Development of 3d micro-nano hybrid patterns using anodized aluminum and micro-indentation

International Nuclear Information System (INIS)

Shin, Hong Gue; Kwon, Jong Tae; Seo, Young Ho; Kim, Byeong Hee

2008-01-01

We developed a simple and cost-effective method of fabricating 3D micro-nano hybrid patterns in which micro-indentation is applied on the anodized aluminum substrate. Nano-patterns were formed first on the aluminum substrate, and then micro-patterns were fabricated by deforming the nano-patterned aluminum substrate. Hemispherical nano-patterns with a 150 nm-diameter on an aluminum substrate were fabricated by anodizing and alumina removing process. Then, micro-pyramid patterns with a side-length of 50 μm were formed on the nano-patterns using micro-indentation. To verify 3D micro-nano hybrid patterns, we replicated 3D micro-nano hybrid patterns by a hot-embossing process. 3D micro-nano hybrid patterns may be used in nano-photonic devices and nano-biochips applications

Development of 3d micro-nano hybrid patterns using anodized aluminum and micro-indentation

Energy Technology Data Exchange (ETDEWEB)

Shin, Hong Gue; Kwon, Jong Tae [Division of Mechanical Engineering and Mechatronics, Kangwon National University, 1 Kangwondaehakgil, Chunchon, Gangwon-do, 200-701 (Korea, Republic of); Seo, Young Ho [Division of Mechanical Engineering and Mechatronics, Kangwon National University, 1 Kangwondaehakgil, Chunchon, Gangwon-do, 200-701 (Korea, Republic of)], E-mail: mems@kangwon.ac.kr; Kim, Byeong Hee [Division of Mechanical Engineering and Mechatronics, Kangwon National University, 1 Kangwondaehakgil, Chunchon, Gangwon-do, 200-701 (Korea, Republic of)

2008-07-31

We developed a simple and cost-effective method of fabricating 3D micro-nano hybrid patterns in which micro-indentation is applied on the anodized aluminum substrate. Nano-patterns were formed first on the aluminum substrate, and then micro-patterns were fabricated by deforming the nano-patterned aluminum substrate. Hemispherical nano-patterns with a 150 nm-diameter on an aluminum substrate were fabricated by anodizing and alumina removing process. Then, micro-pyramid patterns with a side-length of 50 {mu}m were formed on the nano-patterns using micro-indentation. To verify 3D micro-nano hybrid patterns, we replicated 3D micro-nano hybrid patterns by a hot-embossing process. 3D micro-nano hybrid patterns may be used in nano-photonic devices and nano-biochips applications.

3D tomography of cells in micro-channels

Science.gov (United States)

Quint, S.; Christ, A. F.; Guckenberger, A.; Himbert, S.; Kaestner, L.; Gekle, S.; Wagner, C.

2017-09-01

We combine confocal imaging, microfluidics, and image analysis to record 3D-images of cells in flow. This enables us to recover the full 3D representation of several hundred living cells per minute. Whereas 3D confocal imaging has thus far been limited to steady specimens, we overcome this restriction and present a method to access the 3D shape of moving objects. The key of our principle is a tilted arrangement of the micro-channel with respect to the focal plane of the microscope. This forces cells to traverse the focal plane in an inclined manner. As a consequence, individual layers of passing cells are recorded, which can then be assembled to obtain the volumetric representation. The full 3D information allows for a detailed comparison with theoretical and numerical predictions unfeasible with, e.g., 2D imaging. Our technique is exemplified by studying flowing red blood cells in a micro-channel reflecting the conditions prevailing in the microvasculature. We observe two very different types of shapes: "croissants" and "slippers." Additionally, we perform 3D numerical simulations of our experiment to confirm the observations. Since 3D confocal imaging of cells in flow has not yet been realized, we see high potential in the field of flow cytometry where cell classification thus far mostly relies on 1D scattering and fluorescence signals.

3D physical modeling for patterning process development

Science.gov (United States)

Sarma, Chandra; Abdo, Amr; Bailey, Todd; Conley, Will; Dunn, Derren; Marokkey, Sajan; Talbi, Mohamed

2010-03-01

In this paper we will demonstrate how a 3D physical patterning model can act as a forensic tool for OPC and ground-rule development. We discuss examples where the 2D modeling shows no issues in printing gate lines but 3D modeling shows severe resist loss in the middle. In absence of corrective measure, there is a high likelihood of line discontinuity post etch. Such early insight into process limitations of prospective ground rules can be invaluable for early technology development. We will also demonstrate how the root cause of broken poly-line after etch could be traced to resist necking in the region of STI step with the help of 3D models. We discuss different cases of metal and contact layouts where 3D modeling gives an early insight in to technology limitations. In addition such a 3D physical model could be used for early resist evaluation and selection for required ground-rule challenges, which can substantially reduce the cycle time for process development.

Comparison of two structured illumination techniques based on different 3D illumination patterns

Science.gov (United States)

Shabani, H.; Patwary, N.; Doblas, A.; Saavedra, G.; Preza, C.

2017-02-01

Manipulating the excitation pattern in optical microscopy has led to several super-resolution techniques. Among different patterns, the lateral sinusoidal excitation was used for the first demonstration of structured illumination microscopy (SIM), which provides the fastest SIM acquisition system (based on the number of raw images required) compared to the multi-spot illumination approach. Moreover, 3D patterns that include lateral and axial variations in the illumination have attracted more attention recently as they address resolution enhancement in three dimensions. A threewave (3W) interference technique based on coherent illumination has already been shown to provide super-resolution and optical sectioning in 3D-SIM. In this paper, we investigate a novel tunable technique that creates a 3D pattern from a set of multiple incoherently illuminated parallel slits that act as light sources for a Fresnel biprism. This setup is able to modulate the illumination pattern in the object space both axially and laterally with adjustable modulation frequencies. The 3D forward model for the new system is developed here to consider the effect of the axial modulation due to the 3D patterned illumination. The performance of 3D-SIM based on 3W interference and the tunable system are investigated in simulation and compared based on two different criteria. First, restored images obtained for both 3D-SIM systems using a generalized Wiener filter are compared to determine the effect of the illumination pattern on the reconstruction. Second, the effective frequency response of both systems is studied to determine the axial and lateral resolution enhancement that is obtained in each case.

3D Cell Culture in Alginate Hydrogels

Directory of Open Access Journals (Sweden)

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.

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

International Nuclear Information System (INIS)

Gruene, M; Deiwick, A; Koch, L; Schlie, S; Unger, C; Chichkov, B N; Pflaum, M; Wilhelmi, M; Haverich, A

2011-01-01

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.

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

Energy Technology Data Exchange (ETDEWEB)

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.

3D capillary valves for versatile capillary patterning of channel walls

NARCIS (Netherlands)

Papadimitriou, Vasileios; van den Berg, Albert; Eijkel, Jan C.T.

2016-01-01

We demonstrate passive capillary patterning of channel walls with a liquid in situ. Patterning is performed using a novel 3D capillary valve system combining three standard capillary stop valves. A range of different patterns is demonstrated in three channel walls. Capillary patterning was designed

3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.

Science.gov (United States)

Matsusaki, Michiya; Hikimoto, Daichi; Nishiguchi, Akihiro; Kadowaki, Koji; Ohura, Kayoko; Imai, Teruko; Akashi, Mitsuru

2015-02-13

Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Definition of new 3D invariants. Applications to pattern recognition problems with neural networks

International Nuclear Information System (INIS)

Proriol, J.

1996-01-01

We propose a definition of new 3D invariants. Usual pattern recognition methods use 2D descriptions of 3D objects, we propose a 2D approximation of the defined 3D invariants which can be used with neural networks to solve pattern recognition problems. We describe some methods to use the 2 D approximants. This work is an extension of previous 3D invariants used to solve some high energy physics problems. (author)

Laser printing of cells into 3D scaffolds

International Nuclear Information System (INIS)

Ovsianikov, A; Gruene, M; Koch, L; Maiorana, F; Chichkov, B; Pflaum, M; Wilhelmi, M; Haverich, A

2010-01-01

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.

3D NAND Flash Based on Planar Cells

Directory of Open Access Journals (Sweden)

Andrea Silvagni

2017-10-01

Full Text Available In this article, the transition from 2D NAND to 3D NAND is first addressed, and the various 3D NAND architectures are compared. The article carries out a comparison of 3D NAND architectures that are based on a “punch-and-plug” process—with gate-all-around (GAA cell devices—against architectures that are based on planar cell devices. The differences and similarities between the two classes of architectures are highlighted. The differences between architectures using floating-gate (FG and charge-trap (CT devices are also considered. Although the current production of 3D NAND is based on GAA cell devices, it is suggested that architectures with planar cell devices could also be viable for mass production.

Unit cell geometry of 3-D braided structures

Science.gov (United States)

Du, Guang-Wu; Ko, Frank K.

1993-01-01

The traditional approach used in modeling of composites reinforced by three-dimensional (3-D) braids is to assume a simple unit cell geometry of a 3-D braided structure with known fiber volume fraction and orientation. In this article, we first examine 3-D braiding methods in the light of braid structures, followed by the development of geometric models for 3-D braids using a unit cell approach. The unit cell geometry of 3-D braids is identified and the relationship of structural parameters such as yarn orientation angle and fiber volume fraction with the key processing parameters established. The limiting geometry has been computed by establishing the point at which yarns jam against each other. Using this factor makes it possible to identify the complete range of allowable geometric arrangements for 3-D braided preforms. This identified unit cell geometry can be translated to mechanical models which relate the geometrical properties of fabric preforms to the mechanical responses of composite systems.

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

International Nuclear Information System (INIS)

Kassegne, Sam; Moon, Kee; Martín-Ramos, Pablo; Majzoub, Mohammad; Őzturk, Gunay; Desai, Krishna; Parikh, Mihir; Nguyen, Bao; Khosla, Ajit; Chamorro-Posada, Pedro

2012-01-01

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)

High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

Science.gov (United States)

Chen, Yu-Chih; Yoon, Euisik

2017-01-01

Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

Development, Characterization and Cell Cultural Response of 3D Biocompatible Micro-Patterned Poly-ε-Caprolactone Scaffolds Designed and Fabricated Integrating Lithography and Micromolding Fabrication Techniques

KAUST Repository

Limongi, Tania; Miele, Ermanno; Shalabaeva, Victoria; Rocca, Rosanna La; Schipani, Rossana; Malara, Natalia; Angelis, Francesco de; Giugni, Andrea; Di Fabrizio, Enzo M.

2014-01-01

Scaffold design and fabrication are very important subjects for biomaterial, tissue engineering and regenerative medicine research playing a unique role in tissue regeneration and repair. Among synthetic biomaterials Poly-ε- Caprolactone (PCL) is very attractive bioresorbable polyester due to its high permeability, biodegradability and capacity to be blended with other biopolymers. Thanks to its ability to naturally degrade in tissues, PCL has a great potential as a new material for implantable biomedical micro devices. This work focuses on the establishment of a micro fabrication process, by integrating lithography and micromolding fabrication techniques, for the realization of 3D microstructure PCL devices. Scaffold surface exhibits a combination in the patterned length scale; cylindrical pillars of 10 μm height and 10 μm diameter are arranged in a hexagonal lattice with periodicity of 30 μm and their sidewalls are nano-sculptured, with a regular pattern of grooves leading to a spatial modulation in the z direction. In order to demonstrate that these biocompatible pillared PCL substrates are suitable for a proper cell growth, NIH/3T3 mouse embryonic fibroblasts were seeded on them and cells key adhesion parameters were evaluated. Scanning Electron Microscopy and immunofluorescence analysis were carried out to check cell survival, proliferation and adhesion; cells growing on the PCL substrates appeared healthy and formed a well-developed network in close contact with the micro and nano features of the pillared surface. Those 3D scaffolds could be a promising solution for a wide range of applications within tissue engineering and regenerative medicine applications.

Development, Characterization and Cell Cultural Response of 3D Biocompatible Micro-Patterned Poly-ε-Caprolactone Scaffolds Designed and Fabricated Integrating Lithography and Micromolding Fabrication Techniques

KAUST Repository

Limongi, Tania

2014-12-12

Scaffold design and fabrication are very important subjects for biomaterial, tissue engineering and regenerative medicine research playing a unique role in tissue regeneration and repair. Among synthetic biomaterials Poly-ε- Caprolactone (PCL) is very attractive bioresorbable polyester due to its high permeability, biodegradability and capacity to be blended with other biopolymers. Thanks to its ability to naturally degrade in tissues, PCL has a great potential as a new material for implantable biomedical micro devices. This work focuses on the establishment of a micro fabrication process, by integrating lithography and micromolding fabrication techniques, for the realization of 3D microstructure PCL devices. Scaffold surface exhibits a combination in the patterned length scale; cylindrical pillars of 10 μm height and 10 μm diameter are arranged in a hexagonal lattice with periodicity of 30 μm and their sidewalls are nano-sculptured, with a regular pattern of grooves leading to a spatial modulation in the z direction. In order to demonstrate that these biocompatible pillared PCL substrates are suitable for a proper cell growth, NIH/3T3 mouse embryonic fibroblasts were seeded on them and cells key adhesion parameters were evaluated. Scanning Electron Microscopy and immunofluorescence analysis were carried out to check cell survival, proliferation and adhesion; cells growing on the PCL substrates appeared healthy and formed a well-developed network in close contact with the micro and nano features of the pillared surface. Those 3D scaffolds could be a promising solution for a wide range of applications within tissue engineering and regenerative medicine applications.

Printing of Patterned, Engineered E. coli Biofilms with a Low-Cost 3D Printer.

Science.gov (United States)

Schmieden, Dominik T; Basalo Vázquez, Samantha J; Sangüesa, Héctor; van der Does, Marit; Idema, Timon; Meyer, Anne S

2018-05-18

Biofilms can grow on virtually any surface available, with impacts ranging from endangering the lives of patients to degrading unwanted water contaminants. Biofilm research is challenging due to the high degree of biofilm heterogeneity. A method for the production of standardized, reproducible, and patterned biofilm-inspired materials could be a boon for biofilm research and allow for completely new engineering applications. Here, we present such a method, combining 3D printing with genetic engineering. We prototyped a low-cost 3D printer that prints bioink, a suspension of bacteria in a solution of alginate that solidifies on a calcium-containing substrate. We 3D-printed Escherichia coli in different shapes and in discrete layers, after which the cells survived in the printing matrix for at least 1 week. When printed bacteria were induced to form curli fibers, the major proteinaceous extracellular component of E. coli biofilms, they remained adherent to the printing substrate and stably spatially patterned even after treatment with a matrix-dissolving agent, indicating that a biofilm-mimicking structure had formed. This work is the first demonstration of patterned, biofilm-inspired living materials that are produced by genetic control over curli formation in combination with spatial control by 3D printing. These materials could be used as living, functional materials in applications such as water filtration, metal ion sequestration, or civil engineering, and potentially as standardizable models for certain curli-containing biofilms.

Simultaneous cell tracking and image alignment in 3D CLSM imagery of growing arabidopsis thaliana sepals

NARCIS (Netherlands)

Fick, R.H.J.; Fedorov, D.; Roeder, A.H.K.; Manjunath, B.S.

2013-01-01

In this research we propose a combined cell matching and image alignment method for tracking cells based on their nuclear locations in 3D fluorescent Confocal Laser Scanning Microscopy (CLSM) image sequences. We then apply it to study the cell division pattern in the developing sepal of the small

Three-dimensional cell manipulation and patterning using dielectrophoresis via a multi-layer scaffold structure.

Science.gov (United States)

Chu, H K; Huan, Z; Mills, J K; Yang, J; Sun, D

2015-02-07

Cell manipulation is imperative to the areas of cellular biology and tissue engineering, providing them a useful tool for patterning cells into cellular patterns for different analyses and applications. This paper presents a novel approach to perform three-dimensional (3D) cell manipulation and patterning with a multi-layer engineered scaffold. This scaffold structure employed dielectrophoresis as the non-contact mechanism to manipulate cells in the 3D domain. Through establishing electric fields via this multi-layer structure, the cells in the medium became polarized and were attracted towards the interior part of the structure, forming 3D cellular patterns. Experiments were conducted to evaluate the manipulation and the patterning processes with the proposed structure. Results show that with the presence of a voltage input, this multi-layer structure was capable of manipulating different types of biological cells examined through dielectrophoresis, enabling automatic cell patterning in the time-scale of minutes. The effects of the voltage input on the resultant cellular pattern were examined and discussed. Viability test was performed after the patterning operation and the results confirmed that majority of the cells remained viable. After 7 days of culture, 3D cellular patterns were observed through SEM. The results suggest that this scaffold and its automated dielectrophoresis-based patterning mechanism can be used to construct artificial tissues for various tissue engineering applications.

Toward single cell traction microscopy within 3D collagen matrices

International Nuclear Information System (INIS)

Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

2013-01-01

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels

Toward single cell traction microscopy within 3D collagen matrices

Energy Technology Data Exchange (ETDEWEB)

Hall, Matthew S. [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States); Long, Rong [Department of Mechanical Engineering, University of Alberta, Edmonton, AB, Canada T6G 2G8 (Canada); Feng, Xinzeng [Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853 (United States); Huang, YuLing [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States); Hui, Chung-Yuen [Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853 (United States); Wu, Mingming, E-mail: mw272@cornell.edu [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States)

2013-10-01

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

Semaphorin 3G Provides a Repulsive Guidance Cue to Lymphatic Endothelial Cells via Neuropilin-2/PlexinD1.

Science.gov (United States)

Liu, Xinyi; Uemura, Akiyoshi; Fukushima, Yoko; Yoshida, Yutaka; Hirashima, Masanori

2016-11-22

The vertebrate circulatory system is composed of closely related blood and lymphatic vessels. It has been shown that lymphatic vascular patterning is regulated by blood vessels during development, but its molecular mechanisms have not been fully elucidated. Here, we show that the artery-derived ligand semaphorin 3G (Sema3G) and the endothelial cell receptor PlexinD1 play a role in lymphatic vascular patterning. In mouse embryonic back skin, genetic inactivation of Sema3G or PlexinD1 results in abnormal artery-lymph alignment and reduced lymphatic vascular branching. Conditional ablation in mice demonstrates that PlexinD1 is primarily required in lymphatic endothelial cells (LECs). In vitro analyses show that Sema3G binds to neuropilin-2 (Nrp2), which forms a receptor complex with PlexinD1. Sema3G induces cell collapse in an Nrp2/PlexinD1-dependent manner. Our findings shed light on a molecular mechanism by which LECs are distributed away from arteries and form a branching network during lymphatic vascular development. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

Xue Gang; Ren Zhenxin; Chen Yaxiong; Zhu Jiayun; Du Yarong; Pan Dong; Li Xiaoman; Hu Burong; Grabham, Peter W.

2015-01-01

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)

Agarose hydrogel induced MCF-7 and BMG-1 cell line progressive 3D and 3D revert cultures.

Science.gov (United States)

Subramaniyan, Aishwarya; Ravi, Maddaly

2018-04-01

3D culture systems have enhanced the utility of cancer cell lines as they are considered closer to the in vivo systems. A variety of changes are induced in cells cultured in 3D systems; an apparent and striking feature being the spontaneous acquisition of distinct morphological entities. 3D reverts (3DRs) can be obtained by introducing 3D aggregates in scaffold/matrix-free culture units. It could be seen that the two cell lines used in this study exhibited differences in 3DR structures, though both were cultured on agarose hydrogels. Also, differences in 3DR formation, growth and survival were different. While 3D aggregates of several cell lines have been reported for a variety of studies, there are no studies that describe or utilize 3DRs. 3DRs can provide insights into complex events that can occur in cancer cells; especially as material to study metastasis, migration, and invasion. © 2017 Wiley Periodicals, Inc.

Novel Compound-Forming Technology Using Bioprinting and Electrospinning for Patterning a 3D Scaffold Construct with Multiscale Channels

Directory of Open Access Journals (Sweden)

Yuanshao Sun

2016-12-01

Full Text Available One of the biggest challenges for tissue engineering is to efficiently provide oxygen and nutrients to cells on a three-dimensional (3D engineered scaffold structure. Thus, achieving sufficient vascularization of the structure is a critical problem in tissue engineering. This facilitates the need to develop novel methods to enhance vascularization. Use of patterned hydrogel structures with multiscale channels can be used to achieve the required vascularization. Patterned structures need to be biocompatible and biodegradable. In this study, gelatin was used as the main part of a hydrogel to prepare a biological structure with 3D multiscale channels using bioprinting combined with selection of suitable materials and electrostatic spinning. Human umbilical vein endothelial cells (HUVECs were then used to confirm efficacy of the structure, inferred from cell viability on different engineered construct designs. HUVECs were seeded on the surface of channels and cultured in vitro. HUVECs showed high viability and diffusion within the construct. This method can be used as a practical platform for the fabrication of engineered construct for vascularization.

AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

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Kong, Jun; Wang, Fusheng; Teodoro, George; Liang, Yanhui; Zhu, Yangyang; Tucker-Burden, Carol; Brat, Daniel J

2015-04-01

A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.

Modeling the formation of cell-matrix adhesions on a single 3D matrix fiber.

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Escribano, J; Sánchez, M T; García-Aznar, J M

2015-11-07

Cell-matrix adhesions are crucial in different biological processes like tissue morphogenesis, cell motility, and extracellular matrix remodeling. These interactions that link cell cytoskeleton and matrix fibers are built through protein clutches, generally known as adhesion complexes. The adhesion formation process has been deeply studied in two-dimensional (2D) cases; however, the knowledge is limited for three-dimensional (3D) cases. In this work, we simulate different local extracellular matrix properties in order to unravel the fundamental mechanisms that regulate the formation of cell-matrix adhesions in 3D. We aim to study the mechanical interaction of these biological structures through a three dimensional discrete approach, reproducing the transmission pattern force between the cytoskeleton and a single extracellular matrix fiber. This numerical model provides a discrete analysis of the proteins involved including spatial distribution, interaction between them, and study of the different phenomena, such as protein clutches unbinding or protein unfolding. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of 3D Modeling Software Usage Patterns for K-12 Students

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Wu, Yi-Chieh; Liao, Wen-Hung; Chi, Ming-Te; Li, Tsai-Yen

2016-01-01

In response to the recent trend in maker movement, teachers are learning 3D techniques actively and bringing 3D printing into the classroom to enhance variety and creativity in designing lectures. This study investigates the usage pattern of a 3D modeling software, Qmodel Creator, which is targeted at K-12 students. User logs containing…

Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells.

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Ortinau, Stefanie; Schmich, Jürgen; Block, Stephan; Liedmann, Andrea; Jonas, Ludwig; Weiss, Dieter G; Helm, Christiane A; Rolfs, Arndt; Frech, Moritz J

2010-11-11

3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenviroment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them. In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay. Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures. Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3

Automation of 3D cell culture using chemically defined hydrogels.

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Rimann, Markus; Angres, Brigitte; Patocchi-Tenzer, Isabel; Braum, Susanne; Graf-Hausner, Ursula

2014-04-01

Drug development relies on high-throughput screening involving cell-based assays. Most of the assays are still based on cells grown in monolayer rather than in three-dimensional (3D) formats, although cells behave more in vivo-like in 3D. To exemplify the adoption of 3D techniques in drug development, this project investigated the automation of a hydrogel-based 3D cell culture system using a liquid-handling robot. The hydrogel technology used offers high flexibility of gel design due to a modular composition of a polymer network and bioactive components. The cell inert degradation of the gel at the end of the culture period guaranteed the harmless isolation of live cells for further downstream processing. Human colon carcinoma cells HCT-116 were encapsulated and grown in these dextran-based hydrogels, thereby forming 3D multicellular spheroids. Viability and DNA content of the cells were shown to be similar in automated and manually produced hydrogels. Furthermore, cell treatment with toxic Taxol concentrations (100 nM) had the same effect on HCT-116 cell viability in manually and automated hydrogel preparations. Finally, a fully automated dose-response curve with the reference compound Taxol showed the potential of this hydrogel-based 3D cell culture system in advanced drug development.

Reconstruction of incomplete cell paths through a 3D-2D level set segmentation

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Hariri, Maia; Wan, Justin W. L.

2012-02-01

Segmentation of fluorescent cell images has been a popular technique for tracking live cells. One challenge of segmenting cells from fluorescence microscopy is that cells in fluorescent images frequently disappear. When the images are stacked together to form a 3D image volume, the disappearance of the cells leads to broken cell paths. In this paper, we present a segmentation method that can reconstruct incomplete cell paths. The key idea of this model is to perform 2D segmentation in a 3D framework. The 2D segmentation captures the cells that appear in the image slices while the 3D segmentation connects the broken cell paths. The formulation is similar to the Chan-Vese level set segmentation which detects edges by comparing the intensity value at each voxel with the mean intensity values inside and outside of the level set surface. Our model, however, performs the comparison on each 2D slice with the means calculated by the 2D projected contour. The resulting effect is to segment the cells on each image slice. Unlike segmentation on each image frame individually, these 2D contours together form the 3D level set function. By enforcing minimum mean curvature on the level set surface, our segmentation model is able to extend the cell contours right before (and after) the cell disappears (and reappears) into the gaps, eventually connecting the broken paths. We will present segmentation results of C2C12 cells in fluorescent images to illustrate the effectiveness of our model qualitatively and quantitatively by different numerical examples.

Contributions of 3D Cell Cultures for Cancer Research.

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Ravi, Maddaly; Ramesh, Aarthi; Pattabhi, Aishwarya

2017-10-01

Cancer cell lines have contributed immensely in understanding the complex physiology of cancers. They are excellent material for studies as they offer homogenous samples without individual variations and can be utilised with ease and flexibility. Also, the number of assays and end-points one can study is almost limitless; with the advantage of improvising, modifying or altering several variables and methods. Literally, a new dimension to cancer research has been achieved by the advent of 3Dimensional (3D) cell culture techniques. This approach increased many folds the ways in which cancer cell lines can be utilised for understanding complex cancer biology. 3D cell culture techniques are now the preferred way of using cancer cell lines to bridge the gap between the 'absolute in vitro' and 'true in vivo'. The aspects of cancer biology that 3D cell culture systems have contributed include morphology, microenvironment, gene and protein expression, invasion/migration/metastasis, angiogenesis, tumour metabolism and drug discovery, testing chemotherapeutic agents, adaptive responses and cancer stem cells. We present here, a comprehensive review on the applications of 3D cell culture systems for these aspects of cancers. J. Cell. Physiol. 232: 2679-2697, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mesoderm Lineage 3D Tissue Constructs Are Produced at Large-Scale in a 3D Stem Cell Bioprocess.

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Cha, Jae Min; Mantalaris, Athanasios; Jung, Sunyoung; Ji, Yurim; Bang, Oh Young; Bae, Hojae

2017-09-01

Various studies have presented different approaches to direct pluripotent stem cell differentiation such as applying defined sets of exogenous biochemical signals and genetic/epigenetic modifications. Although differentiation to target lineages can be successfully regulated, such conventional methods are often complicated, laborious, and not cost-effective to be employed to the large-scale production of 3D stem cell-based tissue constructs. A 3D-culture platform that could realize the large-scale production of mesoderm lineage tissue constructs from embryonic stem cells (ESCs) is developed. ESCs are cultured using our previously established 3D-bioprocess platform which is amenable to mass-production of 3D ESC-based tissue constructs. Hepatocarcinoma cell line conditioned medium is introduced to the large-scale 3D culture to provide a specific biomolecular microenvironment to mimic in vivo mesoderm formation process. After 5 days of spontaneous differentiation period, the resulting 3D tissue constructs are composed of multipotent mesodermal progenitor cells verified by gene and molecular expression profiles. Subsequently the optimal time points to trigger terminal differentiation towards cardiomyogenesis or osteogenesis from the mesodermal tissue constructs is found. A simple and affordable 3D ESC-bioprocess that can reach the scalable production of mesoderm origin tissues with significantly improved correspondent tissue properties is demonstrated. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Classifying and Analyzing 3d Cell Motion in Jammed Microgels

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Bhattacharjee, Tapomoy; Sawyer, W. Gregory; Angelini, Thomas

Soft granular polyelectrolyte microgels swell in liquid cell growth media to form a continuous elastic solid that can easily transition between solid to fluid state under a low shear stress. Such Liquid-like solids (LLS) have recently been used to create 3D cellular constructs as well as to support, culture and harvest cells in 3D. Current understanding of cell migration mechanics in 3D was established from experiments performed in natural and synthetic polymer networks. Spatial variation in network structure and the transience of degradable gels limit their usefulness in quantitative cell mechanics studies. By contrast, LLS growth media approximates a homogeneous continuum, enabling tractable cell mechanics measurements to be performed in 3D. Here, we introduce a process to understand and classify cytotoxic T cell motion in 3D by studying cellular motility in LLS media. General classification of T cell motion can be achieved with a very traditional statistical approach: the cell's mean squared displacement (MSD) as a function of delay time. We will also use Langevin approaches combined with the constitutive equations of the LLS medium to predict the statistics of T cell motion. National Science Foundation under Grant No. DMR-1352043.

Perceived crosstalk assessment on patterned retarder 3D display

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Zou, Bochao; Liu, Yue; Huang, Yi; Wang, Yongtian

2014-03-01

CONTEXT: Nowadays, almost all stereoscopic displays suffer from crosstalk, which is one of the most dominant degradation factors of image quality and visual comfort for 3D display devices. To deal with such problems, it is worthy to quantify the amount of perceived crosstalk OBJECTIVE: Crosstalk measurements are usually based on some certain test patterns, but scene content effects are ignored. To evaluate the perceived crosstalk level for various scenes, subjective test may bring a more correct evaluation. However, it is a time consuming approach and is unsuitable for real­ time applications. Therefore, an objective metric that can reliably predict the perceived crosstalk is needed. A correct objective assessment of crosstalk for different scene contents would be beneficial to the development of crosstalk minimization and cancellation algorithms which could be used to bring a good quality of experience to viewers. METHOD: A patterned retarder 3D display is used to present 3D images in our experiment. By considering the mechanism of this kind of devices, an appropriate simulation of crosstalk is realized by image processing techniques to assign different values of crosstalk to each other between image pairs. It can be seen from the literature that the structures of scenes have a significant impact on the perceived crosstalk, so we first extract the differences of the structural information between original and distorted image pairs through Structural SIMilarity (SSIM) algorithm, which could directly evaluate the structural changes between two complex-structured signals. Then the structural changes of left view and right view are computed respectively and combined to an overall distortion map. Under 3D viewing condition, because of the added value of depth, the crosstalk of pop-out objects may be more perceptible. To model this effect, the depth map of a stereo pair is generated and the depth information is filtered by the distortion map. Moreover, human attention

Stem/Progenitor Cell Proteoglycans Decorated with 7-D-4, 4-C-3 and 3-B-3(-) Chondroitin Sulphate Motifs Are Morphogenetic Markers Of Tissue Development.

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Hayes, Anthony J; Smith, Susan M; Caterson, Bruce; Melrose, James

2018-06-11

This study reviewed the occurrence of chondroitin sulphate (CS) motifs 4-C-3, 7-D-4 and 3-B-3(-) which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulphation motifs 7-D-4, 4-C-3 and 3-B-3 (-) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

A 3D Human Renal Cell Carcinoma-on-a-Chip for the Study of Tumor Angiogenesis.

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Miller, Chris P; Tsuchida, Connor; Zheng, Ying; Himmelfarb, Jonathan; Akilesh, Shreeram

2018-06-01

Tractable human tissue-engineered 3D models of cancer that enable fine control of tumor growth, metabolism, and reciprocal interactions between different cell types in the tumor microenvironment promise to accelerate cancer research and pharmacologic testing. Progress to date mostly reflects the use of immortalized cancer cell lines, and progression to primary patient-derived tumor cells is needed to realize the full potential of these platforms. For the first time, we report endothelial sprouting induced by primary patient tumor cells in a 3D microfluidic system. Specifically, we have combined primary human clear cell renal cell carcinoma (ccRCC) cells from six independent donors with human endothelial cells in a vascularized, flow-directed, 3D culture system ("ccRCC-on-a-chip"). The upregulation of key angiogenic factors in primary human ccRCC cells, which exhibited unique patterns of donor variation, was further enhanced when they were cultured in 3D clusters. When embedded in the matrix surrounding engineered human vessels, these ccRCC tumor clusters drove potent endothelial cell sprouting under continuous flow, thus recapitulating the critical angiogenic signaling axis between human ccRCC cells and endothelial cells. Importantly, this phenotype was driven by a primary tumor cell-derived biochemical gradient of angiogenic growth factor accumulation that was subject to pharmacological blockade. Our novel 3D system represents a vascularized tumor model that is easy to image and quantify and is fully tunable in terms of input cells, perfusate, and matrices. We envision that this ccRCC-on-a-chip will be valuable for mechanistic studies, for studying tumor-vascular cell interactions, and for developing novel and personalized antitumor therapies. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Apple derived cellulose scaffolds for 3D mammalian cell culture.

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Daniel J Modulevsky

Full Text Available There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads.

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Wang, Lin; Acosta, Miguel A; Leach, Jennie B; Carrier, Rebecca L

2013-04-21

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems.

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads

OpenAIRE

Wang, Lin; Acosta, Miguel A.; Leach, Jennie B.; Carrier, Rebecca L.

2013-01-01

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and ...

1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells.

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Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N; Glenn, Sean T; Liu, Song; Trump, Donald L; Johnson, Candace S

2015-04-01

Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. miRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253J and 253J-BV cells express endogenous vitamin D receptor (VDR), which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Copyright © 2014 Elsevier Ltd. All rights reserved.

Adaptive fringe-pattern projection for image saturation avoidance in 3D surface-shape measurement.

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Li, Dong; Kofman, Jonathan

2014-04-21

In fringe-projection 3D surface-shape measurement, image saturation results in incorrect intensities in captured images of fringe patterns, leading to phase and measurement errors. An adaptive fringe-pattern projection (AFPP) method was developed to adapt the maximum input gray level in projected fringe patterns to the local reflectivity of an object surface being measured. The AFPP method demonstrated improved 3D measurement accuracy by avoiding image saturation in highly-reflective surface regions while maintaining high intensity modulation across the entire surface. The AFPP method can avoid image saturation and handle varying surface reflectivity, using only two prior rounds of fringe-pattern projection and image capture to generate the adapted fringe patterns.

3D Protein Dynamics in the Cell Nucleus.

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Singh, Anand P; Galland, Rémi; Finch-Edmondson, Megan L; Grenci, Gianluca; Sibarita, Jean-Baptiste; Studer, Vincent; Viasnoff, Virgile; Saunders, Timothy E

2017-01-10

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

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Sun, Xiuzhi S.; Nguyen, Thu A.

2013-01-01

Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing. PMID:23527204

Dual-Stage Crosslinking of a Gel-Phase Bioink Improves Cell Viability and Homogeneity for 3D Bioprinting.

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Dubbin, Karen; Hori, Yuki; Lewis, Kazuomori K; Heilshorn, Sarah C

2016-10-01

Current bioinks for cell-based 3D bioprinting are not suitable for technology scale-up due to the challenges of cell sedimentation, cell membrane damage, and cell dehydration. A novel bioink hydrogel is presented with dual-stage crosslinking specifically designed to overcome these three major hurdles. This bioink enables the direct patterning of highly viable, multicell type constructs with long-term spatial fidelity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering Breast Cancer Microenvironments and 3D Bioprinting

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Jorge A. Belgodere

2018-05-01

Full Text Available The extracellular matrix (ECM is a critical cue to direct tumorigenesis and metastasis. Although two-dimensional (2D culture models have been widely employed to understand breast cancer microenvironments over the past several decades, the 2D models still exhibit limited success. Overwhelming evidence supports that three dimensional (3D, physiologically relevant culture models are required to better understand cancer progression and develop more effective treatment. Such platforms should include cancer-specific architectures, relevant physicochemical signals, stromal–cancer cell interactions, immune components, vascular components, and cell-ECM interactions found in patient tumors. This review briefly summarizes how cancer microenvironments (stromal component, cell-ECM interactions, and molecular modulators are defined and what emerging technologies (perfusable scaffold, tumor stiffness, supporting cells within tumors and complex patterning can be utilized to better mimic native-like breast cancer microenvironments. Furthermore, this review emphasizes biophysical properties that differ between primary tumor ECM and tissue sites of metastatic lesions with a focus on matrix modulation of cancer stem cells, providing a rationale for investigation of underexplored ECM proteins that could alter patient prognosis. To engineer breast cancer microenvironments, we categorized technologies into two groups: (1 biochemical factors modulating breast cancer cell-ECM interactions and (2 3D bioprinting methods and its applications to model breast cancer microenvironments. Biochemical factors include matrix-associated proteins, soluble factors, ECMs, and synthetic biomaterials. For the application of 3D bioprinting, we discuss the transition of 2D patterning to 3D scaffolding with various bioprinting technologies to implement biophysical cues to model breast cancer microenvironments.

Differences of statin activity in 2D and 3D pancreatic cancer cell cultures

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Paškevičiūtė M

2017-11-01

Full Text Available Miglė Paškevičiūtė,1 Vilma Petrikaitė1,21Department of Drug Chemistry, Faculty of Pharmacy, Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Department of Biothermodynamics and Drug Design, Vilnius University Institute of Biotechnology, Vilnius, LithuaniaPurpose: To evaluate the anticancer activity of lovastatin (LOVA, mevastatin (MEVA, pitavastatin (PITA, and simvastatin (SIMVA in 2D and 3D models of three human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, and PANC-1.Methods: The effect of statins on cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide test. The activity of statins in 3D pancreatic cancer cell cultures was examined by measuring the size change of spheroids. The type of cell death was identified by cell staining with Hoechst 33342 and propidium iodide. The activity of statins on the clonogenicity of cancer cells was tested by evaluating the effect on the colony-forming ability of cells.Results: The rank order of the activity of tested statins on cell viability was as follows: PITA > SIMVA > LOVA > MEVA. Among the tested statins, PITA had the greatest effect on cell viability (half maximal effective concentration values after 72 h on BxPC-3, MIA PaCa-2, and PANC-1 cells were 1.4±0.4 µM, 1.0±0.2 µM, and 1.0±0.5 µM, respectively. PITA also showed the strongest effect on tumor spheroid growth. Statins suppressed the colony formation of cancer cells. PITA demonstrated the greatest reduction in colony size and number. Apoptosis and necrosis assay results showed that at lower concentrations statins mostly induced cell death through apoptosis, whereas higher concentrations of compounds activated also necrotic processes.Conclusion: Statins, especially PITA, demonstrate an anticancer activity against pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 in both 2D and 3D models.Keywords: HMG-CoA reductase, cell viability, spheroid, apoptosis

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

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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.

Voronoi cell patterns: Theoretical model and applications

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González, Diego Luis; Einstein, T. L.

2011-11-01

We use a simple fragmentation model to describe the statistical behavior of the Voronoi cell patterns generated by a homogeneous and isotropic set of points in 1D and in 2D. In particular, we are interested in the distribution of sizes of these Voronoi cells. Our model is completely defined by two probability distributions in 1D and again in 2D, the probability to add a new point inside an existing cell and the probability that this new point is at a particular position relative to the preexisting point inside this cell. In 1D the first distribution depends on a single parameter while the second distribution is defined through a fragmentation kernel; in 2D both distributions depend on a single parameter. The fragmentation kernel and the control parameters are closely related to the physical properties of the specific system under study. We use our model to describe the Voronoi cell patterns of several systems. Specifically, we study the island nucleation with irreversible attachment, the 1D car-parking problem, the formation of second-level administrative divisions, and the pattern formed by the Paris Métro stations.

Direct Investment Casting For Pattern Developed By Desktop 3D Printer

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Marwah O.M.F.

2017-01-01

Full Text Available Development of RP technologies has encouraged rapid study on portable 3D Printer in which there are varieties of portable 3D printer machines in market. Nevertheless, less reports regarding its consumption of fabricated pattern to be used in direct investment casting. This study has focused on development of ABS P400 pattern in terms of collapsibility behaviour which has capability to be used as sacrificial pattern in direct investment casting. In addition, the internal built structures have been built into two designs such as square and polygon patterns respectively. The patterns were constructed in semi cylindrical geometry which comes with one side opened and one side closed together wrapped with 4 mm of ceramic shell. This experimental were conducted with variation temperature starting from 30°C until 150°C with increment of 5°C per minutes while for the numerical simulation, the temperature selected was between 30 °C to 120 °C with 10 °C increment per minutes. Moreover, the observation was made that glass transition temperature, Tg happened near 110°C. It was observed that the shell cracking happened on the ceramic shell. Other than that, the polygon pattern tends to collapsed inwardly rather than square pattern during the burnout process. A result also shows that, there is significant amount of stress reduction on both square and polygon which was 45 % respectively. Besides that, the amount of strain on pattern itself has shown about 9% reduction. Moreover, there is greater difference in terms of ceramic shell strain reduction which was 38% for square and polygon patterns respectively. Lastly, there is 11 % reduction of strain when compared square and polygon in terms of axial strain on ceramic shell

One-Year stable perovskite solar cells by 2D/3D interface engineering

Science.gov (United States)

Grancini, G.; Roldán-Carmona, C.; Zimmermann, I.; Mosconi, E.; Lee, X.; Martineau, D.; Narbey, S.; Oswald, F.; de Angelis, F.; Graetzel, M.; Nazeeruddin, Mohammad Khaja

2017-06-01

Despite the impressive photovoltaic performances with power conversion efficiency beyond 22%, perovskite solar cells are poorly stable under operation, failing by far the market requirements. Various technological approaches have been proposed to overcome the instability problem, which, while delivering appreciable incremental improvements, are still far from a market-proof solution. Here we show one-year stable perovskite devices by engineering an ultra-stable 2D/3D (HOOC(CH2)4NH3)2PbI4/CH3NH3PbI3 perovskite junction. The 2D/3D forms an exceptional gradually-organized multi-dimensional interface that yields up to 12.9% efficiency in a carbon-based architecture, and 14.6% in standard mesoporous solar cells. To demonstrate the up-scale potential of our technology, we fabricate 10 × 10 cm2 solar modules by a fully printable industrial-scale process, delivering 11.2% efficiency stable for >10,000 h with zero loss in performances measured under controlled standard conditions. This innovative stable and low-cost architecture will enable the timely commercialization of perovskite solar cells.

Microfluidic 3D cell culture: potential application for tissue-based bioassays

Science.gov (United States)

Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

2014-01-01

Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

Patterned cell arrays and patterned co-cultures on polydopamine-modified poly(vinyl alcohol) hydrogels

International Nuclear Information System (INIS)

Beckwith, Kai M; Sikorski, Pawel

2013-01-01

Live cell arrays are an emerging tool that expand traditional 2D in vitro cell culture, increasing experimental precision and throughput. A patterned cell system was developed by combining the cell-repellent properties of polyvinyl alcohol hydrogels with the cell adhesive properties of self-assembled films of dopamine (polydopamine). It was shown that polydopamine could be patterned onto spin-cast polyvinyl alcohol hydrogels by microcontact printing, which in turn effectively patterned the growth of several cell types (HeLa, human embryonic kidney, human umbilical vein endothelial cells (HUVEC) and prostate cancer). The cells could be patterned in geometries down to single-cell confinement, and it was demonstrated that cell patterns could be maintained for at least 3 weeks. Furthermore, polydopamine could be used to modify poly(vinyl alcohol) in situ using a cell-compatible deposition buffer (1 mg mL −1 dopamine in 25 mM tris with a physiological salt balance). The treatment switched the PVA hydrogel from cell repellent to cell adhesive. Finally, by combining microcontact printing and in situ deposition of polydopamine, patterned co-cultures of the same cell type (HeLa/HeLa) and dissimilar cell types (HeLa/HUVEC) were realized through simple chemistry and could be studied over time. The combination of polyvinyl alcohol and polydopamine was shown to be an attractive route to versatile, patterned cell culture experiments with minimal infrastructure requirements and low complexity. (paper)

Effect of 3D Cultivation Conditions on the Differentiation of Endodermal Cells

Science.gov (United States)

Petrakova, O. S.; Ashapkin, V. V.; Voroteliak, E. A.; Bragin, E. Y.; Shtratnikova, V. Y.; Chernioglo, E. S.; Sukhanov, Y. V.; Terskikh, V. V.; Vasiliev, A. V.

2012-01-01

Cellular therapy of endodermal organs is one of the most important issues in modern cellular biology and biotechnology. One of the most promising directions in this field is the study of the transdifferentiation abilities of cells within the same germ layer. A method for anin vitroinvestigation of the cell differentiation potential (the cell culture in a three-dimensional matrix) is described in this article. Cell cultures of postnatal salivary gland cells and postnatal liver progenitor cells were obtained; their comparative analysis under 2D and 3D cultivation conditions was carried out. Both cell types have high proliferative abilities and can be cultivated for more than 20 passages. Under 2D cultivation conditions, the cells remain in an undifferentiated state. Under 3D conditions, they undergo differentiation, which was confirmed by a lower cell proliferation and by an increase in the differentiation marker expression. Salivary gland cells can undergo hepatic and pancreatic differentiation under 3D cultivation conditions. Liver progenitor cells also acquire a pancreatic differentiation capability under conditions of 3D cultivation. Thus, postnatal salivary gland cells exhibit a considerable differentiation potential within the endodermal germ layer and can be used as a promising source of endodermal cells for the cellular therapy of liver pathologies. Cultivation of cells under 3D conditions is a useful model for thein vitroanalysis of the cell differentiation potential. PMID:23346379

Economic 3D-printing approach for transplantation of human stem cell-derived β-like cells.

Science.gov (United States)

Song, Jiwon; Millman, Jeffrey R

2016-12-01

Transplantation of human pluripotent stem cells (hPSC) differentiated into insulin-producing β cells is a regenerative medicine approach being investigated for diabetes cell replacement therapy. This report presents a multifaceted transplantation strategy that combines differentiation into stem cell-derived β (SC-β) cells with 3D printing. By modulating the parameters of a low-cost 3D printer, we created a macroporous device composed of polylactic acid (PLA) that houses SC-β cell clusters within a degradable fibrin gel. Using finite element modeling of cellular oxygen diffusion-consumption and an in vitro culture system that allows for culture of devices at physiological oxygen levels, we identified cluster sizes that avoid severe hypoxia within 3D-printed devices and developed a microwell-based technique for resizing clusters within this range. Upon transplantation into mice, SC-β cell-embedded 3D-printed devices function for 12 weeks, are retrievable, and maintain structural integrity. Here, we demonstrate a novel 3D-printing approach that advances the use of differentiated hPSC for regenerative medicine applications and serves as a platform for future transplantation strategies.

Point Cluster Analysis Using a 3D Voronoi Diagram with Applications in Point Cloud Segmentation

Directory of Open Access Journals (Sweden)

Shen Ying

2015-08-01

Full Text Available Three-dimensional (3D point analysis and visualization is one of the most effective methods of point cluster detection and segmentation in geospatial datasets. However, serious scattering and clotting characteristics interfere with the visual detection of 3D point clusters. To overcome this problem, this study proposes the use of 3D Voronoi diagrams to analyze and visualize 3D points instead of the original data item. The proposed algorithm computes the cluster of 3D points by applying a set of 3D Voronoi cells to describe and quantify 3D points. The decompositions of point cloud of 3D models are guided by the 3D Voronoi cell parameters. The parameter values are mapped from the Voronoi cells to 3D points to show the spatial pattern and relationships; thus, a 3D point cluster pattern can be highlighted and easily recognized. To capture different cluster patterns, continuous progressive clusters and segmentations are tested. The 3D spatial relationship is shown to facilitate cluster detection. Furthermore, the generated segmentations of real 3D data cases are exploited to demonstrate the feasibility of our approach in detecting different spatial clusters for continuous point cloud segmentation.

A 3D Monte Carlo model of radiation affecting cells, and its application to neuronal cells and GCR irradiation

Science.gov (United States)

Ponomarev, Artem; Sundaresan, Alamelu; Kim, Angela; Vazquez, Marcelo E.; Guida, Peter; Kim, Myung-Hee; Cucinotta, Francis A.

A 3D Monte Carlo model of radiation transport in matter is applied to study the effect of heavy ion radiation on human neuronal cells. Central nervous system effects, including cognitive impairment, are suspected from the heavy ion component of galactic cosmic radiation (GCR) during space missions. The model can count, for instance, the number of direct hits from ions, which will have the most affect on the cells. For comparison, the remote hits, which are received through δ-rays from the projectile traversing space outside the volume of the cell, are also simulated and their contribution is estimated. To simulate tissue effects from irradiation, cellular matrices of neuronal cells, which were derived from confocal microscopy, were simulated in our model. To produce this realistic model of the brain tissue, image segmentation was used to identify cells in the images of cells cultures. The segmented cells were inserted pixel by pixel into the modeled physical space, which represents a volume of interacting cells with periodic boundary conditions (PBCs). PBCs were used to extrapolate the model results to the macroscopic tissue structures. Specific spatial patterns for cell apoptosis are expected from GCR, as heavy ions produce concentrated damage along their trajectories. The apoptotic cell patterns were modeled based on the action cross sections for apoptosis, which were estimated from the available experimental data. The cell patterns were characterized with an autocorrelation function, which values are higher for non-random cell patterns, and the values of the autocorrelation function were compared for X rays and Fe ion irradiations. The autocorrelation function indicates the directionality effects present in apoptotic neuronal cells from GCR.

3D Printing of Living Responsive Materials and Devices.

Science.gov (United States)

Liu, Xinyue; Yuk, Hyunwoo; Lin, Shaoting; Parada, German Alberto; Tang, Tzu-Chieh; Tham, Eléonore; de la Fuente-Nunez, Cesar; Lu, Timothy K; Zhao, Xuanhe

2018-01-01

3D printing has been intensively explored to fabricate customized structures of responsive materials including hydrogels, liquid-crystal elastomers, shape-memory polymers, and aqueous droplets. Herein, a new method and material system capable of 3D-printing hydrogel inks with programed bacterial cells as responsive components into large-scale (3 cm), high-resolution (30 μm) living materials, where the cells can communicate and process signals in a programmable manner, are reported. The design of 3D-printed living materials is guided by quantitative models that account for the responses of programed cells in printed microstructures of hydrogels. Novel living devices are further demonstrated, enabled by 3D printing of programed cells, including logic gates, spatiotemporally responsive patterning, and wearable devices. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Voronoi Cell Patterns: theoretical model and application to submonolayer growth

Science.gov (United States)

González, Diego Luis; Einstein, T. L.

2012-02-01

We use a simple fragmentation model to describe the statistical behavior of the Voronoi cell patterns generated by a homogeneous and isotropic set of points in 1D and in 2D. In particular, we are interested in the distribution of sizes of these Voronoi cells. Our model is completely defined by two probability distributions in 1D and again in 2D, the probability to add a new point inside an existing cell and the probability that this new point is at a particular position relative to the preexisting point inside this cell. In 1D the first distribution depends on a single parameter while the second distribution is defined through a fragmentation kernel; in 2D both distributions depend on a single parameter. The fragmentation kernel and the control parameters are closely related to the physical properties of the specific system under study. We apply our model to describe the Voronoi cell patterns of island nucleation for critical island sizes i=0,1,2,3. Experimental results for the Voronoi cells of InAs/GaAs quantum dots are also described by our model.

Positive Oct -3/4 and D2-40 Immunohistochemical Expression in Germ Cells and Suspected Histology Pattern of Intratubular Germ Cell Neoplasia in Boys with Cryptorchidism Vanish after the Age of 2 Years

DEFF Research Database (Denmark)

Thorup, Jorgen; Clasen-Linde, Erik; Cortes, Dina

2017-01-01

of repeat biopsy with anti-stem cell factor (SCF) receptor.  Results  The prevalence of Oct-3/4 and D2-40-positive staining of germ cells in testicular biopsies were in age groups less than 6 months, 100% and 50%; 6-12 months, 60% and 17%; and 1-2 years, 12% and 4%. A 1 year, 1-month-old boy with Prader-Willi...... syndrome treated with growth hormone had ITGCN in both cryptorchid testes. In another three bilateral nonsyndromic cases, 8 months, 8 months and 1-year-old, a histological pattern in accordance with ITGCN was found. These three boys had a repeat biopsy from both testes performed at the age of 3 years, 4......, but no increased risk of malignancy.  Materials and Methods  Histology sections from 373 testicular biopsies from 289 boys aged 1 month to 2 years operated for cryptorchidism were incubated with primary antibodies including anti-placental-like-alkaline phosphatase, antiOct-3/4, anti-C-kit, anti-D2-40, and in case...

3D Plasma Nanotextured® Polymeric Surfaces for Protein or Antibody Arrays, and Biomolecule and Cell Patterning.

Science.gov (United States)

Tsougeni, Katerina; Ellinas, Kosmas; Koukouvinos, George; Petrou, Panagiota S; Tserepi, Angeliki; Kakabakos, Sotirios E; Gogolides, Evangelos

2018-01-01

Plasma micro-nanotexturing is a generic technology for topographical and chemical modification of surfaces and their implementation in microfluidics and microarrays. Nanotextured surfaces with desirable chemical functionality (and wetting behavior) have shown excellent biomolecule immobilization and cell adhesion. Specifically, nanotextured hydrophilic areas show (a) strong binding of biomolecules and (b) strong adhesion of cells, while nanotextured superhydrophobic areas show null adsorption of (a) proteins and (b) cells. Here we describe the protocols for (a) biomolecule adsorption control on nanotextured surfaces for microarray fabrication and (b) cell adhesion on such surfaces. 3D plasma nanotextured® substrates are commercialized through Nanoplasmas private company, a spin-off of the National Centre for Scientific Research Demokritos.

Nano-scale microfluidics to study 3D chemotaxis at the single cell level.

Directory of Open Access Journals (Sweden)

Corina Frick

Full Text Available Directed migration of cells relies on their ability to sense directional guidance cues and to interact with pericellular structures in order to transduce contractile cytoskeletal- into mechanical forces. These biomechanical processes depend highly on microenvironmental factors such as exposure to 2D surfaces or 3D matrices. In vivo, the majority of cells are exposed to 3D environments. Data on 3D cell migration are mostly derived from intravital microscopy or collagen-based in vitro assays. Both approaches offer only limited controllability of experimental conditions. Here, we developed an automated microfluidic system that allows positioning of cells in 3D microenvironments containing highly controlled diffusion-based chemokine gradients. Tracking migration in such gradients was feasible in real time at the single cell level. Moreover, the setup allowed on-chip immunocytochemistry and thus linking of functional with phenotypical properties in individual cells. Spatially defined retrieval of cells from the device allows down-stream off-chip analysis. Using dendritic cells as a model, our setup specifically allowed us for the first time to quantitate key migration characteristics of cells exposed to identical gradients of the chemokine CCL19 yet placed on 2D vs in 3D environments. Migration properties between 2D and 3D migration were distinct. Morphological features of cells migrating in an in vitro 3D environment were similar to those of cells migrating in animal tissues, but different from cells migrating on a surface. Our system thus offers a highly controllable in vitro-mimic of a 3D environment that cells traffic in vivo.

Patterns of failure in children with medulloblastoma treated with 3D conformal radiotherapy

International Nuclear Information System (INIS)

Skowronska-Gardas, Anna; Chojnacka, Marzanna; Morawska-Kaczynska, Marzena; Perek, Danuta; Perek-Polnik, Marta

2007-01-01

Background and purpose: Craniospinal irradiation for medulloblastoma is one of the most complex techniques employed in radiotherapy. Many reports stress the impact of irradiation quality on survival in these patients. Our report presents the outcome and patterns of failure for 95 patients treated with 3D conformal radiotherapy (3D-CRT). Materials and methods: From 1998 to 2003, 95 children with medulloblastoma received 3D conformal radiotherapy. All of them were previously treated with surgery and chemotherapy. The brain and upper spinal cord were treated with two lateral 6 MV photon fields. In four patients, the cribriform plate was irradiated by the additional field. For primary tumour bed we applied two or three photon beams. Spinal cord was irradiated either with 18-20 MeV electron fields or with a mixed beam. Results: With a median follow-up of 48 months, 32/95 patients suffered a multifocal (21) or isolated (11) recurrence. We evaluated every primary site of failure. In all patients, the recurrence appeared within the isodose level of 95-100%. Conclusions: Patterns of failure in medulloblastoma patients treated with 3D conformal radiotherapy indicated that the relapse was mainly associated with poor response to pre-irradiation chemotherapy. We believe that 3D conformal radiotherapy allows avoiding failures, related to radiotherapy uncertainties

Regulation of 1,25-dihydroxyvitamin D, receptors by [3H]-1,25-dihydroxyvitamin D3 in cultured cells (T-47D): evidence for receptor upregulation

International Nuclear Information System (INIS)

Reinhardt, T.A.; Horst, R.L.

1986-01-01

The authors examined the effect of 1,25-(OH) 2 D 3 on receptor concentration in cultured cells (T-47D). Two days prior to experiment, cells were fed with RPMI 1640 + 10% serum and 24-32 hours prior to experiment the media was replaced with RPMI 1640 + 25 mM Hepes + 1% serum. [ 3 H]-1,25-(OH) 2 D 3 +/- 100-fold molar excess cold hormone was used to treat the cells. Occupied receptors were measured in freshly prepared cytosols. Total receptors were measured following a 16-hour incubation of cytosols in the presence of 0.6 nM [ 3 H]-1,25-(OH) 2 D 3 +/- 100-fold molar excess of cold hormone at 4 0 C. Treatment of cell cultures for 16-18 hours with 0.5-1.0 nM [ 3 H]-1,25-(OH) 2 D 3 resulted in a 30-40% receptor occupancy by the hormone and a 2- to 3-fold increase in total cell receptor as compared to vehicle-treated controls. Time course studies showed a rapid increase in total receptors up to 16 hours post-treatment in the face of declining receptor occupancy. Actinomycin D blocked the [ 3 H]-1,25-(OH) 2 D 3 -dependent rise in cell receptor. The physiological significance of this receptor upregulation is not known nor is it known whether upregulation results from synthesis of new receptors and/or is the result of the activation of preformed receptors by a inducible activator protein

Development of a microfluidic perfusion 3D cell culture system

Science.gov (United States)

Park, D. H.; Jeon, H. J.; Kim, M. J.; Nguyen, X. D.; Morten, K.; Go, J. S.

2018-04-01

Recently, 3-dimensional in vitro cell cultures have gained much attention in biomedical sciences because of the closer relevance between in vitro cell cultures and in vivo environments. This paper presents a microfluidic perfusion 3D cell culture system with consistent control of long-term culture conditions to mimic an in vivo microenvironment. It consists of two sudden expansion reservoirs to trap incoming air bubbles, gradient generators to provide a linear concentration, and microchannel mixers. Specifically, the air bubbles disturb a flow in the microfluidic channel resulting in the instability of the perfusion cell culture conditions. For long-term stable operation, the sudden expansion reservoir is designed to trap air bubbles by using buoyancy before they enter the culture system. The performance of the developed microfluidic perfusion 3D cell culture system was examined experimentally and compared with analytical results. Finally, it was applied to test the cytotoxicity of cells infected with Ewing’s sarcoma. Cell death was observed for different concentrations of H2O2. For future work, the developed microfluidic perfusion 3D cell culture system can be used to examine the behavior of cells treated with various drugs and concentrations for high-throughput drug screening.

3D Reconstruction of Coronary Artery Vascular Smooth Muscle Cells.

Directory of Open Access Journals (Sweden)

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.

3D morphometry of red blood cells by digital holography.

Science.gov (United States)

Memmolo, Pasquale; Miccio, Lisa; Merola, Francesco; Gennari, Oriella; Netti, Paolo Antonio; Ferraro, Pietro

2014-12-01

Three dimensional (3D) morphometric analysis of flowing and not-adherent cells is an important aspect for diagnostic purposes. However, diagnostics tools need to be quantitative, label-free and, as much as possible, accurate. Recently, a simple holographic approach, based on shape from silhouette algorithm, has been demonstrated for accurate calculation of cells biovolume and displaying their 3D shapes. Such approach has been adopted in combination with holographic optical tweezers and successfully applied to cells with convex shape. Nevertheless, unfortunately, the method fails in case of specimen with concave surfaces. Here, we propose an effective approach to achieve correct 3D shape measurement that can be extended in case of cells having concave surfaces, thus overcoming the limit of the previous technique. We prove the new procedure for healthy red blood cells (RBCs) (i.e., discocytes) having a concave surface in their central region. Comparative analysis of experimental results with a theoretical 3D geometrical model of RBC is discussed in order to evaluate accuracy of the proposed approach. Finally, we show that the method can be also useful to classify, in terms of morphology, different varieties of RBCs. © 2014 International Society for Advancement of Cytometry.

3D bioprinting for engineering complex tissues.

Science.gov (United States)

Mandrycky, Christian; Wang, Zongjie; Kim, Keekyoung; Kim, Deok-Ho

2016-01-01

Bioprinting is a 3D fabrication technology used to precisely dispense cell-laden biomaterials for the construction of complex 3D functional living tissues or artificial organs. While still in its early stages, bioprinting strategies have demonstrated their potential use in regenerative medicine to generate a variety of transplantable tissues, including skin, cartilage, and bone. However, current bioprinting approaches still have technical challenges in terms of high-resolution cell deposition, controlled cell distributions, vascularization, and innervation within complex 3D tissues. While no one-size-fits-all approach to bioprinting has emerged, it remains an on-demand, versatile fabrication technique that may address the growing organ shortage as well as provide a high-throughput method for cell patterning at the micrometer scale for broad biomedical engineering applications. In this review, we introduce the basic principles, materials, integration strategies and applications of bioprinting. We also discuss the recent developments, current challenges and future prospects of 3D bioprinting for engineering complex tissues. Combined with recent advances in human pluripotent stem cell technologies, 3D-bioprinted tissue models could serve as an enabling platform for high-throughput predictive drug screening and more effective regenerative therapies. Copyright © 2015 Elsevier Inc. All rights reserved.

Vitamin D3 targets epidermal and dermal dendritic cells for induction of distinct regulatory T cells

NARCIS (Netherlands)

van der Aar, Angelic M. G.; Sibiryak, Darya S.; Bakdash, Ghaith; van Capel, Toni M. M.; van der Kleij, Hanneke P. M.; Opstelten, Dirk-Jan E.; Teunissen, Marcel B. M.; Kapsenberg, Martien L.; de Jong, Esther C.

2011-01-01

Background: The vitamin D metabolite 1,25(OH) 2D3 (VitD3) is a potent immunosuppressive drug and, among others, is used for topical treatment of psoriasis. A proposed mechanism of VitD3-mediated suppression is priming of dendritic cells (DCs) to induce regulatory T (Treg) cells. Objective:

Single molecule microscopy in 3D cell cultures and tissues.

Science.gov (United States)

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

2014-12-15

From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

Cytotoxicity of TSP in 3D Agarose Gel Cultured Cell.

Directory of Open Access Journals (Sweden)

Song-I Chun

Full Text Available A reference reagent, 3-(trimethylsilyl propionic-2, 2, 3, 3-d4 acid sodium (TSP, has been used frequently in nuclear magnetic resonance (NMR and magnetic resonance spectroscopy (MRS as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantitative and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells.A human osteosarcoma cell line (MG-63 was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conventional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT PicoGreen dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viability/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding.In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quantifying DNA (cell proliferation and cell viability. High concentrations of TSP (from 10 to 30 mM reduced both cell proliferation and viability (to 30% of the control after one week of exposure, but no such effects were found using low concentrations of TSP (0-10 mM.This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure.

Nucleus and nucleus-cytoskeleton connections in 3D cell migration

Energy Technology Data Exchange (ETDEWEB)

Liu, Lingling, E-mail: liulingling2012@163.com; Luo, Qing, E-mail: qing.luo@cqu.edu.cn; Sun, Jinghui, E-mail: sunjhemail@163.com; Song, Guanbin, E-mail: song@cqu.edu.cn

2016-10-15

Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and review how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration. - Graphical abstract: Schematic representations of a cell migrating on a 2D substrate and a cell migrating in a 3D extracellular matrix environment. (A) Nucleus-cytoskeleton connections are essential to 3D migration. Mechanical signals are transduced by integrins at the cell surface and channeled to cytoskeletal proteins, which generates prestress. The nucleus-cytoskeleton connections can either act as a stable skeleton to anchor the nuclei or provide active force to move the nuclei. The LINC complex is responsible for the nucleo-cytoskeletal coupling. Nesprins connect the cytoskeletal proteins to the inner nuclear membrane proteins SUN1 and SUN2. The SUN proteins connect to the lamins that form the lamina, which attaches to the chromatin. This physical connectivity transmits the mechanical signals from receptors at

Nucleus and nucleus-cytoskeleton connections in 3D cell migration

International Nuclear Information System (INIS)

Liu, Lingling; Luo, Qing; Sun, Jinghui; Song, Guanbin

2016-01-01

Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and review how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration. - Graphical abstract: Schematic representations of a cell migrating on a 2D substrate and a cell migrating in a 3D extracellular matrix environment. (A) Nucleus-cytoskeleton connections are essential to 3D migration. Mechanical signals are transduced by integrins at the cell surface and channeled to cytoskeletal proteins, which generates prestress. The nucleus-cytoskeleton connections can either act as a stable skeleton to anchor the nuclei or provide active force to move the nuclei. The LINC complex is responsible for the nucleo-cytoskeletal coupling. Nesprins connect the cytoskeletal proteins to the inner nuclear membrane proteins SUN1 and SUN2. The SUN proteins connect to the lamins that form the lamina, which attaches to the chromatin. This physical connectivity transmits the mechanical signals from receptors at

Fabrication of highly modulable fibrous 3D extracellular microenvironments

KAUST Repository

Zhang, Xixiang; Han, Fangfei; Syed, Ahad; Bukhari, Ebtihaj M.; Siang, Basil Chew Joo; Yang, Shan; Zhou, Bingpu; Wen, Wei-jia; Jiang, Dechen

2017-01-01

Three-dimensional (3D) in vitro scaffolds that mimic the irregular fibrous structures of in vivo extracellular matrix (ECM) are critical for many important biological applications. However, structural properties modulation of fibrous 3D scaffolds remains a challenge. Here, we report the first highly modulable 3D fibrous scaffolds self-assembled by high-aspect-ratio (HAR) microfibers. The scaffolds structural properties can be easily tailored to incorporate various physical cues, including geometry, stiffness, heterogeneity and nanotopography. Moreover, the fibrous scaffolds are readily and accurately patterned on desired locations of the substrate. Cell culture exhibits that our scaffolds can elicit strong bidirectional cell-material interactions. Furthermore, a functional disparity between the two-dimensional substrate and our 3D scaffolds is identified by cell spreading and proliferation data. These results prove the potential of the proposed scaffold as a biomimetic extracellular microenvironment for cell study.

Fabrication of highly modulable fibrous 3D extracellular microenvironments

KAUST Repository

Zhang, Xixiang

2017-06-13

Three-dimensional (3D) in vitro scaffolds that mimic the irregular fibrous structures of in vivo extracellular matrix (ECM) are critical for many important biological applications. However, structural properties modulation of fibrous 3D scaffolds remains a challenge. Here, we report the first highly modulable 3D fibrous scaffolds self-assembled by high-aspect-ratio (HAR) microfibers. The scaffolds structural properties can be easily tailored to incorporate various physical cues, including geometry, stiffness, heterogeneity and nanotopography. Moreover, the fibrous scaffolds are readily and accurately patterned on desired locations of the substrate. Cell culture exhibits that our scaffolds can elicit strong bidirectional cell-material interactions. Furthermore, a functional disparity between the two-dimensional substrate and our 3D scaffolds is identified by cell spreading and proliferation data. These results prove the potential of the proposed scaffold as a biomimetic extracellular microenvironment for cell study.

A 3D-printed microbial cell culture platform with in situ PEGDA hydrogel barriers for differential substrate delivery.

Science.gov (United States)

Kadilak, Andrea L; Rehaag, Jessica C; Harrington, Cameron A; Shor, Leslie M

2017-09-01

Additive manufacturing, or 3D-printing techniques have recently begun to enable simpler, faster, and cheaper production of millifluidic devices at resolutions approaching 100-200  μ m. At this resolution, cell culture devices can be constructed that more accurately replicate natural environments compared with conventional culturing techniques. A number of microfluidics researchers have begun incorporating additive manufacturing into their work, using 3D-printed devices in a wide array of chemical, fluidic, and even some biological applications. Here, we describe a 3D-printed cell culture platform and demonstrate its use in culturing Pseudomonas putida KT2440 bacteria for 44 h under a differential substrate gradient. Polyethylene glycol diacrylate (PEGDA) hydrogel barriers are patterned in situ within a 3D-printed channel. Transport of the toluidine blue tracer dye through the hydrogel barriers is characterized. Nutrients and oxygen were delivered to cells in the culture region by diffusion through the PEGDA hydrogel barriers from adjacent media or saline perfusion channels. Expression of green fluorescent protein by P. putida KT2440 enabled real time visualization of cell density within the 3D-printed channel, and demonstrated cells were actively expressing protein over the course of the experiment. Cells were observed clustering near hydrogel barrier boundaries where fresh substrate and oxygen were being delivered via diffusive transport, but cells were unable to penetrate the barrier. The device described here provides a versatile and easy to implement platform for cell culture in readily controlled gradient microenvironments. By adjusting device geometry and hydrogel properties, this platform could be further customized for a wide variety of biological applications.

Advances in 3D neuronal cell culture

NARCIS (Netherlands)

Frimat, Jean Philippe; Xie, Sijia; Bastiaens, Alex; Schurink, Bart; Wolbers, Floor; Den Toonder, Jaap; Luttge, Regina

2015-01-01

In this contribution, the authors present our advances in three-dimensional (3D) neuronal cell culture platform technology contributing to controlled environments for microtissue engineering and analysis of cellular physiological and pathological responses. First, a micromachined silicon sieving

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

Lifescience Database Archive (English)

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 ...

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

Lifescience Database Archive (English)

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 ...

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

Science.gov (United States)

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. © The Author(s) 2016.

3D mapping of individual cells using a proton microbeam

International Nuclear Information System (INIS)

Michelet, C.; Moretto, Ph.

1999-01-01

Various imaging techniques carried out with a nuclear microprobe make it possible to reveal by 2D mapping, the internal structure of isolated cells. An improvement of those techniques allows today 3D mapping of cells. STIM- and PIXE-Tomography have been recently implemented on the CENBG microbeam line. The performance offered by these methods, which are capable of resolving objects having diameters less then 100 μm, has been validated on reference specimens and on human cells from cultures. In addition to the fineness of the resolution, these techniques offer the advantage of performing volume analyses without prior cutting of the samples. The ultimate aim of this program of research is to perform 3D elemental chemical analysis of individual cells in the field of biomedicine

Axial tomography in 3D live cell microscopy

Science.gov (United States)

Richter, Verena; Bruns, Sarah; Bruns, Thomas; Piper, Mathis; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

2017-07-01

A miniaturized setup for sample rotation on a microscope stage has been developed, combined with light sheet, confocal or structured illumination microscopy and applied to living cells as well as to small organisms. This setup permits axial tomography with improved visualization of single cells or small cell clusters as well as an enhanced effective 3D resolution upon sample rotation.

Practical 3-D Beam Pattern Based Channel Modeling for Multi-Polarized Massive MIMO Systems.

Science.gov (United States)

Aghaeinezhadfirouzja, Saeid; Liu, Hui; Balador, Ali

2018-04-12

In this paper, a practical non-stationary three-dimensional (3-D) channel models for massive multiple-input multiple-output (MIMO) systems, considering beam patterns for different antenna elements, is proposed. The beam patterns using dipole antenna elements with different phase excitation toward the different direction of travels (DoTs) contributes various correlation weights for rays related towards/from the cluster, thus providing different elevation angle of arrivals (EAoAs) and elevation angle of departures (EAoDs) for each antenna element. These include the movements of the user that makes our channel to be a non-stationary model of clusters at the receiver (RX) on both the time and array axes. In addition, their impacts on 3-D massive MIMO channels are investigated via statistical properties including received spatial correlation. Additionally, the impact of elevation/azimuth angles of arrival on received spatial correlation is discussed. Furthermore, experimental validation of the proposed 3-D channel models on azimuth and elevation angles of the polarized antenna are specifically evaluated and compared through simulations. The proposed 3-D generic models are verified using relevant measurement data.

3D Texture Analysis in Renal Cell Carcinoma Tissue Image Grading

Science.gov (United States)

Cho, Nam-Hoon; Choi, Heung-Kook

2014-01-01

One of the most significant processes in cancer cell and tissue image analysis is the efficient extraction of features for grading purposes. This research applied two types of three-dimensional texture analysis methods to the extraction of feature values from renal cell carcinoma tissue images, and then evaluated the validity of the methods statistically through grade classification. First, we used a confocal laser scanning microscope to obtain image slices of four grades of renal cell carcinoma, which were then reconstructed into 3D volumes. Next, we extracted quantitative values using a 3D gray level cooccurrence matrix (GLCM) and a 3D wavelet based on two types of basis functions. To evaluate their validity, we predefined 6 different statistical classifiers and applied these to the extracted feature sets. In the grade classification results, 3D Haar wavelet texture features combined with principal component analysis showed the best discrimination results. Classification using 3D wavelet texture features was significantly better than 3D GLCM, suggesting that the former has potential for use in a computer-based grading system. PMID:25371701

3D Texture Analysis in Renal Cell Carcinoma Tissue Image Grading

Directory of Open Access Journals (Sweden)

Tae-Yun Kim

2014-01-01

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

Biophysical force regulation in 3D tumor cell invasion

Science.gov (United States)

Wu, Mingming

When embedded within 3D extracellular matrices (ECM), animal cells constantly probe and adapt to the ECM locally (at cell length scale) and exert forces and communicate with other cells globally (up to 10 times of cell length). It is now well accepted that mechanical crosstalk between animal cells and their microenvironment critically regulate cell function such as migration, proliferation and differentiation. Disruption of the cell-ECM crosstalk is implicated in a number of pathologic processes including tumor progression and fibrosis. Central to the problem of cell-ECM crosstalk is the physical force that cells generate. By measuring single cell generated force within 3D collagen matrices, we revealed a mechanical crosstalk mechanism between the tumor cells and the ECM. Cells generate sufficient force to stiffen collagen fiber network, and stiffer matrix, in return promotes larger cell force generation. Our work highlights the importance of fibrous nonlinear elasticity in regulating tumor cell-ECM interaction, and results may have implications in the rapid tissue stiffening commonly found in tumor progression and fibrosis. This work is partially supported by NIH Grants R21RR025801 and R21GM103388.

Pattern recognition: invariants in 3D

International Nuclear Information System (INIS)

Proriol, J.

1992-01-01

In e + e - events, the jets have a spherical 3D symmetry. A set of invariants are defined for 3D objects with a spherical symmetry. These new invariants are used to tag the number of jets in e + e - events. (K.A.) 3 refs

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

Directory of Open Access Journals (Sweden)

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.

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells

DEFF Research Database (Denmark)

Chandrasekaran, Abinaya; Avci, Hasan; Ochalek, Anna

2017-01-01

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency......), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells...... the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6+/NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural...

Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

International Nuclear Information System (INIS)

Lambros, Maria Polikandritou; Parsa, Cyrus; Mulamalla, HariChandana; Orlando, Robert; Lau, Bernard; Huang, Ying; Pon, Doreen; Chow, Moses

2011-01-01

Research highlights: → We irradiated a 3-D human oral cell culture of keratinocytes and fibroblasts with 12 and 2 Gy. → 6 h after irradiation the histopathology and apoptosis of the 3-D culture were evaluated. Microarrays were used to assess the gene expression in the irradiated 3-D tissue. → 12 Gy induced significant histopathologic changes and cellular apoptosis. → 12 Gy significantly affected genes of the NF-kB pathway, inflammatory cytokines and DAMPs. -- Abstract: Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first

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

Directory of Open Access Journals (Sweden)

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

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

Science.gov (United States)

Wang, Tao; Green, Ryan; Nair, Rajesh Ramakrishnan; Howell, Mark; Mohapatra, Subhra; Guldiken, Rasim; Mohapatra, Shyam Sundar

2015-12-19

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 3D cell

[D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer cells in vitro.

OpenAIRE

Woll, P J; Rozengurt, E

1988-01-01

In the search for a more potent bombesin antagonist, we found [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P to be effective in mouse fibroblasts and to inhibit the growth of small cell lung cancer, a tumor that secretes bombesin-like peptides that may act as autocrine growth factors. In murine Swiss 3T3 cells, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P proved to be a bombesin antagonist as judged by the following criteria: (i) inhibition of DNA synthesis induced by gastrin-releasing peptide and ot...

Cell migration through connective tissue in 3-D

Science.gov (United States)

Fabry, Ben

2008-03-01

A prerequisite for metastasis formation is the ability of tumor cells to invade and migrate through connective tissue. Four key components endow tumor cells with this ability: secretion of matrix-degrading enzymes, firm but temporary adhesion onto connective tissue fibers, contractile force generation, and rapid remodeling of cytoskeletal structures. Cell adhesion, contraction, and cytoskeletal remodeling are biomechanical parameter that can be measured on single cells using a panel of biophysical methods. We use 2-D and 3-D traction microscopy to measure contractile forces; magnetic tweezer microrheology to estimate adhesion strengths, cytoskeletal stiffness and molecular turn-over rates; and nanoscale particle tracking to measure cytoskeletal remodeling. On a wide range of tumor cell lines we could show that cell invasiveness correlates with increased expression of integrin adhesion receptors, increased contractile force generation, and increased speed of cytoskeletal reorganization. Each of those biomechanical parameters, however, varied considerably between cell lines of similar invasivity, suggesting that tumor cells employ multiple invasion strategies that cannot be unambiguously characterized using a single assay.

3D cancer cell migration in a confined matrix

Science.gov (United States)

Alobaidi, Amani; Sun, Bo

Cancer cell migration is widely studied in 2D motion, which does not mimic the invasion processes in vivo. More recently, 3D cell migration studies have been performed. The ability of cancer cells to migrate within the extracellular matrix depends on the physical and biochemical features of the extracellular matrix. We present a model of cell motility in confined matrix geometry. The aim of the study is to study cancer migration in collagen matrix, as a soft tissue, to investigate their motility within the confined and surrounding collagen environment. Different collagen concentrations have been used to show the ability of these cancer cells to move through such a complex structure by measuring Cancer cell migration velocity as well as the displacement. Graduate student physics department.

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

International Nuclear Information System (INIS)

Borau, C; García-Aznar, J M; Kamm, R D

2011-01-01

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

Hyperbolically Patterned 3D Graphene Metamaterial with Negative Poisson's Ratio and Superelasticity.

Science.gov (United States)

Zhang, Qiangqiang; Xu, Xiang; Lin, Dong; Chen, Wenli; Xiong, Guoping; Yu, Yikang; Fisher, Timothy S; Li, Hui

2016-03-16

A hyperbolically patterned 3D graphene metamaterial (GM) with negative Poisson's ratio and superelasticity is highlighted. It is synthesized by a modified hydrothermal approach and subsequent oriented freeze-casting strategy. GM presents a tunable Poisson's ratio by adjusting the structural porosity, macroscopic aspect ratio (L/D), and freeze-casting conditions. Such a GM suggests promising applications as soft actuators, sensors, robust shock absorbers, and environmental remediation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

Science.gov (United States)

Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A; Davidson, Michael W; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M; Fabry, Ben

2015-11-01

Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation. © FASEB.

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.

Science.gov (United States)

Chandrasekaran, Abinaya; Avci, Hasan X; Ochalek, Anna; Rösingh, Lone N; Molnár, Kinga; László, Lajos; Bellák, Tamás; Téglási, Annamária; Pesti, Krisztina; Mike, Arpad; Phanthong, Phetcharat; Bíró, Orsolya; Hall, Vanessa; Kitiyanant, Narisorn; Krause, Karl-Heinz; Kobolák, Julianna; Dinnyés, András

2017-12-01

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6 + /NESTIN + cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

Science.gov (United States)

Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

2017-02-28

Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

BAFF induces spleen CD4+ T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

International Nuclear Information System (INIS)

Ji, Fang; Chen, Rongjing; Liu, Baojun; Zhang, Xiaoping; Han, Junli; Wang, Haining; Shen, Gang; Tao, Jiang

2012-01-01

Highlights: ► Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4 + T cells. ► Carrying out siRNA technology to study FOXO3A protein function. ► Helpful to understand the T cell especially CD4 + T cell‘s role in immunological reaction. -- Abstract: The TNF ligand family member “B cell-activating factor belonging to the TNF family” (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4 + spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4 + T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4 + spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4 + T cell proliferation.

Perfusion directed 3D mineral formation within cell-laden hydrogels.

Science.gov (United States)

Sawyer, Stephen William; Shridhar, Shivkumar Vishnempet; Zhang, Kairui; Albrecht, Lucas; Filip, Alex; Horton, Jason; Soman, Pranav

2018-06-08

Despite the promise of stem cell engineering and the new advances in bioprinting technologies, one of the major challenges in the manufacturing of large scale bone tissue scaffolds is the inability to perfuse nutrients throughout thick constructs. Here, we report a scalable method to create thick, perfusable bone constructs using a combination of cell-laden hydrogels and a 3D printed sacrificial polymer. Osteoblast-like Saos-2 cells were encapsulated within a gelatin methacrylate (GelMA) hydrogel and 3D printed polyvinyl alcohol (PVA) pipes were used to create perfusable channels. A custom-built bioreactor was used to perfuse osteogenic media directly through the channels in order to induce mineral deposition which was subsequently quantified via microCT. Histological staining was used to verify mineral deposition around the perfused channels, while COMSOL modeling was used to simulate oxygen diffusion between adjacent channels. This information was used to design a scaled-up construct containing a 3D array of perfusable channels within cell-laden GelMA. Progressive matrix mineralization was observed by cells surrounding perfused channels as opposed to random mineral deposition in static constructs. MicroCT confirmed that there was a direct relationship between channel mineralization within perfused constructs and time within the bioreactor. Furthermore, the scalable method presented in this work serves as a model on how large-scale bone tissue replacement constructs could be made using commonly available 3D printers, sacrificial materials, and hydrogels. © 2018 IOP Publishing Ltd.

Nanoscale Analysis of a Hierarchical Hybrid Solar Cell in 3D.

Science.gov (United States)

Divitini, Giorgio; Stenzel, Ole; Ghadirzadeh, Ali; Guarnera, Simone; Russo, Valeria; Casari, Carlo S; Bassi, Andrea Li; Petrozza, Annamaria; Di Fonzo, Fabio; Schmidt, Volker; Ducati, Caterina

2014-05-01

A quantitative method for the characterization of nanoscale 3D morphology is applied to the investigation of a hybrid solar cell based on a novel hierarchical nanostructured photoanode. A cross section of the solar cell device is prepared by focused ion beam milling in a micropillar geometry, which allows a detailed 3D reconstruction of the titania photoanode by electron tomography. It is found that the hierarchical titania nanostructure facilitates polymer infiltration, thus favoring intermixing of the two semiconducting phases, essential for charge separation. The 3D nanoparticle network is analyzed with tools from stochastic geometry to extract information related to the charge transport in the hierarchical solar cell. In particular, the experimental dataset allows direct visualization of the percolation pathways that contribute to the photocurrent.

Proposal for the development of 3D Vertically Integrated Pattern Recognition Associative Memory (VIPRAM)

Energy Technology Data Exchange (ETDEWEB)

Deptuch, Gregory; Hoff, Jim; Kwan, Simon; Lipton, Ron; Liu, Ted; Ramberg, Erik; Todri, Aida; Yarema, Ray; /Fermilab; Demarteua, Marcel,; Drake, Gary; Weerts, Harry; /Argonne /Chicago U. /Padua U. /INFN, Padua

2010-10-01

Future particle physics experiments looking for rare processes will have no choice but to address the demanding challenges of fast pattern recognition in triggering as detector hit density becomes significantly higher due to the high luminosity required to produce the rare process. The authors propose to develop a 3D Vertically Integrated Pattern Recognition Associative Memory (VIPRAM) chip for HEP applications, to advance the state-of-the-art for pattern recognition and track reconstruction for fast triggering.

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

Directory of Open Access Journals (Sweden)

Christoph eSchmitz

2014-05-01

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

A Versatile Method of Patterning Proteins and Cells.

Science.gov (United States)

Shrirao, Anil B; Kung, Frank H; Yip, Derek; Firestein, Bonnie L; Cho, Cheul H; Townes-Anderson, Ellen

2017-02-26

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

Caveolin-1 mediated radioresistance of 3D grown pancreatic cancer cells

International Nuclear Information System (INIS)

Hehlgans, Stephanie; Eke, Iris; Storch, Katja; Haase, Michael; Baretton, Gustavo B.; Cordes, Nils

2009-01-01

Background and purpose: Resistance of pancreatic ductal adenocarcinoma (PDAC) to chemo- and radiotherapy is a major obstacle. The integral membrane protein Caveolin-1 (Cav-1) has been suggested as a potent target in human pancreatic carcinoma cells. Materials and methods: Human pancreatic tumor cells were examined in a three-dimensional (3D) cell culture model with regard to clonogenic survival, apoptosis, radiogenic DNA-double strand breaks and protein expression and phosphorylation under siRNA-mediated knockdown of Cav-1 without and in combination with irradiation (X-rays, 0-6 Gy). Immunohistochemistry was used to assess Cav-1 expression in biopsies from patients with PDAC. Results: Tumor cells in PDAC showed significantly higher Cav-1 expression relative to tumor stroma. Cav-1 knockdown significantly reduced β1 integrin expression and Akt phosphorylation, induced Caspase 3- and Caspase 8-dependent apoptosis and enhanced the radiosensitivity of 3D cell cultures. While cell cycling and Cav-1 promoter activity remained stable, Cav-1 knockdown-induced radiosensitization correlated with elevated numbers of residual DNA-double strand breaks. Conclusions: Our data strongly support the concept of Cav-1 as a potent target in pancreatic carcinoma cells due to radiosensitization and Cav-1 overexpression in tumor cells of PDAC. 3D cell cultures are powerful and useful tools for the testing of novel targeting strategies to optimize conventional radio- and chemotherapy regimes for PDAC.

Podosomes, But Not the Maturation Status, Determine the Protease-Dependent 3D Migration in Human Dendritic Cells.

Science.gov (United States)

Cougoule, Céline; Lastrucci, Claire; Guiet, Romain; Mascarau, Rémi; Meunier, Etienne; Lugo-Villarino, Geanncarlo; Neyrolles, Olivier; Poincloux, Renaud; Maridonneau-Parini, Isabelle

2018-01-01

Dendritic cells (DC) are professional Antigen-Presenting Cells scattered throughout antigen-exposed tissues and draining lymph nodes, and survey the body for pathogens. Their ability to migrate through tissues, a 3D environment, is essential for an effective immune response. Upon infection, recognition of Pathogen-Associated Molecular Patterns (PAMP) by Toll-like receptors (TLR) triggers DC maturation. Mature DC (mDC) essentially use the protease-independent, ROCK-dependent amoeboid mode in vivo , or in collagen matrices in vitro . However, the mechanisms of 3D migration used by human immature DC (iDC) are still poorly characterized. Here, we reveal that human monocyte-derived DC are able to use two migration modes in 3D. In porous matrices of fibrillar collagen I, iDC adopted the amoeboid migration mode. In dense matrices of gelled collagen I or Matrigel, iDC used the protease-dependent, ROCK-independent mesenchymal migration mode. Upon TLR4 activation by LPS, mDC-LPS lose the capacity to form podosomes and degrade the matrix along with impaired mesenchymal migration. TLR2 activation by Pam 3 CSK 4 resulted in DC maturation, podosome maintenance, and efficient mesenchymal migration. Under all these conditions, when DC used the mesenchymal mode in dense matrices, they formed 3D podosomes at the tip of cell protrusions. Using PGE 2 , known to disrupt podosomes in DC, we observed that the cells remained in an immature status and the mesenchymal migration mode was abolished. We also observed that, while CCL5 (attractant of iDC) enhanced both amoeboid and mesenchymal migration of iDC, CCL19 and CCL21 (attractants of mDC) only enhanced mDC-LPS amoeboid migration without triggering mesenchymal migration. Finally, we examined the migration of iDC in tumor cell spheroids, a tissue-like 3D environment. We observed that iDC infiltrated spheroids of tumor cells using both migration modes. Altogether, these results demonstrate that human DC adopt the mesenchymal mode to

Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix.

Science.gov (United States)

Wang, Xuanzhi; Dai, Xingliang; Zhang, Xinzhi; Li, Xinda; Xu, Tao; Lan, Qing

2018-04-15

Cancer stem cells (CSCs), being tumor-initiating with self-renewal capacity and heterogeneity, are most likely the cause of tumor resistance, reoccurrence and metastasis. To further investigate the role of CSCs in tumor biology, there is a need to develop an effective culture system to grow, maintain and enrich CSCs. Three-dimensional (3D) cell culture model has been widely used in tumor research and drug screening. Recently, researchers have begun to utilize 3D models to culture cancer cells for CSCs enrichment. In this study, glioma cell line was cultured with 3D porous chitosan (CS) scaffolds or chitosan-hyaluronic acid (CS-HA) scaffolds to explore the possibility of glioma stem cells (GSCs)-like cells enrichment, to study the morphology, gene expression, and in vivo tumorigenicity of 3D scaffolds cells, and to compare results to 2D controls. Results showed that glioma cells on both CS and CS-HA scaffolds could form tumor cell spheroids and increased the expression of GSCs biomarkers compared to conventional 2D monolayers. Furthermore, cells in CS-HA scaffolds had higher expression levels of epithelial-to-mesenchymal transition (EMT)-related gene. Specifically, the in vivo tumorigenicity capability of CS-HA scaffold cultured cells was greater than 2D cells or CS scaffold cultured cells. It is indicated that the chemical composition of scaffold plays an important role in the enrichment of CSCs. Our results suggest that CS-HA scaffolds have a better capability to enrich GSCs-like cells and can serve as a simple and effective way to cultivate and enrich CSCs in vitro to support the study of CSCs biology and development of novel anti-cancer therapies. Copyright © 2018 Elsevier Inc. All rights reserved.

Galectin-3 and cyclin D1 expression in non-small cell lung cancer

Directory of Open Access Journals (Sweden)

Gołecki Marcin

2011-10-01

Full Text Available Abstract Introduction Lung cancer is a major cause of mortality and morbidity worldwide. Galectin-3 is multifunctional protein, which is involved in regulation of cell growth, cell adhesion, cell proliferation, angiogenesis and apoptosis. Cyclin D1 together with other cyclin plays an important role in cell cycle control. Cyclin D1 regulates the G1-to-S phase transition. The aim of this study was the evaluation of correlations between clinicopathological findings and cyclin D1 and galectin-3 expression in non-small cell lung cancer (NSCLC. We wanted also to analyze the prognostic value of cyclin D1 and galectin-3 expression. Moreover we tried to evaluate the correlations between galectin-3 and cyclin D1 expression in tumor tissue. Materials and methods We used the immunochemistry method to investigate the expression of galectin-3 and cyclin D1 in the paraffin-embedded tumor tissue of 47 patients (32 men and 15 women; mean age 59.34 ± 8.90. years. We used monoclonal antibodies to cyclin D1 (NCL-L-cyclin D1-GM clone P2D11F11 NOVO CASTRA and to galectin-3 (mouse monoclonal antibody NCL-GAL3 NOVO CASTRA. Results Galectin-3 expression was positive in 18 cases (38.29% and cyclin D1 in 39 (82.97%. We showed only weak trend, that galectin-3 expression was lower in patients without lymph node involvement (p = 0.07 and cyclin D1 expression was higher in this group (p = 0.080. We didn't reveal differences in cyclin D1 and galectin-3 expression in SCC and adenocarcinoma patients. We didn't demonstrated also differences in galectin-3 and cyclin D1 expression depending on disease stage. Moreover we analyzed the prognostic value of cyclin D1 expression and galectin-3 in all examinated patients and separately in SCC and in adenocarcinoma and in all stages, but we didn't find any statistical differences. We demonstrated that in galectin-3 positive tumors cyclin D1 expression was higher (96.55% vs 61.11%, Chi2 Yatesa 7.53, p = 0.0061 and we revealed negative

Cell patterning through inkjet printing of one cell per droplet

International Nuclear Information System (INIS)

Yamaguchi, Shuichi; Akiyama, Yoshitake; Morishima, Keisuke; Ueno, Akira

2012-01-01

The inkjet ejection technology used in printers has been adopted and research has been conducted on manufacturing artificial tissue by patterning cells through micronozzle ejection of small droplets containing multiple cells. However, stable injection of cells has proven difficult, owing to the frequent occurrence of nozzle clogging. In this paper, a piezoelectric inkjet head constructed with a glass capillary that enabled viewing of the nozzle section was developed, the movement of cells ejected from the nozzle tip was analyzed, and a method for stably ejecting cells was verified. A pull–push ejection method was compared with a push–pull ejection method regarding the voltage waveform applied to the piezoelectric element of the head. The push–pull method was found to be more suitable for stable ejection. Further, ejection of one cell per droplet was realized by detecting the position of the cell in the nozzle section and utilizing these position data. Thus, a method for more precise patterning of viable cells at desired position and number was established. This method is very useful and promising not only for biofabrication, 3D tissue construction, cell printing, but also for a number of biomedical application, such as bioMEMS, lab on a chip research field. (paper)

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) Signaling Capacity and the Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer (NSCLC): Implications for Use of 1,25(OH)2D3 in NSCLC Treatment

International Nuclear Information System (INIS)

Upadhyay, Santosh Kumar; Verone, Alissa; Shoemaker, Suzanne; Qin, Maochun; Liu, Song; Campbell, Moray; Hershberger, Pamela A.

2013-01-01

1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) exerts anti-proliferative activity by binding to the vitamin D receptor (VDR) and regulating gene expression. We previously reported that non-small cell lung cancer (NSCLC) cells which harbor epidermal growth factor receptor (EGFR) mutations display elevated VDR expression (VDR high ) and are vitamin D-sensitive. Conversely, those with K-ras mutations are VDR low and vitamin D-refractory. Because EGFR mutations are found predominately in NSCLC cells with an epithelial phenotype and K-ras mutations are more common in cells with a mesenchymal phenotype, we investigated the relationship between vitamin D signaling capacity and the epithelial mesenchymal transition (EMT). Using NSCLC cell lines and publically available lung cancer cell line microarray data, we identified a relationship between VDR expression, 1,25(OH) 2 D 3 sensitivity, and EMT phenotype. Further, we discovered that 1,25(OH) 2 D 3 induces E-cadherin and decreases EMT-related molecules SNAIL, ZEB1, and vimentin in NSCLC cells. 1,25(OH) 2 D 3 -mediated changes in gene expression are associated with a significant decrease in cell migration and maintenance of epithelial morphology. These data indicate that 1,25(OH) 2 D 3 opposes EMT in NSCLC cells. Because EMT is associated with increased migration, invasion, and chemoresistance, our data imply that 1,25(OH) 2 D 3 may prevent lung cancer progression in a molecularly defined subset of NSCLC patients

3D Printing of Thermo-Responsive Methylcellulose Hydrogels for Cell-Sheet Engineering.

Science.gov (United States)

Cochis, Andrea; Bonetti, Lorenzo; Sorrentino, Rita; Contessi Negrini, Nicola; Grassi, Federico; Leigheb, Massimiliano; Rimondini, Lia; Farè, Silvia

2018-04-10

A possible strategy in regenerative medicine is cell-sheet engineering (CSE), i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS). The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC)-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na₂SO₄ and PBS). MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3) and endothelial murine cells (MS1), and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues.

3D Printing of Thermo-Responsive Methylcellulose Hydrogels for Cell-Sheet Engineering

Directory of Open Access Journals (Sweden)

Andrea Cochis

2018-04-01

Full Text Available A possible strategy in regenerative medicine is cell-sheet engineering (CSE, i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS. The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na2SO4 and PBS. MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3 and endothelial murine cells (MS1, and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues.

3D-Printed external light traps for solar cells

NARCIS (Netherlands)

van Dijk, L.; Paetzold, U.W.; Blab, Gerhard; Marcus, E.A.P.; Oostra, A.J.; van de Groep, J.; Polman, A.; Schropp, R.E.I.; Di Vece, M.

2015-01-01

We demonstrate a universally applicable 3D-printed external light trap for solar cells. We placed a macroscopic external light trap made of smoothened, silver coated plastic at the sun-facing surface of different types of solar cells. The trap consists of a reflective parabolic concentrator on top

3D tissue formation by stacking detachable cell sheets formed on nanofiber mesh.

Science.gov (United States)

Kim, Min Sung; Lee, Byungjun; Kim, Hong Nam; Bang, Seokyoung; Yang, Hee Seok; Kang, Seong Min; Suh, Kahp-Yang; Park, Suk-Hee; Jeon, Noo Li

2017-03-23

We present a novel approach for assembling 3D tissue by layer-by-layer stacking of cell sheets formed on aligned nanofiber mesh. A rigid frame was used to repeatedly collect aligned electrospun PCL (polycaprolactone) nanofiber to form a mesh structure with average distance between fibers 6.4 µm. When human umbilical vein endothelial cells (HUVECs), human foreskin dermal fibroblasts, and skeletal muscle cells (C2C12) were cultured on the nanofiber mesh, they formed confluent monolayers and could be handled as continuous cell sheets with areas 3 × 3 cm 2 or larger. Thicker 3D tissues have been formed by stacking multiple cell sheets collected on frames that can be nested (i.e. Matryoshka dolls) without any special tools. When cultured on the nanofiber mesh, skeletal muscle, C2C12 cells oriented along the direction of the nanofibers and differentiated into uniaxially aligned multinucleated myotube. Myotube cell sheets were stacked (upto 3 layers) in alternating or aligned directions to form thicker tissue with ∼50 µm thickness. Sandwiching HUVEC cell sheets with two dermal fibroblast cell sheets resulted in vascularized 3D tissue. HUVECs formed extensive networks and expressed CD31, a marker of endothelial cells. Cell sheets formed on nanofiber mesh have a number of advantages, including manipulation and stacking of multiple cell sheets for constructing 3D tissue and may find applications in a variety of tissue engineering applications.

Lensfree diffractive tomography for the imaging of 3D cell cultures

Science.gov (United States)

Berdeu, Anthony; Momey, Fabien; Dinten, Jean-Marc; Gidrol, Xavier; Picollet-D'hahan, Nathalie; Allier, Cédric

2017-02-01

New microscopes are needed to help reaching the full potential of 3D organoid culture studies by gathering large quantitative and systematic data over extended periods of time while preserving the integrity of the living sample. In order to reconstruct large volumes while preserving the ability to catch every single cell, we propose new imaging platforms based on lens-free microscopy, a technic which is addressing these needs in the context of 2D cell culture, providing label-free and non-phototoxic acquisition of large datasets. We built lens-free diffractive tomography setups performing multi-angle acquisitions of 3D organoid cultures embedded in Matrigel and developed dedicated 3D holographic reconstruction algorithms based on the Fourier diffraction theorem. Nonetheless, holographic setups do not record the phase of the incident wave front and the biological samples in Petri dish strongly limit the angular coverage. These limitations introduce numerous artefacts in the sample reconstruction. We developed several methods to overcome them, such as multi-wavelength imaging or iterative phase retrieval. The most promising technic currently developed is based on a regularised inverse problem approach directly applied on the 3D volume to reconstruct. 3D reconstructions were performed on several complex samples such as 3D networks or spheroids embedded in capsules with large reconstructed volumes up to 25 mm3 while still being able to identify single cells. To our knowledge, this is the first time that such an inverse problem approach is implemented in the context of lens-free diffractive tomography enabling to reconstruct large fully 3D volumes of unstained biological samples.

BAFF induces spleen CD4{sup +} T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

Energy Technology Data Exchange (ETDEWEB)

Ji, Fang; Chen, Rongjing [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Liu, Baojun [Laboratory of Lung, Inflammation and Cancers, Huashan Hospital, Fudan University, Shanghai (China); Zhang, Xiaoping [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Han, Junli; Wang, Haining [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Shen, Gang [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Tao, Jiang, E-mail: taojiang2012@yahoo.cn [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China)

2012-09-07

Highlights: Black-Right-Pointing-Pointer Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4{sup +} T cells. Black-Right-Pointing-Pointer Carrying out siRNA technology to study FOXO3A protein function. Black-Right-Pointing-Pointer Helpful to understand the T cell especially CD4{sup +} T cell's role in immunological reaction. -- Abstract: The TNF ligand family member 'B cell-activating factor belonging to the TNF family' (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4{sup +} spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4{sup +} T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4{sup +} spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4{sup +} T cell proliferation.

Measurement of cell motility on proton beam micromachined 3D scaffolds

International Nuclear Information System (INIS)

Zhang, F.; Sun, F.; Kan, J.A. van; Shao, P.G.; Zheng, Z.; Ge, R.W.; Watt, F.

2005-01-01

Tissue engineering is a rapidly developing and highly interdisciplinary field that applies the principles of cell biology, engineering and material science. In natural tissues, the cells are arranged in a three-dimensional (3D) matrix which provides the appropriate functional, nutritional and spatial conditions. In scaffold guided tissue engineering 3D scaffolds provide the critical function of acting as extracellular matrices onto which cells can attach, grow, and form new tissue. The main focus of this paper is to understand cell behavior on micro-grooved and ridged substrates and to study the effects of geometrical constraints on cell motility and cell function. In this study, we found that BAE (Bovine Aortic Endothelial) cells naturally align with and are guided along 3D ridges and grooves machined into polymethylmethacrylate (PMMA) substrates. Average cell speed on micro-grooves and ridges ranged from 0.015 μm/s (for 12 μm wide and 10 μm deep ridges) to 0.025 μm/s (for 20 μm wide and 10 μm deep ridges). This compares with the cell motility rate on a flat PMMA surface where the average cell speed is around 0.012 μm/s. In this work we used scaffolds which were directly written with a focused proton beam, typically 1 MeV protons with a beam spot size of 1 x 1 μm 2

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

International Nuclear Information System (INIS)

Shen, Chong; Meng, Qin; Zhang, Guoliang

2015-01-01

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

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

OpenAIRE

M?ller, Thomas; Amoroso, Matteo; H?gg, Daniel; Brantsing, Camilla; Rotter, Nicole; Apelgren, Peter; Lindahl, Anders; K?lby, Lars; Gatenholm, Paul

2017-01-01

Background: The three-dimensional (3D) bioprinting technology allows creation of 3D constructs in a layer-by-layer fashion utilizing biologically relevant materials such as biopolymers and cells. The aim of this study is to investigate the use of 3D bioprinting in a clinically relevant setting to evaluate the potential of this technique for in vivo chondrogenesis. Methods: Thirty-six nude mice (Balb-C, female) received a 5- ? 5- ? 1-mm piece of bioprinted cell-laden nanofibrillated cellulose/...

Fabrication of 3D SiO x structures using patterned PMMA sacrificial layer

Science.gov (United States)

Li, Zhiqin; Xiang, Quan; Zheng, Mengjie; Bi, Kaixi; Chen, Yiqin; Chen, Keqiu; Duan, Huigao

2018-02-01

Three-dimensional (3D) nanofabrication based on electron-beam lithography (EBL) has drawn wide attention for various applications with its high patterning resolution and design flexibility. In this work, we present a bilayer EBL process to obtain 3D freestanding SiO x structures via the release of the bottom sacrificial layer. This new kind of bilayer process enables us to define various 3D freestanding SiO x structures with high resolution and low edge roughness. As a proof of concept for applications, metal-coated freestanding SiO x microplates with an underlying air gap were fabricated to form asymmetric Fabry-Perot resonators, which can be utilized for colorimetric refractive index sensing and thus also have application potential for biochemical detection, anti-counterfeiting and smart active nano-optical devices.

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

Science.gov (United States)

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

2017-02-01

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

Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions.

Science.gov (United States)

Kamei, Ken-Ichiro; Koyama, Yoshie; Tokunaga, Yumie; Mashimo, Yasumasa; Yoshioka, Momoko; Fockenberg, Christopher; Mosbergen, Rowland; Korn, Othmar; Wells, Christine; Chen, Yong

2016-11-01

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Linking stem cell function and growth pattern of intestinal organoids.

Science.gov (United States)

Thalheim, Torsten; Quaas, Marianne; Herberg, Maria; Braumann, Ulf-Dietrich; Kerner, Christiane; Loeffler, Markus; Aust, Gabriela; Galle, Joerg

2018-01-15

Intestinal stem cells (ISCs) require well-defined signals from their environment in order to carry out their specific functions. Most of these signals are provided by neighboring cells that form a stem cell niche, whose shape and cellular composition self-organize. Major features of this self-organization can be studied in ISC-derived organoid culture. In this system, manipulation of essential pathways of stem cell maintenance and differentiation results in well-described growth phenotypes. We here provide an individual cell-based model of intestinal organoids that enables a mechanistic explanation of the observed growth phenotypes. In simulation studies of the 3D structure of expanding organoids, we investigate interdependences between Wnt- and Notch-signaling which control the shape of the stem cell niche and, thus, the growth pattern of the organoids. Similar to in vitro experiments, changes of pathway activities alter the cellular composition of the organoids and, thereby, affect their shape. Exogenous Wnt enforces transitions from branched into a cyst-like growth pattern; known to occur spontaneously during long term organoid expansion. Based on our simulation results, we predict that the cyst-like pattern is associated with biomechanical changes of the cells which assign them a growth advantage. The results suggest ongoing stem cell adaptation to in vitro conditions during long term expansion by stabilizing Wnt-activity. Our study exemplifies the potential of individual cell-based modeling in unraveling links between molecular stem cell regulation and 3D growth of tissues. This kind of modeling combines experimental results in the fields of stem cell biology and cell biomechanics constituting a prerequisite for a better understanding of tissue regeneration as well as developmental processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Conducting Polymer Scaffolds for Hosting and Monitoring 3D Cell Culture

KAUST Repository

Inal, Sahika

2017-05-03

This work reports the design of a live-cell monitoring platform based on a macroporous scaffold of a conducting polymer, poly(3,4-ethylene dioxythiophene):poly(styrenesulfonate). The conducting polymer scaffolds support 3D cell cultures due to their biocompatibility and tissue-like elasticity, which can be manipulated by inclusion of biopolymers such as collagen. Integration of a media perfusion tube inside the scaffold enables homogenous cell spreading and fluid transport throughout the scaffold, ensuring long term cell viability. This also allows for co-culture of multiple cell types inside the scaffold. The inclusion of cells within the porous architecture affects the impedance of the electrically conducting polymer network and, thus, is utilized as an in situ tool to monitor cell growth. Therefore, while being an integral part of the 3D tissue, the conducting polymer is an active component, enhancing the tissue function, and forming the basis for a bioelectronic device with integrated sensing capability.

Regulation of cholesterol 25-hydroxylase expression by vitamin D3 metabolites in human prostate stromal cells

International Nuclear Information System (INIS)

Wang, J.-H.; Tuohimaa, Pentti

2006-01-01

Vitamin D 3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D 3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1α,25(OH) 2 D 3 and 500 nM 25OHD 3 upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1α,25(OH) 2 D 3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as 1α,25(OH) 2 D 3 induced CH25H mRNA expression. 1α,25(OH) 2 D 3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D 3 treated cells. Our data suggest that ch25h could be a vitamin D 3 target gene and may partly mediate anti-proliferative action of vitamin D 3 in human primary prostate stromal cells

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

DEFF Research Database (Denmark)

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

2015-01-01

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...... 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...... the tested biomimetic environment, paving the way to further developments in bioimpedance tracking of 3D cell cultures and tissue engineering....

Biomaterials-based 3D cell printing for next-generation therapeutics and diagnostics.

Science.gov (United States)

Jang, Jinah; Park, Ju Young; Gao, Ge; Cho, Dong-Woo

2018-02-01

Building human tissues via 3D cell printing technology has received particular attention due to its process flexibility and versatility. This technology enables the recapitulation of unique features of human tissues and the all-in-one manufacturing process through the design of smart and advanced biomaterials and proper polymerization techniques. For the optimal engineering of tissues, a higher-order assembly of physiological components, including cells, biomaterials, and biomolecules, should meet the critical requirements for tissue morphogenesis and vascularization. The convergence of 3D cell printing with a microfluidic approach has led to a significant leap in the vascularization of engineering tissues. In addition, recent cutting-edge technology in stem cells and genetic engineering can potentially be adapted to the 3D tissue fabrication technique, and it has great potential to shift the paradigm of disease modeling and the study of unknown disease mechanisms required for precision medicine. This review gives an overview of recent developments in 3D cell printing and bioinks and provides technical requirements for engineering human tissues. Finally, we propose suggestions on the development of next-generation therapeutics and diagnostics. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2015-11-02

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

Cell cycle sensitivity of HL-60 cells to the differentiation-inducing effects of 1-alpha,25-dihydroxyvitamin D3

International Nuclear Information System (INIS)

Studzinski, G.P.; Bhandal, A.K.; Brelvi, Z.S.

1985-01-01

A recently described system for monocyte-like differentiation of HL-60 cells was utilized to determine if the initiation of this pathway can be linked to a set of replicative cellular events. The standard induction system consisted of a 4-h exposure to 100 nM 1-alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] followed by determination of nonspecific esterase and phagocytic activity 24 h later. The cell cycle status was ascertained by the incorporation of [ 3 H]thymidine and autoradiography. Studies in which cell cycle block in the G1/S phase boundary region was produced by a partial inhibition of DNA synthesis with thymidine, or sodium butyrate, showed that the exposure of such semisynchronous cultures to 1,25(OH)2D3 resulted in an increased proportion of differentiated cells. Conversely, blocking the cell cycle with vinblastine (G2/M block) or theobromine (mid-G1 block) inhibited the initiation of differentiation by 1,25(OH)2D3. Experiments in which the differentiated cells were examined for the cell cycle position at the time of the exposure to 1,25(OH)2D3 by [ 3 H]thymidine labeling and autoradiography confirmed that the late G1 and early S phase cells are those which predominate in the differentiated fraction of 1,25(OH)2D3-treated HL-60 cultures. These results link pre- and early replicative cellular events to the induction of monocytic differentiation by 1,25(OH)2D3

Vorinostat differentially alters 3D nuclear structure of cancer and non-cancerous esophageal cells.

Science.gov (United States)

Nandakumar, Vivek; Hansen, Nanna; Glenn, Honor L; Han, Jessica H; Helland, Stephanie; Hernandez, Kathryn; Senechal, Patti; Johnson, Roger H; Bussey, Kimberly J; Meldrum, Deirdre R

2016-08-09

The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an 'epigenetic' drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat's differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.

SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture.

Science.gov (United States)

Arhoma, A; Chantry, A D; Haywood-Small, S L; Cross, N A

2017-11-15

Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL-induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate-based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter-Glo luminescence assay, Caspase-8 and -9 activities were measured by Caspase-Glo™ assay kit. TRAIL-resistant cells were generated by culture of RPMI 8226 and NCI-H929 by acute exposure to TRAIL followed by selection of TRAIL-resistant cells. TRAIL significantly induced apoptosis in a dose-dependent manner in OPM-2, RPMI 8226, NCI-H929, U266, JJN-3 MM cell lines and ADC-1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM-2, and enhanced TRAIL responses were both via Caspase-8 and -9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co-treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI-H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL-sensitive cell

1,25D3 differentially suppresses bladder cancer cell migration and invasion through the induction of miR-101-3p.

Science.gov (United States)

Ma, Yingyu; Luo, Wei; Bunch, Brittany L; Pratt, Rachel N; Trump, Donald L; Johnson, Candace S

2017-09-01

Metastasis is the major cause of bladder cancer death. 1,25D 3 , the active metabolite of vitamin D, has shown anti-metastasis activity in several cancer model systems. However, the role of 1,25D 3 in migration and invasion in bladder cancer is unknown. To investigate whether 1,25D 3 affects migration and invasion, four human bladder cell lines with different reported invasiveness were selected: low-invasive T24 and 253J cells and highly invasive 253J-BV and TCCSUP cells. All of the four bladder cancer cells express endogenous and inducible vitamin D receptor (VDR) as examined by immunoblot analysis. 1,25D 3 had no effect on the proliferation of bladder cancer cells as assessed by MTT assay. In contrast, 1,25D 3 suppressed migration and invasion in the more invasive 253J-BV and TCCSUP cells, but not in the low-invasive 253J and T24 cells using "wound" healing, chemotactic migration and Matrigel-based invasion assays. 1,25D 3 promoted the expression of miR-101-3p and miR-126-3p in 253J-BV cells as examined by qRT-PCR. miR-101-3p inhibitor partially abrogated and pre-miR-101-3p further suppressed the inhibition of 1,25D 3 on migration and invasion in 253J-BV cells. Further, 1,25D 3 enhanced VDR recruitment to the promoter region of miR-101-3p using ChIP-qPCR assay. 1,25D 3 enhanced the promoter activity of miR-101-3p as evaluated by luciferase reporter assay. Taken together, 1,25D 3 suppresses bladder cancer cell migration and invasion in two invasive/migration competent lines but not in two less invasive/motile lines, which is partially through the induction of miR-101-3p expression at the transcriptional level.

Vitamin D3 regulates cell viability in gastric cancer and cholangiocarcinoma

OpenAIRE

Baek, Sungmin; Lee, Young-Suk; Shim, Hye-Eun; Yoon, Sik; Baek, Sun-Yong; Kim, Bong-Seon; Oh, Sae-Ock

2011-01-01

A low serum level of vitamin D has been associated with an increased incidence of gastrointestinal tract cancers. However, the effects of vitamin D3 have not been investigated in gastric cancer and cholangiocarcinoma. In the present study, we found that vitamin D3 treatment significantly suppressed the viability of gastric cancer and cholangiocarcinoma cells. Moreover, vitamin D3 had a synergistic effect with other anti-cancer drugs, such as paclitaxel, adriamycin, and vinblastine, for suppre...

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

Science.gov (United States)

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…

Stereolithographic hydrogel printing of 3D microfluidic cell culture chips

DEFF Research Database (Denmark)

Zhang, Rujing

that support the required freedom in design, detail and chemistry for fabricating truly 3D constructs have remained limited. Here, we report a stereolithographic high-resolution 3D printing technique utilizing poly(ethylene glycol) diacrylate (PEGDA, MW 700) to manufacture diffusion-open and mechanically...... and material flexibility by embedding a highly compliant cell-laden gelatin hydrogel within the confines of a 3D printed resilient PEGDA hydrogel chip of intermediate compliance. Overall, our proposed strategy represents an automated, cost-effective and high resolution technique to manufacture complex 3D...... epoxy component as structural supports interfacing the external world as well as compliant PEGDA component as microfluidic channels have been manufactured and perfused. Although still in the preliminary stage, this dual-material printing approach shows the potential for constructing complex 3D...

Differences in gene expression of cells growing in conventional 2D versus 3D cell culture

International Nuclear Information System (INIS)

Zschenker, Oliver; Cordes, Nils; Streichert, Thomas

2009-01-01

Full text: Telomeres are DNA protein complexes on the ends of chromosomes that distinguish the ends of chromosomes from double strand breaks and prevent degradation or fusion by nonhomologous end-joining. The loss of telomeres is associated with a loss of heterochromatic features leading to a less compact chromatin structure which allows e.g. DNA repair proteins to get better access to the site of the DNA damage and facilitate chromosome fusions. Telomerase is an enzyme that can counteract the loss of telomeres by adding telomeric repeats on the ends of chromosomes. Since telomerase is active in most tumor cells, telomerase is suggested to be the reason for the unlimited number of cell divisions of cancer cells. TRF2 is one of the most important proteins of the Shelterin complex protecting the telomeres from shortening by inhibiting ATM which is up-stream of the DNA repair mechanisms. Thus, we are concentrating on TRF2 and telomerase to investigate the differences in DNA repair in telomeric (heterochromatic) versus euchromatic regions. Human cancer cells with differences in status of p53 and telomerase like A549, UT-SCC15 and FaDu cells are used. Without any treatment, FaDu cells express high levels of telomerase and TRF2 in conventional 2D cell culture which is in contrast to e.g. A549. We found that telomerase is even higher expressed in 3D than in 2D cell culture. To connect telomere associated processes to both repair of radiogenic DNA damage/lesions and to cell-extracellular matrix interactions, we performed whole genome microarray analysis. By comparing the differential expression of genes associated with these three cell functions, we intend to yield new molecular insight into radiotherapy relevant tumor characteristics, particularly radioresistance and DNA damage response network processing. (author)

Mesenchymal stem cells enhance the metastasis of 3D-cultured hepatocellular carcinoma cells

International Nuclear Information System (INIS)

Liu, Chang; Liu, Yang; Xu, Xiao-xi; Guo, Xin; Sun, Guang-wei; Ma, Xiao-jun

2016-01-01

Accumulating evidences have demonstrated that mesenchymal stem cells (MSC) could be recruited to the tumor microenvironment. Umbilical cord mesenchymal stem cells (UCMSC) were attractive vehicles for delivering therapeutic agents against cancer. Nevertheless, the safety of UCMSC in the treatment of tumors including hepatocellular carcinoma (HCC) was still undetermined. In this study, an in vitro co-culture system was established to evaluate the effect of UCMSC on the cell growth, cancer stem cell (CSC) characteristics, drug resistance, metastasis of 3D-cultured HCC cells, and the underlying mechanism was also investigated. It was found that after co-cultured with UCMSC, the metastatic ability of 3D-cultured HCC cells was significantly enhanced as indicated by up-regulation of matrix metalloproteinase (MMP), epithelial-mesenchymal transition (EMT)-related genes, and migration ability. However, cell growth, drug resistance and CSC-related gene expression of HCC cells were not affected by UCMSC. Moreover, EMT was reversed, MMP-2 expression was down-regulated, and migration ability of HCC cell was significantly inhibited when TGF-β receptor inhibitor SB431542 was added into the co-culture system. Therefore, these data indicated that UCMSC could significantly enhance the tumor cell metastasis, which was due to the EMT of HCC cells induced by TGF-β. The online version of this article (doi:10.1186/s12885-016-2595-4) contains supplementary material, which is available to authorized users

Regulation of Motility, Invasion and Metastatic Potential of Squamous Cell Carcinoma by 1,25D3

Science.gov (United States)

Ma, Yingyu; Yu, Wei-Dong; Su, Bing; Seshadri, Mukund; Luo, Wei; Trump, Donald L.; Johnson, Candace S.

2012-01-01

BACKGROUND 1,25D3, the active metabolite of vitamin D, has been shown to exhibit broad spectrum anti-tumor activity in xenograft animal models. However, its activity against metastatic disease has not been extensively investigated. METHODS Squamous cell carcinoma (SCC) or 1,25D3-resistant variant SCC-DR cells were treated with 1,25D3. Actin organization was examined by immunofluorescence assay. Cell migration was assessed by “wound” healing and chemotactic migration assay. Cell invasion was assessed by Matrigel-based invasion assay and in situ zymography. MMP-2 and MMP-9 expression and secretion was examined by immunoblot analysis and ELISA, respectively. E-cadherin expression was assessed by flow cytometry, immunoblot analysis and immunohistochemistry. Knockdown of E-cadherin was achieved by siRNA. Experimental metastasis mouse model was done by intravenous injection of tumor cells. Lung tumor development was assessed by magnetic resonance imaging, gross observation and histology. RESULTS SCC cellular morphology and actin organization were altered by 10 nM of 1,25D3. 1,25D3 inhibited SCC cell motility and invasion, which was associated with reduced expression and secretion of MMP-2 and MMP-9. 1,25D3 promoted the expression of E-cadherin. These findings were not observed in SCC-DR cells. Knock down of E-cadherin rescued 1,25D3-inhibited cell migration. Intravenous injection of SCC or SCC-DR cells resulted in the establishment of extensive pulmonary lesions in saline-treated C3H mice. Treatment with 1,25D3 resulted in a marked reduction in the formation of lung tumor colonies in animals injected with SCC but not SCC-DR cells. CONCLUSIONS 1,25D3 suppresses SCC cell motility, invasion and metastasis, partially through the promotion of E-cadherin-mediated cell-cell adhesion. PMID:22833444

Computational model-informed design and bioprinting of cell-patterned constructs for bone tissue engineering.

Science.gov (United States)

Carlier, Aurélie; Skvortsov, Gözde Akdeniz; Hafezi, Forough; Ferraris, Eleonora; Patterson, Jennifer; Koç, Bahattin; Van Oosterwyck, Hans

2016-05-17

Three-dimensional (3D) bioprinting is a rapidly advancing tissue engineering technology that holds great promise for the regeneration of several tissues, including bone. However, to generate a successful 3D bone tissue engineering construct, additional complexities should be taken into account such as nutrient and oxygen delivery, which is often insufficient after implantation in large bone defects. We propose that a well-designed tissue engineering construct, that is, an implant with a specific spatial pattern of cells in a matrix, will improve the healing outcome. By using a computational model of bone regeneration we show that particular cell patterns in tissue engineering constructs are able to enhance bone regeneration compared to uniform ones. We successfully bioprinted one of the most promising cell-gradient patterns by using cell-laden hydrogels with varying cell densities and observed a high cell viability for three days following the bioprinting process. In summary, we present a novel strategy for the biofabrication of bone tissue engineering constructs by designing cell-gradient patterns based on a computational model of bone regeneration, and successfully bioprinting the chosen design. This integrated approach may increase the success rate of implanted tissue engineering constructs for critical size bone defects and also can find a wider application in the biofabrication of other types of tissue engineering constructs.

Looking into the Future: Toward Advanced 3D Biomaterials for Stem-Cell-Based Regenerative Medicine.

Science.gov (United States)

Liu, Zhongmin; Tang, Mingliang; Zhao, Jinping; Chai, Renjie; Kang, Jiuhong

2018-04-01

Stem-cell-based therapies have the potential to provide novel solutions for the treatment of a variety of diseases, but the main obstacles to such therapies lie in the uncontrolled differentiation and functional engraftment of implanted tissues. The physicochemical microenvironment controls the self-renewal and differentiation of stem cells, and the key step in mimicking the stem cell microenvironment is to construct a more physiologically relevant 3D culture system. Material-based 3D assemblies of stem cells facilitate the cellular interactions that promote morphogenesis and tissue organization in a similar manner to that which occurs during embryogenesis. Both natural and artificial materials can be used to create 3D scaffolds, and synthetic organic and inorganic porous materials are the two main kinds of artificial materials. Nanotechnology provides new opportunities to design novel advanced materials with special physicochemical properties for 3D stem cell culture and transplantation. Herein, the advances and advantages of 3D scaffold materials, especially with respect to stem-cell-based therapies, are first outlined. Second, the stem cell biology in 3D scaffold materials is reviewed. Third, the progress and basic principles of developing 3D scaffold materials for clinical applications in tissue engineering and regenerative medicine are reviewed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei

Directory of Open Access Journals (Sweden)

M Cremer

2009-06-01

Full Text Available Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has been linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly causally involved in the formation of cell type specific heterochromatin compartments, composed of (pericentromeric regions and chromosomal subregions from neighboring chromosome territories, which contain silent genes.

Pattern selection near the onset of convection in binary mixtures in cylindrical cells

International Nuclear Information System (INIS)

Alonso, Arantxa; Mercader, Isabel; Batiste, Oriol

2014-01-01

We report numerical investigations of three-dimensional pattern formation of binary mixtures in a vertical cylindrical container heated from below. Negative separation ratio mixtures, for which the onset of convection occurs via a subcritical Hopf bifurcation, are considered. We focus on the dynamics in the neighbourhood of the initial oscillatory instability and analyze the spatio-temporal properties of the patterns for different values of the aspect ratio of the cell, 0.25≲Γ≲11 (Γ≡R/d, where R is the radius of the cell and d its height). Despite the oscillatory nature of the primary instability, for highly constrained geometries, Γ≲2.5, only pure thermal stationary modes are selected after long transients. As the aspect ratio of the cell increases, for intermediate aspect ratio cells such as Γ=3, multistability and coexistence of stationary and time-dependent patterns is observed. In highly extended cylinders, Γ≈11, the dynamics near the onset is completely different from the pure fluid case, and a startling diversity of confined patterns is observed. Many of these patterns are consistent with experimental observations. Remarkably, though, we have obtained persistent large amplitude highly localized states not reported previously. (paper)

Pattern selection near the onset of convection in binary mixtures in cylindrical cells

Energy Technology Data Exchange (ETDEWEB)

Alonso, Arantxa; Mercader, Isabel; Batiste, Oriol, E-mail: arantxa@fa.upc.edu [Departament de Física Aplicada, Universitat Politècnica de Catalunya, Mòdul B4, 08034 Barcelona (Spain)

2014-08-01

We report numerical investigations of three-dimensional pattern formation of binary mixtures in a vertical cylindrical container heated from below. Negative separation ratio mixtures, for which the onset of convection occurs via a subcritical Hopf bifurcation, are considered. We focus on the dynamics in the neighbourhood of the initial oscillatory instability and analyze the spatio-temporal properties of the patterns for different values of the aspect ratio of the cell, 0.25≲Γ≲11 (Γ≡R/d, where R is the radius of the cell and d its height). Despite the oscillatory nature of the primary instability, for highly constrained geometries, Γ≲2.5, only pure thermal stationary modes are selected after long transients. As the aspect ratio of the cell increases, for intermediate aspect ratio cells such as Γ=3, multistability and coexistence of stationary and time-dependent patterns is observed. In highly extended cylinders, Γ≈11, the dynamics near the onset is completely different from the pure fluid case, and a startling diversity of confined patterns is observed. Many of these patterns are consistent with experimental observations. Remarkably, though, we have obtained persistent large amplitude highly localized states not reported previously. (paper)

Fast computation of hologram patterns of a 3D object using run-length encoding and novel look-up table methods.

Science.gov (United States)

Kim, Seung-Cheol; Kim, Eun-Soo

2009-02-20

In this paper we propose a new approach for fast generation of computer-generated holograms (CGHs) of a 3D object by using the run-length encoding (RLE) and the novel look-up table (N-LUT) methods. With the RLE method, spatially redundant data of a 3D object are extracted and regrouped into the N-point redundancy map according to the number of the adjacent object points having the same 3D value. Based on this redundancy map, N-point principle fringe patterns (PFPs) are newly calculated by using the 1-point PFP of the N-LUT, and the CGH pattern for the 3D object is generated with these N-point PFPs. In this approach, object points to be involved in calculation of the CGH pattern can be dramatically reduced and, as a result, an increase of computational speed can be obtained. Some experiments with a test 3D object are carried out and the results are compared to those of the conventional methods.

Patterned three-dimensional encapsulation of embryonic stem cells using dielectrophoresis and stereolithography.

Science.gov (United States)

Bajaj, Piyush; Marchwiany, Daniel; Duarte, Carlos; Bashir, Rashid

2013-03-01

Controlling the assembly of cells in three dimensions is very important for engineering functional tissues, drug screening, probing cell-cell/cell-matrix interactions, and studying the emergent behavior of cellular systems. Although the current methods of cell encapsulation in hydrogels can distribute them in three dimensions, these methods typically lack spatial control of multi-cellular organization and do not allow for the possibility of cell-cell contacts as seen for the native tissue. Here, we report the integration of dielectrophoresis (DEP) with stereolithography (SL) apparatus for the spatial patterning of cells on custom made gold micro-electrodes. Afterwards, they are encapsulated in poly (ethylene glycol) diacrylate (PEGDA) hydrogels of different stiffnesses. This technique can mimic the in vivo microscale tissue architecture, where the cells have a high degree of three dimensional (3D) spatial control. As a proof of concept, we show the patterning and encapsulation of mouse embryonic stem cells (mESCs) and C2C12 skeletal muscle myoblasts. mESCs show high viability in both the DEP (91.79% ± 1.4%) and the no DEP (94.27% ± 0.5%) hydrogel samples. Furthermore, we also show the patterning of mouse embryoid bodies (mEBs) and C2C12 spheroids in the hydrogels, and verify their viability. This robust and flexible in vitro platform can enable various applications in stem cell differentiation and tissue engineering by mimicking elements of the native 3D in vivo cellular micro-environment. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Micropatterning of 3D Microenvironments for Living Biosensor Applications

Directory of Open Access Journals (Sweden)

William F. Hynes

2014-02-01

Full Text Available Micro-scale printing and patterning of living cells has multiple applications including tissue engineering, cell signaling assays, and the fabrication of cell-based biosensors. In this work, a molecular printing instrument, the Bioforce Nano eNabler, was modified to enable micron-scale “quill-pen” based printing of mammalian cells in a 3D hyaluronan/gelatin based hydrogel. Specifically, photo-initiated “thiol-ene” click chemistry was used to couple the thiol groups of thiolated hyaluronan/thiolated gelatin to the alkene groups of 4-arm polyethylene glycol (PEG-norbornene molecules. Rapid photopolymerization enabled direct printing and controlled curing of living cells within the hydrogel matrix. The resulting hydrogels were biocompatible with human adipose-derived stem cells, NIH-3T3 cells, and mouse embryonic stem cells. The utility of this printing approach was also explored for cell-based biosensors. Micro-printed cells expressing a redox sensitive variant of the green fluorescent protein (roGFP-R12 showed a measurable fluorescent response to addition of oxidizing and then reducing agents. This work represents a novel approach to micron-scale cell patterning, and its potential for living, cell-based biosensors.

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

NARCIS (Netherlands)

Higuera Sierra, Gustavo

2010-01-01

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

Analysis of interactions of Salmonella type three secretion mutants with 3-D intestinal epithelial cells.

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Andrea L Radtke

Full Text Available The prevailing paradigm of Salmonella enteropathogenesis based on monolayers asserts that Salmonella pathogenicity island-1 Type Three Secretion System (SPI-1 T3SS is required for bacterial invasion into intestinal epithelium. However, little is known about the role of SPI-1 in mediating gastrointestinal disease in humans. Recently, SPI-1 deficient nontyphoidal Salmonella strains were isolated from infected humans and animals, indicating that SPI-1 is not required to cause enteropathogenesis and demonstrating the need for more in vivo-like models. Here, we utilized a previously characterized 3-D organotypic model of human intestinal epithelium to elucidate the role of all characterized Salmonella enterica T3SSs. Similar to in vivo reports, the Salmonella SPI-1 T3SS was not required to invade 3-D intestinal cells. Additionally, Salmonella strains carrying single (SPI-1 or SPI-2, double (SPI-1/2 and complete T3SS knockout (SPI-1/SPI-2: flhDC also invaded 3-D intestinal cells to wildtype levels. Invasion of wildtype and TTSS mutants was a Salmonella active process, whereas non-invasive bacterial strains, bacterial size beads, and heat-killed Salmonella did not invade 3-D cells. Wildtype and T3SS mutants did not preferentially target different cell types identified within the 3-D intestinal aggregates, including M-cells/M-like cells, enterocytes, or Paneth cells. Moreover, each T3SS was necessary for substantial intracellular bacterial replication within 3-D cells. Collectively, these results indicate that T3SSs are dispensable for Salmonella invasion into highly differentiated 3-D models of human intestinal epithelial cells, but are required for intracellular bacterial growth, paralleling in vivo infection observations and demonstrating the utility of these models in predicting in vivo-like pathogenic mechanisms.

Dynamic heterogeneity of DNA methylation and hydroxymethylation in embryonic stem cell populations captured by single-cell 3D high-content analysis

Energy Technology Data Exchange (ETDEWEB)

Tajbakhsh, Jian, E-mail: tajbakhshj@cshs.org [Chromatin Biology Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Translational Cytomics Group, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Stefanovski, Darko [Translational Cytomics Group, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19348 (United States); Tang, George [Chromatin Biology Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Translational Cytomics Group, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Wawrowsky, Kolja [Translational Cytomics Group, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Liu, Naiyou; Fair, Jeffrey H. [Department of Surgery and UF Health Comprehensive Transplant Center, University of Florida College of Medicine, Gainesville, FL 32608 (United States)

2015-03-15

Cell-surface markers and transcription factors are being used in the assessment of stem cell fate and therapeutic safety, but display significant variability in stem cell cultures. We assessed nuclear patterns of 5-hydroxymethylcytosine (5hmC, associated with pluripotency), a second important epigenetic mark, and its combination with 5-methylcytosine (5mC, associated with differentiation), also in comparison to more established markers of pluripotency (Oct-4) and endodermal differentiation (FoxA2, Sox17) in mouse embryonic stem cells (mESC) over a 10-day differentiation course in vitro: by means of confocal and super-resolution imaging together with 3D high-content analysis, an essential tool in single-cell screening. In summary: 1) We did not measure any significant correlation of putative markers with global 5mC or 5hmC. 2) While average Oct-4 levels stagnated on a cell-population base (0.015 lnIU/day), Sox17 and FoxA2 increased 22-fold and 3-fold faster, respectively (Sox17: 0.343 lnIU/day; FoxA2: 0.046 lnIU/day). In comparison, global DNA methylation levels increased 4-fold faster (0.068 lnIU/day), and global hydroxymethylation declined at 0.046 lnIU/day, both with a better explanation of the temporal profile. 3) This progression was concomitant with the occurrence of distinct nuclear codistribution patterns that represented a heterogeneous spectrum of states in differentiation; converging to three major coexisting 5mC/5hmC phenotypes by day 10: 5hmC{sup +}/5mC{sup −}, 5hmC{sup +}/5mC{sup +}, and 5hmC{sup −}/5mC{sup +} cells. 4) Using optical nanoscopy we could delineate the respective topologies of 5mC/5hmC colocalization in subregions of nuclear DNA: in the majority of 5hmC{sup +}/5mC{sup +} cells 5hmC and 5mC predominantly occupied mutually exclusive territories resembling euchromatic and heterochromatic regions, respectively. Simultaneously, in a smaller subset of cells we observed a tighter colocalization of the two cytosine variants, presumably

Cell adhesion pattern created by OSTE polymers.

Science.gov (United States)

Liu, Wenjia; Li, Yiyang; Ding, Xianting

2017-04-24

Engineering surfaces with functional polymers is a crucial issue in the field of micro/nanofabrication and cell-material interface studies. For many applications of surface patterning, it does not need cells to attach on the whole surface. Herein, we introduce a novel polymer fabrication protocol of off-stoichiometry thiol-ene (OSTE) polymers to create heterogeneity on the surface by utilizing 3D printing and soft-lithography. By choosing two OSTE polymers with different functional groups, we create a pattern where only parts of the surface can facilitate cell adhesion. We also study the hydrophilic property of OSTE polymers by mixing poly(ethylene glycol) (PEG) directly with pre-polymers and plasma treatments afterwards. Moreover, we investigate the effect of functional groups' excess ratio and hydrophilic property on the cell adhesion ability of OSTE polymers. The results show that the cell adhesion ability of OSTE materials can be tuned within a wide range by the coupling effect of functional groups' excess ratio and hydrophilic property. Meanwhile, by mixing PEG with pre-polymers and undergoing oxygen plasma treatment afterward can significantly improve the hydrophilic property of OSTE polymers.

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

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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.

Current State-of-the-Art 3D Tissue Models and Their Compatibility with Live Cell Imaging.

Science.gov (United States)

Bardsley, Katie; Deegan, Anthony J; El Haj, Alicia; Yang, Ying

2017-01-01

Mammalian cells grow within a complex three-dimensional (3D) microenvironment where multiple cells are organized and surrounded by extracellular matrix (ECM). The quantity and types of ECM components, alongside cell-to-cell and cell-to-matrix interactions dictate cellular differentiation, proliferation and function in vivo. To mimic natural cellular activities, various 3D tissue culture models have been established to replace conventional two dimensional (2D) culture environments. Allowing for both characterization and visualization of cellular activities within possibly bulky 3D tissue models presents considerable challenges due to the increased thickness and subsequent light scattering features of such 3D models. In this chapter, state-of-the-art methodologies used to establish 3D tissue models are discussed, first with a focus on both scaffold-free and scaffold-based 3D tissue model formation. Following on, multiple 3D live cell imaging systems, mainly optical imaging modalities, are introduced. Their advantages and disadvantages are discussed, with the aim of stimulating more research in this highly demanding research area.

When fast atom diffraction turns 3D

International Nuclear Information System (INIS)

Zugarramurdi, Asier; Borisov, Andrei G.

2013-01-01

Fast atom diffraction at surfaces (FAD) in grazing incidence geometry is characterized by the slow motion in the direction perpendicular to the surface and fast motion parallel to the surface plane along a low index direction. It is established experimentally that for the typical surfaces the FAD reveals the 2D diffraction patterns associated with exchange of the reciprocal lattice vector perpendicular to the direction of fast motion. The reciprocal lattice vector exchange along the direction of fast motion is negligible. The usual approximation made in the description of the experimental data is then to assume that the effective potential leading to the diffraction results from the averaging of the 3D surface potential along the atomic strings forming the axial channel. In this work we use full quantum wave packet propagation calculations to study theoretically the possibility to observe the 3D diffraction in FAD experiments. We show that for the surfaces with large unit cell, such as can be the case for reconstructed or vicinal surfaces, the 3D diffraction can be observed. The reciprocal lattice vector exchange along the direction of fast motion leads to several Laue circles in the diffraction pattern

Determination of displacements and their derivatives from 3D fringe patterns via extended monogenic phasor method

Science.gov (United States)

Sciammarella, Cesar A.; Lamberti, Luciano

2018-05-01

For 1D signals, it is necessary to resort to a 2D abstract space because the concept of phase utilized in the retrieval of fringe pattern analysis information relies on the use of a vectorial function. Fourier and Hilbert transforms provide in-quadrature signals that lead to the very important basic concept of local phase. A 3D abstract space must hence be generated in order to analyze 2D signals. A 3D vector space in a Cartesian complex space is graphically represented by a Poincare sphere. In this study, the extension of the associated spaces is extended to 3D. A 4D hypersphere is defined for that purpose. The proposed approach is illustrated by determining the deformations of the heart left ventricle.

Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond.

Science.gov (United States)

Liu, Chun; Oikonomopoulos, Angelos; Sayed, Nazish; Wu, Joseph C

2018-03-08

The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimic in vivo tissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single '4D multi-organ system'. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling. © 2018. Published by The Company of Biologists Ltd.

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

Energy Technology Data Exchange (ETDEWEB)

Grebennikov, A.N.; Zhitnik, A.K.; Zvenigorodskaya, O.A. [and others

1995-12-31

In conformity with the protocol of the Workshop under Contract {open_quotes}Assessment of RBMK reactor safety using modern Western Codes{close_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.

Single-walled carbon nanotubes alter Schwann cell behavior differentially within 2D and 3D environments.

Science.gov (United States)

Behan, Brenda L; DeWitt, Daniel G; Bogdanowicz, Danielle R; Koppes, Abigail N; Bale, Shyam S; Thompson, Deanna M

2011-01-01

Both spinal cord injury (SCI) and large-gap peripheral nerve defects can be debilitating affecting a patient's long-term quality of life and presently, there is no suitable treatment for functional regeneration of these injured tissues. A number of works have suggested the benefits of electrical stimulation to promote both glial migration and neuronal extension. In this work, an electrically conductive hydrogel containing single-walled carbon nanotubes (SWCNT) for neural engineering applications is presented and the Schwann cell (SC) response to SWCNT is examined in both 2D and 3D microenvironments. Results from clonogenic and alamarBlue® assays in 2D indicate that SWCNT (10-50 μg mL(-1)) inhibit SC proliferation but do not affect cell viability. Following SWCNT exposure in 2D, changes in SC morphology can be observed with the nanomaterial attached to the cell membrane at concentrations as low as 10 μg mL(-1). In contrast to the results gathered in 2D, SC embedded within the 3D hydrogel loaded with 10-50 μg mL(-1) of SWCNT exhibited little or no measurable change in cell proliferation, viability, or morphology as assessed using a digestion assay, alamarBlue, and confocal microscopy. Collectively, this highlights that an electrically-conductive SWCNT collagen I-Matrigel™ biomaterial may be suitable for neural tissue engineering and is able to sustain populations of SC. Findings suggest that 2D nanoparticle toxicity assays may not be accurate predictors of the 3D response, further motivating the examination of these materials in a more physiologically relevant environment. Copyright © 2010 Wiley Periodicals, Inc.

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

Directory of Open Access Journals (Sweden)

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.

The role of monocytes and T cells in 1,25-dihydroxyvitamin D3 mediated inhibition of B cell function in vitro

DEFF Research Database (Denmark)

Müller, K; Heilmann, C; Poulsen, L K

1991-01-01

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) inhibits immunoglobulin production by human mononuclear cells (MNC) in vitro. The present study was undertaken to evaluate the role of T cells and monocytes in 1,25-(OH)2D3 induced suppression of B cell functions. The synthetic vitamin D3 analogue MC 903...... was examined in parallel. 1,25-(OH)2D3 and MC 903 showed a dose-related inhibition of IgM, IgG and IgA plaque-forming cells in poke-weed mitogen (PWM) activated cultures of MNC. This effect was most likely mediated through impairment of T cell and monocyte functions. First, the inhibitory effect was seen after...

Proof-of-concept: 3D bioprinting of pigmented human skin constructs.

Science.gov (United States)

Ng, Wei Long; Qi, Jovina Tan Zhi; Yeong, Wai Yee; Naing, May Win

2018-01-23

Three-dimensional (3D) pigmented human skin constructs have been fabricated using a 3D bioprinting approach. The 3D pigmented human skin constructs are obtained from using three different types of skin cells (keratinocytes, melanocytes and fibroblasts from three different skin donors) and they exhibit similar constitutive pigmentation (pale pigmentation) as the skin donors. A two-step drop-on-demand bioprinting strategy facilitates the deposition of cell droplets to emulate the epidermal melanin units (pre-defined patterning of keratinocytes and melanocytes at the desired positions) and manipulation of the microenvironment to fabricate 3D biomimetic hierarchical porous structures found in native skin tissue. The 3D bioprinted pigmented skin constructs are compared to the pigmented skin constructs fabricated by conventional a manual-casting approach; in-depth characterization of both the 3D pigmented skin constructs has indicated that the 3D bioprinted skin constructs have a higher degree of resemblance to native skin tissue in term of the presence of well-developed stratified epidermal layers and the presence of a continuous layer of basement membrane proteins as compared to the manually-cast samples. The 3D bioprinting approach facilitates the development of 3D in vitro pigmented human skin constructs for potential toxicology testing and fundamental cell biology research.

Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake.

Science.gov (United States)

Stamati, Katerina; Priestley, John V; Mudera, Vivek; Cheema, Umber

2014-09-10

Angiogenesis is an essential neovascularisation process, which if recapitulated in 3D in vitro, will provide better understanding of endothelial cell (EC) behaviour. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 key components. We were able to control the aggregation pattern of ECs in 3D collagen hydrogels, by varying the matrix composition and/or having a source of cells signalling angiogenic proteins. These aggregation patterns reflect the different developmental pathways that ECs take to form different sized tubular structures. Cultures with added laminin and thus increased expression of α6 integrin showed a significant increase (p3D. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Vitamin D enhances omega-3 polyunsaturated fatty acids-induced apoptosis in breast cancer cells.

Science.gov (United States)

Yang, Jing; Zhu, Shenglong; Lin, Guangxiao; Song, Ci; He, Zhao

2017-08-01

Breast cancer is a leading type of cancer in women and generally classified into three subtypes of ER + /PR + , HER2 + and triple negative. Both omega-3 polyunsaturated fatty acids and vitamin D 3 play positive role in the reduction of breast cancer incidence. However, whether combination of omega-3 polyunsaturated fatty acids and vitamin D 3 has stronger protective effect on breast carcinogenesis still remains unknown. In this study, we show that the combination of ω-3 free fatty acids (ω-3 FFAs) and 1α, 25-dihydroxy-vitamin D 3 (VD 3 ) dramatically enhances cell apoptosis among three subtypes of breast cancer cell lines. Bcl-2 and total PARP protein levels are decreased in combined treatment MCF-7 and SK-BR-3 cells. Caspase signals play a vital role in cell apoptosis induced by combination. Moreover, Raf-MAPK signaling pathway is involved in the apoptosis induction by combination of ω-3 FFAs+VD 3 . These results demonstrate that the induction of cell apoptosis by combined treatment is dependent on different signaling pathways in three subtypes of breast cancer cell lines. © 2017 International Federation for Cell Biology.

Cancer cell migration within 3D layer-by-layer microfabricated photocrosslinked PEG scaffolds with tunable stiffness.

Science.gov (United States)

Soman, Pranav; Kelber, Jonathan A; Lee, Jin Woo; Wright, Tracy N; Vecchio, Kenneth S; Klemke, Richard L; Chen, Shaochen

2012-10-01

Our current understanding of 3-dimensional (3D) cell migration is primarily based on results from fibrous scaffolds with randomly organized internal architecture. Manipulations that change the stiffness of these 3D scaffolds often alter other matrix parameters that can modulate cell motility independently or synergistically, making observations less predictive of how cells behave when migrating in 3D. In order to decouple microstructural influences and stiffness effects, we have designed and fabricated 3D polyethylene glycol (PEG) scaffolds that permit orthogonal tuning of both elastic moduli and microstructure. Scaffolds with log-pile architectures were used to compare the 3D migration properties of normal breast epithelial cells (HMLE) and Twist-transformed cells (HMLET). Our results indicate that the nature of cell migration is significantly impacted by the ability of cells to migrate in the third dimension. 2D ECM-coated PEG substrates revealed no statistically significant difference in cell migration between HMLE and HMLET cells among substrates of different stiffness. However, when cells were allowed to move along the third dimension, substantial differences were observed for cell displacement, velocity and path straightness parameters. Furthermore, these differences were sensitive to both substrate stiffness and the presence of the Twist oncogene. Importantly, these 3D modes of migration provide insight into the potential for oncogene-transformed cells to migrate within and colonize tissues of varying stiffness. Copyright © 2012 Elsevier Ltd. All rights reserved.

From 2D to 3D: The morphology, proliferation and differentiation of MC3T3-E1 on silk fibroin/chitosan matrices.

Science.gov (United States)

Li, Da-Wei; He, Feng-Li; He, Jin; Deng, Xudong; Liu, Ya-Li; Liu, Yang-Yang; Ye, Ya-Jing; Yin, Da-Chuan

2017-12-15

It has been widely accepted that cell culture in two-dimensional (2D) conditions may not be able to represent growth in three-dimensional (3D) conditions. Systematic comparisons between 2D and 3D cell cultures are needed to appropriately use the existing 2D results. In this work, we conducted a comparative study between 2D and 3D cell cultures of MC3T3-E1 using the same type of material (a mixture of silk fibroin (SF) and chitosan (CS)). Our results showed 3D SF/CS scaffold exhibited different effects on cell culture compared with the 2D cases. 1) The cells grown in 3D scaffold showed multiple morphologies. 2) The proliferation of cells in 3D scaffold was long-term and sustainable. 3) Cell differentiation occurred throughout the entire 3D scaffold. The results showed that cell culture in 3D SF/CS scaffold exhibited different features than 2D cases and 3D SF/CS scaffold could be a promising material for 3D cell culture. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immunological compatibility status of placenta-derived stem cells is mediated by scaffold 3D structure.

Science.gov (United States)

Azizian, Sara; Khatami, Fatemeh; Modaresifar, Khashayar; Mosaffa, Nariman; Peirovi, Habibollah; Tayebi, Lobat; Bahrami, Soheyl; Redl, Heinz; Niknejad, Hassan

2018-02-23

Placenta-derived amniotic epithelial cells (AECs), a great cell source for tissue engineering and stem cell therapy, are immunologically inert in their native state; however, immunological changes in these cells after culture and differentiation have challenged their applications. The aim of this study was to investigate the effect of 2D and 3D scaffolds on human lymphocyte antigens (HLA) expression by AECs. The effect of different preparation parameters including pre-freezing time and temperature was evaluated on 3D chitosan-gelatine scaffolds properties. Evaluation of MHC class I, HLA-DR and HLA-G expression in AECs after 7 d culture on 2D bed and 3D scaffold of chitosan-gelatine showed that culture of AECs on the 2D substrate up-regulated MHC class I and HLA-DR protein markers on AECs surface and down-regulated HLA-G protein. In contrast, 3D scaffold did not increase protein expression of MHC class I and HLA-DR. Moreover, HLA-G protein expression remained unchanged in 3D culture. These results confirm that 3D scaffold can remain AECs in their native immunological state and modification of physical properties of the scaffold is a key regulator of immunological markers at the gene and protein expression levels; a strategy which circumvents rejection challenge of amniotic stem cells to be translated into the clinic.

Jamming and liquidity in 3D cancer cell aggregates

Science.gov (United States)

Oswald, Linda; Grosser, Steffen; Lippoldt, Jürgen; Pawlizak, Steve; Fritsch, Anatol; KäS, Josef A.

Traditionally, tissues are treated as simple liquids, which holds for example for embryonic tissue. However, recent experiments have shown that this picture is insufficient for other tissue types, suggesting possible transitions to solid-like behavior induced by cellular jamming. The coarse-grained self-propelled Voronoi (SPV) model predicts such a transition depending on cell shape which is thought to arise from an interplay of cell-cell adhesion and cortical tension. We observe non-liquid behavior in 3D breast cancer spheroids of varying metastatic potential and correlate single cell shapes, single cell dynamics and collective dynamic behavior of fusion and segregation experiments via the SPV model.

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

Science.gov (United States)

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.

GSK-3 Inhibition Sensitizes Acute Myeloid Leukemia Cells to 1,25D-Mediated Differentiation

Science.gov (United States)

Gupta, Kalpana; Stefan, Tammy; Ignatz-Hoover, James; Moreton, Stephen; Parizher, Gary; Saunthararajah, Yogen; Wald, David N.

2017-01-01

1,25-dihydroxyvitamin D3 (1,25D), the biologically active form of vitamin D, is widely considered a promising therapy for acute myeloid leukemia (AML) based on its ability to drive differentiation of leukemic cells. However, clinical trials have been disappointing in part to dose-limiting hypercalcemia. Here we show how inhibiting glycogen synthase kinase 3 (GSK3) can improve the differentiation response of AML cells to 1,25D-mediated differentiation. GSK3 inhibition in AML cells enhanced the differentiating effects of low concentrations of 1,25D. In addition, GSK3 inhibition augmented the ability of 1,25D to induce irreversible growth inhibition and slow the progression of AML in mouse models. Mechanistic studies revealed that GSK3 inhibition led to the hyperphosphorylation of the vitamin D receptor (VDR), enabling an interaction between VDR and the coactivator, SRC-3 (NCOA3), thereby increasing transcriptional activity. We also found that activation of JNK-mediated pathways in response to GSK3 inhibition contributed to the potentiation of 1,25D-induced differentiation. Taken together, our findings offer a preclinical rationale to explore the repositioning of GSK3 inhibitors to enhance differentiation-based therapy for AML treatment. PMID:26964622

Spatial Patterning of Newly-Inserted Material during Bacterial Cell Growth

Science.gov (United States)

Ursell, Tristan

2012-02-01

In the life cycle of a bacterium, rudimentary microscopy demonstrates that cell growth and elongation are essential characteristics of cellular reproduction. The peptidoglycan cell wall is the main load-bearing structure that determines both cell shape and overall size. However, simple imaging of cellular growth gives no indication of the spatial patterning nor mechanism by which material is being incorporated into the pre-existing cell wall. We employ a combination of high-resolution pulse-chase fluorescence microscopy, 3D computational microscopy, and detailed mechanistic simulations to explore how spatial patterning results in uniform growth and maintenance of cell shape. We show that growth is happening in discrete bursts randomly distributed over the cell surface, with a well-defined mean size and average rate. We further use these techniques to explore the effects of division and cell wall disrupting antibiotics, like cephalexin and A22, respectively, on the patterning of cell wall growth in E. coli. Finally, we explore the spatial correlation between presence of the bacterial actin-like cytoskeletal protein, MreB, and local cell wall growth. Together these techniques form a powerful method for exploring the detailed dynamics and involvement of antibiotics and cell wall-associated proteins in bacterial cell growth.[4pt] In collaboration with Kerwyn Huang, Stanford University.

Femtosecond Laser Direct Write Integration of Multi-Protein Patterns and 3D Microstructures into 3D Glass Microfluidic Devices

Directory of Open Access Journals (Sweden)

Daniela Serien

2018-01-01

Full Text Available Microfluidic devices and biochips offer miniaturized laboratories for the separation, reaction, and analysis of biochemical materials with high sensitivity and low reagent consumption. The integration of functional or biomimetic elements further functionalizes microfluidic devices for more complex biological studies. The recently proposed ship-in-a-bottle integration based on laser direct writing allows the construction of microcomponents made of photosensitive polymer inside closed microfluidic structures. Here, we expand this technology to integrate proteinaceous two-dimensional (2D and three-dimensional (3D microstructures with the aid of photo-induced cross-linking into glass microchannels. The concept is demonstrated with bovine serum albumin and enhanced green fluorescent protein, each mixed with photoinitiator (Sodium 4-[2-(4-Morpholino benzoyl-2-dimethylamino] butylbenzenesulfonate. Unlike the polymer integration, fabrication over the entire channel cross-section is challenging. Two proteins are integrated into the same channel to demonstrate multi-protein patterning. Using 50% w/w glycerol solvent instead of 100% water achieves almost the same fabrication resolution for in-channel fabrication as on-surface fabrication due to the improved refractive index matching, enabling the fabrication of 3D microstructures. A glycerol-water solvent also reduces the risk of drying samples. We believe this technology can integrate diverse proteins to contribute to the versatility of microfluidics.

Investigation of non-thermal plasma effects on lung cancer cells within 3D collagen matrices

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Karki, Surya B.; Thapa Gupta, Tripti; Yildirim-Ayan, Eda; Eisenmann, Kathryn M.; Ayan, Halim

2017-08-01

Recent breakthroughs in plasma medicine have identified a potential application for the non-thermal plasma in cancer therapy. Most studies on the effects of non-thermal plasma on cancer cells have used traditional two-dimensional (2D) monolayer cell culture. However, very few studies are conducted employing non-thermal plasma in animal models. Two dimensional models do not fully mimic the three-dimensional (3D) tumor microenvironment and animal models are expensive and time-consuming. Therefore, we used 3D collagen matrices that closely resemble the native geometry of cancer tissues and provide more physiologically relevant results than 2D models, while providing a more cost effective and efficient precursor to animal studies. We previously demonstrated a role for non-thermal plasma application in promoting apoptotic cell death and reducing the viability of A549 lung adenocarcinoma epithelial cells cultured upon 2D matrices. In this study, we wished to determine the efficacy of non-thermal plasma application in driving apoptotic cell death of A549 lung cancer cells encapsulated within a 3D collagen matrix. The percentage of apoptosis increased as treatment time increased and was time dependent. In addition, the anti-viability effect of plasma was demonstrated. Twenty-four hours post-plasma treatment, 38% and 99% of cell death occurred with shortest (15 s) and longest treatment time (120 s) respectively at the plasma-treated region. We found that plasma has a greater effect on the viability of A549 lung cancer cells on the superficial surface of 3D matrices and has diminishing effects as it penetrates the 3D matrix. We also identified the nitrogen and oxygen species generated by plasma and characterized their penetration in vertical and lateral directions within the 3D matrix from the center of the plasma-treated region. Therefore, the utility of non-thermal dielectric barrier discharge plasma in driving apoptosis and reducing the viability of lung cancer cells

Investigation of non-thermal plasma effects on lung cancer cells within 3D collagen matrices

International Nuclear Information System (INIS)

Karki, Surya B; Gupta, Tripti Thapa; Yildirim-Ayan, Eda; Ayan, Halim; Eisenmann, Kathryn M

2017-01-01

Recent breakthroughs in plasma medicine have identified a potential application for the non-thermal plasma in cancer therapy. Most studies on the effects of non-thermal plasma on cancer cells have used traditional two-dimensional (2D) monolayer cell culture. However, very few studies are conducted employing non-thermal plasma in animal models. Two dimensional models do not fully mimic the three-dimensional (3D) tumor microenvironment and animal models are expensive and time-consuming. Therefore, we used 3D collagen matrices that closely resemble the native geometry of cancer tissues and provide more physiologically relevant results than 2D models, while providing a more cost effective and efficient precursor to animal studies. We previously demonstrated a role for non-thermal plasma application in promoting apoptotic cell death and reducing the viability of A549 lung adenocarcinoma epithelial cells cultured upon 2D matrices. In this study, we wished to determine the efficacy of non-thermal plasma application in driving apoptotic cell death of A549 lung cancer cells encapsulated within a 3D collagen matrix. The percentage of apoptosis increased as treatment time increased and was time dependent. In addition, the anti-viability effect of plasma was demonstrated. Twenty-four hours post-plasma treatment, 38% and 99% of cell death occurred with shortest (15 s) and longest treatment time (120 s) respectively at the plasma-treated region. We found that plasma has a greater effect on the viability of A549 lung cancer cells on the superficial surface of 3D matrices and has diminishing effects as it penetrates the 3D matrix. We also identified the nitrogen and oxygen species generated by plasma and characterized their penetration in vertical and lateral directions within the 3D matrix from the center of the plasma-treated region. Therefore, the utility of non-thermal dielectric barrier discharge plasma in driving apoptosis and reducing the viability of lung cancer cells

Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling

Science.gov (United States)

Madl, Christopher M.; Lesavage, Bauer L.; Dewi, Ruby E.; Dinh, Cong B.; Stowers, Ryan S.; Khariton, Margarita; Lampe, Kyle J.; Nguyen, Duong; Chaudhuri, Ovijit; Enejder, Annika; Heilshorn, Sarah C.

2017-12-01

Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ~0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell-cell contact and promote β-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.

A simple hanging drop cell culture protocol for generation of 3D spheroids.

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Foty, Ramsey

2011-05-06

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels or in biomaterial scaffolds. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of

Gelatin methacrylamide hydrogel with graphene nanoplatelets for neural cell-laden 3D bioprinting.

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Wei Zhu; Harris, Brent T; Zhang, Lijie Grace

2016-08-01

Nervous system is extremely complex which leads to rare regrowth of nerves once injury or disease occurs. Advanced 3D bioprinting strategy, which could simultaneously deposit biocompatible materials, cells and supporting components in a layer-by-layer manner, may be a promising solution to address neural damages. Here we presented a printable nano-bioink composed of gelatin methacrylamide (GelMA), neural stem cells, and bioactive graphene nanoplatelets to target nerve tissue regeneration in the assist of stereolithography based 3D bioprinting technique. We found the resultant GelMA hydrogel has a higher compressive modulus with an increase of GelMA concentration. The porous GelMA hydrogel can provide a biocompatible microenvironment for the survival and growth of neural stem cells. The cells encapsulated in the hydrogel presented good cell viability at the low GelMA concentration. Printed neural construct exhibited well-defined architecture and homogenous cell distribution. In addition, neural stem cells showed neuron differentiation and neurites elongation within the printed construct after two weeks of culture. These findings indicate the 3D bioprinted neural construct has great potential for neural tissue regeneration.

Direct 3D cell-printing of human skin with functional transwell system.

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Kim, Byoung Soo; Lee, Jung-Seob; Gao, Ge; Cho, Dong-Woo

2017-06-06

Three-dimensional (3D) cell-printing has been emerging as a promising technology with which to build up human skin models by enabling rapid and versatile design. Despite the technological advances, challenges remain in the development of fully functional models that recapitulate complexities in the native tissue. Moreover, although several approaches have been explored for the development of biomimetic human skin models, the present skin models based on multistep fabrication methods using polydimethylsiloxane chips and commercial transwell inserts could be tackled by leveraging 3D cell-printing technology. In this paper, we present a new 3D cell-printing strategy for engineering a 3D human skin model with a functional transwell system in a single-step process. A hybrid 3D cell-printing system was developed, allowing for the use of extrusion and inkjet modules at the same time. We began by revealing the significance of each module in engineering human skin models; by using the extrusion-dispensing module, we engineered a collagen-based construct with polycaprolactone (PCL) mesh that prevented the contraction of collagen during tissue maturation; the inkjet-based dispensing module was used to uniformly distribute keratinocytes. Taking these features together, we engineered a human skin model with a functional transwell system; the transwell system and fibroblast-populated dermis were consecutively fabricated by using the extrusion modules. Following this process, keratinocytes were uniformly distributed onto the engineered dermis by the inkjet module. Our transwell system indicates a supportive 3D construct composed of PCL, enabling the maturation of a skin model without the aid of commercial transwell inserts. This skin model revealed favorable biological characteristics that included a stabilized fibroblast-stretched dermis and stratified epidermis layers after 14 days. It was also observed that a 50 times reduction in cost was achieved and 10 times less medium was

1,25-Dihydroxyvitamin D{sub 3} (1,25(OH){sub 2}D{sub 3}) Signaling Capacity and the Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer (NSCLC): Implications for Use of 1,25(OH){sub 2}D{sub 3} in NSCLC Treatment

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Upadhyay, Santosh Kumar; Verone, Alissa; Shoemaker, Suzanne [Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263 (United States); Qin, Maochun; Liu, Song [Department of Biostatistics and Bioinformatics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263 (United States); Campbell, Moray; Hershberger, Pamela A., E-mail: pamela.hershberger@roswellpark.org [Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263 (United States)

2013-11-08

1,25-dihydroxyvitamin D{sub 3} (1,25(OH){sub 2}D{sub 3}) exerts anti-proliferative activity by binding to the vitamin D receptor (VDR) and regulating gene expression. We previously reported that non-small cell lung cancer (NSCLC) cells which harbor epidermal growth factor receptor (EGFR) mutations display elevated VDR expression (VDR{sup high}) and are vitamin D-sensitive. Conversely, those with K-ras mutations are VDR{sup low} and vitamin D-refractory. Because EGFR mutations are found predominately in NSCLC cells with an epithelial phenotype and K-ras mutations are more common in cells with a mesenchymal phenotype, we investigated the relationship between vitamin D signaling capacity and the epithelial mesenchymal transition (EMT). Using NSCLC cell lines and publically available lung cancer cell line microarray data, we identified a relationship between VDR expression, 1,25(OH){sub 2}D{sub 3} sensitivity, and EMT phenotype. Further, we discovered that 1,25(OH){sub 2}D{sub 3} induces E-cadherin and decreases EMT-related molecules SNAIL, ZEB1, and vimentin in NSCLC cells. 1,25(OH){sub 2}D{sub 3}-mediated changes in gene expression are associated with a significant decrease in cell migration and maintenance of epithelial morphology. These data indicate that 1,25(OH){sub 2}D{sub 3} opposes EMT in NSCLC cells. Because EMT is associated with increased migration, invasion, and chemoresistance, our data imply that 1,25(OH){sub 2}D{sub 3} may prevent lung cancer progression in a molecularly defined subset of NSCLC patients.

Vitamin D-binding protein controls T cell responses to vitamin D

DEFF Research Database (Denmark)

Kongsbak, Martin; von Essen, Marina Rode; Levring, Trine Bøegh

2014-01-01

BACKGROUND: In vitro studies have shown that the active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), can regulate differentiation of CD4+ T cells by inhibiting Th1 and Th17 cell differentiation and promoting Th2 and Treg cell differentiation. However, the serum concentration of 1...... that activated T cells express the 25(OH)D-1α-hydroxylase CYP27B1 that converts 25(OH)D3 to 1,25(OH)2D3, it is still controversial whether activated T cells have the capacity to produce sufficient amounts of 1,25(OH)2D3 to affect vitamin D-responsive genes. Furthermore, it is not known how the vitamin D......-binding protein (DBP) found in high concentrations in serum affects T cell responses to 25(OH)D3. RESULTS: We found that activated T cells express CYP27B1 and have the capacity to produce sufficient 1,25(OH)2D3 to affect vitamin D-responsive genes when cultured with physiological concentrations of 25(OH)D3...

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton's jelly stem cells (hWJSCs).

Science.gov (United States)

Mostafavi-Pour, Zohreh; Ashrafi, Mohammad Reza; Talaei-Khozani, Tahereh

2018-06-01

Human Wharton's jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P < 0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.

3D Printing and 3D Bioprinting in Pediatrics.

Science.gov (United States)

Vijayavenkataraman, Sanjairaj; Fuh, Jerry Y H; Lu, Wen Feng

2017-07-13

Additive manufacturing, commonly referred to as 3D printing, is a technology that builds three-dimensional structures and components layer by layer. Bioprinting is the use of 3D printing technology to fabricate tissue constructs for regenerative medicine from cell-laden bio-inks. 3D printing and bioprinting have huge potential in revolutionizing the field of tissue engineering and regenerative medicine. This paper reviews the application of 3D printing and bioprinting in the field of pediatrics.

3D Printing and 3D Bioprinting in Pediatrics

OpenAIRE

Vijayavenkataraman, Sanjairaj; Fuh, Jerry Y H; Lu, Wen Feng

2017-01-01

Additive manufacturing, commonly referred to as 3D printing, is a technology that builds three-dimensional structures and components layer by layer. Bioprinting is the use of 3D printing technology to fabricate tissue constructs for regenerative medicine from cell-laden bio-inks. 3D printing and bioprinting have huge potential in revolutionizing the field of tissue engineering and regenerative medicine. This paper reviews the application of 3D printing and bioprinting in the field of pediatrics.

Potential gene regulatory role for cyclin D3 in muscle cells

Indian Academy of Sciences (India)

2015-06-27

Jun 27, 2015 ... D3-expressing cells on induction of differentiation. 2. Materials and .... 2 –ΔΔCt method was used for quantification and each gene ..... of pluripotency genes known to be silenced by deposition of ..... embryonic stem cells.

A 3D human neural cell culture system for modeling Alzheimer’s disease

Science.gov (United States)

Kim, Young Hye; Choi, Se Hoon; D’Avanzo, Carla; Hebisch, Matthias; Sliwinski, Christopher; Bylykbashi, Enjana; Washicosky, Kevin J.; Klee, Justin B.; Brüstle, Oliver; Tanzi, Rudolph E.; Kim, Doo Yeon

2015-01-01

Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer’s disease (AD) because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel three-dimensional (3D) culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of β-amyloid and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix, and the analysis of AD pathogenesis. The 3D culture generation takes 1–2 days. The aggregation of β-amyloid is observed after 6-weeks of differentiation followed by robust tau pathology after 10–14 weeks. PMID:26068894

Ordered patterns of cell shape and orientational correlation during spontaneous cell migration.

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Yusuke T Maeda

Full Text Available BACKGROUND: In the absence of stimuli, most motile eukaryotic cells move by spontaneously coordinating cell deformation with cell movement in the absence of stimuli. Yet little is known about how cells change their own shape and how cells coordinate the deformation and movement. Here, we investigated the mechanism of spontaneous cell migration by using computational analyses. METHODOLOGY: We observed spontaneously migrating Dictyostelium cells in both a vegetative state (round cell shape and slow motion and starved one (elongated cell shape and fast motion. We then extracted regular patterns of morphological dynamics and the pattern-dependent systematic coordination with filamentous actin (F-actin and cell movement by statistical dynamic analyses. CONCLUSIONS/SIGNIFICANCE: We found that Dictyostelium cells in both vegetative and starved states commonly organize their own shape into three ordered patterns, elongation, rotation, and oscillation, in the absence of external stimuli. Further, cells inactivated for PI3-kinase (PI3K and/or PTEN did not show ordered patterns due to the lack of spatial control in pseudopodial formation in both the vegetative and starved states. We also found that spontaneous polarization was achieved in starved cells by asymmetric localization of PTEN and F-actin. This breaking of the symmetry of protein localization maintained the leading edge and considerably enhanced the persistence of directed migration, and overall random exploration was ensured by switching among the different ordered patterns. Our findings suggest that Dictyostelium cells spontaneously create the ordered patterns of cell shape mediated by PI3K/PTEN/F-actin and control the direction of cell movement by coordination with these patterns even in the absence of external stimuli.

Developement of 3D Vertically Integrated Pattern Recognition Associative Memory (VIPRAM)

International Nuclear Information System (INIS)

Deputch, G.; Hoff, J.; Lipton, R.; Liu, T.; Olsen, J.; Ramberg, E.; Wu, Jin-Yuan; Yarema, R.; Shochet, M.; Tang, F.; Demarteau, M.

2011-01-01

Many next-generation physics experiments will be characterized by the collection of large quantities of data, taken in rapid succession, from which scientists will have to unravel the underlying physical processes. In most cases, large backgrounds will overwhelm the physics signal. Since the quantity of data that can be stored for later analysis is limited, real-time event selection is imperative to retain the interesting events while rejecting the background. Scaling of current technologies is unlikely to satisfy the scientific needs of future projects, so investments in transformational new technologies need to be made. For example, future particle physics experiments looking for rare processes will have to address the demanding challenges of fast pattern recognition in triggering as detector hit density becomes significantly higher due to the high luminosity required to produce the rare processes. In this proposal, we intend to develop hardware-based technology that significantly advances the state-of-the-art for fast pattern recognition within and outside HEP using the 3D vertical integration technology that has emerged recently in industry. The ultimate physics reach of the LHC experiments will crucially depend on the tracking trigger's ability to help discriminate between interesting rare events and the background. Hardware-based pattern recognition for fast triggering on particle tracks has been successfully used in high-energy physics experiments for some time. The CDF Silicon Vertex Trigger (SVT) at the Fermilab Tevatron is an excellent example. The method used there, developed in the 1990's, is based on algorithms that use a massively parallel associative memory architecture to identify patterns efficiently at high speed. However, due to much higher occupancy and event rates at the LHC, and the fact that the LHC detectors have a much larger number of channels in their tracking detectors, there is an enormous challenge in implementing pattern recognition

Induction of regulatory dendritic cells by dexamethasone and 1alpha,25-Dihydroxyvitamin D(3)

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Gad, Monika; Walter, Mark R

2004-01-01

D(3) the active form of Vitamin D(3) (D(3)) in combination with dexamethasone (Dex) has a synergistic effect on LPS-induced maturation of DC. Monocyte-derived DCs cultured with D(3) and Dex during LPS-induced maturation have a low stimulatory effect on allogeneic T cells comparable...

Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli.

Science.gov (United States)

Wu, Hong; Chen, Jinchun; Chen, Guo-Qiang

2016-12-01

E. coli JM109∆envC∆nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109∆minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109∆envC∆nlpD and E. coli JM109∆minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109∆minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109∆envC∆nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.

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

International Nuclear Information System (INIS)

Hehlgans, Stephanie; Lange, Inga; Eke, Iris; Cordes, Nils

2009-01-01

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.

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

Science.gov (United States)

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

2016-06-07

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

The effect of miR-338-3p on HBx deletion-mutant (HBx-d382 mediated liver-cell proliferation through CyclinD1 regulation.

Directory of Open Access Journals (Sweden)

Xiaoyu Fu

Full Text Available Hepatitis B Virus (HBV DNA integration and HBV X (HBx deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382-400 can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3'UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells, and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3'UTR region, the effect of miR-338-3p on the second binding site (nt 2397-2403 was required for the inhibition.miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3'UTR region, mainly at nt 2397-2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the

Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time

Directory of Open Access Journals (Sweden)

Baek N

2016-11-01

Full Text Available NamHuk Baek,1,* Ok Won Seo,1,* MinSung Kim,1 John Hulme,2 Seong Soo A An2 1Department of R & D, NanoEntek Inc., Seoul, Republic of Korea; 2Department of BioNano Technology Gachon University, Gyeonggi-do, Republic of Korea *These authors contributed equally to this work Abstract: Recently, increasing numbers of cell culture experiments with 3D spheroids presented better correlating results in vivo than traditional 2D cell culture systems. 3D spheroids could offer a simple and highly reproducible model that would exhibit many characteristics of natural tissue, such as the production of extracellular matrix. In this paper numerous cell lines were screened and selected depending on their ability to form and maintain a spherical shape. The effects of increasing concentrations of doxorubicin (DXR on the integrity and viability of the selected spheroids were then measured at regular intervals and in real-time. In total 12 cell lines, adenocarcinomic alveolar basal epithelial (A549, muscle (C2C12, prostate (DU145, testis (F9, pituitary epithelial-like (GH3, cervical cancer (HeLa, HeLa contaminant (HEp2, embryo (NIH3T3, embryo (PA317, neuroblastoma (SH-SY5Y, osteosarcoma U2OS, and embryonic kidney cells (293T, were screened. Out of the 12, 8 cell lines, NIH3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U2OS formed regular spheroids and the effects of DXR on these structures were measured at regular intervals. Finally, 5 cell lines, A549, HeLa, SH-SY5Y, U2OS, and 293T, were selected for real-time monitoring and the effects of DXR treatment on their behavior were continuously recorded for 5 days. A potential correlation regarding the effects of DXR on spheroid viability and ATP production was measured on days 1, 3, and 5. Cytotoxicity of DXR seemed to occur after endocytosis, since the cellular activities and ATP productions were still viable after 1 day of the treatment in all spheroids, except SH-SY5Y. Both cellular activity and ATP production were

Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.

Science.gov (United States)

Omlor, G W; Nerlich, A G; Tirlapur, U K; Urban, J P; Guehring, T

2014-12-01

Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. 3D

Application of metaheuristics to Loading Pattern Optimization problems based on the IAEA-3D and BIBLIS-2D data

International Nuclear Information System (INIS)

Meneses, Anderson Alvarenga de Moura; Araujo, Lenilson Moreira; Nast, Fernando Nogueira; Da Silva, Patrick Vasconcelos; Schirru, Roberto

2018-01-01

Highlights: •Metaheuristics were applied to Loading Pattern Optimization problems and compared. •The problems are based on data of the benchmarks IAEA and BIBLIS. •The metaheuristics compared were PSO, Cross-Entropy, PBIL and Artificial Bee Colony. •Angra 1 NPP data were also used for further comparison of the algorithms. -- Abstract: The Loading Pattern Optimization (LPO) of a Nuclear Power Plant (NPP), or in-core fuel management optimization, is a real-world and prominent problem in Nuclear Engineering with the goal of finding an optimal (or near-optimal) Loading Pattern (LP), in terms of energy production, within adequate safety margins. Most of the reactor models used in the LPO problem are particular cases, such as research or power reactors with technical data that cannot be made available for several reasons, which makes the reproducibility of tests unattainable. In the present article we report the results of LPO of problems based upon reactor physics benchmarks. Since such data are well-known and widely available in the literature, it is possible to reproduce tests for comparison of techniques. We performed the LPO with the data of the benchmarks IAEA-3D and BIBLIS-2D. The Reactor Physics code RECNOD, which was used in previous works for the optimization of Angra 1 NPP in Brazil, was also used for further comparison. Four Optimization Metaheuristics (OMHs) were applied to those problems: Particle Swarm Optimization (PSO), Cross-Entropy algorithm (CE), Artificial Bee Colony (ABC) and Population-Based Incremental Learning (PBIL). For IAEA-3D, the best algorithm was the ABC. For BIBLIS-2D, PBIL was the best OMH. For Angra 1 / RECNOD optimization problem, PBIL, ABC and CE were the best OMHs.

Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

Energy Technology Data Exchange (ETDEWEB)

Ko, Jae Hyung [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Kim, Yang Hee [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Jeong, Seong Hee; Lee, Song [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Park, Si-Nae [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Shim, In Kyong, E-mail: shimiink@gmail.com [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Kim, Song Cheol, E-mail: drksc@amc.seoul.kr [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Department of Surgery, University of Ulsan College of Medicine & Asan Medical Center, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of)

2015-08-07

Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.

Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

International Nuclear Information System (INIS)

Ko, Jae Hyung; Kim, Yang Hee; Jeong, Seong Hee; Lee, Song; Park, Si-Nae; Shim, In Kyong; Kim, Song Cheol

2015-01-01

Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release

Chicken HOXA3 Gene: Its Expression Pattern and Role in Branchial Nerve Precursor Cell Migration

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Watari-Goshima, Natsuko; Chisaka, Osamu

2011-01-01

In vertebrates, the proximal and distal sensory ganglia of the branchial nerves are derived from neural crest cells (NCCs) and placodes, respectively. We previously reported that in Hoxa3 knockout mouse embryos, NCCs and placode-derived cells of the glossopharyngeal nerve were defective in their migration. In this report, to determine the cell-type origin for this Hoxa3 knockout phenotype, we blocked the expression of the gene with antisense morpholino oligonucleotides (MO) specifically in either NCCs/neural tube or placodal cells of chicken embryos. Our results showed that HOXA3 function was required for the migration of the epibranchial placode-derived cells and that HOXA3 regulated this cell migration in both NCCs/neural tube and placodal cells. We also report that the expression pattern of chicken HOXA3 was slightly different from that of mouse Hoxa3. PMID:21278919

Comparison between 3D and 1D simulations of a regenerative blower for fuel cell applications

International Nuclear Information System (INIS)

Badami, M.; Mura, M.

2012-01-01

Highlights: ► A hydrogen recirculation blower for automotive fuel cells applications is studied. ► A 3D CFD analysis has been carried out to better understand the internal flows of the machine. ► The CFD results are compared to a 1D model set up by the authors in previous works. ► The main hypotheses put forward for the theoretical 1D model are compatible with the 3D analysis. - Abstract: A 3D Computational Fluid Dynamics (CFD) analysis has been carried out to better understand the internal fluid dynamics of a regenerative blower used for hydrogen recirculation in a Proton Exchange Membrane (PEM), Fuel Cell (FC) utilized for automotive applications. The obtained results are used to highlight the motion of the fluid in the vanes and in the side channel of the machine and to verify the main hypotheses put forward concerning the theoretical 1D model set up by the authors in previous works on the basis of the momentum exchange theory. Finally, the CFD analysis has been used to point out the effect of the slope of the vanes on the performance of the regenerative blower, and the results have been compared with those obtained using of the 1D model.

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

Science.gov (United States)

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

2011-01-01

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

Dopamine receptors D3 and D5 regulate CD4(+)T-cell activation and differentiation by modulating ERK activation and cAMP production.

Science.gov (United States)

Franz, Dafne; Contreras, Francisco; González, Hugo; Prado, Carolina; Elgueta, Daniela; Figueroa, Claudio; Pacheco, Rodrigo

2015-07-15

Dopamine receptors have been described in T-cells, however their signalling pathways coupled remain unknown. Since cAMP and ERKs play key roles regulating T-cell physiology, we aim to determine whether cAMP and ERK1/2-phosphorylation are modulated by dopamine receptor 3 (D3R) and D5R, and how this modulation affects CD4(+) T-cell activation and differentiation. Our pharmacologic and genetic evidence shows that D3R-stimulation reduced cAMP levels and ERK2-phosphorylation, consequently increasing CD4(+) T-cell activation and Th1-differentiation, respectively. Moreover, D5R expression reinforced TCR-triggered ERK1/2-phosphorylation and T-cell activation. In conclusion, these findings demonstrate how D3R and D5R modulate key signalling pathways affecting CD4(+) T-cell activation and Th1-differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Cancer Cell Migration within 3D Layer-By-Layer Microfabricated Photocrosslinked PEG Scaffolds with Tunable Stiffness

OpenAIRE

Soman, Pranav; Kelber, Jonathan A.; Lee, Jin Woo; Wright, Tracy; Vecchio, Kenneth S.; Klemke, Richard L.; Chen, Shaochen

2012-01-01

Our current understanding of 3-dimensional (3D) cell migration is primarily based on results from fibrous scaffolds with randomly organized internal architecture. Manipulations that change the stiffness of these 3D scaffolds often alter other matrix parameters that can modulate cell motility independently or synergistically, making observations less predictive of how cells behave when migrating in 3D. In order to decouple microstructural influences and stiffness effects, we have designed and ...

A Dual Band Additively Manufactured 3D Antenna on Package with Near-Isotropic Radiation Pattern

KAUST Repository

Su, Zhen

2018-04-06

Internet of things (IoT) applications need wireless connectivity on devices with very small footprints, and in RF obscure environments. The antenna for such applications must work on multiple GSM bands (preferred choice for network connectivity), provide near isotropic radiation pattern to maintain orientation insensitive communication, be small in size so that it can be integrated with futuristic miniaturized IoT devices, and be low in cost to be implemented on billions of devices. This paper presents a novel 3D dual band near-isotropic wideband GSM antenna to fulfill these requirements. The antenna has been realized on the package of electronics through additive manufacturing to ensure efficient utilization of available space and lower cost. The proposed antenna consists of a meander line antenna that is folded on the faces of a 3D package with two variations, 0.375λ length for narrowband version and 0.67λ length for the wideband version. Theoretical conditions to achieve near isotropic radiation pattern with bent wire antennas on a 3D surface have been derived. The antenna has been optimized to operate with embedded electronics and a large metallic battery. The antenna provides 8.9% and 34.4% bandwidths, at 900 and 1800 MHz respectively with decent near isotropic radiation behavior.

A 3D Microfluidic Model to Recapitulate Cancer Cell Migration and Invasion

Directory of Open Access Journals (Sweden)

Yi-Chin Toh

2018-04-01

Full Text Available We have developed a microfluidic-based culture chip to simulate cancer cell migration and invasion across the basement membrane. In this microfluidic chip, a 3D microenvironment is engineered to culture metastatic breast cancer cells (MX1 in a 3D tumor model. A chemo-attractant was incorporated to stimulate motility across the membrane. We validated the usefulness of the chip by tracking the motilities of the cancer cells in the system, showing them to be migrating or invading (akin to metastasis. It is shown that our system can monitor cell migration in real time, as compare to Boyden chambers, for example. Thus, the chip will be of interest to the drug-screening community as it can potentially be used to monitor the behavior of cancer cell motility, and, therefore, metastasis, in the presence of anti-cancer drugs.

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

Science.gov (United States)

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.

Intradermal application of vitamin D3 increases migration of CD14+ dermal dendritic cells and promotes the development of Foxp3+ regulatory T cells

NARCIS (Netherlands)

Bakdash, Ghaith; Schneider, Laura P.; van Capel, Toni M. M.; Kapsenberg, Martien L.; Teunissen, Marcel B. M.; de Jong, Esther C.

2013-01-01

The active form of vitamin D3 (VitD) is a potent immunosuppressive drug. Its effects are mediated in part through dendritic cells (DCs) that promote the development of regulatory T cells (Tregs). However, it remains elusive how VitD would influence the different human skin DC subsets, e.g.,

Light-absorbent liquid immersion angled exposure for patterning 3D samples with vertical sidewalls

International Nuclear Information System (INIS)

Kumagai, Shinya; Kubo, Hironori; Sasaki, Minoru

2017-01-01

To make photolithography patterns on 3D samples, the angled (inclined) exposure technique has been used so far. However, technological issues have emerged in making photolithography patterns on the surface of trench structures. The surface of the trench structures can be covered with a photoresist film by spray-coating but the photoresist film deposited on the sidewalls and bottom of the trench is generally thin. The thin photoresist film deposited inside the trench has been easily overdosed. Moreover, irregular patterns have frequently been formed by the light reflected inside the trench. In this study, we have developed liquid immersion photolithography using a light-absorbent material. The light-reflection inside the trench was suppressed. Various patterns were transferred in the photoresist film deposited on the trench structures which had an aspect ratio of 0.74. Compared to immersion photolithography using pure water under p -polarization light control, the light-absorbent liquid immersion photolithography developed here patterned well the surfaces of the trench sidewalls and bottom. (paper)

Effects of 1,25-dihydroxyvitamin D3 and vitamin D3 on the expression of the vitamin D receptor in human skeletal muscle cells

Science.gov (United States)

Vitamin D receptor (VDR) expression and action in non-human skeletal muscle have recently been reported in several studies, yet data on the activity and expression of VDR in human muscle cells are scarce. We conducted a series of studies to examine the (1) effect of 1,25-dihydroxyvitamin D3 (1,25(OH...

Osteogenic Differentiation of Miniature Pig Mesenchymal Stem Cells in 2D and 3D Environment

Czech Academy of Sciences Publication Activity Database

Juhásová, Jana; Juhás, Štefan; Klíma, Jiří; Strnádel, Ján; Holubová, Monika; Motlík, Jan

2011-01-01

Roč. 60, č. 3 (2011), s. 559-571 ISSN 0862-8408 R&D Projects: GA MŠk 1M0538; GA MŠk 2B06130 Institutional research plan: CEZ:AV0Z50450515 Keywords : miniature pig * mesenchymal stem cells * cell differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.555, year: 2011

Locoregional deformation pattern of the patellar cartilage after different loading types. High-resolution 3D-MRI volumetry at 3 T in-vivo

International Nuclear Information System (INIS)

Horng, Annie; Raya, J.; Klinikum der Ludwig-Maximilians-Universitaet Muenchen, Grosshadern; Zscharn, M.

2011-01-01

Purpose: To analyze locoregional deformation patterns indicative of contact areas in patellar cartilage after different loading exercises. Materials and Methods: 7 healthy patellae were examined in-vivo before and immediately after standardized loading (kneeling, squatting or knee bends) and after 90 minutes of rest using a sagittal 3D-T1-w FLASH WE sequence (22 msec/ 9.8msec/ 15 / 0.3 x 0.3 x 1.5 mm 3 ) at 3 T. After cartilage segmentation and 3D reconstruction, voxel-based and global precision errors (PR) were calculated. The former were used to determine significant differences in local cartilage thickness. Voxel-based 2σ-thickness difference maps were calculated to visualize locoregional deformation patterns. Global changes in volume (Vol), mean thickness (mTh) and cartilage-bone-interface area (CBIA) were calculated. Results: The voxel-based PR depended on cartilage thickness (D) ranging from 0.12 - 0.35 mm. For D ≥ 1 mm the RF was 3 (2.4 %) for Vol, 0.06 mm (2.0 %) for mTh and 16 mm 2 (1.4 %) for CBIA. The focal cartilage deformation equaled 14 % of the local thickness reduction. The deformation areas were oval and located in the peripheral medial (more vertically oriented, all exercises) and caudo-lateral (more horizontally oriented, kneeling and knee bends) aspects of the patella and were least pronounced in knee bends. Significant changes for Vol/mTh ranged from 2.1 to 3.7 %. Conclusion: This MRI-based study is the first to identify in-vivo voxel-based patellar cartilage deformation patterns indicating contact and loading zones after kneeling and squatting. These zones are anatomically and functionally plausible and may represent areas where stress induced degeneration and subsequent OA can originate. The data may facilitate understanding of individual knee loading properties and help to improve and validate biomechanical models for the knee. (orig.)

Complex furrows in a 2D epithelial sheet code the 3D structure of a beetle horn.

Science.gov (United States)

Matsuda, Keisuke; Gotoh, Hiroki; Tajika, Yuki; Sushida, Takamichi; Aonuma, Hitoshi; Niimi, Teruyuki; Akiyama, Masakazu; Inoue, Yasuhiro; Kondo, Shigeru

2017-10-24

The external organs of holometabolous insects are generated through two consecutive processes: the development of imaginal primordia and their subsequent transformation into the adult structures. During the latter process, many different phenomena at the cellular level (e.g. cell shape changes, cell migration, folding and unfolding of epithelial sheets) contribute to the drastic changes observed in size and shape. Because of this complexity, the logic behind the formation of the 3D structure of adult external organs remains largely unknown. In this report, we investigated the metamorphosis of the horn in the Japanese rhinoceros beetle Trypoxylus dichotomus. The horn primordia is essentially a 2D epithelial cell sheet with dense furrows. We experimentally unfolded these furrows using three different methods and found that the furrow pattern solely determines the 3D horn structure, indicating that horn formation in beetles occurs by two distinct processes: formation of the furrows and subsequently unfolding them. We postulate that this developmental simplicity offers an inherent advantage to understanding the principles that guide 3D morphogenesis in insects.

Bioprinting of 3D Tissue Models Using Decellularized Extracellular Matrix Bioink.

Science.gov (United States)

Pati, Falguni; Cho, Dong-Woo

2017-01-01

Bioprinting provides an exciting opportunity to print and pattern all the components that make up a tissue-cells and extracellular matrix (ECM) material-in three dimensions (3D) to generate tissue analogues. A large number of materials have been used for making bioinks; however, majority of them cannot represent the complexity of natural ECM and thus are unable to reconstitute the intrinsic cellular morphologies and functions. We present here a method for making of bioink from decellularized extracellular matrices (dECMs) and a protocol for bioprinting of cell-laden constructs with this novel bioink. The dECM bioink is capable of providing an optimized microenvironment that is conducive to the growth of 3D structured tissue. We have prepared bioinks from different tissues, including adipose, cartilage and heart tissues and achieved high cell viability and functionality of the bioprinted tissue structures using our novel bioink.

TRPV6 determines the effect of vitamin D3 on prostate cancer cell growth.

Directory of Open Access Journals (Sweden)

V'yacheslav Lehen'kyi

Full Text Available Despite remarkable advances in the therapy and prevention of prostate cancer it is still the second cause of death from cancer in industrialized countries. Many therapies initially shown to be beneficial for the patients were abandoned due to the high drug resistance and the evolution rate of the tumors. One of the prospective therapeutical agents even used in the first stage clinical trials, 1,25-dihydroxyvitamin D3, was shown to be either unpredictable or inefficient in many cases. We have already shown that TRPV6 calcium channel, which is the direct target of 1,25-dihydroxyvitamin D3 receptor, positively controls prostate cancer proliferation and apoptosis resistance (Lehen'kyi et al., Oncogene, 2007. However, how the known 1,25-dihydroxyvitamin D3 antiproliferative effects may be compatible with the upregulation of pro-oncogenic TRPV6 channel remains a mystery. Here we demonstrate that in low steroid conditions 1,25-dihydroxyvitamin D3 upregulates the expression of TRPV6, enhances the proliferation by increasing the number of cells entering into S-phase. We show that these pro-proliferative effects of 1,25-dihydroxyvitamin D3 are directly mediated via the overexpression of TRPV6 channel which increases calcium uptake into LNCaP cells. The apoptosis resistance of androgen-dependent LNCaP cells conferred by TRPV6 channel is drastically inversed when 1,25-dihydroxyvitamin D3 effects were combined with the successful TRPV6 knockdown. In addition, the use of androgen-deficient DU-145 and androgen-insensitive LNCaP C4-2 cell lines allowed to suggest that the ability of 1,25-dihydroxyvitamin D3 to induce the expression of TRPV6 channel is a crucial determinant of the success or failure of 1,25-dihydroxyvitamin D3-based therapies.

Integration of atomic layer deposition CeO2 thin films with functional complex oxides and 3D patterns

International Nuclear Information System (INIS)

Coll, M.; Palau, A.; Gonzalez-Rosillo, J.C.; Gazquez, J.; Obradors, X.; Puig, T.

2014-01-01

We present a low-temperature, < 300 °C, ex-situ integration of atomic layer deposition (ALD) ultrathin CeO 2 layers (3 to 5 unit cells) with chemical solution deposited La 0.7 Sr 0.3 MnO 3 (LSMO) functional complex oxides for multilayer growth without jeopardizing the morphology, microstructure and physical properties of the functional oxide layer. We have also extended this procedure to pulsed laser deposited YBa 2 Cu 3 O 7 (YBCO) thin films. Scanning force microscopy, X-ray diffraction, aberration corrected scanning transmission electron microscopy and macroscopic magnetic measurements were used to evaluate the quality of the perovskite films before and after the ALD process. By means of microcontact printing and ALD we have prepared CeO 2 patterns using an ozone-robust photoresist that will avoid the use of hazardous lithography processes directly on the device components. These bilayers, CeO 2 /LSMO and CeO 2 /YBCO, are foreseen to have special interest for resistive switching phenomena in resistive random-access memory. - Highlights: • Integration of atomic layer deposition (ALD) CeO 2 layers on functional complex oxides • Resistive switching is identified in CeO 2 /La 0.7 Sr 0.3 MnO 3 and CeO 2 /YBa 2 Cu 3 O 7 bilayers. • Study of the robustness of organic polymers for area-selective ALD • Combination of ALD and micro-contact printing to obtain 3D patterns of CeO 2

Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

Science.gov (United States)

Choi, Young-Jun; Kim, Hyemin; Kim, Ji-Woo; Yoon, Seokjoo; Park, Han-Jin

2018-05-01

The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

MAPLE deposition of 3D micropatterned polymeric substrates for cell culture

Energy Technology Data Exchange (ETDEWEB)

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.

3D-Printing Electrolytes for Solid-State Batteries.

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McOwen, Dennis W; Xu, Shaomao; Gong, Yunhui; Wen, Yang; Godbey, Griffin L; Gritton, Jack E; Hamann, Tanner R; Dai, Jiaqi; Hitz, Gregory T; Hu, Liangbing; Wachsman, Eric D

2018-05-01

Solid-state batteries have many enticing advantages in terms of safety and stability, but the solid electrolytes upon which these batteries are based typically lead to high cell resistance. Both components of the resistance (interfacial, due to poor contact with electrolytes, and bulk, due to a thick electrolyte) are a result of the rudimentary manufacturing capabilities that exist for solid-state electrolytes. In general, solid electrolytes are studied as flat pellets with planar interfaces, which minimizes interfacial contact area. Here, multiple ink formulations are developed that enable 3D printing of unique solid electrolyte microstructures with varying properties. These inks are used to 3D-print a variety of patterns, which are then sintered to reveal thin, nonplanar, intricate architectures composed only of Li 7 La 3 Zr 2 O 12 solid electrolyte. Using these 3D-printing ink formulations to further study and optimize electrolyte structure could lead to solid-state batteries with dramatically lower full cell resistance and higher energy and power density. In addition, the reported ink compositions could be used as a model recipe for other solid electrolyte or ceramic inks, perhaps enabling 3D printing in related fields. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Relationship between Infill patterns in 3D printing and Hounsfield Unit

International Nuclear Information System (INIS)

Savi, M.; Cechinel, C.M.; Silveira, L.C.; Soares, F.A.P.; Potiens, M.P.A.

2017-01-01

Introduction: One of the requirements for a phantom to correctly simulate the human body is that the radiation attenuation of the material used is compatible with the values of its corresponding tissues. The aim of this study is to evaluate the values of Hounsfield Unit (HU) in a 3D printed material in order to verify its compatibility, on tomography, with human tissues, so that it can be used as feedstock for simulators constructed in 3D printers. Methods: Cubes with 2cm of edge were printed using ABS filament with 8 different forms of internal filling. The samples were irradiated by a CT scanner, the measured HUs and their results compared to the literature. Results: Modification of the filling form as well as its percentage influenced the HU values that ranged from -133 to -451. Conclusion: The specific variations of internal fill patterns directly influence the interaction of the material used with the radiation, thus altering the HU values. Despite the variation found, the HU values were sufficient to simulate few tissues in the human body, which requires future studies with new materials that further attenuate the radiation and the range of tissues to be expanded. (author)

Relationship between Infill patterns in 3D printing and Hounsfield Unit

Energy Technology Data Exchange (ETDEWEB)

Savi, M.; Cechinel, C.M.; Silveira, L.C.; Soares, F.A.P., E-mail: Matheus.savi@ifsc.edu.br [Instituo Federal de Santa Catarina (IFSC), Florianópolis (Brazil). Departamento Acadêmico de Saúde e Serviços; Potiens, M.P.A. [Instituto de Pesquisas Energéticas Nucleares (IPEN/CNEN-SP), São Paulo, SP (Brazil). Lab. de Calibração de Instrumentos

2017-07-01

Introduction: One of the requirements for a phantom to correctly simulate the human body is that the radiation attenuation of the material used is compatible with the values of its corresponding tissues. The aim of this study is to evaluate the values of Hounsfield Unit (HU) in a 3D printed material in order to verify its compatibility, on tomography, with human tissues, so that it can be used as feedstock for simulators constructed in 3D printers. Methods: Cubes with 2cm of edge were printed using ABS filament with 8 different forms of internal filling. The samples were irradiated by a CT scanner, the measured HUs and their results compared to the literature. Results: Modification of the filling form as well as its percentage influenced the HU values that ranged from -133 to -451. Conclusion: The specific variations of internal fill patterns directly influence the interaction of the material used with the radiation, thus altering the HU values. Despite the variation found, the HU values were sufficient to simulate few tissues in the human body, which requires future studies with new materials that further attenuate the radiation and the range of tissues to be expanded. (author)

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

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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.

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

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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

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. 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. 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 pfibrocystic from the metastatic cell populations. Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.

Growth Inhibition of Osteosarcoma Cell Lines in 3D Cultures: Role of Nitrosative and Oxidative Stress.

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Gorska, Magdalena; Krzywiec, Pawel Bieniasz; Kuban-Jankowska, Alicja; Zmijewski, Michal; Wozniak, Michal; Wierzbicka, Justyna; Piotrowska, Anna; Siwicka, Karolina

2016-01-01

3D cell cultures have revolutionized the understanding of cell behavior, allowing culture of cells with the possibility of resembling in vivo intercellular signaling and cell-extracellular matrix interaction. The effect of limited oxygen penetration into 3D culture of highly metastatic osteosarcoma 143B cells in terms of expression of nitro-oxidative stress markers was investigated and compared to standard 2D cell culture. Human osteosarcoma (143B cell line) cells were cultured as monolayers, in collagen and Matrigel. Cell viability, gene expression of nitro-oxidative stress markers, and vascular endothelial growth factor were determined using Trypan blue assay, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Three-dimensional environments modify nitro-oxidative stress and influence gene expression and cell proliferation of OS 143B cells. Commercial cell lines might not constitute a good model of 3D cultures for bone tissue engineering, as they are highly sensitive to hypoxia, and hypoxic conditions can induce oxidation of the cellular environment. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Establishment and cell cycle distribution pattern of a radioresistant subline from human lung cancer D6 cell line

International Nuclear Information System (INIS)

Wei Qichun; Zheng Shu

2003-01-01

Objective: To establish a radioresistant cell subline from a human D6 lung cancer cell line and investigate the mechanism of radioresistance. Methods: D6 human NSCLC cells were exposed to X-rays generated by a linear accelerator(650 cGy per fraction). After a total exposure dose of 5200 cGy, a monoclone was obtained. The radiosensitivity and cell cycle distribution of this clone, together with its parent D6 cells, were measured by clonogenic assay and flow cytometry. Results: The new clone, namely D 6 -R subline, had a higher D 0 (D 0 =2.08 Gy) and a broader initial shoulder(Dq=1.64 Gy, N=2.20) than those of the parent D6 cell line (D 0 =1.84 Gy, Dq=0.34 Gy, N=1.20), being 1.65-fold increase in radioresistance as regards to the SF 2 . The D6-R subline also showed higher percentage of cells in S phase(53.4% vs 37.8%), but lower percentages in G 1 (44.1% vs 57.2%) and G 2 /M(2.5% vs 5%) phases. Conclusion: The new subline D6-R is more radioresistant as compare to its parent D6 cell line, and has a different cell cycle distribution

Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression.

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Lanuza, Pilar M; Vigueras, Alan; Olivan, Sara; Prats, Anne C; Costas, Santiago; Llamazares, Guillermo; Sanchez-Martinez, Diego; Ayuso, José María; Fernandez, Luis; Ochoa, Ignacio; Pardo, Julián

2018-01-01

Haploidentical Natural Killer (NK) cells have been shown as an effective and safe alternative for the treatment of haematological malignancies with poor prognosis for which traditional therapies are ineffective. In contrast to haematological cancer cells, that mainly grow as single suspension cells, solid carcinomas are characterised by a tridimensional (3D) architecture that provide specific surviving advantages and resistance against chemo- and radiotherapy. However, little is known about the impact of 3D growth on solid cancer immunotherapy especially adoptive NK cell transfer. We have recently developed a protocol to activate ex vivo human primary NK cells using B lymphoblastic cell lines, which generates NK cells able to overcome chemoresistance in haematological cancer cells. Here we have analysed the activity of these allogeneic NK cells against colorectal (CRC) human cell lines growing in 3D spheroid culture and correlated with the expression of some of the main ligands regulating NK cell activity. Our results indicate that activated NK cells efficiently kill colorectal tumour cell spheroids in both 2D and 3D cultures. Notably, although 3D CRC cell cultures favoured the expression of the inhibitory immune checkpoint PD-L1, it did not correlate with increased resistance to NK cells. Finally, we have analysed in detail the infiltration of NK cells in 3D spheroids by microscopy and found that at low NK cell density, cell death is not observed although NK cells are able to infiltrate into the spheroid. In contrast, higher densities promote tumoural cell death before infiltration can be detected. These findings show that highly dense activated human primary NK cells efficiently kill colorectal carcinoma cells growing in 3D cultures independently of PD-L1 expression and suggest that the use of allogeneic activated NK cells could be beneficial for the treatment of colorectal carcinoma.

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

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

2013-12-19

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

3D material cytometry (3DMaC): a very high-replicate, high-throughput analytical method using microfabricated, shape-specific, cell-material niches.

Science.gov (United States)

Parratt, Kirsten; Jeong, Jenny; Qiu, Peng; Roy, Krishnendu

2017-08-08

Studying cell behavior within 3D material niches is key to understanding cell biology in health and diseases, and developing biomaterials for regenerative medicine applications. Current approaches to studying these cell-material niches have low throughput and can only analyze a few replicates per experiment resulting in reduced measurement assurance and analytical power. Here, we report 3D material cytometry (3DMaC), a novel high-throughput method based on microfabricated, shape-specific 3D cell-material niches and imaging cytometry. 3DMaC achieves rapid and highly multiplexed analyses of very high replicate numbers ("n" of 10 4 -10 6 ) of 3D biomaterial constructs. 3DMaC overcomes current limitations of low "n", low-throughput, and "noisy" assays, to provide rapid and simultaneous analyses of potentially hundreds of parameters in 3D biomaterial cultures. The method is demonstrated here for a set of 85 000 events containing twelve distinct cell-biomaterial micro-niches along with robust, customized computational methods for high-throughput analytics with potentially unprecedented statistical power.

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

Directory of Open Access Journals (Sweden)

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.

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

Science.gov (United States)

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

2017-01-01

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

Near-Infrared Light-Sensitive Polyvinyl Alcohol Hydrogel Photoresist for Spatiotemporal Control of Cell-Instructive 3D Microenvironments.

Science.gov (United States)

Qin, Xiao-Hua; Wang, Xiaopu; Rottmar, Markus; Nelson, Bradley J; Maniura-Weber, Katharina

2018-03-01

Advanced hydrogel systems that allow precise control of cells and their 3D microenvironments are needed in tissue engineering, disease modeling, and drug screening. Multiphoton lithography (MPL) allows true 3D microfabrication of complex objects, but its biological application requires a cell-compatible hydrogel resist that is sufficiently photosensitive, cell-degradable, and permissive to support 3D cell growth. Here, an extremely photosensitive cell-responsive hydrogel composed of peptide-crosslinked polyvinyl alcohol (PVA) is designed to expand the biological applications of MPL. PVA hydrogels are formed rapidly by ultraviolet light within 1 min in the presence of cells, providing fully synthetic matrices that are instructive for cell-matrix remodeling, multicellular morphogenesis, and protease-mediated cell invasion. By focusing a multiphoton laser into a cell-laden PVA hydrogel, cell-instructive extracellular cues are site-specifically attached to the PVA matrix. Cell invasion is thus precisely guided in 3D with micrometer-scale spatial resolution. This robust hydrogel enables, for the first time, ultrafast MPL of cell-responsive synthetic matrices at writing speeds up to 50 mm s -1 . This approach should enable facile photochemical construction and manipulation of 3D cellular microenvironments with unprecedented flexibility and precision. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells.

Directory of Open Access Journals (Sweden)

Liang-Hui Chu

Full Text Available Angiogenesis involves stimulation of endothelial cells (EC by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the "angiome" could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A. We used the Short Time-series Expression Miner (STEM to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC and human microvascular EC (MEC. The results show that VEGFR1-VEGFR2 levels are more closely coupled than VEGFR1-VEGFR3 or VEGFR2-VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.

Dopamine receptor D3 expressed on CD4+ T cells favors neurodegeneration of dopaminergic neurons during Parkinson's disease.

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González, Hugo; Contreras, Francisco; Prado, Carolina; Elgueta, Daniela; Franz, Dafne; Bernales, Sebastián; Pacheco, Rodrigo

2013-05-15

Emerging evidence has demonstrated that CD4(+) T cells infiltrate into the substantia nigra (SN) in Parkinson's disease (PD) patients and in animal models of PD. SN-infiltrated CD4(+) T cells bearing inflammatory phenotypes promote microglial activation and strongly contribute to neurodegeneration of dopaminergic neurons. Importantly, altered expression of dopamine receptor D3 (D3R) in PBLs from PD patients has been correlated with disease severity. Moreover, pharmacological evidence has suggested that D3R is involved in IFN-γ production by human CD4(+) T cells. In this study, we examined the role of D3R expressed on CD4(+) T cells in neurodegeneration of dopaminergic neurons in the SN using a mouse model of PD. Our results show that D3R-deficient mice are strongly protected against loss of dopaminergic neurons and microglial activation during 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. Notably, D3R-deficient mice become susceptible to MPTP-induced neurodegeneration and microglial activation upon transfer of wild-type (WT) CD4(+) T cells. Furthermore, RAG1 knockout mice, which are devoid of T cells and are resistant to MPTP-induced neurodegeneration, become susceptible to MPTP-induced loss of dopaminergic neurons when reconstituted with WT CD4(+) T cells but not when transferred with D3R-deficient CD4(+) T cells. In agreement, experiments analyzing activation and differentiation of CD4(+) T cells revealed that D3R favors both T cell activation and acquisition of the Th1 inflammatory phenotype. These findings indicate that D3R expressed on CD4(+) T cells plays a fundamental role in the physiopathology of MPTP-induced PD in a mouse model.

3D/4D multiscale imaging in acute lymphoblastic leukemia cells: visualizing dynamics of cell death

Science.gov (United States)

Sarangapani, Sreelatha; Mohan, Rosmin Elsa; Patil, Ajeetkumar; Lang, Matthew J.; Asundi, Anand

2017-06-01

Quantitative phase detection is a new methodology that provides quantitative information on cellular morphology to monitor the cell status, drug response and toxicity. In this paper the morphological changes in acute leukemia cells treated with chitosan were detected using d'Bioimager a robust imaging system. Quantitative phase image of the cells was obtained with numerical analysis. Results show that the average area and optical volume of the chitosan treated cells is significantly reduced when compared with the control cells, which reveals the effect of chitosan on the cancer cells. From the results it can be attributed that d'Bioimager can be used as a non-invasive imaging alternative to measure the morphological changes of the living cells in real time.