time. This membrane-anchored sensor could also be used for multiple target detection by having different DNA probes inserted, providing potentially useful tools for versatile applications in cell biology, biomedical research, drug discovery, and tissue engineering.

  2. Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

    Lambros, Maria Polikandritou, E-mail: mlambros@westernu.edu [Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Parsa, Cyrus [Department of Clinical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, Pomona, CA 91766 (United States); Mulamalla, HariChandana [Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Orlando, Robert [Department of Clinical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, Pomona, CA 91766 (United States); Lau, Bernard [Center for Advancement of Drug Research and Evaluation (CADRE), Western University of Health Sciences, Pomona, CA 91766 (United States); Huang, Ying [Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Center for Advancement of Drug Research and Evaluation (CADRE), Western University of Health Sciences, Pomona, CA 91766 (United States); Pon, Doreen [Department of Pharmacy Practice and Administration, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Chow, Moses [Department of Pharmacy Practice and Administration, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Center for Advancement of Drug Research and Evaluation (CADRE), Western University of Health Sciences, Pomona, CA 91766 (United States)

    2011-02-04

    Research highlights: {yields} We irradiated a 3-D human oral cell culture of keratinocytes and fibroblasts with 12 and 2 Gy. {yields} 6 h after irradiation the histopathology and apoptosis of the 3-D culture were evaluated. Microarrays were used to assess the gene expression in the irradiated 3-D tissue. {yields} 12 Gy induced significant histopathologic changes and cellular apoptosis. {yields} 12 Gy significantly affected genes of the NF-kB pathway, inflammatory cytokines and DAMPs. -- Abstract: Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this

  3. Role of nanotopography in the development of tissue engineered 3D organs and tissues using mesenchymal stem cells.

    Salmasi, Shima; Kalaskar, Deepak M; Yoon, Wai-Weng; Blunn, Gordon W; Seifalian, Alexander M

    2015-03-26

    Recent regenerative medicine and tissue engineering strategies (using cells, scaffolds, medical devices and gene therapy) have led to fascinating progress of translation of basic research towards clinical applications. In the past decade, great deal of research has focused on developing various three dimensional (3D) organs, such as bone, skin, liver, kidney and ear, using such strategies in order to replace or regenerate damaged organs for the purpose of maintaining or restoring organs' functions that may have been lost due to aging, accident or disease. The surface properties of a material or a device are key aspects in determining the success of the implant in biomedicine, as the majority of biological reactions in human body occur on surfaces or interfaces. Furthermore, it has been established in the literature that cell adhesion and proliferation are, to a great extent, influenced by the micro- and nano-surface characteristics of biomaterials and devices. In addition, it has been shown that the functions of stem cells, mesenchymal stem cells in particular, could be regulated through physical interaction with specific nanotopographical cues. Therefore, guided stem cell proliferation, differentiation and function are of great importance in the regeneration of 3D tissues and organs using tissue engineering strategies. This review will provide an update on the impact of nanotopography on mesenchymal stem cells for the purpose of developing laboratory-based 3D organs and tissues, as well as the most recent research and case studies on this topic.

  4. 3D Plant Cell Architecture of Arabidopsis thaliana (Brassicaceae Using Focused Ion Beam–Scanning Electron Microscopy

    Bhawana

    2014-06-01

    Full Text Available Premise of the study: Focused ion beam–scanning electron microscopy (FIB-SEM combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron-level resolution. We have applied FIB-SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution. Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type. Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue. Discussion: The application of FIB-SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB-SEM for organelle distribution and cell architecture studies.

  5. The fabrication of double layer tubular vascular tissue engineering scaffold via coaxial electrospinning and its 3D cell coculture.

    Ye, Lin; Cao, Jie; Chen, Lamei; Geng, Xue; Zhang, Ai-Ying; Guo, Lian-Rui; Gu, Yong-Quan; Feng, Zeng-Guo

    2015-12-01

    A continuous electrospinning technique was applied to fabricate double layer tubular tissue engineering vascular graft (TEVG) scaffold. The luminal layer was made from poly(ɛ-caprolac-tone)(PCL) ultrafine fibers via common single axial electrospinning followed by the outer layer of core-shell structured nanofibers via coaxial electrospinning. For preparing the outer layernano-fibers, the PCL was electrospun into the shell and both bovine serum albumin (BSA) and tetrapeptide val-gal-pro-gly (VAPG) were encapsulated into the core. The core-shell structure in the outer layer fibers was observed by transmission electron microscope (TEM). The in vitro release tests exhibited the sustainable release behavior of BSA and VAPG so that they provided a better cell growth environment in the interior of tubular scaffold wall. The in vitro culture of smooth muscle cells (SMCs) demonstrated their potential to penetrate into the scaffold wall for the 3D cell culture. Subsequently, 3D cell coculture was conducted. First, SMCs were seeded on the luminal surface of the scaffold and cultured for 5 days, and then endothelial cells (ECs) were also seeded on the luminal surface and cocultured with SMCs for another 2 days. After stained with antibodies, 3D cell distribution on the scaffold was revealed by confocal laser scanning microscopy (CLSM) where ECs were mainly located on the luminal surface whereas SMCs penetrated into the surface and distributed inside the scaffold wall. This double layer tubular scaffold with 3D cell distribution showed the promise to develop it into a novel TEVG for clinical trials in the near future.

  6. Microenvironments and different nanoparticle dynamics in living cells revealed by a standard nanoparticle.

    Pack, Chan Gi; Song, Mi Ryoung; Tae, Eunju Lee; Hiroshima, Michio; Byun, Kyung Hee; Kim, Jun Sung; Sako, Yasushi

    2012-11-10

    For quantitative analysis of nanoparticle diffusions and submicro-environments in living cells, use of newly synthesized silica-based fluorescent nanoparticle (Si-FNP) as a standard nanoprobe is successfully demonstrated. The appropriate characteristics of a standard probe were fully analyzed in vitro by single molecule detection, transmission electron microscopy, and dynamic light scattering. Using fluorescence correlation analysis in single living cells, we quantitatively compared the diffusional properties of the standard Si-FNP with a diameter of 50 nm, peptide coated Si-FNP, streptavidin coated Qdot, and GFP molecule which have different sizes and surface properties. The result demonstrates that the standard Si-FNP without coat is minimally trapped in the vesicles in the process of cellular endocytosis. Interestingly, a large proportion of Si-FNP introduced into the cells by electroporation diffuses freely in the cells during a cell cycle suggesting free diffusing NPs are hardly trapped in the vesicles. The simple but highly sensitive method will provide insight into strategies to understanding the hydrodynamic process of nanoparticle delivery into living cells as well as the cellular microenvironment in the view of submicro-size.

  7. Laser 3D printing with sub-microscale resolution of porous elastomeric scaffolds for supporting human bone stem cells.

    Petrochenko, Peter E; Torgersen, Jan; Gruber, Peter; Hicks, Lucas A; Zheng, Jiwen; Kumar, Girish; Narayan, Roger J; Goering, Peter L; Liska, Robert; Stampfl, Jürgen; Ovsianikov, Aleksandr

    2015-04-01

    A reproducible method is needed to fabricate 3D scaffold constructs that results in periodic and uniform structures with precise control at sub-micrometer and micrometer length scales. In this study, fabrication of scaffolds by two-photon polymerization (2PP) of a biodegradable urethane and acrylate-based photoelastomer is demonstrated. This material supports 2PP processing with sub-micrometer spatial resolution. The high photoreactivity of the biophotoelastomer permits 2PP processing at a scanning speed of 1000 mm s(-1), facilitating rapid fabrication of relatively large structures (>5 mm(3)). These structures are custom printed for in vitro assay screening in 96-well plates and are sufficiently flexible to enable facile handling and transplantation. These results indicate that stable scaffolds with porosities of greater than 60% can be produced using 2PP. Human bone marrow stromal cells grown on 3D scaffolds exhibit increased growth and proliferation compared to smooth 2D scaffold controls. 3D scaffolds adsorb larger amounts of protein than smooth 2D scaffolds due to their larger surface area; the scaffolds also allow cells to attach in multiple planes and to completely infiltrate the porous scaffolds. The flexible photoelastomer material is biocompatible in vitro and is associated with facile handling, making it a viable candidate for further study of complex 3D-printed scaffolds.

  8. Characterizing microscale aluminum composite layer properties on silicon solar cells with hybrid 3D scanning force measurements.

    Bae, Sung-Kuk; Choi, Beomjoon; Chung, Haseung; Shin, Seungwon; Song, Hee-eun; Seo, Jung Hwan

    2016-03-07

    This article presents a novel technique to estimate the mechanical properties of the aluminum composite layer on silicon solar cells by using a hybrid 3-dimensional laser scanning force measurement (3-D LSFM) system. The 3-D LSFM system measures the material properties of sub-layers constituting a solar cell. This measurement is critical for realizing high-efficient ultra-thin solar cells. The screen-printed aluminum layer, which significantly affects the bowing phenomenon, is separated from the complete solar cell by removing the silicon (Si) layer with deep reactive ion etching. An elastic modulus of ~15.1 GPa and a yield strength of ~35.0 MPa for the aluminum (Al) composite layer were obtained by the 3-D LSFM system. In experiments performed for 6-inch Si solar cells, the bowing distances decreased from 12.02 to 1.18 mm while the Si layer thicknesses increased from 90 to 190 μm. These results are in excellent agreement with the theoretical predictions for ultra-thin Si thickness (90 μm) based on the obtained Al composite layer properties.

  9. A 3D Sphere Culture System Containing Functional Polymers for Large-Scale Human Pluripotent Stem Cell Production

    Tomomi G. Otsuji

    2014-05-01

    Full Text Available Utilizing human pluripotent stem cells (hPSCs in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production.

  10. HIF-1α inhibition blocks the cross talk between multiple myeloma plasma cells and tumor microenvironment

    Borsi, Enrica, E-mail: enrica.borsi2@unibo.it [Department of Experimental Diagnostic and Specialty Medicine (DIMES), “L. and A. Seràgnoli”, Bologna University School of Medicine, S. Orsola' s University Hospital (Italy); Perrone, Giulia [Fondazione IRCCS Istituto Nazionale dei Tumori, Hematology Department, Via Venezian 1, 20133 Milano (Italy); Terragna, Carolina; Martello, Marina; Zamagni, Elena; Tacchetti, Paola; Pantani, Lucia; Brioli, Annamaria; Dico, Angela Flores; Zannetti, Beatrice Anna; Rocchi, Serena; Cavo, Michele [Department of Experimental Diagnostic and Specialty Medicine (DIMES), “L. and A. Seràgnoli”, Bologna University School of Medicine, S. Orsola' s University Hospital (Italy)

    2014-11-01

    Multiple myeloma (MM) is a malignant disorder of post-germinal center B cells, characterized by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow (BM). The reciprocal and complex interactions that take place between the different compartments of BM and the MM cells result in tumor growth, angiogenesis, bone disease, and drug resistance. Given the importance of the BM microenvironment in MM pathogenesis, we investigated the possible involvement of Hypoxia-Inducible transcription Factor-1 alpha (HIF-1α) in the PCs-bone marrow stromal cells interplay. To test this hypothesis, we used EZN-2968, a 3rd generation antisense oligonucleotide against HIF-1α, to inhibit HIF-1α functions. Herein, we provide evidence that the interaction between MM cells and BM stromal cells is drastically reduced upon HIF-1α down-modulation. Notably, we showed that upon exposure to HIF-1α inhibitor, neither the incubation with IL-6 nor the co-culture with BM stromal cells were able to revert the anti-proliferative effect induced by EZN-2968. Moreover, we observed a down-modulation of cytokine-induced signaling cascades and a reduction of MM cells adhesion capability to the extracellular matrix proteins in EZN-2968-treated samples. Taken together, these results strongly support the concept that HIF-1α plays a critical role in the interactions between bone BM cells and PCs in Multiple Myeloma. - Highlights: • HIF-1α inhibition induces a mild apoptotic cell death. • Down-modulation of cytokine-induced signaling cascades upon HIF-1α inhibition. • Reduced interaction between MM cells and BMSCs upon HIF-1α down-modulation. • Reduced PCs adhesion to the extracellular matrix protein induced by EZN-2968. • HIF-1α inhibition may be an attractive therapeutic strategy for Multiple Myeloma.

  11. Galectin-3 Determines Tumor Cell Adaptive Strategies in Stressed Tumor Microenvironments

    Cardoso, Ana Carolina Ferreira; Andrade, Luciana Nogueira de Sousa; Bustos, Silvina Odete; Chammas, Roger

    2016-01-01

    Galectin-3 is a member of the β-galactoside-binding lectin family, whose expression is often dysregulated in cancers. While galectin-3 is usually an intracellular protein found in the nucleus and in the cytoplasm, under certain conditions, galectin-3 can be secreted by an yet unknown mechanism. Under stressing conditions (e.g., hypoxia and nutrient deprivation) galectin-3 is upregulated, through the activity of transcription factors, such as HIF-1α and NF-κB. Here, we review evidence that indicates a positive role for galectin-3 in MAPK family signal transduction, leading to cell proliferation and cell survival. Galectin-3 serves as a scaffold protein, which favors the spatial organization of signaling proteins as K-RAS. Upon secretion, extracellular galectin-3 interacts with a variety of cell surface glycoproteins, such as growth factor receptors, integrins, cadherins, and members of the Notch family, among other glycoproteins, besides different extracellular matrix molecules. Through its ability to oligomerize, galectin-3 forms lectin lattices that act as scaffolds that sustain the spatial organization of signaling receptors on the cell surface, dictating its maintenance on the plasma membrane or their endocytosis. Galectin-3 induces tumor cell, endothelial cell, and leukocyte migration, favoring either the exit of tumor cells from a stressed microenvironment or the entry of endothelial cells and leukocytes, such as monocytes/macrophages into the tumor organoid. Therefore, galectin-3 plays homeostatic roles in tumors, as (i) it favors tumor cell adaptation for survival in stressed conditions; (ii) upon secretion, galectin-3 induces tumor cell detachment and migration; and (iii) it attracts monocyte/macrophage and endothelial cells to the tumor mass, inducing both directly and indirectly the process of angiogenesis. The two latter activities are potentially targetable, and specific interventions may be designed to counteract the protumoral role of extracellular

  12. Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE.

    Carmona, G; Perera, U; Gillett, C; Naba, A; Law, A-L; Sharma, V P; Wang, J; Wyckoff, J; Balsamo, M; Mosis, F; De Piano, M; Monypenny, J; Woodman, N; McConnell, R E; Mouneimne, G; Van Hemelrijck, M; Cao, Y; Condeelis, J; Hynes, R O; Gertler, F B; Krause, M

    2016-09-29

    Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.

  13. Starved and asphyxiated: how can CD8+T cells within a tumor microenvironment prevent tumor progression

    Ying eZhang

    2016-02-01

    Full Text Available Although cancer immunotherapy has achieved significant breakthroughs in recent years, its overall efficacy remains limited in the majority of patients. One major barrier is exhaustion of tumor antigen (TA-specific CD8+ tumor-infiltrating lymphocytes (TILs, which conventionally has been attributed to persistent stimulation with antigen within the tumor microenvironment (TME. A series of recent studies have highlighted that the TME poses significant metabolic challenges to TILs, which may contribute to their functional exhaustion. Hypoxia increases the expression of co-inhibitors on activated CD8+T cells, which in general reduces the T cells’ effector functions. It also impairs the cells’ ability to gain energy through oxidative phosphorylation (OXPHOS. Glucose limitation increases expression of programmed cell death protein (PD-1 and reduces functions of activated CD8+T cells. A combination of hypoxia and hypoglycemia, as is common in solid tumors, places CD8+TILs at dual metabolic jeopardy by affecting both major pathways of energy production. Recently, a number of studies addressed the effects of metabolic stress on modulating CD8+T cell metabolism, differentiation and functions. Here we discuss recent findings on how different types of metabolic stress within the TME shape the tumor-killing capacity of CD8+T cells. We propose that manipulating the metabolism of TILs to more efficiently utilize nutrients especially during intermittent periods of hypoxia could maximize their performance, prolong their survival and improve the efficacy of active cancer immunotherapy.

  14. Integration of microfluidic chip with biomimetic hydrogel for 3D controlling and monitoring of cell alignment and migration.

    Lee, Kwang Ho; Lee, Ki Hwa; Lee, Jeonghoon; Choi, Hyuk; Lee, Donghee; Park, Yongdoo; Lee, Sang-Hoon

    2014-04-01

    A biomimetic hydrogel was integrated into microfluidic chips to monitor glioma cell alignment and migration. The extracellular matrix-based biomimetic hydrogel was remodeled by matrix metalloprotease (MMP) secreted by glioma cells and the hydrogel could thus be used to assess cellular behavior. Both static and dynamic cell growth conditions (flow rate of 0.1 mL/h) were used. Cell culture medium with and without vascular endothelial growth factor (VEGF), insensitive VEGF and tissue inhibitor of metalloproteinases (TIMP) were employed to monitor cell behavior. A concentration gradient formed in the hydrogel resulted in differences in cell behavior. Glioma cell viability in the microchannel was 75-85%. Cells in the VEGF-loaded microchannels spread extensively, degrading the MMP-sensitive hydrogel, and achieved cell sizes almost fivefold larger than seen in the control medium. Our integrated system can be used as a model for the study of cellular behavior in a controlled microenvironment generated by fluidic conditions in a biomimetic matrix.

  15. The influence of a human embryonic stem cell-derived microenvironment on targeting of human solid tumor xenografts.

    Tzukerman, Maty; Rosenberg, Tzur; Reiter, Irena; Ben-Eliezer, Shoshana; Denkberg, Galit; Coleman, Raymond; Reiter, Yoram; Skorecki, Karl

    2006-04-01

    The awareness of the important role that the surrounding tissue microenvironment and stromal response play in the process of tumorigenesis has grown as a result of in vivo models of tumor xenograft growth in immunocompromised mice. In the current study, we used human embryonic stem cells in order to study the interactions of tumor cells with the surrounding microenvironment of differentiated human cell tissues and structures. Several cancer cell types stably expressing an H2A-green fluorescence protein fusion protein, which allowed tracking of tumor cells, were injected into mature teratomas and developed into tumors. The salient findings were: (a) the observation of growth of tumor cells with high proliferative capacity within the differentiated microenvironment of the teratoma, (b) the identification of invasion by tumor cells into surrounding differentiated teratoma structures, and (c) the identification of blood vessels of human teratoma origin, growing adjacent to and within the cancer cell-derived tumor. Mouse embryonic stem cell-derived teratomas also supported cancer cell growth, but provided a less suitable model for human tumorigenesis studies. Anticancer immunotherapy treatment directed against A431 epidermoid carcinoma cell-related epitopes induced the complete regression of A431-derived tumor xenografts following direct i.m. injection in immunocompromised mice, as opposed to corresponding tumors growing within a human embryonic stem cell-derived microenvironment, wherein remnant foci of viable tumor cells were detected and resulted in tumor recurrence. We propose using this novel experimental model as a preclinical platform for investigating and manipulating the stromal response in tumor cell growth as an additional tool in cancer research.

  16. Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro.

    Knight, Eleanor; Przyborski, Stefan

    2015-12-01

    Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co-culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue-like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two-dimensional (2D) substrates and review the popular approaches currently available that enable the development of three-dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold-free or scaffold-based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science.

  17. Improving organic tandem solar cells based on water-processed nanoparticles by quantitative 3D nanoimaging

    Pedersen, Emil Bøje Lind; Angmo, Dechan; Dam, Henrik Friis

    2015-01-01

    particle active layer. We extract the layered morphology with structural and density information including the porosity present in the various layers and the silver electrode with high resolution in 3D. The Landfester particle layer is found to have an undesired morphology with negatively correlated top...... and easier access to the printing industry, which has reduced the use of organic solvents since the 1980s. Through ptychographic X-ray computed tomography (PXCT), we image quantitatively a roll-to-roll coated photovoltaic tandem stack consisting of one bulk heterojunction active layer and one Landfester...

  18. Synthesis and Characterization of a Photoelectrode with a Novel 3D Structure for Dye-Sensitized Solar Cells

    Kun-Ching Cho

    2014-01-01

    Full Text Available This study designs a novel dye-sensitized solar cell (DSSC in which the photoanode is derived from its three-dimensional (3D structure. The inside of the cell has a positive illumination structure, with the purposes of increasing the area of photoelectrode thin film and of increasing the illuminated area within a fixed area in order to achieve the objective of enhancing the photoelectric conversion efficiency of cell. For the cell structure experiment, the study uses graphite paper, carbon and platinum as counter electrode materials, and then conducts measurement with cell heights of 3 mm, 5 mm, and 7 mm. The electrolyte used is a gel polymer electrolyte. The assembly of the cell is divided into vertical assembly, inclined assembly, and tandem assembly. In the 3D tandem cell experiment, the counter electrode material is platinum. Experimental results show that when cell height is 7 mm and illuminated area is 0.28 cm2, open-loop voltage is 0.662 V, short-circuit current density is 18.42 mA/cm2, fill factor is 0.31, and the photoelectric conversion efficiency is 3.85%, which is 1.65 times that under vertical assembly (2.34% and 2.15 times that of the flat cell (1.79%.

  19. Mounting Pressure in the Microenvironment: Fluids, Solids, and Cells in Pancreatic Ductal Adenocarcinoma.

    DuFort, Christopher C; DelGiorno, Kathleen E; Hingorani, Sunil R

    2016-06-01

    The microenvironment influences the pathogenesis of solid tumors and plays an outsized role in some. Our understanding of the stromal response to cancers, particularly pancreatic ductal adenocarcinoma, has evolved from that of host defense to tumor offense. We know that most, although not all, of the factors and processes in the microenvironment support tumor epithelial cells. This reappraisal of the roles of stromal elements has also revealed potential vulnerabilities and therapeutic opportunities to exploit. The high concentration in the stroma of the glycosaminoglycan hyaluronan, together with the large gel-fluid phase and pressures it generates, were recently identified as primary sources of treatment resistance in pancreas cancer. Whereas the relatively minor role of free interstitial fluid in the fluid mechanics and perfusion of tumors has been long appreciated, the less mobile, gel-fluid phase has been largely ignored for historical and technical reasons. The inability of classic methods of fluid pressure measurement to capture the gel-fluid phase, together with a dependence on xenograft and allograft systems that inaccurately model tumor vascular biology, has led to an undue emphasis on the role of free fluid in impeding perfusion and drug delivery and an almost complete oversight of the predominant role of the gel-fluid phase. We propose that a hyaluronan-rich, relatively immobile gel-fluid phase induces vascular collapse and hypoperfusion as a primary mechanism of treatment resistance in pancreas cancers. Similar properties may be operant in other solid tumors as well, so revisiting and characterizing fluid mechanics with modern techniques in other autochthonous cancers may be warranted.

  20. Nanoengineered implant as a new platform for regenerative nanomedicine using 3D well-organized human cell spheroids

    Keller, Laetitia; Idoux-Gillet, Ysia; Wagner, Quentin; Eap, Sandy; Brasse, David; Schwinté, Pascale; Arruebo, Manuel; Benkirane-Jessel, Nadia

    2017-01-01

    In tissue engineering, it is still rare today to see clinically transferable strategies for tissue-engineered graft production that conclusively offer better tissue regeneration than the already existing technologies, decreased recovery times, and less risk of complications. Here a novel tissue-engineering concept is presented for the production of living bone implants combining 1) a nanofibrous and microporous implant as cell colonization matrix and 2) 3D bone cell spheroids. This combination, double 3D implants, shows clinical relevant thicknesses for the treatment of an early stage of bone lesions before the need of bone substitutes. The strategy presented here shows a complete closure of a defect in nude mice calvaria after only 31 days. As a novel strategy for bone regenerative nanomedicine, it holds great promises to enhance the therapeutic efficacy of living bone implants. PMID:28138241

  1. 3D exploration of light scattering from live cells in the presence of gold nanomarkers using holographic microscopy

    Joud, Fadwa; Bun, P; Verpillat, Frédéric; Suck, Sarah Y; Tessier, G; Atlan, Michael; Desbiolles, Pierre; Coppey-Moisan, Maite; Abboud, Marie; Gross, Michel

    2011-01-01

    In this paper, we explore the 3D structure of light scattering from dark-field illuminated live 3T3 cells in the presence of 40 nm gold nanomarkers. For this purpose, we use a high resolution holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cell structures, well defined bright scattering patterns that are not angularly titled and clearly located along the optical axis (z). This characterization is an unambiguous signature of the presence of gold biological nanomarkers, and validates the capability of digital holographic microscopy to discriminate them from background signals in live ce

  2. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-12-01

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

  3. Role of nanotopography in the development of tissue engineered 3D organs and tissues using mesenchymal stem cells

    Salmasi, Shima; Kalaskar, Deepak M.; Yoon, Wai-Weng; Blunn, Gordon W.; Seifalian, Alexander M

    2015-01-01

    Recent regenerative medicine and tissue engineering strategies (using cells, scaffolds, medical devices and gene therapy) have led to fascinating progress of translation of basic research towards clinical applications. In the past decade, great deal of research has focused on developing various three dimensional (3D) organs, such as bone, skin, liver, kidney and ear, using such strategies in order to replace or regenerate damaged organs for the purpose of maintaining or restoring organs’ func...

  4. 3D Lifetime Tomography Reveals How CdCl 2 Improves Recombination Throughout CdTe Solar Cells

    Barnard, Edward S. [Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA; PLANT PV, Inc, Alameda CA 94501 USA; Ursprung, Benedikt [PLANT PV, Inc, Alameda CA 94501 USA; Colegrove, Eric [National Renewable Energy Laboratory, Golden CO 80401 USA; Moutinho, Helio R. [National Renewable Energy Laboratory, Golden CO 80401 USA; Borys, Nicholas J. [Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA; Hardin, Brian E. [PLANT PV, Inc, Alameda CA 94501 USA; Peters, Craig H. [PLANT PV, Inc, Alameda CA 94501 USA; Metzger, Wyatt K. [National Renewable Energy Laboratory, Golden CO 80401 USA; Schuck, P. James [Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA

    2016-11-15

    Using two-photon tomography, carrier lifetimes are mapped in polycrystalline CdTe photovoltaic devices. These 3D maps probe subsurface carrier dynamics that are inaccessible with traditional optical techniques. They reveal that CdCl2 treatment of CdTe solar cells suppresses nonradiative recombination and enhances carrier lifetimes throughout the film with substantial improvements particularly near subsurface grain boundaries and the critical buried p-n junction.

  5. Tuning the Mechanical Properties of Poly(Ethylene Glycol) Microgel-Based Scaffolds to Increase 3D Schwann Cell Proliferation.

    Zhou, Wenda; Stukel, Jessica M; Cebull, Hannah L; Willits, Rebecca Kuntz

    2016-04-01

    2D in vitro studies have demonstrated that Schwann cells prefer scaffolds with mechanical modulus approximately 10× higher than the modulus preferred by nerves, limiting the ability of many scaffolds to promote both neuron extension and Schwann cell proliferation. Therefore, the goals of this work are to develop and characterize microgel-based scaffolds that are tuned over the stiffness range relevant to neural tissue engineering and investigate Schwann cell morphology, viability, and proliferation within 3D scaffolds. Using thiol-ene reaction, microgels with surface thiols are produced and crosslinked into hydrogels using a multiarm vinylsulfone (VS). By varying the concentration of VS, scaffold stiffness ranges from 0.13 to 0.76 kPa. Cell morphology in all groups demonstrates that cells are able to spread and interact with the scaffold through day 5. Although the viability in all groups is high, proliferation of Schwann cells within the scaffold of G* = 0.53 kPa is significantly higher than other groups. This result is ≈ 5× lower than previously reported optimal stiffnesses on 2D surfaces, demonstrating the need for correlation of 3D cell response to mechanical modulus. As proliferation is the first step in Schwann cell integration into peripheral nerve conduits, these scaffolds demonstrate that the stiffness is a critical parameter to optimizing the regenerative process.

  6. Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

    Arshi, Armin; Nakashima, Yasuhiro; Nakano, Haruko; Eaimkhong, Sarayoot; Evseenko, Denis; Reed, Jason; Stieg, Adam Z.; Gimzewski, James K.; Nakano, Atsushi

    2013-04-01

    While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

  7. Engineering invitro cellular microenvironment using polyelectrolyte multilayer films to control cell adhesion and for drug delivery applications

    Kidambi, Srivatsan

    Over the past decades, the development of new methods for fabricating thin films that provide precise control of the three-dimensional topography and cell adhesion has generated lots of interest. These films could lead to significant advances in the fields of tissue engineering, drug delivery and biosensors which have become increasingly germane areas of research in the field of chemical engineering. The ionic layer-by-layer (LbL) assembly technique called "Polyelectrolyte Multilayers (PEMs)", introduced by Decher in 1991, has emerged as a versatile and inexpensive method of constructing polymeric thin films, with nanometer-scale control of ionized species. PEMs have long been utilized in such applications as sensors, eletrochromics, and nanomechanical thin films but recently they have also been shown to be excellent candidates for biomaterial applications. In this thesis, we engineered these highly customizable PEM thin films to engineer in vitro cellular microenvironments to control cell adhesion and for drug delivery applications. PEM films were engineered to control the adhesion of primary hepatocytes and primary neurons without the aid of adhesive proteins/ligands. We capitalized upon the differential cell attachment and spreading of primary hepatocytes and neurons on poly(diallyldimethylammoniumchloride) (PDAC) and sulfonated polystyrene (SPS) surfaces to make patterned co-cultures of primary hepatocytes/fibroblasts and primary neurons/astrocytes on the PEM surfaces. In addition, we developed self-assembled monolayer (SAM) patterns of m-d-poly(ethylene glycol) (m-dPEG) acid molecules onto PEMs. The created m-dPEG acid monolayer patterns on PEMs acted as resistive templates, and thus prevented further deposits of consecutive poly(anion)/poly(cation) pairs of charged particles and resulted in the formation of three-dimensional (3-D) patterned PEM films or selective particle depositions atop the original multilayer thin films. These new patterned and structured

  8. Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

    Ko, Jae Hyung [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Kim, Yang Hee [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Jeong, Seong Hee; Lee, Song [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Park, Si-Nae [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Shim, In Kyong, E-mail: shimiink@gmail.com [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Kim, Song Cheol, E-mail: drksc@amc.seoul.kr [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Department of Surgery, University of Ulsan College of Medicine & Asan Medical Center, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of)

    2015-08-07

    Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.

  9. FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells.

    Villinger, Clarissa; Gregorius, Heiko; Kranz, Christine; Höhn, Katharina; Münzberg, Christin; von Wichert, Götz; Mizaikoff, Boris; Wanner, Gerhard; Walther, Paul

    2012-10-01

    Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.

  10. Ammonium accumulation and cell death in a rat 3D brain cell model of glutaric aciduria type I.

    Paris Jafari

    Full Text Available Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i 3-OHGA causes the death of astrocytes, (ii deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.

  11. The Roles of Mesenchymal Stromal/Stem Cells in Tumor Microenvironment Associated with Inflammation

    Drenka Trivanović

    2016-01-01

    Full Text Available State of tumor microenvironment (TME is closely linked to regulation of tumor growth and progression affecting the final outcome, refractoriness, and relapse of disease. Interactions of tumor, immune, and mesenchymal stromal/stem cells (MSCs have been recognized as crucial for understanding tumorigenesis. Due to their outstanding features, stem cell-like properties, capacity to regulate immune response, and dynamic functional phenotype dependent on microenvironmental stimuli, MSCs have been perceived as important players in TME. Signals provided by tumor-associated chronic inflammation educate MSCs to alter their phenotype and immunomodulatory potential in favor of tumor-biased state of MSCs. Adjustment of phenotype to TME and acquisition of tumor-promoting ability by MSCs help tumor cells in maintenance of permissive TME and suppression of antitumor immune response. Potential utilization of MSCs in treatment of tumor is based on their inherent ability to home tumor tissue that makes them suitable delivery vehicles for immune-stimulating factors and vectors for targeted antitumor therapy. Here, we review data regarding intrusive effects of inflammatory TME on MSCs capacity to affect tumor development through modification of their phenotype and interactions with immune system.

  12. 3D collagen type I matrix inhibits the antimigratory effect of doxorubicin

    Millerot-Serrurot Emilie

    2010-08-01

    Full Text Available Abstract Background The cell microenvironment, especially extracellular matrix proteins, plays an important role in tumor cell response to chemotherapeutic drugs. The present study was designed to investigate whether this microenvironment can influence the antimigratory effect of an anthracycline drug, doxorubicin, when tumor cells are grown in a matrix of type I collagen, a three-dimensional (3D context which simulates a natural microenvironment. Methods To this purpose, we studied the migratory parameters, the integrin expression, and the activation state of focal adhesion kinase (FAK and GTPase RhoA involved in the formation of focal adhesions and cell movement. These parameters were evaluated at non toxic concentrations which did not affect HT1080 cell proliferation. Results We show that while doxorubicin decreased cell migration properties by 70% in conventional two-dimensional (2D culture, this effect was completely abolished in a 3D one. Regarding the impact of doxorubicin on the focal adhesion complexes, unlike in 2D systems, the data indicated that the drug neither affected β1 integrin expression nor the state of phosphorylation of FAK and RhoA. Conclusion This study suggests the lack of antiinvasive effect of doxorubicin in a 3D environment which is generally considered to better mimic the phenotypic behaviour of cells in vivo. Consistent with the previously shown resistance to the cytotoxic effect in a 3D context, our results highlight the importance of the matrix configuration on the tumor cell response to antiinvasive drugs.

  13. Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

    Armin Arshi, Yasuhiro Nakashima, Haruko Nakano, Sarayoot Eaimkhong, Denis Evseenko, Jason Reed, Adam Z Stieg, James K Gimzewski and Atsushi Nakano

    2013-01-01

    Full Text Available While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES cells providing an endless reservoir. In addition to secreted factors and cell–cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

  14. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

    Lei, Yuguo; Schaffer, David V.

    2013-12-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 1072-fold expansion over 280 d), yield (∼2.0 × 107 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.

  15. Investigation of cell viability and morphology in 3D bio-printed alginate constructs with tunable stiffness.

    Shi, Pujiang; Laude, Augustinus; Yeong, Wai Yee

    2017-04-01

    In this article, mouse fibroblast cells (L929) were seeded on 2%, 5%, and 10% alginate hydrogels, and they were also bio-printed with 2%, 5%, and 10% alginate solutions individually to form constructs. The elastic and viscous moduli of alginate solutions, their interior structure and stiffness, interactions of cells and alginate, cell viability, migration and morphology were investigated by rheometer, MTT assay, scanning electron microscope (SEM), and fluorescent microscopy. The three types of bio-printed scaffolds of distinctive stiffness were prepared, and the seeded cells showed robust viability either on the alginate hydrogel surfaces or in the 3D bio-printed constructs. Majority of the proliferated cells in the 3D bio-printed constructs weakly attached to the surrounding alginate matrix. The concentration of alginate solution and hydrogel stiffness influenced cell migration and morphology, moreover the cells formed spheroids in the bio-printed 10% alginate hydrogel construct. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1009-1018, 2017.

  16. Probing the tumor microenvironment: collection and induction

    Williams, James K.; Padgen, Michael R.; Wang, Yarong; Entenberg, David; Gertler, Frank; Condeelis, John S.; Castracane, James

    2012-03-01

    The Nano Intravital Device, or NANIVID, is under development as an optically transparent, implantable tool to study the tumor microenvironment. Two etched glass substrates are sealed using a thin polymer membrane to create a reservoir with a single outlet. This reservoir is loaded with a hydrogel blend that contains growth factors or other chemicals to be delivered to the tumor microenvironment. When the device is implanted in the tumor, the hydrogel will swell and release these entrapped molecules, forming a gradient. Validation of the device has been performed in vitro using epidermal growth factor (EGF) and MenaINV, a highly invasive, rat mammary adenocarcinoma cell line. In both 2-D and 3-D environments, cells migrated toward the gradient of EGF released from the device. The chorioallantoic membrane (CAM) of White Leghorn chicken eggs is being utilized to grow xenograft tumors that will be used for ex vivo cell collection. Device optimization is being performed for in vivo use as a tool to collect the invasive cell population. Preliminary cell collection experiments in vivo were performed using a mouse model of breast cancer. As a second application, the device is being explored as a delivery vehicle for chemicals that induce controlled changes in the tumor microenvironment. H2O2 was loaded in the device and generated intracellular reactive oxygen species (ROS) in cells near the device outlet. In the future, other induction targets will be explored, including hypoglycemia and the manipulation of extracellular matrix stiffness.

  17. Prospective dual role of mesenchymal stem cells in breast tumor microenvironment.

    Senst, Christiane; Nazari-Shafti, Timo; Kruger, Stefan; Höner Zu Bentrup, Kirstin; Dupin, Charles L; Chaffin, Abigail E; Srivastav, Sudesh K; Wörner, Philipp M; Abdel-Mageed, Asim B; Alt, Eckhard U; Izadpanah, Reza

    2013-01-01

    Breast cancer tissue is a heterogeneous cellular milieu comprising cancer and host cells. The interaction between breast malignant and non-malignant cells takes place in breast tumor microenvironment (TM), and has a crucial role in breast cancer progression. In addition to cellular component of TM, it mainly consists of cytokines released by tumor cells. The tumor-tropic capacity of mesenchymal stem cells (MSCs) and their interaction with breast TM is an active area of investigation. In the present communication, the interplay between the breast resident adipose tissue-derived MSCs (B-ASCs) and breast TM was studied. It was found that a distinct subset of B-ASCs display a strong affinity for conditioned media (CM) from two breast cancer cell lines, MDA-MB 231 (MDA-CM) and MCF-7 (MCF-CM). The expressions of several cytokines including angiogenin, GM-CSF, IL-6, GRO-α and IL-8 in MDA-CM and MCF-CM have been identified. Upon functional analysis a crucial role for GRO-α and IL-8 in B-ASCs migration was detected. The B-ASC migration was found to be via negative regulation of RECK and enhanced expression of MMPs. Furthermore, transcriptome analysis showed that migratory subpopulation express both pro- and anti-tumorigenic genes and microRNAs (miRNA). Importantly, we observed that the migratory cells exhibit similar gene and miRNA attributes as those seen in B-ASCs of breast cancer patients. These findings are novel and suggest that in breast cancer, B-ASCs migrate to the proximity of tumor foci. Characterization of the molecular mechanisms involved in the interplay between B-ASCs and breast TM will help in understanding the probable role of B-ASCs in breast cancer development, and could pave way for anticancer therapies.

  18. Electricity Generation with the Novel 3D Electrode from Swim Wastewater in a Dual-chamber Microbial Fuel Cell

    Lai Mei-Feng

    2016-01-01

    Full Text Available The swine wastewater has the characteristics of high concentration of organic matter, suspended solids and more high ammonia nitrogen, odor, complex pollution ingredient and large emissions. Microbial fuel cells (MFC is an electrochemical and biological systems related to chemical energy into electrical energy. A two-chambered cubic microbial fuel cell was used to evaluate the effect of a novel 3D electrode which made of iron and copper on the electricity generation. The swine wastewater containing total chemical oxygen demand (TCOD 3300±300 mg/L was used as the feedstock in anode chamber, and the potassium ferricyanide was used as electron acceptor in cathode chamber. The MFC reactor was incubated with the initial pH 7.0 in a air-shaker with a temperature (ca. 35°C and 100 rpm in fed-batch mode. A fixed external resistance (R of 100 Ω was connected between the electrodes and the closed circuit potentials of the MFCs were recorded every 2 min. The results show that using iron 3D electrode has the peak electricity generation of 176 mV at the first two day and maintained the stable electricity voltage of 110 mV during the 5th to 15th days. The COD removal efficiency could reach 80%. Using copper 3D electrode only can generate the peak electricity of 33.1 mV and stable electricity of 27 mV with the COD removal efficiency of 70%.

  19. Super-resolved 3-D imaging of live cells organelles from bright-field photon transmission micrographs

    Rychtarikova, Renata; Shi, Kevin; Malakhova, Daria; Machacek, Petr; Smaha, Rebecca; Urban, Jan; Stys, Dalibor

    2016-01-01

    Current biological and medical research is aimed at obtaining a detailed spatiotemporal map of a live cell's interior to describe and predict cell's physiological state. We present here an algorithm for complete 3-D modelling of cellular structures from a z-stack of images obtained using label-free wide-field bright-field light-transmitted microscopy. The method visualizes 3-D objects with a volume equivalent to the area of a camera pixel multiplied by the z-height. The computation is based on finding pixels of unchanged intensities between two consecutive images of an object spread function. These pixels represent strongly light-diffracting, light-absorbing, or light-emitting objects. To accomplish this, variables derived from R\\'{e}nyi entropy are used to suppress camera noise. Using this algorithm, the detection limit of objects is only limited by the technical specifications of the microscope setup--we achieve the detection of objects of the size of one camera pixel. This method allows us to obtain 3-D re...

  20. 3D printed sample holder for in-operando EPR spectroscopy on high temperature polymer electrolyte fuel cells.

    Niemöller, Arvid; Jakes, Peter; Kayser, Steffen; Lin, Yu; Lehnert, Werner; Granwehr, Josef

    2016-08-01

    Electrochemical cells contain electrically conductive components, which causes various problems if such a cell is analyzed during operation in an EPR resonator. The optimum cell design strongly depends on the application and it is necessary to make certain compromises that need to be individually arranged. Rapid prototyping presents a straightforward option to implement a variable cell design that can be easily adapted to changing requirements. In this communication, it is demonstrated that sample containers produced by 3D printing are suitable for EPR applications, with a particular emphasis on electrochemical applications. The housing of a high temperature polymer electrolyte fuel cell (HT-PEFC) with a phosphoric acid doped polybenzimidazole membrane was prepared from polycarbonate by 3D printing. Using a custom glass Dewar, this fuel cell could be operated at temperatures up to 140°C in a standard EPR cavity. The carbon-based gas diffusion layer showed an EPR signal with a characteristic Dysonian line shape, whose evolution could be monitored in-operando in a non-invasive manner.

  1. 3D printed sample holder for in-operando EPR spectroscopy on high temperature polymer electrolyte fuel cells

    Niemöller, Arvid; Jakes, Peter; Kayser, Steffen; Lin, Yu; Lehnert, Werner; Granwehr, Josef

    2016-08-01

    Electrochemical cells contain electrically conductive components, which causes various problems if such a cell is analyzed during operation in an EPR resonator. The optimum cell design strongly depends on the application and it is necessary to make certain compromises that need to be individually arranged. Rapid prototyping presents a straightforward option to implement a variable cell design that can be easily adapted to changing requirements. In this communication, it is demonstrated that sample containers produced by 3D printing are suitable for EPR applications, with a particular emphasis on electrochemical applications. The housing of a high temperature polymer electrolyte fuel cell (HT-PEFC) with a phosphoric acid doped polybenzimidazole membrane was prepared from polycarbonate by 3D printing. Using a custom glass Dewar, this fuel cell could be operated at temperatures up to 140 °C in a standard EPR cavity. The carbon-based gas diffusion layer showed an EPR signal with a characteristic Dysonian line shape, whose evolution could be monitored in-operando in a non-invasive manner.

  2. The applications of 3D-hexagonal mesoporous SiO2 in proton exchange membranes of fuel cells

    Jiang, B.; Tang, H.; Pan, M.; Chen, L.; Liang, C. [Wuhan Univ. of Technology, Wuhan (China). State Key Laboratory of Advanced Technology for Materials Synthesis and Processing

    2010-07-01

    The advantages of 3D hexagonal ordered mesoporous silica include a homogeneous ordered pore, a large surface area, narrow pore size distribution and excellent thermal stability. Hetropolyacids (HPAs) are high conductivity solid proton conductors that can be loaded onto the channel of mesoporous silica as a carrier for the proton exchange membrane of fuel cells. In this study, an ordered 3D hexagonal structure HPW/SiO{sub 2} nano composite materials was prepared using the sol-gel method with a nonionic surfactant (Brij-76) as the template. The HPW/SiO{sub 2} composite was characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM) and N2 adsorption-desorption. The study showed that the synthesis method can effectively make the HPW disperse uniformly in mesoporous framework, ensuring that the synthesized material has ordered mesoscopic structure. Impedance analysis revealed that the HPW/silica nano-materials have good proton conductivity without humidification at room temperature.

  3. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model.

    Seyed Jamaleddin Mousavi

    Full Text Available Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa, intermediate (20-25 kPa or hard (30-45 kPa substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis.

  4. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model

    Mousavi, Seyed Jamaleddin; Hamdy Doweidar, Mohamed

    2015-01-01

    Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs) can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal) together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa), intermediate (20-25 kPa) or hard (30-45 kPa) substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis. PMID:25933372

  5. Integrin-linked kinase regulates cellular mechanics facilitating the motility in 3D extracellular matrices.

    Kunschmann, Tom; Puder, Stefanie; Fischer, Tony; Perez, Jeremy; Wilharm, Nils; Mierke, Claudia Tanja

    2017-03-01

    The motility of cells plays an important role for many processes such as wound healing and malignant progression of cancer. The efficiency of cell motility is affected by the microenvironment. The connection between the cell and its microenvironment is facilitated by cell-matrix adhesion receptors and upon their activation focal adhesion proteins such as integrin-linked kinase (ILK) are recruited to sites of focal adhesion formation. In particular, ILK connects cell-matrix receptors to the actomyosin cytoskeleton. However, ILK's role in cell mechanics regulating cellular motility in 3D collagen matrices is still not well understood. We suggest that ILK facilitates 3D motility by regulating cellular mechanical properties such as stiffness and force transmission. Thus, ILK wild-type and knock-out cells are analyzed for their ability to migrate on 2D substrates serving as control and in dense 3D extracellular matrices. Indeed, ILK wild-type cells migrated faster on 2D substrates and migrated more numerous and deeper in 3D matrices. Hence, we analyzed cellular deformability, Young's modulus (stiffness) and adhesion forces. We found that ILK wild-type cells are less deformable (stiffer) and produce higher cell-matrix adhesion forces compared to ILK knock-out cells. Finally, ILK is essential for providing cellular mechanical stiffness regulating 3D motility.

  6. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering

    Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan

    A huge shortage of liver organs for transplantation has motivated the research field of tissue engineering to develop bioartificial liver tissue and even a whole liver. The goal of NanoBio4Trans is to create a vascularized bioartificial liver tissue, initially as a liver-support system. Due...... to limitations of primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic...... differentiation of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed....

  7. 3D collagen scaffolds coated with multiwalled carbon nanotubes: initial cell attachment to internal surface.

    Hirata, Eri; Uo, Motohiro; Nodasaka, Yoshinobu; Takita, Hiroko; Ushijima, Natsumi; Akasaka, Tsukasa; Watari, Fumio; Yokoyama, Atsuro

    2010-05-01

    The cell adhesion in a multiwalled carbon nanotube-coated collagen sponge (MWCNT-coated sponge) was investigated. Immediately after seeding, the cells adhered to the inner surface of the MWCNT-coated sponge and a significantly larger number of cells were observed there than for a pure collagen sponge used as control. On the MWCNT-coated sponge, the cells appeared favorable adhesion and spread in the early stages in the center part of the sponge which cells rarely attached without MWCNT-coating. It was suggested that the physical structure of MWCNTs was effective for initial adhesion of cells from the result of serum-free culture. MWCNT-coating makes the material a suitable three-dimensional scaffold for cell culturing, as opposed to other scaffold systems where such an effect is not seen.

  8. A new method to address unmet needs for extracting individual cell migration features from a large number of cells embedded in 3D volumes.

    Ivan Adanja

    Full Text Available BACKGROUND: In vitro cell observation has been widely used by biologists and pharmacologists for screening molecule-induced effects on cancer cells. Computer-assisted time-lapse microscopy enables automated live cell imaging in vitro, enabling cell behavior characterization through image analysis, in particular regarding cell migration. In this context, 3D cell assays in transparent matrix gels have been developed to provide more realistic in vitro 3D environments for monitoring cell migration (fundamentally different from cell motility behavior observed in 2D, which is related to the spread of cancer and metastases. METHODOLOGY/PRINCIPAL FINDINGS: In this paper we propose an improved automated tracking method that is designed to robustly and individually follow a large number of unlabeled cells observed under phase-contrast microscopy in 3D gels. The method automatically detects and tracks individual cells across a sequence of acquired volumes, using a template matching filtering method that in turn allows for robust detection and mean-shift tracking. The robustness of the method results from detecting and managing the cases where two cell (mean-shift trackers converge to the same point. The resulting trajectories quantify cell migration through statistical analysis of 3D trajectory descriptors. We manually validated the method and observed efficient cell detection and a low tracking error rate (6%. We also applied the method in a real biological experiment where the pro-migratory effects of hyaluronic acid (HA were analyzed on brain cancer cells. Using collagen gels with increased HA proportions, we were able to evidence a dose-response effect on cell migration abilities. CONCLUSIONS/SIGNIFICANCE: The developed method enables biomedical researchers to automatically and robustly quantify the pro- or anti-migratory effects of different experimental conditions on unlabeled cell cultures in a 3D environment.

  9. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

  10. New process for fuel cell fabrication. 3D screen printing of metal bipolar plates; Neues Verfahren zur Brennstoffzellenfertigung. 3D-Siebdruck von metallischen Bipolarplatten

    Studnitzky, Thomas [Fraunhofer-Institut fuer Fertigungstechnik und Angewandte Materialforschung (IFAM), Dresden (Germany); Helm, Peter; Heinzel, Angelika [Zentrum fuer BrennstoffzellenTechnik GmbH (ZBT), Duisburg (Germany)

    2011-01-15

    Minimization of space requirements, weight, and production cost is one of the key preconditions for successful launching of the polymer electrolyte membrane fuel cell (PEM). In the stacks constructed from single PEM cells, the bipolar plate is a central component. It determines the weight and volume of the stack and accounts for more than 30 percent of the overall cost, depending on the fabrication process. It is therefore important for producers of fuel cells to develop a process that combines free design, high functionality and low cost in serial production.

  11. Development of a randomized 3D cell model for Monte Carlo microdosimetry simulations

    Douglass, Michael; Bezak, Eva; Penfold, Scott [School of Chemistry and Physics, University of Adelaide, North Terrace, Adelaide 5005, South Australia (Australia) and Department of Medical Physics, Royal Adelaide Hospital, North Terrace, Adelaide 5000, South Australia (Australia)

    2012-06-15

    Purpose: The objective of the current work was to develop an algorithm for growing a macroscopic tumor volume from individual randomized quasi-realistic cells. The major physical and chemical components of the cell need to be modeled. It is intended to import the tumor volume into GEANT4 (and potentially other Monte Carlo packages) to simulate ionization events within the cell regions. Methods: A MATLAB Copyright-Sign code was developed to produce a tumor coordinate system consisting of individual ellipsoidal cells randomized in their spatial coordinates, sizes, and rotations. An eigenvalue method using a mathematical equation to represent individual cells was used to detect overlapping cells. GEANT4 code was then developed to import the coordinate system into GEANT4 and populate it with individual cells of varying sizes and composed of the membrane, cytoplasm, reticulum, nucleus, and nucleolus. Each region is composed of chemically realistic materials. Results: The in-house developed MATLAB Copyright-Sign code was able to grow semi-realistic cell distributions ({approx}2 Multiplication-Sign 10{sup 8} cells in 1 cm{sup 3}) in under 36 h. The cell distribution can be used in any number of Monte Carlo particle tracking toolkits including GEANT4, which has been demonstrated in this work. Conclusions: Using the cell distribution and GEANT4, the authors were able to simulate ionization events in the individual cell components resulting from 80 keV gamma radiation (the code is applicable to other particles and a wide range of energies). This virtual microdosimetry tool will allow for a more complete picture of cell damage to be developed.

  12. Pro-Tumor and Anti-Tumor Functions of IL-17 and of TH17 Cells in Tumor Microenvironment

    Gulubova M.

    2016-10-01

    Full Text Available The current review reveals the seven subclasses of CD4+ T helper cells, i.e. Th1, Th2, Th9, Th17, Th22, regulatory T cells and Tfh, the cytokines produced by them and their role in tumor microenvironment. Main attention was paid to IL-17 and Th17 cells. IL-17-producing cells were described, among which were Treg17 cells and Tc17 cells. The transcription factors, engaged in the activation of Th17 cell differentiation were reviewed. It was shown that Th17 cells might possess regulatory functions in tumor microenvironments that directs toward immunosuppression. The reciprocity between Treg and Th17 cells is realized when the production of a large amount of TGF-β in tumors causes Treg cell differentiation, and the addition of IL-6 shifts the differentiation of naïve T cells to Th17 cells. The main pro-tumor role of IL-17 is the promotion of tumor angiogenesis through stimulation of fibroblasts and endothelial cells. The antitumor functions of IL-17 are associated with enhancement of cytotoxic activity of tumor specific CTL cells and with angiogenesis that provide channels through which immune cells might invade tumor and promote antitumor immunity.

  13. Numerical estimation of 3D mechanical forces exerted by cells on non-linear materials.

    Palacio, J; Jorge-Peñas, A; Muñoz-Barrutia, A; Ortiz-de-Solorzano, C; de Juan-Pardo, E; García-Aznar, J M

    2013-01-04

    The exchange of physical forces in both cell-cell and cell-matrix interactions play a significant role in a variety of physiological and pathological processes, such as cell migration, cancer metastasis, inflammation and wound healing. Therefore, great interest exists in accurately quantifying the forces that cells exert on their substrate during migration. Traction Force Microscopy (TFM) is the most widely used method for measuring cell traction forces. Several mathematical techniques have been developed to estimate forces from TFM experiments. However, certain simplifications are commonly assumed, such as linear elasticity of the materials and/or free geometries, which in some cases may lead to inaccurate results. Here, cellular forces are numerically estimated by solving a minimization problem that combines multiple non-linear FEM solutions. Our simulations, free from constraints on the geometrical and the mechanical conditions, show that forces are predicted with higher accuracy than when using the standard approaches.

  14. CYCD3 D-type cyclins regulate cambial cell proliferation and secondary growth in Arabidopsis.

    Collins, Carl; Maruthi, N M; Jahn, Courtney E

    2015-08-01

    A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-of-function approach was used here to reveal a rate-limiting role for the Arabidopsis CYCLIN D3 (CYCD3) subgroup of cell-cycle genes in the control of cambial cell proliferation and secondary growth, providing conclusive evidence of a direct link between the cell cycle and vascular development. It is shown that all three CYCD3 genes are specifically expressed in the cambium throughout vascular development. Analysis of a triple loss-of-function CYCD3 mutant revealed a requirement for CYCD3 in promoting the cambial cell cycle since mutant stems and hypocotyls showed a marked reduction in diameter linked to reduced mitotic activity in the cambium. Conversely, loss of CYCD3 provoked an increase in xylem cell size and the expression of differentiation markers, showing that CYCD3 is required to restrain the differentiation of xylem precursor cells. Together, our data show that tight control of cambial cell division through developmental- and cell type-specific regulation of CYCD3 is required for normal vascular development, constituting part of a novel mechanism controlling organ growth in higher plants.

  15. A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.

    Sabra, Georges; Vermette, Patrick

    2013-02-01

    The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered.

  16. Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

    Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

    2015-08-01

    In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.

  17. A general approach for time-supersampling of 3D-PIV data by the vortex-in-cell method

    Scarano, Fulvio; Schneiders, Jan; Dwight, Richard; Aerospace Engineering/Aerodynamics Team

    2013-11-01

    Advancements of tomographic PIV [1] have led into 3D time-resolved experiments to study the dynamical evolution of 3D turbulent flows [2]. The known bottleneck of Tomo-PIV is the high laser power required to illuminate large volumes in airflows, which becomes critical beyond 10 kHz. Time-super-sampling is an approach to reduce the sampling rate, proven for frozen turbulence where the advection model yields a significant increase of temporal resolution [3]. Instead, in separated flows, the advection principle yields unacceptable distortions. The use of Navier-Stokes numerical calculations with the vortex-in-cell (VIC) method is proposed herein. The assumption is made of inviscid incompressible flow [4]. The spatial-resolution of the data is exploited to increase the temporal resolution. The dynamical evolution of the vorticity and velocity field between subsequent snapshots in the 3D domain is numerically evaluated. The verification with fully time resolved data of a circular jet indicates a substantial increase of temporal resolution. Interestingly, data sampled below the Nyquist limit could be reconstructed faithfully, indicating the potential of VIC in alleviating requirements on PIV measurement rate. Work supported by the European Research Council grant 202887.

  18. Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

    Jung, Yookyung; Klein, Oliver J.; Wang, Hequn; Evans, Conor L.

    2016-06-01

    Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.

  19. Tumor microenvironment regulates metastasis and metastasis genes of mouse MMTV-PymT mammary cancer cells in vivo.

    Werbeck, J L; Thudi, N K; Martin, C K; Premanandan, C; Yu, L; Ostrowksi, M C; Rosol, T J

    2014-07-01

    Metastasis is the primary cause of death in breast cancer patients, yet there are challenges to modeling this process in vivo. The goal of this study was to analyze the effects of injection site on tumor growth and metastasis and gene expression of breast cancer cells in vivo using the MMTV-PymT breast cancer model (Met-1 cells). Met-1 cells were injected into 5 sites (subcutaneous, mammary fat pad, tail vein, intracardiac, and intratibial), and tumors and metastases were monitored using bioluminescent imaging and confirmed with gross necropsy and histopathology. Met-1 tumors were analyzed based on morphology and changes in gene expression in each tissue microenvironment. There were 6 permissible sites of Met-1 tumor growth (mammary gland, subcutis, lung, adrenal gland, ovary, bone). Met-1 cells grew faster in the subcutis compared to mammary fat pad tumors (highest Ki-67 index). Morphologic differences were evident in each tumor microenvironment. Finally, 7 genes were differentially expressed in the Met-1 tumors in the 6 sites of growth or metastasis. This investigation demonstrates that breast cancer progression and metastasis are regulated by not only the tumor cells but also the experimental model and unique molecular signals from the tumor microenvironment.

  20. Biofabrication of tissue constructs by 3D bioprinting of cell-laden microcarriers.

    Levato, Riccardo; Visser, Jetze; Planell, Josep A; Engel, Elisabeth; Malda, Jos; Mateos-Timoneda, Miguel A

    2014-09-01

    Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.

  1. Biofunctionalized 3-D Carbon Nano-Network Platform for Enhanced Fibroblast Cell Adhesion

    Chowdhury, A. K. M. Rezaul Haque; Tavangar, Amirhossein; Tan, Bo; Venkatakrishnan, Krishnan

    2017-01-01

    Carbon nanomaterials have been investigated for various biomedical applications. In most cases, however, these nanomaterials must be functionalized biologically or chemically due to their biological inertness or possible cytotoxicity. Here, we report the development of a new carbon nanomaterial with a bioactive phase that significantly promotes cell adhesion. We synthesize the bioactive phase by introducing self-assembled nanotopography and altered nano-chemistry to graphite substrates using ultrafast laser. To the best of our knowledge, this is the first time that such a cytophilic bio-carbon is developed in a single step without requiring subsequent biological/chemical treatments. By controlling the nano-network concentration and chemistry, we develop platforms with different degrees of cell cytophilicity. We study quantitatively and qualitatively the cell response to nano-network platforms with NIH-3T3 fibroblasts. The findings from the in vitro study indicate that the platforms possess excellent biocompatibility and promote cell adhesion considerably. The study of the cell morphology shows a healthy attachment of cells with a well-spread shape, overextended actin filaments, and morphological symmetry, which is indicative of a high cellular interaction with the nano-network. The developed nanomaterial possesses great biocompatibility and considerably stimulates cell adhesion and subsequent cell proliferation, thus offering a promising path toward engineering various biomedical devices. PMID:28287138

  2. 3D Printing of Organs-On-Chips

    Hee-Gyeong Yi

    2017-01-01

    Full Text Available Organ-on-a-chip engineering aims to create artificial living organs that mimic the complex and physiological responses of real organs, in order to test drugs by precisely manipulating the cells and their microenvironments. To achieve this, the artificial organs should to be microfabricated with an extracellular matrix (ECM and various types of cells, and should recapitulate morphogenesis, cell differentiation, and functions according to the native organ. A promising strategy is 3D printing, which precisely controls the spatial distribution and layer-by-layer assembly of cells, ECMs, and other biomaterials. Owing to this unique advantage, integration of 3D printing into organ-on-a-chip engineering can facilitate the creation of micro-organs with heterogeneity, a desired 3D cellular arrangement, tissue-specific functions, or even cyclic movement within a microfluidic device. Moreover, fully 3D-printed organs-on-chips more easily incorporate other mechanical and electrical components with the chips, and can be commercialized via automated massive production. Herein, we discuss the recent advances and the potential of 3D cell-printing technology in engineering organs-on-chips, and provides the future perspectives of this technology to establish the highly reliable and useful drug-screening platforms.

  3. An Innovative Hybrid 3D Analytic-Numerical Approach for System Level Modelling of PEM Fuel Cells

    Gregor Tavčar

    2013-10-01

    Full Text Available The PEM fuel cell model presented in this paper is based on modelling species transport and coupling electrochemical reactions to species transport in an innovative way. Species transport is modelled by obtaining a 2D analytic solution for species concentration distribution in the plane perpendicular to the gas-flow and coupling consecutive 2D solutions by means of a 1D numerical gas-flow model. The 2D solution is devised on a jigsaw puzzle of multiple coupled domains which enables the modelling of parallel straight channel fuel cells with realistic geometries. Electrochemical and other nonlinear phenomena are coupled to the species transport by a routine that uses derivative approximation with prediction-iteration. A hybrid 3D analytic-numerical fuel cell model of a laboratory test fuel cell is presented and evaluated against a professional 3D computational fluid dynamic (CFD simulation tool. This comparative evaluation shows very good agreement between results of the presented model and those of the CFD simulation. Furthermore, high accuracy results are achieved at computational times short enough to be suitable for system level simulations. This computational efficiency is owed to the semi-analytic nature of its species transport modelling and to the efficient computational coupling of electrochemical kinetics and species transport.

  4. Interactions of Pluronic nanocarriers with 2D and 3D cell cultures: Effects of PEO block length and aggregation state.

    Arranja, Alexandra; Denkova, Antonia G; Morawska, Karolina; Waton, Gilles; van Vlierberghe, Sandra; Dubruel, Peter; Schosseler, François; Mendes, Eduardo

    2016-02-28

    This work reveals how the physicochemical properties of Pluronic block copolymers influence significantly their interactions with cancer cells, whether in monolayer or spheroid cultures, and how different clinical applications can be foreseen. Two-dimensional (2D) and three-dimensional (3D) cell culture models were used to investigate the interactions of Pluronic carriers with different PEO block length and aggregation state (unimers versus cross-linked micelles) in HeLa and U87 cancer cells. Stabilized micelles of Pluronic P94 or F127 were obtained by polymerization of a crosslinking agent in the micelles hydrophobic core. Nanocarriers were functionalized with a fluorescent probe for visualization, and with a chelator for radiolabeling with Indium-111 and gamma-quantification. The 2D cell models revealed that the internalization pathways and ultimate cellular localization of the Pluronic nanocarriers depended largely on both the PEO block size and aggregation state of the copolymers. The smaller P94 unimers with an average radius of 2.1nm and the shortest PEO block mass (1100gmol(-1)) displayed the highest cellular uptake and retention. 3D tumor spheroids were used to assess the penetration capacity and toxicity potential of the nanocarriers. Results showed that cross-linked F127 micelles were more efficiently delivered across the tumor spheroids, and the penetration depth depends mostly on the transcellular transport of the carriers. The Pluronic P94-based carriers with the shortest PEO block length induced spheroid toxicity, which was significantly influenced by the spheroid cellular type.

  5. Neuronal cells' behavior on polypyrrole coated bacterial nanocellulose three-dimensional (3D) scaffolds.

    Muller, D; Silva, J P; Rambo, C R; Barra, G M O; Dourado, F; Gama, F M

    2013-01-01

    In this work, polypyrrole (PPy) was in situ polymerized onto the surface of bacterial nanocellulose (BNC) produced by Gluconacetobacter xylinus, by chemical oxidation in aqueous medium using ammonium persulfate. Composites (BNC/PPy) were produced with varying concentrations of pyrrole (Py). The produced BNC/PPy membranes were used as a template for the seeding of PC12 rat neuronal cells. Cell suspensions were directly seeded onto the surfaces of the BNC/PPy membranes. The Py concentration affected the behavior of neuronal cells that adhered and grew significantly more on BNC/PPy comparatively to BNC. Scanning electron microscopy (SEM) micrographs revealed that PC12 cells adhered on the surface of the BNC and BNC/PPy membranes. Conductive PPy coatings on nanofibers acting as an active interface for tissue engineering may be used to regulate cell activity through electrical stimulations.

  6. 3D Bearing Capacity of Structured Cells Supported on Cohesive Soil: Simplified Analysis Method

    Martínez-Galván Sergio Antonio

    2013-06-01

    Full Text Available In this paper a simplified analysis method to compute the bearing capacity of structured cell foundations subjected to vertical loading and supported in soft cohesive soil is proposed. A structured cell is comprised by a top concrete slab structurally connected to concrete external walls that enclose the natural soil. Contrary to a box foundation it does not include a bottom slab and hence, the soil within the walls becomes an important component of the structured cell. This simplified method considers the three-dimensional geometry of the cell, the undrained shear strength of cohesive soils and the existence of structural continuity between the top concrete slab and the surrounding walls, along the walls themselves and the walls structural joints. The method was developed from results of numerical-parametric analyses, from which it was found that structured cells fail according to a punching-type mechanism.

  7. Enrichment of diluted cell populations from large sample volumes using 3D carbon-electrode dielectrophoresis.

    Islam, Monsur; Natu, Rucha; Larraga-Martinez, Maria Fernanda; Martinez-Duarte, Rodrigo

    2016-05-01

    Here, we report on an enrichment protocol using carbon electrode dielectrophoresis to isolate and purify a targeted cell population from sample volumes up to 4 ml. We aim at trapping, washing, and recovering an enriched cell fraction that will facilitate downstream analysis. We used an increasingly diluted sample of yeast, 10(6)-10(2) cells/ml, to demonstrate the isolation and enrichment of few cells at increasing flow rates. A maximum average enrichment of 154.2 ± 23.7 times was achieved when the sample flow rate was 10 μl/min and yeast cells were suspended in low electrically conductive media that maximizes dielectrophoresis trapping. A COMSOL Multiphysics model allowed for the comparison between experimental and simulation results. Discussion is conducted on the discrepancies between such results and how the model can be further improved.

  8. Fabrication and characterization of microsieve electrode array (µSEA) enabling cell positioning on 3D electrodes

    Schurink, B.; Tiggelaar, R. M.; Gardeniers, J. G. E.; Luttge, R.

    2017-01-01

    Here the fabrication and characterization of a novel microelectrode array for electrophysiology applications is described, termed a micro sieve electrode array (µSEA). This silicon based µSEA device allows for hydrodynamic parallel positioning of single cells on 3D electrodes realized on the walls of inverted pyramidal shaped pores. To realize the µSEA, a previously realized silicon sieving structure is provided with a patterned boron doped poly-silicon, connecting the contact electrodes with the 3D sensing electrodes in the pores. A LPCVD silicon-rich silicon nitride layer was used as insulation. The selective opening of this insulation layer at the ends of the wiring lines allows to generate well-defined contact and sensing electrodes according to the layout used in commercial microelectrode array readers. The main challenge lays in the simultaneously selective etching of material at both the planar surface (contact electrode) as well as in the sieving structure containing the (3D) pores (sensing electrodes). For the generation of 3D electrodes in the pores a self-aligning technique was developed using the pore geometry to our advantage. This technique, based on sacrificial layer etching, allows for the fine tuning of the sensing electrode surface area and thus supports the positioning and coupling of single cells on the electrode surface in relation to the cell size. Furthermore, a self-aligning silicide is formed on the sensing electrodes to favour the electrical properties. Experiments were performed to demonstrate the working principle of the µSEA using different types of neuronal cells. Capture efficiency in the pores was  >70% with a 70% survival rate of the cell maintained for up to 14 DIV. The TiSi2-boron-doped-poly-silicon sensing electrodes of the µSEA were characterized, which indicated noise levels of  <15 µV and impedance values of 360 kΩ. These findings potentially allow for future electrophysiological measurements using the µSEA.

  9. Cytokine Expression Pattern in Bone Marrow Microenvironment after Allogeneic Stem Cell Transplantation in Primary Myelofibrosis.

    Hussein, Kais; Stucki-Koch, Angelika; Alchalby, Haefaa; Triviai, Ioanna; Kröger, Nicolaus; Kreipe, Hans

    2016-04-01

    The only curative therapy for primary myelofibrosis (PMF) is allogeneic stem cell transplantation (ASCT). However, although we know that patients can benefit from ASCT, we do not know the extent of the changes of the expression profile of cytokines and matrix modulation factors. In this first systematic analysis, we evaluated the expression profile of 103 factors before and after transplantation to identify potential biomarkers. The expression of fibrosis-, inflammation-, and angiogenesis-associated genes was analyzed in a total of 52 bone marrow biopsies: PMF patients (n = 14) before and after ASCT and, for control purposes, post-ASCT multiple myeloma patients (n = 14) and non-neoplastic hematopoiesis (n = 10). In post-ASCT PMF cases, decreased expression of tissue inhibitor of metalloproteinases (TIMP) and platelet-derived growth factor alpha (PDGFA) correlated with bone marrow remodeling and hematological remission. Expression of several other matrix factors remained at high levels and may contribute to post-ASCT remodeling. This is the first systematic analysis of cytokine expression in post-ASCT PMF bone marrow that shows that normalization of bone marrow microenvironment is paralleled by decreased expression of TIMP and PDGFA.

  10. FGFR inhibitors: Effects on cancer cells, tumor microenvironment and whole-body homeostasis (Review).

    Katoh, Masaru

    2016-07-01

    Fibroblast growth factor (FGF)2, FGF4, FGF7 and FGF20 are representative paracrine FGFs binding to heparan-sulfate proteoglycan and fibroblast growth factor receptors (FGFRs), whereas FGF19, FGF21 and FGF23 are endocrine FGFs binding to Klotho and FGFRs. FGFR1 is relatively frequently amplified and overexpressed in breast and lung cancer, and FGFR2 in gastric cancer. BCR-FGFR1, CNTRL-FGFR1, CUX1-FGFR1, FGFR1OP-FGFR1, MYO18A-FGFR1 and ZMYM2-FGFR1 fusions in myeloproliferative neoplasms are non-receptor-type FGFR kinases, whereas FGFR1-TACC1, FGFR2-AFF3, FGFR2-BICC1, FGFR2-PPHLN1, FGFR3-BAIAP2L1 and FGFR3-TACC3 fusions in solid tumors are transmembrane-type FGFRs with C-terminal alterations. AZD4547, BGJ398 (infigratinib), Debio-1347 and dovitinib are FGFR1/2/3 inhibitors; BLU9931 is a selective FGFR4 inhibitor; FIIN-2, JNJ-42756493, LY2874455 and ponatinib are pan-FGFR inhibitors. AZD4547, dovitinib and ponatinib are multi-kinase inhibitors targeting FGFRs, colony stimulating factor 1 receptor (CSF1R), vascular endothelial growth factor (VEGF)R2, and others. The tumor microenvironment consists of cancer cells and stromal/immune cells, such as cancer-associated fibroblasts (CAFs), endothelial cells, M2-type tumor-associating macrophages (M2-TAMs), myeloid-derived suppressor cells (MDSCs) and regulatory T cells. FGFR inhibitors elicit antitumor effects directly on cancer cells, as well as indirectly through the blockade of paracrine signaling. The dual inhibition of FGF and CSF1 or VEGF signaling is expected to enhance the antitumor effects through the targeting of immune evasion and angiogenesis in the tumor microenvironment. Combination therapy using tyrosine kinase inhibitors (FGFR or CSF1R inhibitors) and immune checkpoint blockers (anti-PD-1 or anti-CTLA-4 monoclonal antibodies) may be a promising choice for cancer patients. The inhibition of FGF19-FGFR4 signaling is associated with a risk of liver toxicity, whereas the activation of FGF23-FGFR4 signaling

  11. High resolution 3D imaging of living cells with sub-optical wavelength phonons

    Pérez-Cota, Fernando; Smith, Richard J.; Moradi, Emilia; Marques, Leonel; Webb, Kevin F.; Clark, Matt

    2016-12-01

    Label-free imaging of living cells below the optical diffraction limit poses great challenges for optical microscopy. Biologically relevant structural information remains below the Rayleigh limit and beyond the reach of conventional microscopes. Super-resolution techniques are typically based on the non-linear and stochastic response of fluorescent labels which can be toxic and interfere with cell function. In this paper we present, for the first time, imaging of live cells using sub-optical wavelength phonons. The axial imaging resolution of our system is determined by the acoustic wavelength (λa = λprobe/2n) and not on the NA of the optics allowing sub-optical wavelength acoustic sectioning of samples using the time of flight. The transverse resolution is currently limited to the optical spot size. The contrast mechanism is significantly determined by the mechanical properties of the cells and requires no additional contrast agent, stain or label to image the cell structure. The ability to breach the optical diffraction limit to image living cells acoustically promises to bring a new suite of imaging technologies to bear in answering exigent questions in cell biology and biomedicine.

  12. Extracellular matrix production by nucleus pulposus and bone marrow stem cells in response to altered oxygen and glucose microenvironments.

    Naqvi, Syeda M; Buckley, Conor T

    2015-12-01

    Bone marrow (BM) stem cells may be an ideal source of cells for intervertebral disc (IVD) regeneration. However, the harsh biochemical microenvironment of the IVD may significantly influence the biological and metabolic vitality of injected stem cells and impair their repair potential. This study investigated the viability and production of key matrix proteins by nucleus pulposus (NP) and BM stem cells cultured in the typical biochemical microenvironment of the IVD consisting of altered oxygen and glucose concentrations. Culture-expanded NP cells and BM stem cells were encapsulated in 1.5% alginate and ionically crosslinked to form cylindrical hydrogel constructs. Hydrogel constructs were maintained under different glucose concentrations (1, 5 and 25 mM) and external oxygen concentrations (5 and 20%). Cell viability was measured using the Live/Dead® assay and the production of sulphated glycosaminoglycans (sGAG), and collagen was quantified biochemically and histologically. For BM stem cells, IVD-like micro-environmental conditions (5 mM glucose and 5% oxygen) increased the accumulation of sGAG and collagen. In contrast, low glucose conditions (1 mM glucose) combined with 5% external oxygen concentration promoted cell death, inhibiting proliferation and the accumulation of sGAG and collagen. NP-encapsulated alginate constructs were relatively insensitive to oxygen concentration or glucose condition in that they accumulated similar amounts of sGAG under all conditions. Under IVD-like microenvironmental conditions, NP cells were found to have a lower glucose consumption rate compared with BM cells and may in fact be more suitable to adapt and sustain the harsh microenvironmental conditions. Considering the highly specialised microenvironment of the central NP, these results indicate that IVD-like concentrations of low glucose and low oxygen are critical and influential for the survival and biological behaviour of stem cells. Such findings may promote and accelerate

  13. A novel diamond anvil cell for x-ray diffraction at cryogenic temperatures manufactured by 3D printing

    Jin, H.; Woodall, C. H.; Wang, X.; Parsons, S.; Kamenev, K. V.

    2017-03-01

    A new miniature high-pressure diamond anvil cell was designed and constructed using 3D micro laser sintering technology. This is the first application of the use of rapid prototyping technology to construct high-pressure apparatus. The cell is specifically designed for use as an X-ray diffraction cell that can be used with commercially available diffractometers and open-flow cryogenic equipment to collect data at low temperature and high pressure. The cell is constructed from stainless steel 316L and is about 9 mm in diameter and 7 mm in height, giving it both small dimensions and low thermal mass, and it will fit into the cooling envelope of a standard CryostreamTM cooling system. The cell is clamped using a customized miniature buttress thread of diameter 7 mm and pitch of 0.5 mm enabled by 3D micro laser sintering technology; such dimensions are not attainable using conventional machining. The buttress thread was used as it has favourable uniaxial load properties allowing for higher pressure and better anvil alignment. The clamp can support the load of at least 1.5 kN according to finite element analysis (FEA) simulations. FEA simulations were also used to compare the performance of the standard thread and the buttress thread, and demonstrate that stress is distributed more uniformly in the latter. Rapid prototyping of the pressure cell by the laser sintering resulted in a substantially higher tensile yield strength of the 316L stainless steel (675 MPa compared to 220 MPa for the wrought type of the same material), which increased the upper pressure limit of the cell. The cell is capable of reaching pressures of up to 15 GPa with 600 μm diameter culets of diamond anvils. Sample temperature and pressure changes on cooling were assessed using X-ray diffraction on samples of NaCl and HMT-d12.

  14. A 3D Poly(ethylene glycol)-based Tumor Angiogenesis Model to Study the Influence of Vascular Cells on Lung Tumor Cell Behavior

    Roudsari, Laila C.; Jeffs, Sydney E.; Witt, Amber S.; Gill, Bartley J.; West, Jennifer L.

    2016-09-01

    Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. In vitro models that mimic in vivo tumor neovascularization facilitate exploration of this role. Here we investigated lung adenocarcinoma cancer cells (344SQ) and endothelial and pericyte vascular cells encapsulated in cell-adhesive, proteolytically-degradable poly(ethylene) glycol-based hydrogels. 344SQ in hydrogels formed spheroids and secreted proangiogenic growth factors that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, we engineered a 2-layer hydrogel with 344SQ and vascular cell layers. Large, invasive 344SQ clusters (area > 5,000 μm2, circularity culture system as a platform for studying tumor vascularization.

  15. In-chip fabrication of free-form 3D constructs for directed cell migration analysis

    Olsen, Mark Holm; Hjortø, Gertrud Malene; Hansen, Morten;

    2013-01-01

    through smaller pore sizes made significantly more turns than those through larger pores. The introduction of additional defined barriers in the microporous structure resulted in dendritic cells making more turns while still being able to follow the chemoattractant concentration gradient....

  16. A 3-D Model of Signaling and Transport Pathways in Epithelial Cells

    Quong, A A; Westbrook, C K

    2005-04-01

    A 3-dimensional computer model was developed to simulate the spatial and chemical evolution of calcium ions inside an array of human epithelial kidney cells. This is a prototype model, intended to develop a methodology to incorporate much more complex interactions of metabolic and other processes within many types of cells and lead to increased ability to predict cellular responses to disease as well as to chemical and biological warfare situations. Preliminary tests of the model are described.

  17. Electrosensitization assists cell ablation by nanosecond pulsed electric field in 3D cultures.

    Muratori, Claudia; Pakhomov, Andrei G; Xiao, Shu; Pakhomova, Olga N

    2016-03-18

    Previous studies reported a delayed increase of sensitivity to electroporation (termed "electrosensitization") in mammalian cells that had been subjected to electroporation. Electrosensitization facilitated membrane permeabilization and reduced survival in cell suspensions when the electric pulse treatments were split in fractions. The present study was aimed to visualize the effect of sensitization and establish its utility for cell ablation. We used KLN 205 squamous carcinoma cells embedded in an agarose gel and cell spheroids in Matrigel. A local ablation was created by a train of 200 to 600 of 300-ns pulses (50 Hz, 300-600 V) delivered by a two-needle probe with 1-mm inter-electrode distance. In order to facilitate ablation by engaging electrosensitization, the train was split in two identical fractions applied with a 2- to 480-s interval. At 400-600 V (2.9-4.3 kV/cm), the split-dose treatments increased the ablation volume and cell death up to 2-3-fold compared to single-train treatments. Under the conditions tested, the maximum enhancement of ablation was achieved when two fractions were separated by 100 s. The results suggest that engaging electrosensitization may assist in vivo cancer ablation by reducing the voltage or number of pulses required, or by enabling larger inter-electrode distances without losing the ablation efficiency.

  18. Passivation of ZnO Nanowire Guests and 3D Inverse Opal Host Photoanodes for Dye-Sensitized Solar Cells

    Labouchere, Philippe

    2014-04-23

    A hierarchical host-guest nanostructured photoanode is reported for dye-sensitized solar cells. It is composed of ZnO nanowires grown in situ into the macropores of a 3D ZnO inverse opal structure, which acts both as a seed layer and as a conductive backbone host. Using a combination of self-assembly, hydrothermal or electrodeposition of single crystalline ZnO nanowires and TiO2 passivation, a novel photoanode with scattering capability for optimal light harvesting is fabricated. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. The influence of plasma technology coupled to chemical grafting on the cell growth compliance of 3D hydroxyapatite scaffolds.

    Russo, Laura; Zanini, Stefano; Giannoni, Paolo; Landi, Elena; Villa, Anna; Sandri, Monica; Riccardi, Claudia; Quarto, Rodolfo; Doglia, Silvia M; Nicotra, Francesco; Cipolla, Laura

    2012-11-01

    The development of advanced materials with biomimetic features in order to elicit desired biological responses and to guarantee tissue biocompatibility is recently gaining attention for tissue engineering applications. Bioceramics, such as hydroxyapatite-based biomaterials are now used in a number of different applications throughout the body, covering all areas of the skeleton, due to their biological and chemical similarity to the inorganic phases of bones. When bioactive sintered hydroxyapatite (HA) is desired, biomolecular modification of these materials is needed. In the present work, we investigated the influence of plasma surface modification coupled to chemical grafting on the cell growth compliance of HA 3D scaffolds.

  20. Differentiation capacity and maintenance of differentiated phenotypes of human mesenchymal stromal cells cultured on two distinct types of 3D polymeric scaffolds.

    Leferink, A M; Santos, D; Karperien, M; Truckenmüller, R K; van Blitterswijk, C A; Moroni, L

    2015-12-01

    Many studies have shown the influence of soluble factors and material properties on the differentiation capacity of mesenchymal stromal cells (MSCs) cultured as monolayers. These types of two-dimensional (2D) studies can be used as simplified models to understand cell processes related to stem cell sensing and mechano-transduction in a three-dimensional (3D) context. For several other mechanisms such as cell-cell signaling, cell proliferation and cell morphology, it is well-known that cells behave differently on a planar surface compared to cells in 3D environments. In classical tissue engineering approaches, a combination of cells, 3D scaffolds and soluble factors are considered as the key ingredients for the generation of mechanically stable 3D tissue constructs. However, when MSCs are used for tissue engineering strategies, little is known about the maintenance of their differentiation potential in 3D scaffolds after the removal of differentiation soluble factors. In this study, the differentiation potential of human MSCs (hMSCs) into the chondrogenic and osteogenic lineages on two distinct 3D scaffolds, additive manufactured electrospun scaffolds, was assessed and compared to conventional 2D culture. Human MSCs cultured in the presence of soluble factors in 3D showed to differentiate to the same extent as hMSCs cultured as 2D monolayers or as scaffold-free pellets, indicating that the two scaffolds do not play a consistent role in the differentiation process. In the case of phenotypic changes, the achieved differentiated phenotype was not maintained after the removal of soluble factors, suggesting that the plasticity of hMSCs is retained in 3D cell culture systems. This finding can have implications for future tissue engineering approaches in which the validation of hMSC differentiation on 3D scaffolds will not be sufficient to ensure the maintenance of the functionality of the cells in the absence of appropriate differentiation signals.

  1. Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies

    Wei Liao; Sanjai Sharma

    2016-01-01

    Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells.

  2. Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics

    JFW Greiner

    2011-12-01

    Full Text Available Due to their broad differentiation potential and their persistence into a