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Sample records for 3d pulp cells

  1. Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures

    M. Riccio; Resca, E.; Maraldi, T; Pisciotta, A.; Ferrari, A; Bruzzesi, G.; De Pol, A.

    2010-01-01

    The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During the differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurr...

  2. Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures

    M. Riccio

    2010-11-01

    Full Text Available The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During the differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, MatrigelTM and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in MatrigelTM DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.

  3. The Effect of 5 Proteins On DPPSC (Dental Pulp Pluripotent Stem Cells) For Osteoblast Differentiation Proliferation In 3D

    Chatakun, Punjamaporn

    2014-01-01

    Bone-tissue engineering is a therapeutic target in the field of dental implant and orthopaedic surgery. It is therefore essential to find a microenvironment that enhances the growth and differentiation of osteoblasts both from mesenchymal stem cells (MSCs) and those derived from dental pulp (DPSC). The aim of this research is to determine the relationship among the proteins fibronectin (FN), bone morphogenetic protein 2 (BMP2) osteopontin (OPN), tenascin C (TNC) and bone sialoprotein (BSP) an...

  4. Human tooth pulp anatomy visualization by 3D magnetic resonance microscopy

    Precise assessment of dental pulp anatomy is of an extreme importance for a successful endodontic treatment. As standard radiographs of teeth provide very limited information on dental pulp anatomy, more capable methods are highly appreciated. One of these is 3D magnetic resonance (MR) microscopy of which diagnostic capabilities in terms of a better dental pulp anatomy assessment were evaluated in the study. Twenty extracted human teeth were scanned on a 2.35 T MRI system for MR microscopy using the 3D spin-echo method that enabled image acquisition with isotropic resolution of 100 μm. The 3D images were then post processed by ImageJ program (NIH) to obtain advanced volume rendered views of dental pulps. MR microscopy at 2.35 T provided accurate data on dental pulp anatomy in vitro. The data were presented as a sequence of thin 2D slices through the pulp in various orientations or as volume rendered 3D images reconstructed form arbitrary view-points. Sequential 2D images enabled only an approximate assessment of the pulp, while volume rendered 3D images were more precise in visualization of pulp anatomy and clearly showed pulp diverticles, number of pulp canals and root canal anastomosis. This in vitro study demonstrated that MR microscopy could provide very accurate 3D visualization of dental pulp anatomy. A possible future application of the method in vivo may be of a great importance for the endodontic treatment

  5. 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.

  6. Scaffoldless Tissue-engineered Dental Pulp Cell Constructs for Endodontic Therapy

    Syed-Picard, F.N.; Ray, H.L.; Kumta, P.N.; Sfeir, C.

    2014-01-01

    A major cause of apical periodontitis after endodontic treatment is the bacterial infiltration which could have been challenged by the presence of a vital pulp. In this study, self-assembled, scaffoldless, three-dimensional (3D) tissues were engineered from dental pulp cells (DPCs) and assessed as a device for pulp regeneration. These engineered tissues were placed into the canal space of human tooth root segments that were capped on one end with calcium phosphate cement, and the entire syste...

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

    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. (paper)

  8. 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.

  9. Multizone paper platform for 3D cell cultures.

    Ratmir Derda

    Full Text Available In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc. The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously ("cells-in-gels-in-paper" or CiGiP, this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, "sections" all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.

  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. Stem cell reprogramming: A 3D boost

    Abilez, Oscar J.; Wu, Joseph C.

    2016-03-01

    Biophysical factors in an optimized three-dimensional microenvironment enhance the reprogramming efficiency of human somatic cells into pluripotent stem cells when compared to traditional cell-culture substrates.

  12. Laser printing of cells into 3D scaffolds

    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.

  13. 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. PMID:26464272

  14. Extracellular matrix of dental pulp stem cells: Applications in pulp tissue engineering using somatic MSCs

    Sriram eRavindran

    2014-01-01

    Full Text Available Dental Caries affects approximately 90% of the world’s population. At present, the clinical treatment for dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality. Tissue engineering can potentially solve this problem by enabling regeneration of a functional pulp tissue. Dental pulp stem cells (DPSCs have been shown to be an excellent source for pulp regeneration. However, limited availability of these cells hinders its potential for clinical translation. We have investigated the possibility of using somatic mesenchymal stem cells from other sources for dental pulp tissue regeneration using a biomimetic dental pulp extracellular matrix (ECM incorporated scaffold. Human periodontal ligament stem cells (PDLSCs and human bone marrow stromal cells (HMSCs were investigated for their ability to differentiate towards an odontogenic lineage. In vitro real-time PCR results coupled with histological and immunohistochemical examination of the explanted tissues confirmed the ability of PDLSCs and HMSCs to form a vascularized pulp-like tissue. These findings indicate that the dental pulp stem derived ECM scaffold stimulated odontogenic differentiation of PDLSCs and HMSCs without the need for exogenous addition of growth and differentiation factors. This study represents a translational perspective toward possible therapeutic application of using a combination of somatic stem cells and extracellular matrix for pulp regeneration.

  15. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    Segah Altuntaş; Muhammed Ali Kara; Deniz Selin Aksoy; Zehra Dilşad Çoban; Şefik Güran

    2016-01-01

    Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications hav...

  16. 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.

  17. 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.

  18. Gambaran densitas kamar pulpa gigi sulung menggunakan cone beam CT-3D (Description of pulp chamber density in deciduous teeth using cone beam CT-3D)

    Herdiyati Y; Epsilawati L; Oscandar F; Nurianingsih R

    2013-01-01

    Background: Dental caries is the most common chronic diseases. Detection of caries is needed, especially on the deciduous teeth. An examination such as radiological examination is essential. The radiographic figures distinguish radiolucent of the crown. Digital radiography cone beam computed tomography (CBCT) is able to show a more detailed picture. Purpose: This study was aimed to get value of the density of pulp chamber of caries and non caries deciduous teeth using CBCT radiographs. Method...

  19. 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.

  20. Influence of different types of pulp treatment during isolation in the obtention of human dental pulp stem cells

    Viña-Almunia, Jose; Borras, Consuelo; Gambini, Juan; El Alamy, Marya; Viña, Jose

    2016-01-01

    Background Different methods have been used in order to isolate dental pulp stem cells. The aim of this study was to study the effect of different types of pulp treatment during isolation, under 3% O2 conditions, in the time needed and the efficacy for obtaining dental pulp stem cells. Material and Methods One hundred and twenty dental pulps were used to isolate dental pulp stem cells treating the pulp tissue during isolation using 9 different methods, using digestive, disgregation, or mechanical agents, or combining them. The cells were positive for CD133, Oct4, Nestin, Stro-1, CD34 markers, and negative for the hematopoietic cell marker CD-45, thus confirming the presence of mesenchymal stem cells. The efficacy of dental pulp stem cells obtention and the minimum time needed to obtain such cells comparing the 9 different methods was analyzed. Results Dental pulp stem cells were obtained from 97 of the 120 pulps used in the study, i.e. 80.8% of the cases. They were obtained with all the methods used except with mechanical fragmentation of the pulp, where no enzymatic digestion was performed. The minimum time needed to isolate dental pulp stem cells was 8 hours, digesting with 2mg/ml EDTA for 10 minutes, 4mg/ml of type I collagenase, 4mg/ml of type II dispase for 40 minutes, 13ng/ml of thermolysine for 40 minutes and sonicating the culture for one minute. Conclusions Dental pulp stem cells were obtained in 97 cases from a series of 120 pulps. The time for obtaining dental pulp stem cells was reduced maximally, without compromising the obtention of the cells, by combining digestive, disgregation, and mechanical agents. Key words:Dental pulp stem cells, mesenchymal stem cells, isolation method. PMID:26946201

  1. LPS induces pulp progenitor cell recruitment via complement activation.

    Chmilewsky, F; Jeanneau, C; Laurent, P; About, I

    2015-01-01

    Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS. PMID:25359783

  2. Gambaran densitas kamar pulpa gigi sulung menggunakan cone beam CT-3D (Description of pulp chamber density in deciduous teeth using cone beam CT-3D

    Herdiyati Y

    2013-06-01

    Full Text Available Background: Dental caries is the most common chronic diseases. Detection of caries is needed, especially on the deciduous teeth. An examination such as radiological examination is essential. The radiographic figures distinguish radiolucent of the crown. Digital radiography cone beam computed tomography (CBCT is able to show a more detailed picture. Purpose: This study was aimed to get value of the density of pulp chamber of caries and non caries deciduous teeth using CBCT radiographs. Methods: The study was conducted by using simple descriptive. The samples were all the data CBCT of pediatric patients aged 7-10 years who visited the Dental Hospital of the Faculty of Dentistry, University of Padjadjaran. The samples were teeth with single and double root. Results: The results showed that the value of the normal pulp density is 422.56 Hu, while the condition of caries decreased becomes -77.89 Hu. Conclusion: The tooth with caries showed a lower density than the non caries/tooth.Latar belakang: Karies gigi merupakan penyakit kronis yang sering terjadi. Deteksi terhadap karies sangat diperlukan terutama pada gigi decidius. Pemeriksaan penunjang berupa pemeriksaan radiologis sangat diperlukan. Secara umum gambaran radiografi dapat membedakan karies berupa gambaran radiolusent pada mahkota. Radiografi digital cone beam computed tomografi (CBCT, merupakan jenis radiografi yang mampu memperlihatkan gambaran yang lebih detail. Tujuan: Penelitian ini bertujuan mendapatkan nilai densitas kamar pulpa gigi sulung yang karies dan non karies menggunakan radiografi CBCT. Metode: Penelitian dilakukan dengan metode simple deskriptif. Sampel penelitian adalah semua data CBCT dari pasien anak berusia 7 - 10 tahun yang berkunjung ke RSGM Fakultas Kedokteran Gigi Universitas Padjadjaran. Gigi yang dianalisa meliputi gigi berakar tunggal dan berakar ganda. Hasil: Hasil penelitian menunjukkan bahwa nilai densitas pulpa normal adalah 422,56 Hu, sedangkan pada kondisi

  3. A novel mechanotactic 3D modeling of cell morphology

    Cell morphology plays a critical role in many biological processes, such as cell migration, tissue development, wound healing and tumor growth. Recent investigations demonstrate that, among other stimuli, cells adapt their shapes according to their substrate stiffness. Until now, the development of this process has not been clear. Therefore, in this work, a new three-dimensional (3D) computational model for cell morphology has been developed. This model is based on a previous cell migration model presented by the same authors. The new model considers that during cell–substrate interaction, cell shape is governed by internal cell deformation, which leads to an accurate prediction of the cell shape according to the mechanical characteristic of its surrounding micro-environment. To study this phenomenon, the model has been applied to different numerical cases. The obtained results, which are qualitatively consistent with well-known related experimental works, indicate that cell morphology not only depends on substrate stiffness but also on the substrate boundary conditions. A cell located within an unconstrained soft substrate (several kPa) with uniform stiffness is unable to adhere to its substrate or to send out pseudopodia. When the substrate stiffness increases to tens of kPa (intermediate and rigid substrates), the cell can adequately adhere to its substrate. Subsequently, as the traction forces exerted by the cell increase, the cell elongates and its shape changes. Within very stiff (hard) substrates, the cell cannot penetrate into its substrate or send out pseudopodia. On the other hand, a cell is found to be more elongated within substrates with a constrained surface. However, this elongation decreases when the cell approaches it. It can be concluded that the higher the net traction force, the greater the cell elongation, the larger the cell membrane area, and the less random the cell alignment. (paper)

  4. Self-Assembled Peptide Gels for 3D Cell Culture

    Tang, Claire

    2010-01-01

    Under specific conditions short peptides modified with an N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group can self-assemble into hydrogel scaffolds similar in properties to the natural extracellular matrix. Fmoc-diphenylalanine (Fmoc-FF) for instance, has been shown to form hydrogels at physiological pH that have the ability to support 2D and 3D cell culture. The aim of this investigation is to provide further understanding of the self-assembly mechanism of such systems in order to progre...

  5. Characterization of Progenitor Cells in Pulps of Murine Incisors

    Balic, A.; Mina, M.

    2010-01-01

    The continuous growth of rodent incisors requires the presence of stem cells capable of generating ameloblasts and odontoblasts. While epithelial stem cells giving rise to ameloblasts have been well-characterized, cells giving rise to the odontoblasts in incisors have not been fully characterized. The goal of this study was to gain insight into the potential population in dental pulps of unerupted and erupted incisors that give rise to odontoblasts. We show that pulps from unerupted incisors ...

  6. 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.

  7. Identification and Isolation of Human Dental Pulp Stem Cells

    Xue-Chao YANG; Ming-Wen FAN

    2005-01-01

    @@ 1 Introduction Dentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts,that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue.

  8. Age estimation on the stricture of pulp cavity of the mandibular first premolar by 3D micro-focus X-CT

    To know the effect of aging on pulp cavity volumes, 40 mandibular first premolars obtained from 21-75 years old persons and stored in authors' laboratory were subjected to 3D micro-focus X-CT (SMX-130CT-SV, Shimadzu Works) to determine the volume, and ratios to the root canal pulp volume of the crown (C), cervical 1/3 (R1), central (R2) and apical 1/3 (R3) pulp volumes were calculated. In all ages examined, the ratios were in the order of R1>R2>C>R3, and were decreased by aging with exception for R1. Changes in R1 were significant in thirties to fifties and in R2, in twenties to thirties. Multiple regression analysis for age estimation gave the multiple correlation coefficient of 0.82 and coefficient of determination of 0.67, and the item that the partial correlation coefficient was significantly high was R1 (RPVR1) alone. Regression analysis using RPVR1 as a variable gave the multiple correlation coefficient of 0.81 between actual and estimated ages and estimated error (within ±6 years) by the regression was 42.5%. The method was thus found usefully applied for age estimation in forensic judgment. (S.I.)

  9. 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.

  10. METHOD FOR AUTOMATIC ANALYSIS OF WHEAT STRAW PULP CELL TYPES

    Mikko Karjalainen,

    2012-01-01

    Full Text Available Agricultural residues are receiving increasing interest when studying renewable raw materials for industrial use. Residues, generally referred to as nonwood materials, are usually complex materials. Wheat straw is one of the most abundant agricultural residues around the world and is therefore available for extensive industrial use. However, more information of its cell types is needed to utilize wheat straw efficiently in pulp and papermaking. The pulp cell types and particle dimensions of wheat straw were studied, using an optical microscope and an automatic optical fibre analyzer. The role of various cell types in wheat straw pulp and papermaking is discussed. Wheat straw pulp components were categorized according to particle morphology and categorization with an automatic optical analyzer was used to determine wheat straw pulp cell types. The results from automatic optical analysis were compared to those with microscopic analysis and a good correlation was found. Automatic optical analysis was found to be a promising tool for the in-depth analysis of wheat straw pulp cell types.

  11. Expression of high mobility group box 1 in inflamed dental pulp and its chemotactic effect on dental pulp cells

    Highlights: • HMGB1 translocated from nucleus to cytoplasm during dental pulp inflammation. • HMGB1and its receptor RAGE were up-regulated in hDPCs under LPS stimulation. • HMGB1 enhanced hDPCs migration and induces cytoskeleton reorganization. • HMGB1 may play a critical role in dental pulp repair during inflamed state. - Abstract: High mobility group box 1 protein (HMGB1) is a chromatin protein which can be released extracellularly, eliciting a pro-inflammatory response and promoting tissue repair process. This study aimed to examine the expression and distribution of HMGB1 and its receptor RAGE in inflamed dental pulp tissues, and to assess its effects on proliferation, migration and cytoskeleton of cultured human dental pulp cells (DPCs). Our data demonstrated that cytoplasmic expression of HMGB1 was observed in inflamed pulp tissues, while HMGB1 expression was confined in the nuclei in healthy dental pulp. The mRNA expression of HMGB1 and RAGE were significantly increased in inflamed pulps. In in vitro cultured DPCs, expression of HMGB1 in both protein and mRNA level was up-regulated after treated with lipopolysaccharide (LPS). Exogenous HMGB1 enhanced DPCs migration in a dose-dependent manner and induced the reorganization of f-actin in DPCs. Our results suggests that HMGB1 are not only involved in the process of dental pulp inflammation, but also play an important role in the recruitment of dental pulp stem cells, promoting pulp repair and regeneration

  12. Expression of high mobility group box 1 in inflamed dental pulp and its chemotactic effect on dental pulp cells

    Zhang, Xufang, E-mail: xufang.zhang@student.qut.edu.au [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4059 (Australia); Jiang, Hongwei, E-mail: jianghw@163.com [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Gong, Qimei, E-mail: gongqmei@gmail.com [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Fan, Chen, E-mail: c3.fan@student.qut.edu.au [Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4059 (Australia); Huang, Yihua, E-mail: enu0701@163.com [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Ling, Junqi, E-mail: lingjq@mail.sysu.edu.cn [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China)

    2014-08-08

    Highlights: • HMGB1 translocated from nucleus to cytoplasm during dental pulp inflammation. • HMGB1and its receptor RAGE were up-regulated in hDPCs under LPS stimulation. • HMGB1 enhanced hDPCs migration and induces cytoskeleton reorganization. • HMGB1 may play a critical role in dental pulp repair during inflamed state. - Abstract: High mobility group box 1 protein (HMGB1) is a chromatin protein which can be released extracellularly, eliciting a pro-inflammatory response and promoting tissue repair process. This study aimed to examine the expression and distribution of HMGB1 and its receptor RAGE in inflamed dental pulp tissues, and to assess its effects on proliferation, migration and cytoskeleton of cultured human dental pulp cells (DPCs). Our data demonstrated that cytoplasmic expression of HMGB1 was observed in inflamed pulp tissues, while HMGB1 expression was confined in the nuclei in healthy dental pulp. The mRNA expression of HMGB1 and RAGE were significantly increased in inflamed pulps. In in vitro cultured DPCs, expression of HMGB1 in both protein and mRNA level was up-regulated after treated with lipopolysaccharide (LPS). Exogenous HMGB1 enhanced DPCs migration in a dose-dependent manner and induced the reorganization of f-actin in DPCs. Our results suggests that HMGB1 are not only involved in the process of dental pulp inflammation, but also play an important role in the recruitment of dental pulp stem cells, promoting pulp repair and regeneration.

  13. Results of comparative RBMK neutron computation using VNIIEF codes (cell computation, 3D statics, 3D kinetics). Final report

    In conformity with the protocol of the Workshop under Contract open-quotes Assessment of RBMK reactor safety using modern Western Codesclose quotes VNIIEF performed a neutronics computation series to compare western and VNIIEF codes and assess whether VNIIEF codes are suitable for RBMK type reactor safety assessment computation. The work was carried out in close collaboration with M.I. Rozhdestvensky and L.M. Podlazov, NIKIET employees. The effort involved: (1) cell computations with the WIMS, EKRAN codes (improved modification of the LOMA code) and the S-90 code (VNIIEF Monte Carlo). Cell, polycell, burnup computation; (2) 3D computation of static states with the KORAT-3D and NEU codes and comparison with results of computation with the NESTLE code (USA). The computations were performed in the geometry and using the neutron constants presented by the American party; (3) 3D computation of neutron kinetics with the KORAT-3D and NEU codes. These computations were performed in two formulations, both being developed in collaboration with NIKIET. Formulation of the first problem maximally possibly agrees with one of NESTLE problems and imitates gas bubble travel through a core. The second problem is a model of the RBMK as a whole with imitation of control and protection system controls (CPS) movement in a core

  14. 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 ...

  15. 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 ...

  16. 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 ...

  17. 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 ...

  18. Identification and Isolation of Human Dental Pulp Stem Cells

    2005-01-01

    1 IntroductionDentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts, that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue. Adult pulp stem cells was found in 2000. Now, it become a critical research in the world and a new stem-cells resourse for potential clinical applications. But, specific marker of DPSCs is not clear. STRO-1 was the marker that used universally. But, the expression rate was very low in...

  19. Property Of Human Dental Pulp Stem Cells And Peripheral Blood Hematopoietic Stem Cells That Differentiated Both Group To Cardiac Cells

    Jabari F; Mohammadnejad J; Yavari K

    2013-01-01

    Dental pulp is the soft live tissue inside a tooth. Dental pulp contains stem cells, known as Dental Pulp Stem Cells. The finest Dental Pulp Stem Cells are found in a baby teeth or milk teeth. The stem cells from the milk teeth are ‘mesenchymal’ type of cells. cells that have the ability to generate a wide variety of cell types like chondrocytes, osteoblasts and adipocytes. To isolate high-quality human dental pulp stem cells from accessible resources is an importan...

  20. 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

  1. 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…

  2. Characterization of stem and progenitor cells in the dental pulp of erupted and unerupted murine molars

    Balic, Anamaria; Aguila, H. Leonardo; Caimano, Melissa J.; Francone, Victor P.; Mina, Mina

    2010-01-01

    In the past few years there have been significant advances in the identification of putative stem cells also referred to as “mesenchymal stem cells” (MSC) in dental tissues including the dental pulp. It is thought that MSC in dental pulp share certain similarities with MSC isolated from other tissues. However, cells in dental pulp are still poorly characterized. This study focused on the characterization of progenitor and stem cells in dental pulps of erupted and unerupted mice molars. Our st...

  3. Radiobiology goes 3D: How ECM and cell morphology impact on cell survival after irradiation

    Translational research is essential to find new therapeutic approaches to improve cancer patient survival. Despite extensive efforts in preclinical studies, many novel therapies fail to turn out to be translational from bench to beside. Therefore, new models better reflecting the conditions in vivo are needed to generate results, which transfer reliably into the clinic. The use of three-dimensional (3D) cell culture models has provided new emerging insights into the understanding of cellular behavior upon cancer therapies. Interestingly, cells cultured in a 3D extracellular matrix are more radio- and chemoresistant than cells grown under conventional 2D conditions. In this review, we summarize and discuss underlying mechanisms of this phenomenon including integrin-mediated cell-matrix interactions, cell shape, nuclear organization and chromatin structure. Identifying the molecular differences between 2D and 3D cultured cells will offer the opportunity to improve our research and widen our therapeutic possibilities against cancer.

  4. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

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

    2016-01-01

    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. PMID:27321481

  5. Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

    Hanley Edward N

    2000-10-01

    Full Text Available Abstract Background The relationship between cell shape, proliferation, and extracellular matrix (ECM production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D culture. Results Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1 Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2 Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3 Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth. Conclusions The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

  6. Reprogramming mediated radio-resistance of 3D-grown cancer cells

    In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine. (author)

  7. Hot embossing for fabrication of a microfluidic 3D cell culture platform

    Jeon, Jessie S.; Chung, Seok; Kamm, Roger D.; Charest, Joseph L.

    2011-01-01

    Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying t...

  8. Hot embossing for fabrication of a microfluidic 3D cell culture

    Jeon, Jessie S.; Chung, Seok; Kamm, Roger Dale; Charest, Joseph L.

    2010-01-01

    Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying t...

  9. Fluorapatite-modified Scaffold on Dental Pulp Stem Cell Mineralization

    Guo, T.; Li, Y.; Cao, G.; Zhang, Z; Chang, S.; Czajka-Jakubowska, A.; Nör, J.E.; Clarkson, B.H.; Liu, J.

    2014-01-01

    In previous studies, fluorapatite (FA) crystal-coated surfaces have been shown to stimulate the differentiation and mineralization of human dental pulp stem cells (DPSCs) in two-dimensional cell culture. However, whether the FA surface can recapitulate these properties in three-dimensional culture is still unknown. This study examined the differences in behavior of human DPSCs cultured on electrospun polycaprolactone (PCL) NanoECM nanofibers with or without the FA crystals. Under near-physiol...

  10. 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....

  11. In Vitro Guidance of Dental Pulp Cells by Nd:YAG Laser-Irradiated Endothelial Cells

    Masuda, Yoshiko Murakami; Yamada, Yoshishige; Kimura, Yuichi

    2012-01-01

    Objective: After endothelial cells were ablated by neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation, we investigated the response of pulp cells by examining the expression of transforming growth factor beta-1 (TGF-β1). Background data: The reaction of stimulated blood vessels is related to the initiation of dentinogenesis. After artificial injury of endothelial cells, pulp cells migrate to the site of the injured endothelial cells. Materials and methods: Rat aortic endothelial cel...

  12. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    Segah Altuntaş

    2016-03-01

    Full Text Available Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications have stressed the importance of the expression of pluripotentiality associated markers: the transcription factors Nanog, Sox2, Oct3/4, SSEA4, CD13, Stro1 are indispensable for the stem cells to divide indefinitely without affecting their differentiation potential (self renewal capacity. Progesterone is a steroid hormone leading to menstrual cycle and gestation. There is a widespread rumor among people that pregnancy causes toothy loss. Method: So, progesterone was applied in different concentrations on human dental pulp cells in cell culture. Cell viability assay was applied 24th hour later with trypan blue. RNA isolation, cDNA synthesis and Real Time PCR analysis were applied on selected transcription factors (Nanog and Oct4 (POU5F1 genes which have role on steamness of stem cells. Gene expression analyses results were correlated with the cell viability assay results. Results: Cell viability assay results were 80% viable in control, 82% viable in 7 ml progesterone application, 81% viable in 14 ml progesterone application, 83% viable in 21 ml progesterone application. Due to our findings, progesterone in different concentrations did not chance the cell viability in dental pulpa cells. On gene expression analyses, preliminary results supported that high concentrations of progesterone enhance the gene expressions of steamness genes (Nanog, and Oct4 in dental pulp cells. Conclusions: So, progesterone did not change cell viability in high concentrations. We

  13. 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...

  14. Digital microfabrication of user-defined 3D microstructures in cell-laden hydrogels.

    Soman, Pranav; Chung, Peter H; Zhang, A Ping; Chen, Shaochen

    2013-11-01

    Complex 3D interfacial arrangements of cells are found in several in vivo biosystems such as blood vasculature, renal glomeruli, and intestinal villi. Current tissue engineering techniques fail to develop suitable 3D microenvironments to evaluate the concurrent effects of complex topography and cell encapsulation. There is a need to develop new fabrication approaches that control cell density and distribution within complex 3D features. In this work, we present a dynamic projection printing process that allows rapid construction of complex 3D structures using custom-defined computer-aided-design (CAD) files. Gelatin-methacrylate (GelMA) constructs featuring user-defined spiral, pyramid, flower, and dome micro-geometries were fabricated with and without encapsulated cells. Encapsulated cells demonstrate good cell viability across all geometries both on the scaffold surface and internal to the structures. Cells respond to geometric cues individually as well as collectively throughout the larger-scale patterns. Time-lapse observations also reveal the dynamic nature of mechanical interactions between cells and micro-geometry. When compared to conventional cell-seeding, cell encapsulation within complex 3D patterned scaffolds provides long-term control over proliferation, cell morphology, and geometric guidance. Overall, this biofabrication technique offers a flexible platform to evaluate cell interactions with complex 3D micro-features, with the ability to scale-up towards high-throughput screening platforms. PMID:23686741

  15. 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.

  16. Screening for Stromal and Matrix Effects in 3D Microenvironments of Breast Cancer Cells

    Montanez-Sauri, Sara I.

    Breast cancer progression ensures through the acquisition of genetic mutations, the uncontrollable growth of cells, and their progression to invasion. Studies have shown that the surrounding three-dimensional (3D) microenvironment can also influence breast cancer cell progression by controlling the morphology, differentiation, proliferation, and migration of cells. However, most of the currently available in vitro screening platforms are based on the two-dimensional (2D) culture of cells, and do not provide cells with the complex 3D microenvironment that exists in vivo. Therefore, there is a need for more biologically relevant in vitro platforms to help decipher the complexity of the microenvironment and its influence in breast cancer. In this dissertation we present an automated microfluidic platform that allows to efficiently screen for the effect of multiple matrix and stromal microenvironment in 3D cultures of breast cancer cells. Several extracellular matrix (ECM) compositions and stromal cells are included in the 3D microenvironments to examine their influence on breast cancer cell behavior. The screening results suggest that collagen gels with fibronectin might be influencing paracrine signals between breast cancer cells and stromal cells. The ability of the platform to culture and treat cells in 3D microenvironments offers a powerful screening tool for the identification of compounds and interactions using more in vivo-like 3D microenvironments. The identification of these mechanisms will increase our current understanding of breast cancer, and will aid in the identification of potential therapeutics.

  17. 3D porous biomimetically modified hydrogels supporting stem cells adhesion

    Studenovská, Hana; Vodička, Petr; Proks, Vladimír; Juhásová, Jana; Motlík, Jan; Rypáček, František

    Dublin : National University of Ireland , 2011. s. 119, psiii-630. [Annual Conference of the European Society for Biomaterials /24./. 04.09.2011-08.09.2011, Dublin] R&D Projects: GA MŠk 1M0538 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50450515 Keywords : porous hydrogel * cell adhesion * polypeptide Subject RIV: FH - Neurology

  18. 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...... 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...... 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...

  19. 3D-Modelling of polycrystalline silicon solar cells

    Dugas, J.; Oualid, J.

    1987-01-01

    This work is concerned with the effect of grain boundaries (gb) on the main parameters which characterize polycrystalline silicon solar cells. The variation with illumination of the grain boundary effective recombination velocity has been calculated by means of a self-consistent procedure which takes into account the bending of the minority carrier quasi-Fermi level in the space-charge region and in the gb quasi-neutral region. Abaci have been plotted which allow to predict the photovoltaic p...

  20. Rapid Assembly of Heterogeneous 3D Cell Microenvironments in a Microgel Array.

    Li, Yiwei; Chen, Pu; Wang, Yachao; Yan, Shuangqian; Feng, Xiaojun; Du, Wei; Koehler, Stephan A; Demirci, Utkan; Liu, Bi-Feng

    2016-05-01

    Heterogeneous 3D cell microenvironment arrays are rapidly assembled by combining surface-wettability-guided assembly and microdroplet-array-based operations. This approach enables precise control over individual shapes, sizes, chemical concentrations, cell density, and 3D spatial distribution of multiple components. This technique provides a cost-effective solution to meet the increasing demand of stem cell research, tissue engineering, and drug screening. PMID:26991071

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

    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 (R2 = 0.92) to the human oral adsorption than that of the Transwell culture (R2 = 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. (paper)

  2. Towards the development of a 3D full cell and external busbars thermo-electric model

    Taking advantage of the increasing power of computers, it is now practical to consider building a 3D full cell and external busbars thermo-electric model. In the present study, a 3D full cell quarter thermo-electric model and a 3D cathode half plus liquid zone and busbars thermo-electric model have been developed and solved using a PIll 800 MHz computer. Developing a 3D full cell and external busbars thermo-electric model will constitute a step further towards the development of a fully 'multi-physic' unified aluminium reduction cell model. Already, a full cell thermo-electric model will be able to interact with a MHD model by providing it with accurate liquid zone current density and potshell temperature data and by receiving from it local liquid/ledge interface heat transfer coefficients. (author)

  3. Fibroblasts Lead the Way: A Unified View of 3D Cell Motility.

    Petrie, Ryan J; Yamada, Kenneth M

    2015-11-01

    Primary human fibroblasts are remarkably adaptable, able to migrate in differing types of physiological 3D tissue and on rigid 2D tissue culture surfaces. The crawling behavior of these and other vertebrate cells has been studied intensively, which has helped generate the concept of the cell motility cycle as a comprehensive model of 2D cell migration. However, this model fails to explain how cells force their large nuclei through the confines of a 3D matrix environment and why primary fibroblasts can use more than one mechanism to move in 3D. Recent work shows that the intracellular localization of myosin II activity is governed by cell-matrix interactions to both force the nucleus through the extracellular matrix (ECM) and dictate the type of protrusions used to migrate in 3D. PMID:26437597

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

    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.

  5. 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.

  6. 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. PMID:25058941

  7. XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells.

    Taylor-Kashton, Cheryl; Lichtensztejn, Daniel; Baloglu, Erkan; Senapedis, William; Shacham, Sharon; Kauffman, Michael G; Kotb, Rami; Mai, Sabine

    2016-12-01

    Previous work has shown that the three-dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT-185, KPT-330/selinexor, and KPT-8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment-naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non-lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711-2719, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991404

  8. Characteristics of tumor and host cells in 3-D simulated microgravity environment

    Chopra, V.; Dinh, T.; Wood, T.; Pellis, N.; Hannigan, E.

    Co-cultures of three-dimensional (3-D) constructs of one cell type with dispersed cells of a second cell type in low-shear rotating suspension cultures in simulated microgravity environment have been used to investigate invasive properties of normal and malignant cell types. We have shown that the epithelial and endothelial cells undergo a switch in characteristics when grown in an in vitro 3-D environment, that mimics the in vivo host environment as compared with conventional two-dimensional (2-D) monolayer cultures. Histological preparations and immunohistochemical staining procedures of cocultured harvests demonstrated various markers of interest: like collagen vimentin, mucin, elastin, fibrin, fibrinogen, cytokeratin, adhesion molecules and various angiogenic factors by tumor cells from gynecological cancer patients along with fibroblasts, endothelial cells and patient-derived mononuclear cells (n=8). The growth rate was enhanced 10-15 folds by 3-D cocultures of patient-derived cells as compared with 2-D monolayer cultures and 3-D monocultures. The production of interleukin-2, interleukin-6, interleukin -8, vascular endothelial cell growth factor, basic fibroblast growth factor, and angiogenin was studied by using ELISA and RT- PCR. Human umbilical vein-derived endothelial cell (HUVEC) were used to study the mitogenic response of the conditioned medium collected from 3-D monocultures and cocultures during proliferation and migration assays. The conditioned medium collected from 3-D cocultures of cancer cells also 1) increased the expression of message levels of vascular endothelial growth factor and its receptor flt-1 and KDR was observed by HUVEC, and 2) increased the expression of intracellular and vascular cell adhesion molecules on the surface of HUVEC, when measured by using Live cell ELISA assays and immunofluorescent staining as compared with 3-D monocultures of normal epithelial cells. There was an increase in production of 1) enzymatic activity that

  9. 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.

  10. Cell force measurements in 3D microfabricated environments based on compliant cantilevers.

    Marelli, Mattia; Gadhari, Neha; Boero, Giovanni; Chiquet, Matthias; Brugger, Jürgen

    2014-01-21

    We report the fabrication, functionalization and testing of microdevices for cell culture and cell traction force measurements in three-dimensions (3D). The devices are composed of bent cantilevers patterned with cell-adhesive spots not lying on the same plane, and thus suspending cells in 3D. The cantilevers are soft enough to undergo micrometric deflections when cells pull on them, allowing cell forces to be measured by means of optical microscopy. Since individual cantilevers are mechanically independent of each other, cell traction forces are determined directly from cantilever deflections. This proves the potential of these new devices as a tool for the quantification of cell mechanics in a system with well-defined 3D geometry and mechanical properties. PMID:24217771

  11. 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.

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

    Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

    2016-01-01

    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. PMID:26978075

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

    Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

    2016-01-01

    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. PMID:26978075

  14. 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

    2016-05-01

    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 domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns. PMID:26971819

  15. Cell Proliferation on Macro/Nano Surface Structure and Collagen Immobilization of 3D Polycaprolactone Scaffolds.

    Park, Young-Ouk; Myung, Sung-Woon; Kook, Min-Suk; Jung, Sang-Chul; Kim, Byung-Hoon

    2016-02-01

    In this study, 3D polycaprolactone (PCL) scaffolds were fabricated by 3D printing technique. The macro/nano morphology of, 3D PCL scaffolds surface was etched with oxygen plasma. Acrylic acid (AA) plasma-polymerization was performed to functionalize the macro/nano surface with carboxyl groups and then collagen was immobilized with plasma-polymerized 3D PCL scaffolds. After O2 plasma and AA plasma-polymerization, contact angles were decreased. The FE-SEM and AFM results showed that O2 plasma is increased the surface roughness. The MTT assay results showed that proliferation of the M3CT3-E1 cells increased on the oxygen plasma treated and collagen immobilized 3D PCL scaffolds. PMID:27433597

  16. Micromorph silicon tandem solar cells with fully integrated 3D photonic crystal intermediate reflectors

    Üpping, J.; Bielawny, A.; Fahr, S.; Rockstuhl, C.; Lederer, F.; Steidl, L.; Zentel, R.; Beckers, T.; Lambertz, A.; Carius, R.; Wehrspohn, R. B.

    2010-05-01

    A 3D photonic intermediate reflector for textured micromorph silicon tandem solar cells has been investigated. In thin-film silicon tandem solar cells consisting of amorphous and microcrystalline silicon with two junctions of a-Si/c-Si, efficiency enhancements can be achieved by increasing the current density in the a-Si top cell providing an optimized current matching at high current densities. For an ideal photon-management between top and bottom cell, a spectrally-selective intermediate reflective layer (IRL) is necessary. We present the first fully-integrated 3D photonic thin-film IRL device incorporated on a planar substrate. Using a ZnO inverted opal structure the external quantum efficiency of the top cell in the spectral region of interest could be enhanced. As an outlook we present the design and the preparation of a 3D self organized photonic crystal structure in a textured micromorph tandem solar cell.

  17. 3D photonic crystal interlayers for micromorph thin film silicon tandem cell

    Uepping, Johannes; Bielawny, Andreas; Otto, Martin; Wehrspohn, Ralf B. [Institute of Physics, University of Halle, Wittenberg (Germany); Steidl, Lorenz; Zentel, Rudolf [Dept. of Chemistry, University of Mainz (Germany); Lee, Seung-Mo; Knez, Mato [Max Planck Institute of Microstructure Physics, Halle (Germany); Beckers, Thomas; Carius, Reinhard [Institute of Energy Research, IEF-5 Photovoltaics, Forschungszentrum Juelich GmbH (Germany)

    2010-07-01

    A 3D photonic intermediate reflector for textured micromorph silicon tandem solar cells has been investigated. In thin-film silicon tandem solar cells consisting of amorphous and microcrystalline silicon with two junctions of a-Si/{mu}c-Si, efficiency enhancements can be achieved by increasing the current density in the a-Si top cell. It is one goal to provide an optimized current matching at high current densities. For an ideal photon-management between top and bottom cell, a spectrally selective intermediate reflective layer (IRL) is necessary. We show results toward the first fully integrated 3D photonic thin-film IRL device incorporated in a state-of-the-art textured tandem solar cell. The design and the preparation of a 3D self organized inverted opal photonic crystal structure in a textured micromorph tandem solar cell is presented.

  18. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-11-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

  19. Hot embossing for fabrication of a microfluidic 3D cell culture platform.

    Jeon, Jessie S; Chung, Seok; Kamm, Roger D; Charest, Joseph L

    2011-04-01

    Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying the known commercially-viable manufacturing methods to a cyclic olefin copolymer (COC) material resulted in a microfluidic device with enhanced 3D gel capabilities, controlled surface properties, and improved potential to serve high-volume applications. Hot embossing and roller lamination molded and sealed the microfluidic device. A combination of oxygen plasma and thermal treatments enhanced the sealing, ensured proper placement of the 3D gel, and created controlled and stable surface properties within the device. Culture of cells in the new device indicated no adverse effects of the COC material or processing as compared to previous PDMS devices. The results demonstrate a methodology to transition microfluidic devices for 3D cell culture from scientific research to high-volume applications with broad clinical impact. PMID:21113663

  20. 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.

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

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

    2016-07-22

    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 × 10(2) to 1 × 10(7) 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. PMID:27275952

  2. 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.

  3. MULTILEVEL (3D) MICROFLUIDIC TECHNOLOGY FOR AN INNOVATIVE MAGNETIC CELL SEPARATION PLATFORM

    Fouet, Marc; Cargou, Sébastien; Courson, Rémi; Blatché, Charline; Montrose, A.; Reybier, K; Gué, Anne-Marie

    2014-01-01

    We demonstrate a new concept of devices, which by combining 3D fluid engineering and localized mag-netic actuation enables the full integration of a cell tagging and magnetic separation device. We used a low cost, commercially available dry film (EMS Inc, Ohio, USA) that fits microfluidic requirements and gives the possibility to build easily 3D microfluidic structures. The labelling of blood monocytes with su-perparamagnetic particles was performed "up stream" with the aim of a microparticle...

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

    Highlights: ► Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. ► Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. ► 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 μm 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.

  5. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing

    Akpe Victor; Rydholm Susanna; Liebmann Thomas; Brismar Hjalmar

    2007-01-01

    Abstract Background Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derive...

  6. 3D photonic crystal interlayers for micromorph thin film silicon tandem cell

    Bielawny, Andreas; Uepping, Johannes; Miclea, Paul T.; Wehrspohn, Ralf B. [Institute of Physics, University of Halle, Wittenberg (Germany); Rockstuhl, Carsten; Lederer, Falk [Institue of Physics, Solid States Optics, University of Jena (Germany); Peters, Marius [Freiburg Centre for Material Research, University of Freiburg (Germany); Steidl, Lorenz; Zentel, Rudolf [Dept. of Chemistry, Pharmacy and Earth Science, University of Mainz (Germany); Lee, Seung-Mo; Knez, Mato [Max Planck Institute of Microstructure Physics, Halle (Germany); Lambertz, Andreas; Carius, Reinhard [Institute of Energy Research, IEF-5 Photovoltaics, Forschungszentrum Juelich GmbH (Germany)

    2009-07-01

    The concept of 3D photonic intermediate reflectors for micromorph silicon tandem cells has been investigated toward first prototype cells. The reflector enhances the absorption of spectrally selected light in the top cell and decreases the current mismatch between both junctions. Our device is an inverted opal structure made of ZnO and built using self organized nanoparticles and atomic layer deposition coating methods. This 3D photonic crystal intermediate layer is less dependent of the angle of incidence than other state of the art thickness dependent massive interlayers. We present design rules, preparation and characterization of a 3D photonic thin film device. A first prototype is compared to a state of the art reference silicon tandem cell.

  7. Static Magnetic Field Attenuates Lipopolysaccharide-Induced Inflammation in Pulp Cells by Affecting Cell Membrane Stability

    Sung-Chih Hsieh

    2015-01-01

    Full Text Available One of the causes of dental pulpitis is lipopolysaccharide- (LPS- induced inflammatory response. Following pulp tissue inflammation, odontoblasts, dental pulp cells (DPCs, and dental pulp stem cells (DPSCs will activate and repair damaged tissue to maintain homeostasis. However, when LPS infection is too serious, dental repair is impossible and disease may progress to irreversible pulpitis. Therefore, the aim of this study was to examine whether static magnetic field (SMF can attenuate inflammatory response of dental pulp cells challenged with LPS. In methodology, dental pulp cells were isolated from extracted teeth. The population of DPSCs in the cultured DPCs was identified by phenotypes and multilineage differentiation. The effects of 0.4 T SMF on DPCs were observed through MTT assay and fluorescent anisotropy assay. Our results showed that the SMF exposure had no effect on surface markers or multilineage differentiation capability. However, SMF exposure increases cell viability by 15%. In addition, SMF increased cell membrane rigidity which is directly related to higher fluorescent anisotropy. In the LPS-challenged condition, DPCs treated with SMF demonstrated a higher tolerance to LPS-induced inflammatory response when compared to untreated controls. According to these results, we suggest that 0.4 T SMF attenuates LPS-induced inflammatory response to DPCs by changing cell membrane stability.

  8. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    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

  9. 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...

  10. Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line

    Fey, S. J.; Wrzesinski, Krzysztof

    2012-01-01

    Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this...

  11. 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

  12. 3D Image-Guided Automatic Pipette Positioning for Single Cell Experiments in vivo

    Brian Long; Lu Li; Ulf Knoblich; Hongkui Zeng; Hanchuan Peng

    2015-01-01

    We report a method to facilitate single cell, image-guided experiments including in vivo electrophysiology and electroporation. Our method combines 3D image data acquisition, visualization and on-line image analysis with precise control of physical probes such as electrophysiology microelectrodes in brain tissue in vivo. Adaptive pipette positioning provides a platform for future advances in automated, single cell in vivo experiments.

  13. 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.

  14. Digital Image Analysis of Cells : Applications in 2D, 3D and Time

    Pinidiyaarachchi, Amalka

    2009-01-01

    Light microscopes are essential research tools in biology and medicine. Cell and tissue staining methods have improved immensely over the years and microscopes are now equipped with digital image acquisition capabilities. The image data produced require development of specialized analysis methods. This thesis presents digital image analysis methods for cell image data in 2D, 3D and time sequences. Stem cells have the capability to differentiate into specific cell types. The mechanism behind di...

  15. Development and Validation of a 3D Clinostat for the Study of Cells during Microgravity Simulation.

    Russomano, T; Cardoso, R; Falcao, F; Dalmarco, G; V Dos Santos, C; F Dos Santos, L; G de Azevedo, D; Dos Santos, M; Martinelli, L; Motta, J; Forraz, N; McGuckin, C

    2005-01-01

    The clinostat was originally used to find out why plant roots appear to grow predominantly toward the center of the Earth. Over the last 2-3 decades, slow- and fast-rotating 2D and 3D clinostats have been used to assess cellular adaptation to this environment. A cell culture is placed in a spin module of the clinostat platform and its rotation is set empirically (2-3 rpm). The machine is then allowed to run for a specified period (hours to days) after which the cultures are removed and assayed for specific properties, such as cell growth, size and shape, distribution of receptors, integrity of the cytoskeleton or gene expression. A 3D clinostat was developed by the Microgravity Laboratory/IPCT-PUCRS group and validated by the Stem Cell Group of Kingston University London, which used 4 different types of human cancer cells and cord blood stem cells (CBSC). After rotation for 19h at 37degC, 5%CO2 humidified atmosphere, the 3D clinostat significantly improved proliferation potential of all tested cell populations when compared to static cultures. After only 5 days, high definition microscopic analysis revealed that all CBSC adhered and expanded onto the BDtrade 3D collagen composite scaffolds, and cross-developed into hepatocyte-like cells upon stimulation. PMID:17282243

  16. High Content Imaging (HCI on Miniaturized Three-Dimensional (3D Cell Cultures

    Pranav Joshi

    2015-12-01

    Full Text Available High content imaging (HCI is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy in vivo; thus, there has been an urgent need to perform HCI assays on three-dimensional (3D cell cultures. Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS. One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology.

  17. 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.

  18. Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites

    Jung, Jangwook P.; Bache-Wiig, Meredith K.; Provenzano, Paolo P.; Ogle, Brenda M.

    2016-01-01

    Abstract Extracellular matrix (ECM) proteins are structural elements of tissue and also potent signaling molecules. Previously, our laboratory showed that ECM of 2D coatings can trigger differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into mesodermal lineages in an ECM-specific manner over 14 days, in some cases comparable to chemical induction. To test whether a similar effect was possible in a 3D, tissue-like environment, we designed a synthetic-natural biomaterial composite. The composite can present whole-molecule ECM proteins to cells, even those that do not spontaneously form hydrogels ex vivo, in 3D. To this end, we entrapped collagen type I, laminin-111, or fibronectin in ECM composites with MSCs and directly compared markers of mesodermal differentiation including cardiomyogenic (ACTC1), osteogenic (SPP1), adipogenic (PPARG), and chondrogenic (SOX9) in 2D versus 3D. We found the 3D condition largely mimicked the 2D condition such that the addition of type I collagen was the most potent inducer of differentiation to all lineages tested. One notable difference between 2D and 3D was pronounced adipogenic differentiation in 3D especially in the presence of exogenous collagen type I. In particular, PPARG gene expression was significantly increased ∼16-fold relative to chemical induction, in 3D and not in 2D. Unexpectedly, 3D engagement of ECM proteins also altered immunomodulatory function of MSCs in that expression of IL-6 gene was elevated relative to basal levels in 2D. In fact, levels of IL-6 gene expression in 3D composites containing exogenously supplied collagen type I or fibronectin were statistically similar to levels attained in 2D with tumor necrosis factor-α (TNF-α) stimulation and these levels were sustained over a 2-week period. Thus, this novel biomaterial platform allowed us to compare the biochemical impact of whole-molecule ECM proteins in 2D versus 3D indicating enhanced adipogenic differentiation and IL-6 expression

  19. 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. PMID:23085529

  20. On-chip clearing of arrays of 3-D cell cultures and micro-tissues.

    Grist, S M; Nasseri, S S; Poon, T; Roskelley, C; Cheung, K C

    2016-07-01

    Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 μm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear(T2), and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear(T2); however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This on-chip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications. PMID:27493703

  1. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing

    Liebmann, Thomas; Rydholm, Susanna; Akpe, Victor; Brismar, Hjalmar

    2007-01-01

    Background Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale. Results Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms. Conclusion Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology. PMID:18070345

  2. 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.

  3. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing

    Akpe Victor

    2007-12-01

    Full Text Available Abstract Background Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale. Results Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms. Conclusion Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology.

  4. Mechano-sensing and cell migration: a 3D model approach

    Cell migration is essential for tissue development in different physiological and pathological conditions. It is a complex process orchestrated by chemistry, biological factors, microstructure and surrounding mechanical properties. Focusing on the mechanical interactions, cells do not only exert forces on the matrix that surrounds them, but they also sense and react to mechanical cues in a process called mechano-sensing. Here, we hypothesize the involvement of mechano-sensing in the regulation of directional cell migration through a three-dimensional (3D) matrix. For this purpose, we develop a 3D numerical model of individual cell migration, which incorporates the mechano-sensing process of the cell as the main mechanism regulating its movement. Consistent with this hypothesis, we found that factors, such as substrate stiffness, boundary conditions and external forces, regulate specific and distinct cell movements

  5. Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

    Behnam Ebrahimi; Mohammad Mehdi Yaghoobi; Ali Mohammadi Kamal-abadi; Maryam Raoof

    2011-01-01

    Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-1, jmjd1a, jmjd2c, and cyclin D1. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expres-sion significantly decreased, whereas expression of neuronal markers, such as microtubule asso-ciated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

  6. Genome-wide transcriptomic alterations induced by ethanol treatment in human dental pulp stem cells (DPSCs)

    Omar Khalid; Kim, Jeffrey J.; Lewei Duan; Michael Hoang; David Elashoff; Yong Kim

    2014-01-01

    Human dental pulp stem cells (DPSCs) isolated from adult dental pulp are multipotent mesenchymal stem cells that can be directed to differentiate into osteogenic/odontogenic cells and also trans-differentiate into neuronal cells. The utility of DPSC has been explored in odontogenic differentiation for tooth regeneration. Alcohol abuse appears to lead to periodontal disease, tooth decay and mouth sores that are potentially precancerous. Persons who abuse alcohol are at high risk of having seri...

  7. A 3D GCL compatible cell-centered Lagrangian scheme for solving gas dynamics equations

    Georges, Gabriel; Breil, Jérôme; Maire, Pierre-Henri

    2016-01-01

    Solving the gas dynamics equations under the Lagrangian formalism enables to simulate complex flows with strong shock waves. This formulation is well suited to the simulation of multi-material compressible fluid flows such as those encountered in the domain of High Energy Density Physics (HEDP). These types of flows are characterized by complex 3D structures such as hydrodynamic instabilities (Richtmyer-Meshkov, Rayleigh-Taylor, etc.). Recently, the 3D extension of different Lagrangian schemes has been proposed and appears to be challenging. More precisely, the definition of the cell geometry in the 3D space through the treatment of its non-planar faces and the limiting of a reconstructed field in 3D in the case of a second-order extension are of great interest. This paper proposes two new methods to solve these problems. A systematic and symmetric geometrical decomposition of polyhedral cells is presented. This method enables to define a discrete divergence operator leading to the respect of the Geometric Conservation Law (GCL). Moreover, a multi-dimensional minmod limiter is proposed. This new limiter constructs, from nodal gradients, a cell gradient which enables to ensure the monotonicity of the numerical solution even in presence of strong discontinuity. These new ingredients are employed into a cell-centered Lagrangian scheme. Robustness and accuracy are assessed against various representative test cases.

  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. 3D staggered Lagrangian hydrodynamics scheme with cell-centered Riemann solver-based artificial viscosity

    The aim of the present work is the 3D extension of a general formalism to derive a staggered discretization for Lagrangian hydrodynamics on unstructured grids. The classical compatible discretization is used; namely, momentum equation is discretized using the fundamental concept of subcell forces. Specific internal energy equation is obtained using total energy conservation. The subcell force is derived by invoking the Galilean invariance and thermodynamic consistency. A general form of the subcell force is provided so that a cell entropy inequality is satisfied. The subcell force consists of a classical pressure term plus a tensorial viscous contribution proportional to the difference between the node velocity and the cell-centered velocity. This cell-centered velocity is an extra degree of freedom solved with a cell-centered approximate Riemann solver. The second law of thermodynamics is satisfied by construction of the local positive definite subcell tensor involved in the viscous term. A particular expression of this tensor is proposed. A more accurate extension of this discretization both in time and space is also provided using a piecewise linear reconstruction of the velocity field and a predictor-corrector time discretization. Numerical tests are presented in order to assess the efficiency of this approach in 3D. Sanity checks show that the 3D extension of the 2D approach reproduces 1D and 2D results. Finally, 3D problems such as Sedov, Noh, and Saltzman are simulated. (authors)

  10. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

    Lee, Wonjae; Park, Jon

    2016-01-01

    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. PMID:27381562

  11. Correlative 3D imaging of Whole Mammalian Cells with Light and Electron Microscopy

    Murphy, Gavin E.; Narayan, Kedar; Lowekamp, Bradley C.; Hartnell, Lisa M.; Heymann, Jurgen A. W.; Fu, Jing; Subramaniam, Sriram

    2011-01-01

    We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA–SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ~10 to 20 nm at high contrast and with minimal artifacts from the foc...

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

    Jørgensen, Peter Stanley

    . Alignment of the individual image slices is performed by automatic detection of ducial marks. Uneven illumination is corrected by tting hypersurfaces to the spatial intensity variation in the 3D image data. Routine use of quantitative three dimensional analysis of microstructure is generally restricted by...... for gaining further fundamental understanding of how microstructure affects performance. In this work, methods for automatic 3D characterization of microstructure are studied: from the acquisition of 3D image data by focused ion beam tomography to the extraction of quantitative measures that......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...

  13. MAPLE deposition of 3D micropatterned polymeric substrates for cell culture

    Paun, Irina Alexandra, E-mail: irina.paun@physics.pub.ro [National Institute for Laser, Plasma and Radiation Physics, RO-077125, Magurele, Bucharest (Romania); Faculty of Applied Sciences, University Politehnica of Bucharest, RO-060042, Bucharest (Romania); Mihailescu, Mona [Faculty of Applied Sciences, University Politehnica of Bucharest, RO-060042, Bucharest (Romania); Calenic, Bogdan [Department of Biochemistry, Faculty of Dentistry, UMF Carol Davila, Bucharest (Romania); Luculescu, Catalin Romeo [National Institute for Laser, Plasma and Radiation Physics, RO-077125, Magurele, Bucharest (Romania); Greabu, Maria [Department of Biochemistry, Faculty of Dentistry, UMF Carol Davila, Bucharest (Romania); Dinescu, Maria, E-mail: dinescum@nipne.ro [National Institute for Laser, Plasma and Radiation Physics, RO-077125, Magurele, Bucharest (Romania)

    2013-08-01

    3D micropatterned poly(lactide-co-glycolide)/polyurethane (PLGA/PU) substrates were produced by MAPLE deposition through masks and used for regulating the behavior of oral keratinocyte stem cells in response to topography. Flat PLGA/PU substrates were produced for comparison. 3D imaging of the PLGA/PU substrates and of the cultured cells was performed by Digital Holographic Microscopy. The micropatterns were in the shape of squares of 50 × 50 and 80 × 80 μm{sup 2} areas, ∼1.8 μm in height and separated by 20 μm wide channels. It was found that substrate topography guided the adhesion of the cultured cells: on the smooth substrates the cells adhered randomly and showed no preferred orientation; in contrast, on the micropatterned substrates the cells adhered preferentially onto the squares and not in the separating channels. Furthermore, key properties of the cells (size, viability, proliferation rate and stem cell marker expression) did not show any dependence on substrate topography. The size of the cultured cells, their viability, the proportions of actively/slow proliferating cells, as well as the stem cell markers expressions, were similar for both flat and micropatterned substrates. Finally, it was found that the cells cultured on the PLGA/PU substrates deposited by MAPLE exhibited similar properties as the controls (i.e. cells cultured on glass slides), indicating the capability of the former to preserve the properties of the keratinocyte stem cells.

  14. MAPLE deposition of 3D micropatterned polymeric substrates for cell culture

    3D micropatterned poly(lactide-co-glycolide)/polyurethane (PLGA/PU) substrates were produced by MAPLE deposition through masks and used for regulating the behavior of oral keratinocyte stem cells in response to topography. Flat PLGA/PU substrates were produced for comparison. 3D imaging of the PLGA/PU substrates and of the cultured cells was performed by Digital Holographic Microscopy. The micropatterns were in the shape of squares of 50 × 50 and 80 × 80 μm2 areas, ∼1.8 μm in height and separated by 20 μm wide channels. It was found that substrate topography guided the adhesion of the cultured cells: on the smooth substrates the cells adhered randomly and showed no preferred orientation; in contrast, on the micropatterned substrates the cells adhered preferentially onto the squares and not in the separating channels. Furthermore, key properties of the cells (size, viability, proliferation rate and stem cell marker expression) did not show any dependence on substrate topography. The size of the cultured cells, their viability, the proportions of actively/slow proliferating cells, as well as the stem cell markers expressions, were similar for both flat and micropatterned substrates. Finally, it was found that the cells cultured on the PLGA/PU substrates deposited by MAPLE exhibited similar properties as the controls (i.e. cells cultured on glass slides), indicating the capability of the former to preserve the properties of the keratinocyte stem cells.

  15. Uncovering cancer cell behavioral phenotype in 3-D in vitro metastatic landscapes

    Liu, Liyu; Sun, Bo; Duclos, Guillaume; Kam, Yoonseok; Gatenby, Robert; Stone, Howard; Austin, Robert

    2012-02-01

    One well-known fact is that cancer cell genetics determines cell metastatic potentials. However, from a physics point of view, genetics as cell properties cannot directly act on metastasis. An agent is needed to unscramble the genetics first before generating dynamics for metastasis. Exactly this agent is cell behavioral phenotype, which is rarely studied due to the difficulties of real-time cell tracking in in vivo tissue. Here we have successfully constructed a micro in vitro environment with collagen based Extracellular Matrix (ECM) structures for cell 3-D metastasis. With stable nutrition (glucose) gradient inside, breast cancer cell MDA-MB-231 is able to invade inside the collagen from the nutrition poor site towards the nutrition rich site. Continuous confocal microscopy captures images of the cells every 12 hours and tracks their positions in 3-D space. The micro fluorescent beads pre-mixed inside the ECM demonstrate that invasive cells have altered the structures through mechanics. With the observation and the analysis of cell collective behaviors, we argue that game theory may exist between the pioneering cells and their followers in the metastatic cell group. The cell collaboration may explain the high efficiency of metastasis.

  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. 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.

  18. Organic MEMS/NEMS-based high-efficiency 3D ITO-less flexible photovoltaic cells

    A novel approach based on three-dimensional (3D) architecture for polymeric photovoltaic cells made up of an array of sub-micron and nano-pillars which not only increase the area of the light absorbing surface, but also improve the carrier collection efficiency of bulk-heterojunction organic solar cells is presented. The approach also introduces coating of 3D anodes with a new solution-processable highly conductive transparent polymer (Orgacon™) that replaces expensive vacuum-deposited ITO (indium tin oxide) as well as the additional hole-collecting layer of conventional PEDOT:PSS (poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate)). In addition, the described procedure is well suited to roll-to-roll high-throughput manufacturing. The high aspect-ratio 3D pillars which form the basis for this new architecture are patterned through micro-electromechanical-system- and nano-electromechanical-system-based processes. For the particular case of P3HT (poly(3-hexylthiophene)) and PCBM (phenyl-C61-butyric acid methyl ester) active material, efficiencies in excess of 6% have been achieved for these photovoltaic cells of 3D architecture using ITO-less flexible PET (polyethylene terephthalate) substrates. This increase in efficiency turns out to be more than twice higher than those achieved for their 2D counterparts. (paper)

  19. 3D-printing of Redox flow batteries for energy storage: a rapid prototype laboratory cell

    Arenas-Martinez, L.F.; Walsh, F.C.; Ponce de Leon, C.

    2015-01-01

    Although interest in redox flow batteries (RFBs) for energy storage has grown over the last few years, implementation of RFB technology has been slow and challenging. Recent developments in 3D-printing of materials enable a transforming technology for fast, reproducible and documented cell manufacture. This technology can give an improved engineering approach to cell design and fabrication, needed to fulfil requirements for lower cost, longer lifetime hardware capable of efficient reliable pe...

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

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

    2012-01-01

    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 x 25 x 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical im...

  1. Effects of microenvironment on growth and differentiation of human dental pulp cells

    Datko, Laura Christine

    Dental pulp stem cells (DPSCs) have recently been described as a potential stem cell source for various regenerative medicine and tissue engineering applications. They appear to be multipotent, however more characterization is necessary to determine the true potential of these cells. An important aspect of using DPSCs, or any stem cell type, tissue engineering application is the microenvironment within the construct. The microenvironment could include construct mechanical properties, construct composition, and 3D dynamic conditions in vivo. This work aims to study those specific microenvironment effects on DPSCs. To determine the effects of mechanical properties of the substrate on DPSCs, they were seeded on polyacrylamide (PA) gels of different elastic moduli. These gels ranged from 3 kPa to 75 kPa and a glass coverslip was used as a control. They were also exposed to either standard stem cell media or an osteogenic differentiation media, to determine the potential of the DPSCs for osteogenic/odontogenic differentiation. The cultures were analyzed for morphological changes, osteopontin production, alkaline phosphatase (ALP) production, and mineralization. The results showed that the DPSCs adhered well to the PA gels for the first few days in culture, but by day 7, they were starting to detach from the PA gels and only remain viable in gel defects or along the edges. This selective growth was also reflected in the mineralization, which only occurred in areas of confluence for the cells on the PA gels. Interestingly, all cultures produced osteopontin and ALP, however by the end of the experiment, the cells cultured on glass had the highest ALP production. It appeared that without the addition of growth factors to induce other cell lineages, DPSCs defaulted to an osteogenic/odontogenic lineage. To determine the effect of mineral composition, preliminary studies were done on bone marrow stromal cells (BMSCs) and 7F2 osteoblasts. These two cell types were exposed to

  2. 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

  3. Cell counting in human endobronchial biopsies--disagreement of 2D versus 3D morphometry.

    Vlad A Bratu

    Full Text Available QUESTION: Inflammatory cell numbers are important endpoints in clinical studies relying on endobronchial biopsies. Assumption-based bidimensional (2D counting methods are widely used, although theoretically design-based stereologic three-dimensional (3D methods alone offer an unbiased quantitative tool. We assessed the method agreement between 2D and 3D counting designs in practice when applied to identical samples in parallel. MATERIALS AND METHODS: Biopsies from segmental bronchi were collected from healthy non-smokers (n = 7 and smokers (n = 7, embedded and sectioned exhaustively. Systematic uniform random samples were immunohistochemically stained for macrophages (CD68 and T-lymphocytes (CD3, respectively. In identical fields of view, cell numbers per volume unit (NV were assessed using the physical disector (3D, and profiles per area unit (NA were counted (2D. For CD68+ cells, profiles with and without nucleus were separately recorded. In order to enable a direct comparison of the two methods, the zero-dimensional CD68+/CD3+-ratio was calculated for each approach. Method agreement was tested by Bland-Altmann analysis. RESULTS: In both groups, mean CD68+/CD3+ ratios for NV and NA were significantly different (non-smokers: 0.39 and 0.68, p<0.05; smokers: 0.49 and 1.68, p<0.05. When counting only nucleated CD68+ profiles, mean ratios obtained by 2D and 3D counting were similar, but the regression-based Bland-Altmann analysis indicated a bias of the 2D ratios proportional to their magnitude. This magnitude dependent deviation differed between the two groups. CONCLUSIONS: 2D counts of cell and nuclear profiles introduce a variable size-dependent bias throughout the measurement range. Because the deviation between the 3D and 2D data was different in the two groups, it precludes establishing a 'universal conversion formula'.

  4. 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.

  5. 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

  6. Human dental pulp stem cells cultured in serum-free supplemented medium

    Bonnamain, Virginie; Thinard, Reynald; Sergent-Tanguy, Solène; Huet, Pascal; Bienvenu, Géraldine; Naveilhan, Philippe; Farges, Jean-Christophe; Alliot-Licht, Brigitte

    2013-01-01

    Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) ...

  7. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  8. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  9. A 3-D Model of a Perennial Ryegrass Primary Cell Wall and Its Enzymatic Degradation

    Indrakumar Vetharaniam; Kelly, William J.; Graeme T. Attwood; Harris, Philip J.

    2014-01-01

    We have developed a novel 3-D, agent-based model of cell-wall digestion to improve our understanding of ruminal cell-wall digestion. It offers a capability to study cell walls and their enzymatic modification, by providing a representation of cellulose microfibrils and non-cellulosic polysaccharides and by simulating their spatial and catalytic interactions with enzymes. One can vary cell-wall composition and the types and numbers of enzyme molecules, allowing the model to be applied to a ran...

  10. 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.

  11. 3D cell culture systems modeling tumor growth determinants in cancer target discovery.

    Thoma, Claudio R; Zimmermann, Miriam; Agarkova, Irina; Kelm, Jens M; Krek, Wilhelm

    2014-04-01

    Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models. PMID:24636868

  12. 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.

  13. Spontaneous Differentiation of Dental Pulp stem cells on Dental polymers

    Bherwani, Aneel; Suarato, Giulia; Qin, Sisi; Chang, Chung-Cheh; Akhavan, Aaron; Spiegel, Joseph; Jurukovski, Vladimir; Rafailovich, Miriam; Simon, Marcia

    2012-02-01

    Dental pulp stem cells were plated on two dentally relevant materials i.e. PMMA commonly used for denture and Titanium used for implants. In both cases, we probed for the role of surface interaction and substrate morphology. Different films of PMMA were spun cast directly onto Si wafers; PMMA fibers of different diameters were electro spun onto some of these substrates. Titanium metal was evaporated onto Si surfaces using an electron beam evaporator. In addition, on some surfaces, P4VP nanofibers were spun cast. DPSC were grown in alpha-MEM supplemented with 10% fetal bovine serum, 0.2mM L-ascorbic acid 2-phosphate, 2mm glutamine and 10mM beta-glycerol phosphate either with or without 10nM dexamethasone. After 21 days samples were examined using confocal microscopy of cells and by scanning electron microscopy (SEM) and Energy dispersive X-ray Analysis (EDAX). In the case of Titanium biomineralization was observed independent of dexamethasone, where the deposits were templated along the fibers. Minimal biomineralization was observed on flat Titanium and PMMA samples. Markers of osteogenesis and specific signaling pathways are being evaluated by RT-PCR, which are up regulated on each surface, to understand the fundamental manner in which surfaces interact with cell differentiation.

  14. 3D photonic crystal intermediate reflector for micromorph thin-film tandem solar cell

    Uepping, Johannes; Miclea, Paul T.; Wehrspohn, Ralf B. [Institute of Physics, Martin-Luther-University of Halle-Wittenberg, Heinrich-Damerow-Str. 4, 06120 Halle (Germany); Rockstuhl, Carsten; Lederer, Falk [Institute of Condensed Matter Theory and Solid States Optics, Friedrich Schiller University Jena, 07743 Jena (Germany); Peters, Marius [Freiburg Centre for Material Research, University of Freiburg, 79104 Freiburg (Germany); Steidl, Lorenz; Zentel, Rudolf [Dept. of Chemistry, Pharmacy and Earth Science, Johannes Gutenberg University of Mainz, Duesbergweg 10-14 (Germany); Lee, Seung-Mo; Knez, Mato [Max Planck Institute of Microstructure Physics, Weinberg 2, 06120 Halle (Germany); Lambertz, Andreas; Carius, Reinhard [Institute of Energy Research, IEF-5 Photovoltaics, Forschungszentrum Juelich GmbH, 52425 Juelich (Germany); Bielawny, Andreas

    2008-12-15

    The concept of 3D photonic intermediate reflectors for micromorph silicon tandem solar cells has been investigated. In thin-film silicon tandem solar cells consisting of amorphous and microcrystalline silicon with two junctions of a-Si/{mu}c-Si, efficiency enhancements can be achieved by increasing the current density in the a-Si top cell. It is one goal to provide an optimized current matching at high current densities. For an ideal photon-management between top and bottom cell, a spectrally selective intermediate reflective layer (IRL) is necessary, which is less dependent of the angle of incidence than state-of-the-art thickness dependent massive interlayers. The design, preparation and characterization of a 3D photonic thin-film filter device for this purpose has been pursued straight forward in simulation and experimental realization. The inverted opal is capable of providing a suitable optical band stop with high reflectance and the necessary long wavelength transmittance as well and provides further options for improved light trapping. We have determined numerically the relative efficiency enhancement of an a-Si/{mu}c-Si tandem solar cell using a conductive 3D-photonic crystal. We have further fabricated such structures by ZnO-replication of polymeric opals using chemical vapour deposition and atomic layer deposition techniques and present the results of their characterization. Thin film photonic IRL have been prepared at the rear side of a-Si solar cells. Completed with a back contact, this is the first step to integrate this novel technology into an a-Si/{mu}c-Si tandem solar cell process. The spectral response of the cell is presented and compared with reference cells. (copyright 2008 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  15. 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.

  16. Determination of key parameters of SEU occurrence using 3-D full cell SRAM simulations

    A 3-D entire SRAM cell, based on a 0.35-microm current CMOS technology, is simulated in this work with a DEVICE simulator. The transient current, resulting from a heavy ion strike in the most sensitive region of the cell, is studied as a function of the LET value, the cell layout and the ion penetration depth. A definition of the critical charge is proposed and two new methods are presented to compute this basic amount of charge only using SPICE simulations. Numerical applications are performed with two different generations of submicron CMOS technologies, including the determination of the sensitive thicknesses

  17. 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.

  18. In-chip fabrication of free-form 3D constructs for directed cell migration analysis

    Olsen, Mark Holm; Hjortø, Gertrud Malene; Hansen, Morten;

    2013-01-01

    Free-form constructs with three-dimensional (3D) microporosity were fabricated by two-photon polymerization inside the closed microchannel of an injection-molded, commercially available polymer chip for analysis of directed cell migration. Acrylate constructs were produced as woodpile topologies...... with a range of pore sizes from 5 × 5 μm to 15 × 15 μm and prefilled with fibrillar collagen. Dendritic cells seeded into the polymer chip in a concentration gradient of the chemoattractant CCL21 efficiently negotiated the microporous maze structure for pore sizes of 8 × 8 μm or larger. The cells...

  19. 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.

  20. Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D

    MultiCellular Tumor Spheroid (MCTS) mimics the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. Here we report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization and the activation of cell cycle checkpoints in 3D. Cell cycle and proliferation parameters were investigated in Capan-2 spheroids by immunofluorescence staining, EdU incorporation and using cells engineered to express Fucci-red and -green reporters. We describe in details the changes in proliferation and cell cycle parameters during spheroid growth and regionalization. We report the kinetics and regionalized aspects of cell cycle arrest in response to checkpoint activation induced by EGF starvation, lovastatin treatment and etoposide-induced DNA damage. Our data present the power and the limitation of spheroids made of genetically modified cells to explore cell cycle checkpoints. This study paves the way for the investigation of molecular aspects and dynamic studies of the response to novel antiproliferative agents in 3D models

  1. Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

    Chuang, Han-Ning; Lohaus, Raphaela; Hanisch, Uwe-Karsten; Binder, Claudia

    2013-01-01

    Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7. Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8. For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain

  2. Digital holography for recovering 3D shape of red blood cells

    Memmolo, P.; Miccio, L.; Merola, F.; Gennari, O.; Netti, P.; Ferraro, Pietro

    2015-07-01

    Full morphometric data analysis and 3D rendering of Red Blood Cells (RBCs) is provided by means of Digital Holography (DH) in combination with Optical Tweezers (OT). The proposed method is compared with a geometrical model of RBC in order to evaluate its accuracy and tested for many kinds of RBCs, from healthy ones with double-concavity to that with abnormal shapes. Applications in diagnostics are foreseen.

  3. Non-crimp 3D woven composites unit cell: from geometric modelling to damage simulation

    Bedogni, Enrico

    2013-01-01

    In the last twenty years, the research on composite materials has increased and many progresses have been made. However, there are still unresolved issues concerning the geometric modelling of a material at the meso-level (i.e. on a unit cell) and its damage simulation. In particular, the complexity of the internal geometry of some composite materials, such as 3D textiles, yields to new challenges for the research community. A correct definition of the internal structure in all the important ...

  4. Perfusion Stirred-Tank Bioreactors for 3D Differentiation of Human Neural Stem Cells.

    Simão, Daniel; Arez, Francisca; Terasso, Ana P; Pinto, Catarina; Sousa, Marcos F Q; Brito, Catarina; Alves, Paula M

    2016-01-01

    Therapeutic breakthroughs in neurological disorders have been hampered by the lack of accurate central nervous system (CNS) models. The development of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of new therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmental, anatomic, and physiological) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity, etc.). Recapitulation of CNS phenotypic and functional features in vitro requires the implementation of advanced culture strategies, such as 3D culture systems, which enable to mimic the in vivo structural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. The development of robust and scalable processes for the 3D differentiation of hNSC can improve the accuracy of early stage development in preclinical research. In this context, the use of software-controlled stirred-tank bioreactors (STB) provides an efficient technological platform for hNSC aggregation and differentiation. This system enables to monitor and control important physicochemical parameters for hNSC culture, such as dissolved oxygen. Importantly, the adoption of a perfusion operation mode allows a stable flow of nutrients and differentiation/neurotrophic factors, while clearing the toxic by-products. This contributes to a setting closer to the physiological, by mimicking the in vivo microenvironment. In this chapter, we address the technical requirements and procedures for the implementation of 3D differentiation strategies of hNSC, by operating STB under perfusion mode for long-term cultures. This strategy is suitable

  5. Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

    Jensen Jonas; Tvedesøe Claus; Rölfing Jan Hendrik Duedal; Foldager Casper Bindzus; Lysdahl Helle; Kraft David Christian Evar; Chen Muwan; Baas Jorgen; Le Dang Quang Svend; Bünger Cody Eric

    2016-01-01

    Introduction: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) was compared with that of dental pulp-derived stromal cells (DPSCs) in vitro and in a pig calvaria critical-size bone defect model. Methods: BMSCs and DPSCs were extracted from the tibia bone marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D) polycaprolactone (PCL) – hyaluronic acid – tricalcium phos...

  6. 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.

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

    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 E2 (PGE2) 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.

  8. Probabilistic Voxel-Fe model for single cell motility in 3D

    Borau, Carlos; Polacheck, William J; Kamm, Roger D; García-Aznar, José Manuel

    2015-01-01

    Background Cells respond to a variety of external stimuli regulated by the environment conditions. Mechanical, chemical and biological factors are of great interest and have been deeply studied. Furthermore, mathematical and computational models have been rapidly growing over the past few years, permitting researches to run complex scenarios saving time and resources. Usually these models focus on specific features of cell migration, making them only suitable to study restricted phenomena. Methods Here we present a versatile finite element (FE) cell-scale 3D migration model based on probabilities depending in turn on ECM mechanical properties, chemical, fluid and boundary conditions. Results With this approach we are able to capture important outcomes of cell migration such as: velocities, trajectories, cell shape and aspect ratio, cell stress or ECM displacements. Conclusions The modular form of the model will allow us to constantly update and redefine it as advancements are made in clarifying how cellular events take place. PMID:26290806

  9. Effect of Tc99m Labeling on The Survival Rate of Dental Pulp Stem Cells

    Jabari F

    2014-02-01

    Full Text Available Background and Aim: Human dental pulp stem cells have substantial proliferative and differentiation potential. The isolated stem cells or progenitor cells of the pulp can differentiate into odontoblasts or /and osteoblast-like cells and aid in repair as well as reconstruction of tooth structure. Several ways have been introduced for isolation and tracing of these cells. The aim of this study was to isolate mesenchymal stem cells from deciduous dental pulps as well as labeling them with Technetium (Tc99m to investigate the effect of Tc labeling on the survival rate of stem cells. Materials and Methods: In this experimental study, exfoliated deciduous teeth of 6-11 year old children without any history of systemic disease were collected. Enzymatic and non-enzymatic methods were used to expedite cell isolation and isolated cells (10000 from dental pulp were mixed with 25 millicurie of Tc for tracing purposes. Individual cell activity as well as culture medium activation was evaluated separately afterwards. Data was analyzed using ANOVA statistical test. Results: Isolated dental pulp cells formed single cell derived colonies which showed fibroblast-like growth with solo cloning morphology. Specific staining of the cells indicated them to be stem cells and confirmed their differentiation into bone and fat. Moreover, Technetium significantly decreased the activity of cells. The survival rates of the cells in the period of 1,3,6,24,48 hours were reported to be 95.5%, 85.5%, 77.4%, 68.4%, and 57.3% respectively. Conclusion: The dental pulp stem cells have a significant capacity to differentiate into bone and fat. Tracing the cells with Tc M99 will reduce their survival rate over time.

  10. 3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

    Kim, Yeonhee; Rajagopalan, Padmavathy

    2010-01-01

    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, detoxification and

  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. 3D X-rays application for precision measurement of the cell structure of extruded polystyrene

    Lim, J. Y.; Kim, K. Y.; Shin, H. S.; Yeom, S.; Lee, S. E.

    2015-12-01

    While the thermal performance of existing insulation materials have been determined by blister gases, the thermal performance of future insulation materials will be dependent on the cell size and independent foam content as we use eco-friendly blister gases with a higher thermal conductivity. However, with the current technology we are only able to guess the whole cell size and independent foam content through SEM applied 2D fragmentary scanning but are still far from the level of accurate cell structure data extraction. Under this situation, we utilized X-ray CT scanned 3D images to identify and shape the cell structure and proposed a method of inferring the whole distribution and independent foam content as accurately as possible. According to X-ray CT scanning images and SEM images, the shape was similar but according to tracer applied CT scanning images, the cell size distribution was 380∼400 pm within the range of the general insulation diameter distribution which had the highest reliability. As for extrusion foaming polystyrene, we need additional image processing to identify the independent foam content as its density is too low. So, it is recommended to raise the 3D cell structure completeness of XPS by improving the scanning accuracy.

  13. 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.

  14. Mechanical Properties of 3-D Printed Cellular Foams with triangular cells

    Bunga, Pratap Kumar

    In the present work, poly lactic acid (PLA) is used as a model system to investigate the mechanical behavior of 3-D printed foams with triangular cells. Solid PLA tension and compression specimens and foams made of PLA were fabricated using fused deposition 3-D printing technique. The solid PLA tension specimens were characterized for their densities and found to be about 10% lower in density as compared to their bulk counter parts. The triangular foams had a relative density of about 64%. The relationships between the structure of the foams and its deformation behavior under compression along two in-plane directions were characterized. Furthermore, simple finite element models were developed to understand the observed deformation behavior of triangular foams.

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

    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. 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. 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. 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 tumor cell invasiveness

  16. 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.

  17. 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

  18. Estimation of single cell volume from 3D confocal images using automatic data processing

    Chorvatova, A.; Cagalinec, M.; Mateasik, A.; Chorvat, D., Jr.

    2012-06-01

    Cardiac cells are highly structured with a non-uniform morphology. Although precise estimation of their volume is essential for correct evaluation of hypertrophic changes of the heart, simple and unified techniques that allow determination of the single cardiomyocyte volume with sufficient precision are still limited. Here, we describe a novel approach to assess the cell volume from confocal microscopy 3D images of living cardiac myocytes. We propose a fast procedure based on segementation using active deformable contours. This technique is independent on laser gain and/or pinhole settings and it is also applicable on images of cells stained with low fluorescence markers. Presented approach is a promising new tool to investigate changes in the cell volume during normal, as well as pathological growth, as we demonstrate in the case of cell enlargement during hypertension in rats.

  19. Drug penetration and metabolism in 3D cell cultures treated in a 3D printed fluidic device: assessment of irinotecan via MALDI imaging mass spectrometry.

    LaBonia, Gabriel J; Lockwood, Sarah Y; Heller, Andrew A; Spence, Dana M; Hummon, Amanda B

    2016-06-01

    Realistic in vitro models are critical in the drug development process. In this study, a novel in vitro platform is employed to assess drug penetration and metabolism. This platform, which utilizes a 3D printed fluidic device, allows for dynamic dosing of three dimensional cell cultures, also known as spheroids. The penetration of the chemotherapeutic irinotecan into HCT 116 colon cancer spheroids was examined with MALDI imaging mass spectrometry (IMS). The active metabolite of irinotecan, SN-38, was also detected. After twenty-four hours of treatment, SN-38 was concentrated to the outside of the spheroid, a region of actively dividing cells. The irinotecan prodrug localization contrasted with SN-38 and was concentrated to the necrotic core of the spheroids, a region containing mostly dead and dying cells. These results demonstrate that this unique in vitro platform is an effective means to assess drug penetration and metabolism in 3D cell cultures. This innovative system can have a transformative impact on the preclinical evaluation of drug candidates due to its cost effectiveness and high throughput. PMID:27198560

  20. Many-faced cells and many-edged faces in 3D Poisson–Voronoi tessellations

    Motivated by recent new Monte Carlo data we investigate a heuristic asymptotic theory that applies to n-faced 3D Poisson–Voronoi cells in the limit of large n. We show how this theory may be extended to n-edged cell faces. It predicts the leading order large-n behavior of the average volume and surface area of the n-faced cell, and of the average area and perimeter of the n-edged face. Such a face is shown to be surrounded by a toroidal region of volume n/λ (with λ the seed density) that is void of seeds. Two neighboring cells sharing an n-edged face are found to have their seeds at a typical distance that scales as n−1/6 and whose probability law we determine. We present a new data set of 4 × 109 Monte Carlo generated 3D Poisson–Voronoi cells, larger than any before. Full compatibility is found between the Monte Carlo data and the theory. Deviations from the asymptotic predictions are explained in terms of subleading corrections whose powers in n we estimate from the data. (paper)

  1. Many-faced cells and many-edged faces in 3D Poisson-Voronoi tessellations

    Hilhorst, H. J.; Lazar, E. A.

    2014-10-01

    Motivated by recent new Monte Carlo data we investigate a heuristic asymptotic theory that applies to n-faced 3D Poisson-Voronoi cells in the limit of large n. We show how this theory may be extended to n-edged cell faces. It predicts the leading order large-n behavior of the average volume and surface area of the n-faced cell, and of the average area and perimeter of the n-edged face. Such a face is shown to be surrounded by a toroidal region of volume n/λ (with λ the seed density) that is void of seeds. Two neighboring cells sharing an n-edged face are found to have their seeds at a typical distance that scales as n-1/6 and whose probability law we determine. We present a new data set of 4 × 109 Monte Carlo generated 3D Poisson-Voronoi cells, larger than any before. Full compatibility is found between the Monte Carlo data and the theory. Deviations from the asymptotic predictions are explained in terms of subleading corrections whose powers in n we estimate from the data.

  2. 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.

  3. Low-level laser therapy in 3D cell culture model using gingival fibroblasts.

    Basso, Fernanda G; Soares, Diana G; de Souza Costa, Carlos Alberto; Hebling, Josimeri

    2016-07-01

    Besides extensive data about the effects of low-level laser therapy (LLLT) on different cell types, so far, these results were obtained from monolayer cell culture models, which have limitations in terms of cell morphology and phenotype expression. Therefore, for better in vitro evaluation of the effects of LLLT, this study was performed with a 3D cell culture model, where gingival fibroblasts were seeded in collagen matrix. Cells isolated from a healthy patient were seeded in wells of 24-well plates with culture medium (DMEM) supplemented with 10 % fetal bovine serum and collagen type I solution. After 5 days, a serum-free DMEM was added to the matrices with cells that were subjected or not to three consecutive irradiations of LLLT by means of the LaserTABLE diode device (780 nm, 25 mW) at 0.5, 1.5, and 3 J/cm(2). Twenty-four hours after the last irradiation, cell viability and morphology as well as gene expression of growth factors were assessed. Histological evaluation of matrices demonstrated uniform distribution and morphology of gingival fibroblasts within the collagen matrix. LLLT at 3 J/cm(2) increased gingival fibroblast viability. Enhanced gene expression of hCOL-I and hEGF was observed for 0.5 J/cm(2), while no significant changes were detected for the other irradiation densities tested. In conclusion, LLLT promoted biostimulation of gingival fibroblasts seeded in a 3D cell culture model, demonstrating that this model can be applied for phototherapy studies and that LLLT could penetrate the collagen matrix to increase cell functions related to tissue repair. PMID:27126408

  4. Challenges of stem cell-based pulp and dentin regeneration: a clinical perspective

    Huang, George T.-J.; Al-Habib, Mey; Gauthier, Philippe

    2013-01-01

    There are two types of approaches to regenerate tissues: cell-based and cell-free. The former approach is to introduce exogenous cells into the host to regenerate tissues, and the latter is to use materials other than cells in an attempt to regenerate tissues. There has been a significant advancement in stem cell-based pulp and dentin regeneration research in the past few years. Studies in small and large animals have demonstrated that pulp/dentin-like tissues can be regenerated partially or ...

  5. Direct 3D-printing of cell-laden constructs in microfluidic architectures.

    Liu, Justin; Hwang, Henry H; Wang, Pengrui; Whang, Grace; Chen, Shaochen

    2016-04-21

    Microfluidic platforms have greatly benefited the biological and medical fields, however standard practices require a high cost of entry in terms of time and energy. The utilization of three-dimensional (3D) printing technologies has greatly enhanced the ability to iterate and build functional devices with unique functions. However, their inability to fabricate within microfluidic devices greatly increases the cost of producing several different devices to examine different scientific questions. In this work, a variable height micromixer (VHM) is fabricated using projection 3D-printing combined with soft lithography. Theoretical and flow experiments demonstrate that altering the local z-heights of VHM improved mixing at lower flow rates than simple geometries. Mixing of two fluids occurs as low as 320 μL min(-1) in VHM whereas the planar zigzag region requires a flow rate of 2.4 mL min(-1) before full mixing occurred. Following device printing, to further demonstrate the ability of this projection-based method, complex, user-defined cell-laden scaffolds are directly printed inside the VHM. The utilization of this unique ability to produce 3D tissue models within a microfluidic system could offer a unique platform for medical diagnostics and disease modeling. PMID:26980159

  6. Effects of electromagnetic field frequencies on chondrocytes in 3D cell-printed composite constructs.

    Yi, Hee-Gyeong; Kang, Kyung Shin; Hong, Jung Min; Jang, Jinah; Park, Moon Nyeo; Jeong, Young Hun; Cho, Dong-Woo

    2016-07-01

    In cartilage tissue engineering, electromagnetic field (EMF) therapy has been reported to have a modest effect on promoting cartilage regeneration. However, these studies were conducted using different frequencies of EMF to stimulate chondrocytes. Thus, it is necessary to investigate the effect of EMF frequency on cartilage formation. In addition to the stimulation, a scaffold is required to satisfy the characteristics of cartilage such as its hydrated and dense extracellular matrix, and a mechanical resilience to applied loads. Therefore, we 3D-printed a composite construct composed of a polymeric framework and a chondrocyte-laden hydrogel. Here, we observed frequency-dependent positive and negative effects on chondrogenesis using a 3D cell-printed cartilage tissue. We found that a frequency of 45 Hz promoted gene expression and secretion of extracellular matrix molecules of chondrocytes. In contrast, a frequency of 7.5 Hz suppressed chondrogenic differentiation in vitro. Additionally, the EMF-treated composite constructs prior to implantation showed consistent results with those of in vitro, suggesting that in vitro pre-treatment with different EMF frequencies provides different capabilities for the enhancement of cartilage formation in vivo. This correlation between EMF frequency and 3D-printed chondrocytes suggests the necessity for optimization of EMF parameters when this physical stimulus is applied to engineered cartilage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1797-1804, 2016. PMID:26991030

  7. 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...

  8. 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. PMID:25609254

  9. Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells

    Ikbale El-Ayachi

    2016-03-01

    Full Text Available These data relate to the differentiation of human dental pulp stem cells (DPSC and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells. The data augment another study to characterize immortalized DPSC for the study of neurogenetic “Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders” [1]. Two copies of one typical control cell line (technical replicates were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown.

  10. 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. PMID:27552143

  11. Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells

    Gruene, M; Deiwick, A; Koch, L; Schlie, S; Unger, C; Chichkov, B N [Nanotechnology Department, Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover (Germany); Pflaum, M; Wilhelmi, M; Haverich, A, E-mail: m.gruene@lzh.de [Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover (Germany)

    2011-03-15

    Laser-assisted bioprinting (LaBP) allows the realization of computer-generated 3D tissue grafts consisting of cells embedded in a hydrogel environment. In this study, human adipose-derived stem cells (hASCs) were printed in a free-scalable 3D grid pattern by means of LaBP. We demonstrate that neither the proliferation ability nor the differentiation behaviour of the stem cells was affected by the LaBP procedure. Furthermore, the 3D grafts were differentiated down the adipogenic lineage pathway for 10 days. We verify by quantitative assessments of adipogenic markers that the 3D grafts resemble cell lineages present in natural adipose tissue. Additionally, we provide the proof that even pre-differentiated hASCs could be utilized for the generation of 3D tissue grafts. These results indicate that the biofabrication of living grafts resembling their complex native origin is within reach.

  12. Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells

    Laser-assisted bioprinting (LaBP) allows the realization of computer-generated 3D tissue grafts consisting of cells embedded in a hydrogel environment. In this study, human adipose-derived stem cells (hASCs) were printed in a free-scalable 3D grid pattern by means of LaBP. We demonstrate that neither the proliferation ability nor the differentiation behaviour of the stem cells was affected by the LaBP procedure. Furthermore, the 3D grafts were differentiated down the adipogenic lineage pathway for 10 days. We verify by quantitative assessments of adipogenic markers that the 3D grafts resemble cell lineages present in natural adipose tissue. Additionally, we provide the proof that even pre-differentiated hASCs could be utilized for the generation of 3D tissue grafts. These results indicate that the biofabrication of living grafts resembling their complex native origin is within reach.

  13. Isotropic 3D Nuclear Morphometry of Normal, Fibrocystic and Malignant Breast Epithelial Cells Reveals New Structural Alterations

    Nandakumar, Vivek; Kelbauskas, Laimonas; Hernandez, Kathryn F.; Lintecum, Kelly M.; Senechal, Patti; Bussey, Kimberly J.; Davies, Paul C.W.; Johnson, Roger H.; Meldrum, Deirdre R.

    2012-01-01

    Background Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading ...

  14. 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 of......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...

  15. 3-D slug flow heat transfer analysis of coupled coolant cells in finite LMFBR bundles

    A three-dimensional single region slug flow heat transfer analysis for finite LMFBR rod bundles using a classical analytical solution method has been performed. According to the isolated single cell analysis, the results show that the peripheral clad temperature variation as well as the thermal entrance length are strongly dependent upon the degree of irregularity displayed by various coolant geometries. Since under the present LMFBR conditions, fully-developed temperature fields may hardly be established in such characteristic rod bundle regions, a 3-D heat transfer analysis seems to be mandatory. This implies that the results of fully developed heat transfer analyses are by far too conservative

  16. A harmonic polynomial cell (HPC) method for 3D Laplace equation with application in marine hydrodynamics

    We propose a new efficient and accurate numerical method based on harmonic polynomials to solve boundary value problems governed by 3D Laplace equation. The computational domain is discretized by overlapping cells. Within each cell, the velocity potential is represented by the linear superposition of a complete set of harmonic polynomials, which are the elementary solutions of Laplace equation. By its definition, the method is named as Harmonic Polynomial Cell (HPC) method. The characteristics of the accuracy and efficiency of the HPC method are demonstrated by studying analytical cases. Comparisons will be made with some other existing boundary element based methods, e.g. Quadratic Boundary Element Method (QBEM) and the Fast Multipole Accelerated QBEM (FMA-QBEM) and a fourth order Finite Difference Method (FDM). To demonstrate the applications of the method, it is applied to some studies relevant for marine hydrodynamics. Sloshing in 3D rectangular tanks, a fully-nonlinear numerical wave tank, fully-nonlinear wave focusing on a semi-circular shoal, and the nonlinear wave diffraction of a bottom-mounted cylinder in regular waves are studied. The comparisons with the experimental results and other numerical results are all in satisfactory agreement, indicating that the present HPC method is a promising method in solving potential-flow problems. The underlying procedure of the HPC method could also be useful in other fields than marine hydrodynamics involved with solving Laplace equation

  17. In-cell maintenance by manipulator arm with 3D workspace information recreated by laser rangefinder

    Highlights: → We developed a remote control system for maintenance of in-cell type fuel fabrication equipment. → The system display recreated three-dimensional information of the workspace from data obtained by laser rangefinder and conventional cameras. It has allowed us to operate a manipulator arm remotely with several control modes. → We implemented remote handling experiments using mock up equipment. Performance was compared for remote operation conducted using several different display and operation modes. → It was observed that integration of 3D information from the laser rangefinder reduced operation time and reinforced visual information during remote operation. - Abstract: We developed a remote control system for maintenance of in-cell type fuel fabrication equipment. The system display recreated three-dimensional information of the workspace from data obtained by laser rangefinder and conventional cameras. It has allowed us to operate a manipulator arm remotely with several control modes. In order to evaluate the effectiveness and usefulness of developed system, we implemented remote handling experiments using mock up equipment. Performance was compared for remote operation conducted using several different display and operation modes. We confirmed that the system is able to maintain in-cell fuel fabrication equipment in each display and operation mode. Times required to complete the remote operations were collected and compared in each mode. It was observed that integration of 3D information from the laser rangefinder reduced operation time and reinforced visual information during remote operation.

  18. A harmonic polynomial cell (HPC) method for 3D Laplace equation with application in marine hydrodynamics

    Shao, Yan-Lin, E-mail: yanlin.shao@dnvgl.com; Faltinsen, Odd M.

    2014-10-01

    We propose a new efficient and accurate numerical method based on harmonic polynomials to solve boundary value problems governed by 3D Laplace equation. The computational domain is discretized by overlapping cells. Within each cell, the velocity potential is represented by the linear superposition of a complete set of harmonic polynomials, which are the elementary solutions of Laplace equation. By its definition, the method is named as Harmonic Polynomial Cell (HPC) method. The characteristics of the accuracy and efficiency of the HPC method are demonstrated by studying analytical cases. Comparisons will be made with some other existing boundary element based methods, e.g. Quadratic Boundary Element Method (QBEM) and the Fast Multipole Accelerated QBEM (FMA-QBEM) and a fourth order Finite Difference Method (FDM). To demonstrate the applications of the method, it is applied to some studies relevant for marine hydrodynamics. Sloshing in 3D rectangular tanks, a fully-nonlinear numerical wave tank, fully-nonlinear wave focusing on a semi-circular shoal, and the nonlinear wave diffraction of a bottom-mounted cylinder in regular waves are studied. The comparisons with the experimental results and other numerical results are all in satisfactory agreement, indicating that the present HPC method is a promising method in solving potential-flow problems. The underlying procedure of the HPC method could also be useful in other fields than marine hydrodynamics involved with solving Laplace equation.

  19. Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts.

    Wu, Yang; Sriram, Gopu; Fawzy, Amr S; Fuh, Jerry Yh; Rosa, Vinicius; Cao, Tong; Wong, Yoke San

    2016-08-01

    Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future. PMID:27252227

  20. Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy.

    Chang, Lingqian; Gallego-Perez, Daniel; Zhao, Xi; Bertani, Paul; Yang, Zhaogang; Chiang, Chi-Ling; Malkoc, Veysi; Shi, Junfeng; Sen, Chandan K; Odonnell, Lynn; Yu, Jianhua; Lu, Wu; Lee, L James

    2015-08-01

    Current transfection technologies lead to significant inter-clonal variations. Previously we introduced a unique electrotransfection technology, Nanochannel-Electroporation (NEP), which can precisely and benignly transfect small cell populations (~100-200 cells) with single-cell resolution. Here we report on the development of a novel 3D NEP system for large scale transfection. A properly-engineered array of nanochannels, capable of handling/transfecting ~60 000 cells cm(-2), was fabricated using cleanroom technologies. Positive dielectrophoresis was used to selectively position cells on the nanochannels, thus allowing highly efficient transfection. Single-cell dosage control was demonstrated using both small and large molecules, and different cell types. The potential clinical relevance of this system was tested with difficult-to-transfect natural killer cell suspensions, and plasmids encoding for the chimeric antigen receptor (CAR), a model of high relevance for adoptive immunotherapy. Our results show significantly higher CAR transfection efficiencies for the DEP-NEP system (>70% vs. <30%), as well as enhanced cell viabilities. PMID:26105628

  1. Engineering a perfusable 3D human liver platform from iPS cells.

    Schepers, Arnout; Li, Cheri; Chhabra, Arnav; Seney, Benjamin Tschudy; Bhatia, Sangeeta

    2016-07-01

    In vitro models of human tissue are crucial to our ability to study human disease as well as develop safe and effective drug therapies. Models of single organs in static and microfluidic culture have been established and shown utility for modeling some aspects of health and disease; however, these systems lack multi-organ interactions that are critical to some aspects of drug metabolism and toxicity. Thus, as part of a consortium of researchers, we have developed a liver chip that meets the following criteria: (1) employs human iPS cells from a patient of interest, (2) cultures cells in perfusable 3D organoids, and (3) is robust to variations in perfusion rate so as to be compatible in series with other specialized tissue chips (e.g. heart, lung). In order to achieve this, we describe methods to form hepatocyte aggregates from primary and iPS-derived cells, alone and in co-culture with support cells. This necessitated a novel culture protocol for the interrupted differentiation of iPS cells that permits their removal from a plated surface and aggregation while maintaining phenotypic hepatic functions. In order to incorporate these 3D aggregates in a perfusable platform, we next encapsulated the cells in a PEG hydrogel to prevent aggregation and overgrowth once on chip. We adapted a C-trap chip architecture from the literature that enabled robust loading with encapsulated organoids and culture over a range of flow rates. Finally, we characterize the liver functions of this iHep organoid chip under perfusion and demonstrate a lifetime of at least 28 days. We envision that such this strategy can be generalized to other microfluidic tissue models and provides an opportunity to query patient-specific liver responses in vitro. PMID:27296616

  2. 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

    2016-01-01

    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

  3. Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells.

    Duncan, Henry F

    2012-03-01

    Histone deacetylase inhibitors (HDACis) alter the homeostatic balance between 2 groups of cellular enzymes, histone deacetylases (HDACs) and histone acetyltransferases (HATs), increasing transcription and influencing cell behavior. This study investigated the potential of 2 HDACis, valproic acid (VPA) and trichostatin A (TSA), to promote reparative processes in pulp cells as assayed by viability, cell cycle, and mineralization analyses.

  4. 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.

  5. A Hyaluronan-Based Scaffold for the in Vitro Construction of Dental Pulp-Like Tissue

    Letizia Ferroni; Chiara Gardin; Stefano Sivolella; Giulia Brunello; Mario Berengo; Adriano Piattelli; Eriberto Bressan; Barbara Zavan

    2015-01-01

    Dental pulp tissue supports the vitality of the tooth, but it is particularly vulnerable to external insults, such as mechanical trauma, chemical irritation or microbial invasion, which can lead to tissue necrosis. In the present work, we present an endodontic regeneration method based on the use of a tridimensional (3D) hyaluronan scaffold and human dental pulp stem cells (DPSCs) to produce a functional dental pulp-like tissue in vitro. An enriched population of DPSCs was seeded onto hyalur...

  6. Has 3-D conformal radiotherapy (3D CRT) improved the local tumour control for stage I non-small cell lung cancer?

    Aims and background: The high local failure rates observed after radiotherapy in stage I non-small cell lung cancer (NSCLC) may be improved by the use of 3-dimensional conformal radiotherapy (3D CRT). Materials and methods: The case-records of 113 patients who were treated with curative 3D CRT between 1991 and 1999 were analysed. No elective nodal irradiation was performed, and doses of 60 Gy or more, in once-daily fractions of between 2 and 3 Gy, were prescribed. Results: The median actuarial survival of patients was 20 months, with 1-, 3- and 5-year survival of 71, 25 and 12%, respectively. Local disease progression was the cause of death in 30% of patients, and 22% patients died from distant metastases. Grade 2-3 acute radiation pneumonitis (SWOG) was observed in 6.2% of patients. The median actuarial local progression-free survival (LPFS) was 27 months, with 85 and 43% of patients free from local progression at 1 and 3 years, respectively. Endobronchial tumour extension significantly influenced LPFS, both on univariate (P=0.023) and multivariate analysis (P=0.023). The median actuarial cause-specific survival (CSS) was 19 months, and the respective 1- and 3-year rates were 72 and 30%. Multivariate analysis showed T2 classification (P=0.017) and the presence of endobronchial tumour extension (P=0.029) to be adverse prognostic factors for CSS. On multivariate analysis, T-stage significantly correlated with distant failure (P=0.005). Conclusions: Local failure rates remain substantial despite the use of 3D CRT for stage I NSCLC. Additional improvements in local control can come about with the use of radiation dose escalation and approaches to address the problem of tumour mobility

  7. Adaptive geometric tessellation for 3D reconstruction of anisotropically developing cells in multilayer tissues from sparse volumetric microscopy images.

    Anirban Chakraborty

    Full Text Available The need for quantification of cell growth patterns in a multilayer, multi-cellular tissue necessitates the development of a 3D reconstruction technique that can estimate 3D shapes and sizes of individual cells from Confocal Microscopy (CLSM image slices. However, the current methods of 3D reconstruction using CLSM imaging require large number of image slices per cell. But, in case of Live Cell Imaging of an actively developing tissue, large depth resolution is not feasible in order to avoid damage to cells from prolonged exposure to laser radiation. In the present work, we have proposed an anisotropic Voronoi tessellation based 3D reconstruction framework for a tightly packed multilayer tissue with extreme z-sparsity (2-4 slices/cell and wide range of cell shapes and sizes. The proposed method, named as the 'Adaptive Quadratic Voronoi Tessellation' (AQVT, is capable of handling both the sparsity problem and the non-uniformity in cell shapes by estimating the tessellation parameters for each cell from the sparse data-points on its boundaries. We have tested the proposed 3D reconstruction method on time-lapse CLSM image stacks of the Arabidopsis Shoot Apical Meristem (SAM and have shown that the AQVT based reconstruction method can correctly estimate the 3D shapes of a large number of SAM cells.

  8. 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

    2016-02-01

    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. PMID:26626828

  9. Temperature distributions in the laser-heated diamond anvil cell from 3-D numerical modeling

    We present TempDAC, a 3-D numerical model for calculating the steady-state temperature distribution for continuous wave laser-heated experiments in the diamond anvil cell. TempDAC solves the steady heat conduction equation in three dimensions over the sample chamber, gasket, and diamond anvils and includes material-, temperature-, and direction-dependent thermal conductivity, while allowing for flexible sample geometries, laser beam intensity profile, and laser absorption properties. The model has been validated against an axisymmetric analytic solution for the temperature distribution within a laser-heated sample. Example calculations illustrate the importance of considering heat flow in three dimensions for the laser-heated diamond anvil cell. In particular, we show that a “flat top” input laser beam profile does not lead to a more uniform temperature distribution or flatter temperature gradients than a wide Gaussian laser beam

  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. 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

    2016-06-01

    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). PMID:27219645

  12. 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.

  13. A Parallelized 3D Particle-In-Cell Method With Magnetostatic Field Solver And Its Applications

    Hsu, Kuo-Hsien; Chen, Yen-Sen; Wu, Men-Zan Bill; Wu, Jong-Shinn

    2008-10-01

    A parallelized 3D self-consistent electrostatic particle-in-cell finite element (PIC-FEM) code using an unstructured tetrahedral mesh was developed. For simulating some applications with external permanent magnet set, the distribution of the magnetostatic field usually also need to be considered and determined accurately. In this paper, we will firstly present the development of a 3D magnetostatic field solver with an unstructured mesh for the flexibility of modeling objects with complex geometry. The vector Poisson equation for magnetostatic field is formulated using the Galerkin nodal finite element method and the resulting matrix is solved by parallel conjugate gradient method. A parallel adaptive mesh refinement module is coupled to this solver for better resolution. Completed solver is then verified by simulating a permanent magnet array with results comparable to previous experimental observations and simulations. By taking the advantage of the same unstructured grid format of this solver, the developed PIC-FEM code could directly and easily read the magnetostatic field for particle simulation. In the upcoming conference, magnetron is simulated and presented for demonstrating the capability of this code.

  14. 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.

  15. Complete factorial design experiment for 3D load cell instrumented crank validation.

    Omar, Valle-Casas; Rafael, Dalazen; Vinicius, Cene; Alexandre, Balbinot

    2015-08-01

    Developing of instrumentation systems for sport medicine is a promising area, that's why this research evaluates the design of a new instrumented crank arm prototype for a race bicycle projecting an experiment for indoor - outdoor comparison. This study investigated the viability of an instrumentation 3D load cell for force measurement crank, implementing a design of experiment. A Complete factorial design experiment was developed for data validation, with an Analysis of Variance (ANOVA) throwing significant results for controlled factors with response variables rms, mean and variance. A software routine allowed to obtained system variables metrics for Symmetry and Cadence analysis, which came out from Effective force bilateral comparing and speed computation. Characterization allowed achieving calibration curves that were used for data conversion in force projection channels with a linearity error of 0.29% (perpendicular), 0.55% (parallel) and 0.10% (lateral). Interactions of factors resulted significant mainly for indoor tests in symmetry and cadence was significant in interactions generally for outdoor tests. Implemented system was able to generate Effective Force graph for 3D plot symmetry analysis, torque and power symmetry for specialist's analysis. PMID:26737085

  16. 3-D-conformal radiation therapy for pediatric giant cell tumors of the skull base

    Hug, E.B. [Massachusetts General Hospital, Boston, MA (United States). Dept. of Radiation Oncology; Harvard Univ., Cambridge, MA (United States). Cyclotron Lab.; Dartmouth Hitchcock Medical Center, Lebanon, NH (United States). Section of Radiation Oncology; Muenter, M.W.; Vries, A. de [Massachusetts General Hospital, Boston, MA (United States). Dept. of Radiation Oncology; Adams, J.A.; Munzenrider, J.E. [Massachusetts General Hospital, Boston, MA (United States). Dept. of Radiation Oncology; Harvard Univ., Cambridge, MA (United States). Cyclotron Lab.; Rosenberg, A.E. [Massachusetts General Hospital, Boston, MA (United States). Dept. of Pathology

    2002-05-01

    Background: Giant cell tumors (GCT) of the base of skull are rare neoplasms. This report reviews the treatment of four pediatric patients presenting with aggressive giant cell tumor, using fractionated and combined, conformal proton and photon radiation therapy at Massachusetts General Hospital and Harvard Cyclotron Laboratory. Patients and Methods: Three female patients and one adolescent male, ages 10-15 years, had undergone prior, extensive surgical resection(s) and were treated for either primary (two patients) or recurrent (two patients) disease. Gross residual tumor was evident in three patients and microscopic disease suspected in one patient. Combined proton and photon radiation theory was based on three-dimensional (3-D) planning, consisting of fractionated treatment, one fraction per day at 1.8 CGE (cobalt-gray equivalent) to total target doses of 57.6, 57.6, 59.4, and 61.2 Gy/CGE. Results: With observation times of 3.1 years, 3.3, 5.3, and 5.8 years, all four patients were alive and well and remained locally controlled without evidence of recurrent disease. Except for one patient with partial pituitary insufficiency following radiotherapy for sellar recurrent disease, thus far no late effects attributable to radiation therapy have been observed. Conclusions: 3-D conformal radiation therapy offers a realistic chance of tumor control for aggressive giant cell tumor in the skull base, either postoperatively or at time of recurrence. Conformal treatment techniques allow the safe delivery of relatively high radiation doses in the pediatric patient without apparent increase of side effects. (orig.)

  17. 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.

  18. Dual targeting of EGFR and focal adhesion kinase in 3D grown HNSCC cell cultures

    Purpose: Epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) show frequent overexpression and hyperactivity in various human malignancies including head and neck squamous cell carcinomas (HNSCC). To examine effects of dual EGFR/FAK inhibition on cellular radiosensitivity of HNSCC cells in a more physiological environment, we employed a previously established laminin-rich extracellular matrix (lrECM) based three-dimensional (3D) cell culture model. Materials and methods: UTSCC15 and SAS HNSCC cell lines stably transfected with EGFR-CFP or CFP were used. Single or combined EGFR (Cetuximab, siRNA) and FAK (TAE226, siRNA) inhibition were accomplished prior to measuring clonogenic survival and protein expression and phosphorylation. Immunofluorescence enabled visualization of EGFR-CFP and FAK. Results: Cetuximab resulted in higher radiosensitization in EGFR-CFP overexpressing cell lines than CFP controls. Single EGFR or FAK inhibition mediated radiosensitization, while dual EGFR/FAK targeting further augmented this effect. Despite signaling alterations upon Cetuximab and siRNA knockdown, analysis of protein expression and phosphorylation indicates EGFR and FAK signaling coexistence without obvious overlap. Conclusions: Combined EGFR/FAK targeting yielded stronger radiosensitization than either approach alone, which might be based on non-overlapping downstream signaling. Whether dual targeting of EGFR and FAK can reasonably be combined with radiotherapy and chemotherapy needs clarification.

  19. Mechanical Changes in Human Dental Pulp Stem Cells during Early Odontogenic Differentiation

    Jones, Taneka D.; Naimipour, Hamed; Sun, Shan; Cho, Michael; Alapati, Satish B.

    2014-01-01

    Cell adhesion and migration in bioactive scaffolds require actin cytoskeleton remodeling and focal adhesion formation. Additionally, human dental pulp stem cells (hDPSCs) undergo several changes in their mechanical properties during odontogenic differentiation. The effect of factors essential for odontogenesis on the actin stress fiber elasticity and focal adhesion formation is not known.

  20. Chitosan-collagen biomembrane embedded with calcium-aluminate enhances dentinogenic potential of pulp cells.

    Soares, Diana Gabriela; Rosseto, Hebert Luís; Basso, Fernanda Gonçalves; Scheffel, Débora Salles; Hebling, Josimeri; Costa, Carlos Alberto de Souza

    2016-01-01

    The development of biomaterials capable of driving dental pulp stem cell differentiation into odontoblast-like cells able to secrete reparative dentin is the goal of current conservative dentistry. In the present investigation, a biomembrane (BM) composed of a chitosan/collagen matrix embedded with calcium-aluminate microparticles was tested. The BM was produced by mixing collagen gel with a chitosan solution (2:1), and then adding bioactive calcium-aluminate cement as the mineral phase. An inert material (polystyrene) was used as the negative control. Human dental pulp cells were seeded onto the surface of certain materials, and the cytocompatibility was evaluated by cell proliferation and cell morphology, assessed after 1, 7, 14 and 28 days in culture. The odontoblastic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, total protein production, gene expression of DMP-1/DSPP and mineralized nodule deposition. The pulp cells were able to attach onto the BM surface and spread, displaying a faster proliferative rate at initial periods than that of the control cells. The BM also acted on the cells to induce more intense ALP activity, protein production at 14 days, and higher gene expression of DSPP and DMP-1 at 28 days, leading to the deposition of about five times more mineralized matrix than the cells in the control group. Therefore, the experimental biomembrane induced the differentiation of pulp cells into odontoblast-like cells featuring a highly secretory phenotype. This innovative bioactive material can drive other protocols for dental pulp exposure treatment by inducing the regeneration of dentin tissue mediated by resident cells. PMID:27119587

  1. Metabolic response of lung cancer cells to radiation in a paper-based 3D cell culture system.

    Simon, Karen A; Mosadegh, Bobak; Minn, Kyaw Thu; Lockett, Matthew R; Mohammady, Marym R; Boucher, Diane M; Hall, Amy B; Hillier, Shawn M; Udagawa, Taturo; Eustace, Brenda K; Whitesides, George M

    2016-07-01

    This work demonstrates the application of a 3D culture system-Cells-in-Gels-in-Paper (CiGiP)-in evaluating the metabolic response of lung cancer cells to ionizing radiation. The 3D tissue-like construct-prepared by stacking multiple sheets of paper containing cell-embedded hydrogels-generates a gradient of oxygen and nutrients that decreases monotonically in the stack. Separating the layers of the stack after exposure enabled analysis of the cellular response to radiation as a function of oxygen and nutrient availability; this availability is dictated by the distance between the cells and the source of oxygenated medium. As the distance between the cells and source of oxygenated media increased, cells show increased levels of hypoxia-inducible factor 1-alpha, decreased proliferation, and reduced sensitivity to ionizing radiation. Each of these cellular responses are characteristic of cancer cells observed in solid tumors. With this setup we were able to differentiate three isogenic variants of A549 cells based on their metabolic radiosensitivity; these three variants have known differences in their metastatic behavior in vivo. This system can, therefore, capture some aspects of radiosensitivity of populations of cancer cells related to mass-transport phenomenon, carry out systematic studies of radiation response in vitro that decouple effects from migration and proliferation of cells, and regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiation. PMID:27116031

  2. 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 sc...

  3. Biocompatibility and bioactivity of calcium silicate-based endodontic sealers in human dental pulp cells

    Leticia Boldrin MESTIERI; GOMES-CORNÉLIO, Ana Lívia; RODRIGUES, Elisandra Márcia; SALLES, Loise Pedrosa; BOSSO-MARTELO, Roberta; Juliane Maria GUERREIRO-TANOMARU; TANOMARU-FILHO, Mário

    2015-01-01

    Mineral Trioxide Aggregate (MTA) is a calcium silicate-based material. New sealers have been developed based on calcium silicate as MTA Fillapex and MTA Plus. Objective The aim of this study was to evaluate biocompatibility and bioactivity of these two calcium silicate-based sealers in culture of human dental pulp cells (hDPCs). Material and Methods The cells were isolated from third molars extracted from a 16-year-old patient. Pulp tissue was sectioned into fragments with approximately 1 mm3...

  4. Dental pulp stem cells as a multifaceted tool for bioengineering and the regeneration of craniomaxillofacial tissues

    Aurrekoetxea, Maitane; Garcia-Gallastegui, Patricia; Irastorza, Igor; Luzuriaga, Jon; Uribe-Etxebarria, Verónica; Unda, Fernando; Ibarretxe, Gaskon

    2015-01-01

    Dental pulp stem cells, or DPSC, are neural crest-derived cells with an outstanding capacity to differentiate along multiple cell lineages of interest for cell therapy. In particular, highly efficient osteo/dentinogenic differentiation of DPSC can be achieved using simple in vitro protocols, making these cells a very attractive and promising tool for the future treatment of dental and periodontal diseases. Among craniomaxillofacial organs, the tooth and salivary gland are two such cases in wh...

  5. Dental Pulp Stem Cells as a multifaceted tool for bioengineering and the regeneration of craniomaxillofacial tissues

    Maitane eAurrekoetxea; Patricia eGarcía-Gallastegui; Igor eIrastorza; Jon eLuzuriaga; Verónica eUribe-Etxebarria; Fernando eUnda; Gaskon eIbarretxe

    2015-01-01

    Dental pulp stem cells, or DPSC, are neural crest-derived cells with an outstanding capacity to differentiate along multiple cell lineages of interest for cell therapy. In particular, highly efficient osteo/dentinogenic differentiation of DPSC can be achieved using simple in vitro protocols, making these cells a very attractive and promising tool for the future treatment of dental and periodontal diseases. Among craniomaxillofacial organs, the tooth and salivary gland are two such cases in wh...

  6. Human Serum Promotes Osteogenic Differentiation of Human Dental Pulp Stem Cells In Vitro and In Vivo

    Alessandra Pisciotta; Massimo Riccio; Gianluca Carnevale; Francesca Beretti; Lara Gibellini; Tullia Maraldi; Gian Maria Cavallini; Adriano Ferrari; Giacomo Bruzzesi; Anto De Pol

    2012-01-01

    Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachmen...

  7. Isotropic 3D nuclear morphometry of normal, fibrocystic and malignant breast epithelial cells reveals new structural alterations.

    Vivek Nandakumar

    Full Text Available BACKGROUND: Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria. METHODOLOGY: We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure. PRINCIPAL FINDINGS: We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations. CONCLUSIONS: Our results provide a new perspective on nuclear

  8. Parallel 3D Finite Element Particle-in-Cell Simulations with Pic3P

    Candel, A.; Kabel, A.; Lee, L.; Li, Z.; Ng, C.; Schussman, G.; Ko, K.; /SLAC; Ben-Zvi, I.; Kewisch, J.; /Brookhaven

    2009-06-19

    SLAC's Advanced Computations Department (ACD) has developed the parallel 3D Finite Element electromagnetic Particle-In-Cell code Pic3P. Designed for simulations of beam-cavity interactions dominated by space charge effects, Pic3P solves the complete set of Maxwell-Lorentz equations self-consistently and includes space-charge, retardation and boundary effects from first principles. Higher-order Finite Element methods with adaptive refinement on conformal unstructured meshes lead to highly efficient use of computational resources. Massively parallel processing with dynamic load balancing enables large-scale modeling of photoinjectors with unprecedented accuracy, aiding the design and operation of next-generation accelerator facilities. Applications include the LCLS RF gun and the BNL polarized SRF gun.

  9. Atypical pyramidal cells in epileptic human cortex: CFLS and 3-D reconstructions.

    Belichencko, P; Dahlström, A; von Essen, C; Lindström, S; Nordborg, C; Sourander, P

    1992-09-01

    Epileptic temporal cortices, removed from 3 patients with intractable partial epilepsy (IPE) during neurosurgery, were studied. Pyramidal neurons (40-50 per slice) in laminae III, V and white matter, were injected with lucifer yellow. Samples were examined in a confocal laser scanning microscope (Biorad 600) and individual cells scanned at 0.1-1 microns incremental levels. 2-D maximal linear projection was used for overview. Frames (50-60) of scanned neurons were transformed into 3-D volumes, using VoxelView software on a Silicone Graphics workstation and rotated. All samples contained neurons with duplicated apical dendrites, additional basal dendrites or were misplaced in a horizontal position in the white matter. The relation between these preliminary observations and the disease is discussed. PMID:1421134

  10. Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model

    Gagliano, Nicoletta; Celesti, Giuseppe; Tacchini, Lorenza; Pluchino, Stefano; Sforza, Chiarella; Rasile, Marco; Valerio, Vincenza; Laghi, Luigi; Conte, Vincenzo; Procacci, Patrizia

    2016-01-01

    AIM: To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. METHODS: HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, β-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS: The E-cadherin/β-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/β-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression. CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids. PMID:27182158

  11. Novel carbocyclic curcumin analog CUR3d modulates genes involved in multiple apoptosis pathways in human hepatocellular carcinoma cells.

    Bhullar, Khushwant S; Jha, Amitabh; Rupasinghe, H P Vasantha

    2015-12-01

    Anticancer activity of a novel curcumin analog (E)-2-(4-hydroxy-3-methoxybenzylidene)-5-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)cyclopentanone (CUR3d) was studied using a human hepatocellular carcinoma cell line (HepG2). The results showed that CUR3d completely inhibits the tumor cell proliferation in a dose- and time-dependent manner. CUR3d at 100 μmol/L activated the pro-apoptotic caspase-3 along with downregulation of anti-apoptotic BIRC5 and Bcl2. CUR3d treatment controlled the cancer cell growth by downregulating the expression of PI3K/Akt (Akt1, Akt2) pathway along with NF-κB. CUR3d down-regulated the members of epidermal growth receptor family (EGFR, ERBB3, ERBB2) and insulin like growth receptors (IGF1, IGF-1R, IGF2). This correlated with the downregulation of G-protein (RHOA, RHOB) and RAS (ATF2, HRAS, KRAS, NRAS) pathway signaling. CUR3d also arrested cell cycle via inhibition of CDK2, CDK4, CDK5, CDK9, MDM2, MDM4 and TERT genes. Cell cycle essential aurora kinases (AURKα, AURKβ) and polo-like kinases (PLK1, PLK2, PLK3) were also modulated by CUR3d. Topoisomerases (TOP2α, TOP2β), important factors in cancer cell immortality, as well as HIF-1α were downregulated following CUR3d treatment. The expression of protein kinase-C family (PRKC-A, PRKC-D, PRKC-E) was also attenuated by CUR3d. The downregulation of histone deacetylases (Class I, II, IV) and PARP I further strengthened the anticancer efficacy of CUR3d. Downregulation of carcinogenic cathepsins (CTSB, CTSD) and heat shock proteins exhibited CUR3d's potency as a potential immunological adjuvant. Finally, the non-toxic manifestation of CUR3d in healthy liver and lung cells along with downregulation of drug resistant gene ABCC1 further warrant need for advance investigations. PMID:26409325

  12. Proteomic analysis of mesenchymal stem cells from normal and deep carious dental pulp.

    Dandan Ma

    Full Text Available Dental pulp stem cells (DPSCs, precursor cells of odontoblasts, are ideal seed cells for tooth tissue engineering and regeneration. Our previous study has demonstrated that stem cells exist in dental pulp with deep caries and are called carious dental pulp stem cells (CDPSCs. The results indicated that CDPSCs had a higher proliferative and stronger osteogenic differentiation potential than DPSCs. However, the molecular mechanisms responsible for the biological differences between DPSCs and CDPSCs are poorly understood. The aim of this study was to define the molecular features of DPSCs and CDPSCs by comparing the proteomic profiles using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS. Our results revealed that there were 18 protein spots differentially expressed between DPSCs and CDPSCs in a narrow pH range of 4 to 7. These differently expressed proteins are mostly involved in the regulation of cell proliferation, differentiation, cell cytoskeleton and motility. In addition, our results suggested that CDPSCs had a higher expression of antioxidative proteins that might protect CDPSCs from oxidative stress. This study explores some potential proteins responsible for the biological differences between DPSCs and CDPSCs and expands our understanding on the molecular mechanisms of mineralization of DPSCs in the formation of the dentin-pulp complex.

  13. Antibody-free isolation of rare cancer cells from blood based on 3D lateral dielectrophoresis.

    Cheng, I-Fang; Huang, Wei-Lun; Chen, Tzu-Ying; Liu, Chien-Wei; Lin, Yu-De; Su, Wu-Chou

    2015-07-21

    We present an antibody-free approach for the high-purity and high-throughput dielectrophoretic (DEP) isolation of circulating tumour cells (CTCs) from blood in a microfluidic chip. A hydrodynamic sheath flow is designed upstream in the chip to direct the suspension samples to the channel side walls, thus providing a queue to allow DEP-induced lateral displacements. High-throughput continuous cancer cell sorting (maximum flow rate: ~2.4 mL h(-1), linear velocity: ~4 mm s(-1)) is achieved with a sustained 3D lateral DEP (LDEP) particle force normal to the continuous through-flow. This design allows the continuous fractionation of micro/nanosized particles into different downstream subchannels based on the differences in their different critical negative DEP strengths/mobilities. The main advantage of this separation strategy is that increasing the channel length can effectively increase the throughput proportionally. The effective separation of rare cancer cells (<0.001%) from diluted human blood in a handheld chip is demonstrated. An enrichment factor of 10(5) and a recovery rate of ~85% from a 0.001% cancer cell sample are achieved at an optimal flow rate of 20 μL min(-1) passing through a 6 cm long LDEP channel with an appropriate voltage at a frequency of 10 kHz. A higher throughput of 2.4 mL h(-1) is also achieved with a 13 cm long metal-based microchannel. PMID:26085231

  14. 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.

  15. Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways.

    Hyung-Mun Yun

    Full Text Available Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs and polycaprolactone (PCL, and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by in vitro wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin, and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, β1 and β3 and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering.

  16. Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways

    Kim, Mi-joo; Kim, Jung-Ju; Lee, Jung-Hwan; Lee, Hae-Hyoung; Park, Kyung-Ran; Yi, Jin-Kyu; Kim, Hae-Won; Kim, Eun-cheol

    2015-01-01

    Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs) and polycaprolactone (PCL), and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs) were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt) supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by in vitro wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin), and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, β1 and β3) and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering. PMID:26382272

  17. 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/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. PMID:27597635

  18. 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.

  19. Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate hepatic-like differentiation.

    Chen, Y K; Huang, Anderson H C; Chan, Anthony W S; Lin, L M

    2016-06-01

    Reviewing the literature, hepatic differentiation of human dental pulp stem cells (hDPSCs) from cryopreserved dental pulp tissues of vital extracted teeth with disease has not been studied. This study is aimed to evaluate the hypothesis that hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease could possess potential hepatic differentiation. Forty vital extracted teeth with disease recruited for hDPSCs isolation, stem cell characterization and hepatic differentiation were randomly and equally divided into group A (liquid nitrogen-stored dental pulp tissues) and group B (freshly derived dental pulp tissues). Samples of hDPSCs isolated from groups A and B but without hepatic growth factors formed negative controls. A well-differentiated hepatocellular carcinoma cell line was employed as a positive control. All the isolated hDPSCs from groups A and B showed hepatic-like differentiation with morphological change from a spindle-shaped to a polygonal shape and normal karyotype. Differentiated hDPSCs and the positive control expressed hepatic metabolic function genes and liver-specific genes. Glycogen storage of differentiated hDPSCs was noted from day 7 of differentiation-medium culture. Positive immunofluorescence staining of low-density lipoprotein and albumin was observed from day 14 of differentiation-medium culture; urea production in the medium was noted from week 6. No hepatic differentiation was observed for any of the samples of the negative controls. We not only demonstrated the feasibility of hepatic-like differentiation of hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease but also indicated that the differentiated cells possessed normal karyotype and were functionally close to normal hepatic-like cells. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23950016

  20. 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.

  1. Improving organic tandem solar cells based on water-processed nanoparticles by quantitative 3D nanoimaging.

    Pedersen, E B L; Angmo, D; Dam, H F; Thydén, K T S; Andersen, T R; Skjønsfjell, E T B; Krebs, F C; Holler, M; Diaz, A; Guizar-Sicairos, M; Breiby, D W; Andreasen, J W

    2015-08-28

    Organic solar cells have great potential for upscaling due to roll-to-roll processing and a low energy payback time, making them an attractive sustainable energy source for the future. Active layers coated with water-dispersible Landfester particles enable greater control of the layer formation 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 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 bottom interfaces, wide thickness distribution and only partial surface coverage causing electric short circuits through the layer. By top coating a polymer material onto the Landfester nanoparticles we eliminate the structural defects of the layer such as porosity and roughness, and achieve the increased performance larger than 1 V expected for a tandem cell. This study highlights that quantitative imaging of weakly scattering stacked layers of organic materials has become feasible by PXCT, and that this information cannot be obtained by other methods. In the present study, this technique specifically reveals the need to improve the coatability and layer formation of Landfester nanoparticles, thus allowing improved solar cells to be produced. PMID:26220159

  2. X-ray tomography: Biological cells in 3-D at better than 50 nm resolution

    Full text: X-ray microscopy can be used to image whole, hydrated, specimens with a spatial resolution 5-10 times better than that obtained using visible light microscopy. X-ray imaging at photon energies below the K- absorption edge of oxygen, referred to as the water window, exploits the strong natural contrast for organic material embedded in a mostly water matrix. With a transmission X-ray microscope using Fresnel zone plate optics, specimens up to 10 microns thick can be examined. The highest X-ray transmission in hydrated samples is obtained at a wavelength of 2.4 nm but, due to the low numerical aperture of zone plate lenses operated in st order diffraction mode the structures resolved are much larger than the X-ray wavelength. Because of the low NA of X-ray lenses (NA=0.05), combined with the effect of polychromatic illumination and a wavelength dependant focal length, the effective depth of ld is large (6-10 microns). The experiments presented here were performed at the Advanced Light Source using the full ld transmission X-ray microscope, XM-1. This microscope employs a bend magnet X-ray source and zone plate condenser and objective lenses. The condenser zone plate acts as a monochromator and the X-ray images are recorded directly on a cooled, back-thinned 1024x1024 pixel CCD camera. The sample holder was a rotationally symmetric glass tube; the region containing the sample was 10 microns in diameter with a wall thickness of 200 nm. Live yeast cells were loaded into the tube, rapidly frozen by a blast of liquid nitrogen-cooled helium gas, and maintained at 140 deg C by a steady flow of cold helium. The image sequence spanned 180 deg and consisted of 45 images spaced by 4 deg. The images were aligned to a common axis and computed tomographic reconstruction was used to obtain the 3-D X-ray linear absorption coefficient. Volume rendering and animation of reconstructed data was performed using the 3-D program, Amira. Acquisition time for 90 images was 3 min

  3. Genome-wide transcriptomic alterations induced by ethanol treatment in human dental pulp stem cells (DPSCs).

    Khalid, Omar; Kim, Jeffrey J; Duan, Lewei; Hoang, Michael; Elashoff, David; Kim, Yong

    2014-12-01

    Human dental pulp stem cells (DPSCs) isolated from adult dental pulp are multipotent mesenchymal stem cells that can be directed to differentiate into osteogenic/odontogenic cells and also trans-differentiate into neuronal cells. The utility of DPSC has been explored in odontogenic differentiation for tooth regeneration. Alcohol abuse appears to lead to periodontal disease, tooth decay and mouth sores that are potentially precancerous. Persons who abuse alcohol are at high risk of having seriously deteriorated teeth, gums and compromised oral health in general. It is currently unknown if alcohol exposure has any impact on adult stem cell maintenance, stem cell fate determination and plasticity, and stem cell niche environment. Here we provide detailed experimental methods, analysis and information associated with our data deposited into Gene Expression Omnibus (GEO) under GSE57255. Our data provide transcriptomic changes that are occurring by EtOH treatment of DPSCs at 24-hour and 48-hour time point. PMID:25045622

  4. Genome-wide transcriptomic alterations induced by ethanol treatment in human dental pulp stem cells (DPSCs

    Omar Khalid

    2014-12-01

    Full Text Available Human dental pulp stem cells (DPSCs isolated from adult dental pulp are multipotent mesenchymal stem cells that can be directed to differentiate into osteogenic/odontogenic cells and also trans-differentiate into neuronal cells. The utility of DPSC has been explored in odontogenic differentiation for tooth regeneration. Alcohol abuse appears to lead to periodontal disease, tooth decay and mouth sores that are potentially precancerous. Persons who abuse alcohol are at high risk of having seriously deteriorated teeth, gums and compromised oral health in general. It is currently unknown if alcohol exposure has any impact on adult stem cell maintenance, stem cell fate determination and plasticity, and stem cell niche environment. Here we provide detailed experimental methods, analysis and information associated with our data deposited into Gene Expression Omnibus (GEO under GSE57255. Our data provide transcriptomic changes that are occurring by EtOH treatment of DPSCs at 24-hour and 48-hour time point.

  5. Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

    Preethy SP

    2010-01-01

    Full Text Available BACKGROUND: Recent studies have shown that mesenchymal stem cells isolated from post natal human dental pulp, (Dental pulp stem cells-DPSCs which is from permanent teeth and SHED (stem cells from human exfoliated deciduous teeth,the Periodontal ligament stem cells (PDLSC and Stem cells from root Apical papilla(SCAPhave the potential to differentiate into cells of a variety of tissues including heart, muscle, cartilage, bone, nerve, salivary glands, teeth etc(1,2,3,4.This multipotential ability of DPSCs is being researched for clinical application for treating a variety of diseases like myocardial infarction, muscular dystrophy, neuro-degenerative disorders, cartilage replacement, tooth regeneration and for repair of bone defects to mention a few. Moreover, the isolation of stem cells from teeth is minimally invasive, readily accessible and the non immunogenic characteristic of dental stem cells has paved the way for efforts to store the exfoliated deciduous teeth or milk teeth which is usually discarded, for use in the future. In this study we have isolated and expanded in vitro, the cells obtained from human dental pulp. MATERIALS AND METHODS: After obtaining written informed consent, 24 teeth that were extracted for therapeutic or cosmetic reasons from 16 patients were used in this study. The specimens were transported from the clinic to NCRM lab taking 6 to 48 Hrs. For removal of the pulp tissue, the teeth were split obliquely at the Cementoenamel junction and the pulp tissue was isolated using brooches. The extracted pulp tissues were subjected to digestion using Collagenase type-I and type II at 37˚C for 15- 30 minutes. The digested cells were filtered with 70µm filter and centrifuged at 1800 rpm for 10 minutes. The pellet was then suspended in Dulbecco’s modified Eagle’s medium (DMEM/Ham’s F12 supplemented with 15% fetal bovine serum , 100 U/ml penicillin, 100 µg/ml streptomycin,2 m M L -glutamine, and 2 m M nonessential amino

  6. Bioengineering a 3D integumentary organ system from iPS cells using an in vivo transplantation model.

    Takagi, Ryoji; Ishimaru, Junko; Sugawara, Ayaka; Toyoshima, Koh-Ei; Ishida, Kentaro; Ogawa, Miho; Sakakibara, Kei; Asakawa, Kyosuke; Kashiwakura, Akitoshi; Oshima, Masamitsu; Minamide, Ryohei; Sato, Akio; Yoshitake, Toshihiro; Takeda, Akira; Egusa, Hiroshi; Tsuji, Takashi

    2016-04-01

    The integumentary organ system is a complex system that plays important roles in waterproofing, cushioning, protecting deeper tissues, excreting waste, and thermoregulation. We developed a novel in vivo transplantation model designated as a clustering-dependent embryoid body transplantation method and generated a bioengineered three-dimensional (3D) integumentary organ system, including appendage organs such as hair follicles and sebaceous glands, from induced pluripotent stem cells. This bioengineered 3D integumentary organ system was fully functional following transplantation into nude mice and could be properly connected to surrounding host tissues, such as the epidermis, arrector pili muscles, and nerve fibers, without tumorigenesis. The bioengineered hair follicles in the 3D integumentary organ system also showed proper hair eruption and hair cycles, including the rearrangement of follicular stem cells and their niches. Potential applications of the 3D integumentary organ system include an in vitro assay system, an animal model alternative, and a bioengineered organ replacement therapy. PMID:27051874

  7. Pin cell discontinuity factors in the transient 3-D discrete ordinates code TORT-TD

    Even with the rapid increase of computing power, whole core transient and accident analyses based on the direct solution of the 3-D neutron transport equation with a large number of energy groups and a detailed heterogeneous description of the core still remain computationally challenging. Current industrial methods for reactor core calculations therefore involve a two step approach in which lattice (assembly) depletion transport methods are used to prepare energy collapsed and fuel assembly or pin cell homogenized cross sections for subsequent whole core transport calculations. Spatial homogenization, in principal, requires the knowledge of both the actual boundary condition (local core environment) of the fuel assembly and the exact heterogeneous flux distribution (reference solution) of the whole core problem within that fuel assembly. Since, in particular, the latter is not known a priori, an infinite medium (zero net current) condition is used in the lattice calculations. It is well known that this approximation may lead to undesirable errors in cores in which large flux gradients are present across the fuel assemblies. This is the case in cores that have high heterogeneity and/or strong local absorbers, e.g. PWRs with partial MOX loading and inserted control rod clusters. There are two major approaches to mitigate spatial homogenization errors, superhomogenization (SPH) factors, and discontinuity factors within the scope of equivalence theory (ET) and generalized equivalence theory (GET). Although discontinuity factors are usually applied at the level of fuel assembly node size (assembly discontinuity factors, ADF), the methodology can be extended to pin cell homogenized whole core calculations involving pin cell discontinuity factors (PDF). There are also further developments for both the diffusion and the simplified transport (SP3) equation. In this paper, PDFs are introduced into the time-dependent 3-D discrete ordinates code TORT-TD in order to reduce the

  8. 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'.

  9. Tuning Cell Differentiation into a 3D Scaffold Presenting a Pore Shape Gradient for Osteochondral Regeneration.

    Di Luca, Andrea; Lorenzo-Moldero, Ivan; Mota, Carlos; Lepedda, Antonio; Auhl, Dietmar; Van Blitterswijk, Clemens; Moroni, Lorenzo

    2016-07-01

    Osteochondral regeneration remains nowadays a major problem since the outcome of current techniques is not satisfactory in terms of functional tissue formation and development. A possible solution is the combination of human mesenchymal stem cells (hMSCs) with additive manufacturing technologies to fabricate scaffolds with instructive properties. In this study, the differentiation of hMSCs within a scaffold presenting a gradient in pore shape is presented. The variation in pore shape is determined by varying the angle formed by the fibers of two consequent layers. The fiber deposition patterns are 0-90, which generate squared pores, 0-45, 0-30, and 0-15, that generate rhomboidal pores with an increasing major axis as the deposition angle decreases. Within the gradient construct, squared pores support a better chondrogenic differentiation whereas cells residing in the rhomboidal pores display a better osteogenic differentiation. When cultured under osteochondral conditions the trend in both osteogenic and chondrogenic markers is maintained. Engineering the pore shape, thus creating axial gradients in structural properties, seems to be an instructive strategy to fabricate functional 3D scaffolds that are able to influence hMSCs differentiation for osteochondral tissue regeneration. PMID:27109461

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

    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'

  11. Imperative role of dental pulp stem cells in regenerative therapies: A systematic review

    Ramchandra Kabir

    2014-01-01

    Full Text Available Stem cells are primitive cells that can differentiate and regenerate organs in different parts of the body such as heart, bones, muscles and nervous system. This has been a field of great clinical interest with immense possibilities of using the stem cells in regeneration of human organ those are damaged due to disease, developmental defects and accident. The knowledge of stem cell technology is increasing quickly in all medical specialties and in dental field too. Stem cells of dental origin appears to hold the key to various cell-based therapies in regenerative medicine, but most avenues are in experimental stages and many procedures are undergoing standardization and validation. Long-term preservation of SHED cells or DPSC is becoming a popular consideration, similar to the banking of umbilical cord blood. Dental pulp stem cells (DPSCs are the adult multipotent cells that reside in the cell rich zone of the dental pulp. The multipotent nature of these DPSCs may be utilized in both dental and medical applications. A systematic review of the literature was performed using various internet based search engines (PubMed, Medline Plus, Cochrane, Medknow, Ebsco, Science Direct, Hinari, WebMD, IndMed, Embase using keywords like "dental pulp stem cells", "regeneration", "medical applications", "tissue engineering". DPSCs appears to be a promising innovation for the re-growth of tissues however, long term clinical studies need to be carried out that could establish some authentic guidelines in this perspective.

  12. Functional 3D Neural Mini-Tissues from Printed Gel-Based Bioink and Human Neural Stem Cells.

    Gu, Qi; Tomaskovic-Crook, Eva; Lozano, Rodrigo; Chen, Yu; Kapsa, Robert M; Zhou, Qi; Wallace, Gordon G; Crook, Jeremy M

    2016-06-01

    Direct-write printing of stem cells within biomaterials presents an opportunity to engineer tissue for in vitro modeling and regenerative medicine. Here, a first example of constructing neural tissue by printing human neural stem cells that are differentiated in situ to functional neurons and supporting neuroglia is reported. The supporting biomaterial incorporates a novel clinically relevant polysaccharide-based bioink comprising alginate, carboxymethyl-chitosan, and agarose. The printed bioink rapidly gels by stable cross-linking to form a porous 3D scaffold encapsulating stem cells for in situ expansion and differentiation. Differentiated neurons form synaptic contacts, establish networks, are spontaneously active, show a bicuculline-induced increased calcium response, and are predominantly gamma-aminobutyric acid expressing. The 3D tissues will facilitate investigation of human neural development, function, and disease, and may be adaptable for engineering other 3D tissues from different stem cell types. PMID:27028356

  13. 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.

  14. Analyzing Biological Performance of 3D-Printed, Cell-Impregnated Hybrid Constructs for Cartilage Tissue Engineering.

    Izadifar, Zohreh; Chang, Tuanjie; Kulyk, William; Chen, Xiongbiao; Eames, B Frank

    2016-03-01

    Three-dimensional (3D) bioprinting of hybrid constructs is a promising biofabrication method for cartilage tissue engineering because a synthetic polymer framework and cell-impregnated hydrogel provide structural and biological features of cartilage, respectively. During bioprinting, impregnated cells may be subjected to high temperatures (caused by the adjacent melted polymer) and process-induced mechanical forces, potentially compromising cell function. This study addresses these biofabrication issues, evaluating the heat distribution of printed polycaprolactone (PCL) strands and the rheological property and structural stability of alginate hydrogels at various temperatures and concentrations. The biocompatibility of parameters from these studies was tested by culturing 3D hybrid constructs bioprinted with primary cells from embryonic chick cartilage. During initial two-dimensional culture expansion of these primary cells, two morphologically and molecularly distinct cell populations ("rounded" and "fibroblastic") were isolated. The biological performance of each population was evaluated in 3D hybrid constructs separately. The cell viability, proliferation, and cartilage differentiation were observed at high levels in hybrid constructs of both cell populations, confirming the validity of these 3D bioprinting parameters for effective cartilage tissue engineering. Statistically significant performance variations were observed, however, between the rounded and fibroblastic cell populations. Molecular and morphological data support the notion that such performance differences may be attributed to the relative differentiation state of rounded versus fibroblastic cells (i.e., differentiated chondrocytes vs. chondroprogenitors, respectively), which is a relevant issue for cell-based tissue engineering strategies. Taken together, our study demonstrates that bioprinting 3D hybrid constructs of PCL and cell-impregnated alginate hydrogel is a promising approach for

  15. DNA degradation within mouse brain and dental pulp cells 72 hours postmortem

    Jilong Zheng; Xiaona Li; Di Shan; Han Zhang; Dawei Guan

    2012-01-01

    In this study, we sought to elucidate the process of DNA degradation in brain and dental pulp cells of mice, within postmortem 0-72 hours, by using the single cell gel electrophoresis assay and professional comet image analysis and processing techniques. The frequency of comet-like cells, the percentage of tail DNA, tail length, tail moment, Olive moment and tail area increased in tandem with increasing postmortem interval. In contrast, the head radius, the percentage of head DNA and head area showed a decreasing trend. Linear regression analysis revealed a high correlation between these parameters and the postmortem interval. The findings suggest that the single cell gel electrophoresis assay is a quick and sensitive method to detect DNA degradation in brain and dental pulp cells, providing an objective and accurate new way to estimate postmortem interval.

  16. Dental pulp stem cells (DPSCs) differentiation study by confocal Raman microscopy

    Salehi, H.; Collart-Dutilleul, P.-Y.; Gergely, C.; Cuisinier, F. J. G.

    2014-03-01

    Regenerative medicine brings a huge application for Mesenchymal stem cells such as Dental Pulp Stem Cells (DPSCs). Confocal Raman microscopy, a non-invasive, label free , real time and high spatial resolution imaging technique is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800-3000 cm-1 region (C-H stretching) and 960 cm-1 peak (phosphate PO4 3-) were collected. In Dental Pulp Stem Cells 21st day differentiated in buffer solution, phosphate peaks ν1 PO4 3- (first vibrational mode) at 960cm-1 and ν2 PO4 3- at 430cm-1 and ν4 PO4 3- at 585cm-1 are obviously present. Confocal Raman microscopy enables the detection of cell differentiation and it can be used to investigate clinical stem cell research.

  17. Surface Acoustic Waves (SAW-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures

    Tao Wang

    2015-12-01

    Full Text Available Detection and quantification of cell viability and growth in two-dimensional (2D and three-dimensional (3D cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose–response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in

  18. Cast and 3D printed ion exchange membranes for monolithic microbial fuel cell fabrication

    Philamore, Hemma; Rossiter, Jonathan; Walters, Peter; Winfield, Jonathan; Ieropoulos, Ioannis

    2015-09-01

    We present novel solutions to a key challenge in microbial fuel cell (MFC) technology; greater power density through increased relative surface area of the ion exchange membrane that separates the anode and cathode electrodes. The first use of a 3D printed polymer and a cast latex membrane are compared to a conventionally used cation exchange membrane. These new techniques significantly expand the geometric versatility available to ion exchange membranes in MFCs, which may be instrumental in answering challenges in the design of MFCs including miniaturisation, cost and ease of fabrication. Under electrical load conditions selected for optimal power transfer, peak power production (mean 10 batch feeds) was 11.39 μW (CEM), 10.51 μW (latex) and 0.92 μW (Tangoplus). Change in conductivity and pH of anolyte were correlated with MFC power production. Digital and environmental scanning electron microscopy show structural changes to and biological precipitation on membrane materials following long term use in an MFC. The cost of the novel membranes was lower than the conventional CEM. The efficacy of two novel membranes for ion exchange indicates that further characterisation of these materials and their fabrication techniques, shows great potential to significantly increase the range and type of MFCs that can be produced.

  19. Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells

    Positioning of genes relative to nuclear heterochromatic compartments is thought to help regulate their transcriptional activity. Given that human subtelomeric regions are rich in highly expressed genes, we asked whether human telomeres are related to transcription-permissive nuclear compartments. To address this question, we investigated in the nuclei of normal human lymphocytes the spatial relations of two constitutively expressed genes (ACTB and RARA) and three nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity and transcription levels. We observed that genes and gene transcripts locate close to telomere clusters and away from chromocenters upon activation of transcription. These findings, together with the observation that SC35 domains, which are enriched in pre-mRNA processing factors, are in close proximity to telomeres, indicate that telomere-neighboring regions are permissive to gene expression in human cells. Therefore, the associations of telomeres observed in the interphase nucleus might contribute, as opposed to chromocenters, for the establishment of transcription-permissive 3D nuclear compartments

  20. Digital holography as a method for 3D imaging and estimating the biovolume of motile cells.

    Merola, F; Miccio, L; Memmolo, P; Di Caprio, G; Galli, A; Puglisi, R; Balduzzi, D; Coppola, G; Netti, P; Ferraro, P

    2013-12-01

    Sperm morphology is regarded as a significant prognostic factor for fertilization, as abnormal sperm structure is one of the most common factors in male infertility. Furthermore, obtaining accurate morphological information is an important issue with strong implications in zoo-technical industries, for example to perform sorting of species X from species Y. A challenging step forward would be the availability of a fast, high-throughput and label-free system for the measurement of physical parameters and visualization of the 3D shape of such biological specimens. Here we show a quantitative imaging approach to estimate simply and quickly the biovolume of sperm cells, combining the optical tweezers technique with digital holography, in a single and integrated set-up for a biotechnology assay process on the lab-on-a-chip scale. This approach can open the way for fast and high-throughput analysis in label-free microfluidic based "cytofluorimeters" and prognostic examination based on sperm morphology, thus allowing advancements in reproductive science. PMID:24129638

  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. Prospective use of the 3D printing technology for the microstructural engineering of Solid Oxide Fuel Cell components

    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)

  3. 3D cell cultures of human head and neck squamous cell carcinoma cells are radiosensitized by the focal adhesion kinase inhibitor TAE226

    Background and purpose: Focal adhesion kinase (FAK), a main player in integrin signaling and survival, is frequently overexpressed in human cancers and therefore postulated as potential target in cancer therapy. The aim of this study was to evaluate the radiosensitizing potential of the FAK inhibitor TAE226 in three-dimensional (3D) tumor cell cultures. Materials and methods: Head and neck squamous cell carcinoma (HNSCC) cells (FaDu, UT-SCC15, UT-SCC45), lung cancer cells (A549), colorectal carcinoma cells (DLD-1, HCT-116) and pancreatic tumor cells (MiaPaCa2, Panc1) were treated with different concentrations of TAE226 (0-1 μm; 1 or 24 h) without or in combination with irradiation (0-6 Gy, X-ray, single dose). Subsequently, 3D clonogenic survival assays (laminin-rich extracellular matrix) and Western blotting (expression/phosphorylation, e.g. FAK, Akt, ERK1/2) were performed. Results: All investigated 3D cell cultures showed a dose-dependent reduction in clonogenic survival by TAE226. Intriguingly, TAE226 only significantly radiosensitized 3D HNSCC cell cultures accompanied by a pronounced dephosphorylation of FAK, Akt and ERK1/2. Conclusions: Our data demonstrate TAE226 as potent FAK inhibitor that enhances the cellular radiosensitivity particularly of HNSCC cells grown in a 3D cell culture model. Future in vitro and in vivo investigations will clarify, to which extent this approach might be clinically relevant for radiotherapy of HNSCC.

  4. Immunogenicity and T cell recognition in swine of foot-and-mouth disease virus polymerase 3D

    Immunization of domestic pigs with a vaccinia virus (VV) recombinant expressing foot-and-mouth disease virus (FMDV) 3D protein conferred partial protection against challenge with infectious virus. The severity reduction of the clinical symptoms developed by the challenged animals occurred in the absence of significant levels of anti-3D circulating antibodies. This observation suggested that the partial protection observed was mediated by the induction of a 3D-specific cellular immune response. To gain information on the T cell recognition of FMDV 3D protein, we conducted in vitro proliferative assays using lymphocytes from outbred pigs experimentally infected with FMDV and 90 overlapping peptides spanning the complete 3D sequence. The use of pools of two to three peptides allowed the identification of T cell epitopes that were efficiently recognized by lymphocytes from at least four of the five animals analyzed. This recognition was heterotypic because anti-peptide responses increased upon reinfection of animals with a FMDV isolate from a different serotype. The results obtained with individual peptides confirmed the antigenicity observed with peptide pools. Detection of cytokine mRNAs by RT-PCR in lymphocytes stimulated in vitro by individual 3D peptides revealed that IFN-γ mRNA was the most consistently induced, suggesting that the activated T cells belong to the Th 1 subset. These results indicate that 3D protein contains epitopes that can be efficiently recognized by porcine T lymphocytes from different infected animals, both upon primary and secondary (heterotypic) FMDV infection. These epitopes can extend the repertoire of viral T cell epitopes to be included in subunit and synthetic FMD vaccines

  5. Differentiation potential of STRO-1+ dental pulp stem cells changes during cell passaging

    Wang Ruoning

    2010-05-01

    Full Text Available Abstract Background Dental pulp stem cells (DPSCs can be driven into odontoblast, osteoblast, and chondrocyte lineages in different inductive media. However, the differentiation potential of naive DPSCs after serial passaging in the routine culture system has not been fully elucidated. Results DPSCs were isolated from human/rat dental pulps by the magnetic activated cell sorting based on STRO-1 expression, cultured and passaged in the conventional culture media. The biological features of STRO-1+ DPSCs at the 1st and 9th passages were investigated. During the long-term passage, the proliferation ability of human STRO-1+ DPSCs was downregulated as indicated by the growth kinetics. When compared with STRO-1+ DPSCs at the 1st passage (DPSC-P1, the expression of mature osteoblast-specific genes/proteins (alkaline phosphatase, bone sialoprotein, osterix, and osteopontin, odontoblast-specific gene/protein (dentin sialophosphoprotein and dentin sialoprotein, and chondrocyte-specific gene/protein (type II collagen was significantly upregulated in human STRO-1+ DPSCs at the 9th passage (DPSC-P9. Furthermore, human DPSC-P9 cells in the mineralization-inducing media presented higher levels of alkaline phosphatase at day 3 and day 7 respectively, and produced more mineralized matrix than DPSC-P9 cells at day 14. In vivo transplantation results showed that rat DPSC-P1 cell pellets developed into dentin, bone and cartilage structures respectively, while DPSC-P9 cells can only generate bone tissues. Conclusions These findings suggest that STRO-1+ DPSCs consist of several interrelated subpopulations which can spontaneously differentiate into odontoblasts, osteoblasts, and chondrocytes. The differentiation capacity of these DPSCs changes during cell passaging, and DPSCs at the 9th passage restrict their differentiation potential to the osteoblast lineage in vivo.

  6. Human dental pulp stem cell is a promising autologous seed cell for bone tissue engineering

    LI Jing-hui; LIU Da-yong; ZHANG Fang-ming; WANG Fan; ZHANG Wen-kui; ZHANG Zhen-ting

    2011-01-01

    Background The seed cell is a core problem in bone tissue engineering research.Recent research indicates that human dental pulp stem cells (hDPSCs) can differentiate into osteoblasts in vitro,which suggests that they may become a new kind of seed cells for bone tissue engineering.The aim of this study was to evaluate the osteogenic differentiation of hDPSCs in vitro and bone-like tissue formation when transplanted with three-dimensional gelatin scaffolds in vivo,and hDPSCs may become appropriate seed cells for bone tissue engineering.Methods We have utilized enzymatic digestion to obtain hDPSCs from dental pulp tissue extracted during orthodontic treatment.After culturing and expansion to three passages,the cells were seeded in 6-well plates or on three-dimensional gelatin scaffolds and cultured in osteogenic medium.After 14 days in culture,the three-dimensional gelatin scaffolds were implanted subcutaneously in nude mice for 4 weeks.In 6-well plate culture,osteogenesis was assessed by alkaline phosphatase staining,Von Kossa staining,and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the osteogenesis-specific genes type I collagen (COL l),bone sialoprotein (BSP),osteocalcin (OCN),RUNX2,and osterix (OSX).In three-dimensional gelatin scaffold culture,X-rays,hematoxylin/eosin staining,and immunohistochemical staining were used to examine bone formation.Results In vitro studies revealed that hDPSCs do possess osteogenic differentiation potential.In vivo studies revealed that hDPSCs seeded on gelatin scaffolds can form bone structures in heterotopic sites of nude mice.Conclusions These findings suggested that hDPSCs may be valuable as seed cells for bone tissue engineering.As a special stem cell source,hDPSCs may blaze a new path for bone tissue engineering.

  7. Calcium Electroporation: Evidence for Differential Effects in Normal and Malignant Cell Lines, Evaluated in a 3D Spheroid Model

    Frandsen, Stine Krog; Gibot, Laure; Madi, Moinecha; Gehl, Julie; Rols, Marie-Pierre

    2015-01-01

    Background Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill efficacy–and normal cell sensitivity. Methods Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different h...

  8. Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates.

    Franken, Lars; Klein, Marika; Spasova, Marina; Elsukova, Anna; Wiedwald, Ulf; Welz, Meike; Knolle, Percy; Farle, Michael; Limmer, Andreas; Kurts, Christian

    2015-01-01

    A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcription factor SpiC, which lack red pulp macrophages, liver Kupffer cells take over the task of erythrocyte degradation and become superparamagnetic. We describe a simple additional magnetic separation step that avoids this problem and substantially improves purity of magnetic cell isolates from the spleen. PMID:26260698

  9. Culture medium modulates the behaviour of human dental pulp-derived cells: Technical Note

    S Lopez-Cazaux

    2006-02-01

    Full Text Available In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8mM Ca and 1mM Pi and RPMI 1640 (0.8mM Ca and 5mM Pi on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of -smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

  10. Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids.

    Beaumont, Kimberley A; Anfosso, Andrea; Ahmed, Farzana; Weninger, Wolfgang; Haass, Nikolas K

    2015-01-01

    Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions. PMID:26779761