Статті в журналах: "3-dimensional cell culture"

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KODAMA, Makoto. "New System of 3 Dimensional Cell Culture." Journal of the Society of Mechanical Engineers 119, no. 1169 (2016): 218–21. http://dx.doi.org/10.1299/jsmemag.119.1169_218.

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Ravi, Maddaly, Aishwarya Pargaonkar, Anuradha Ramesh, Gatika Agrawal, Jennifer Sally, SriVijayaGanapathy Srinivasan, and Abhishek Kalra. "Three-dimensional prints from 3-dimensional cell culture aggregates of human cancer cell lines." Sri Ramachandra Journal of Health Sciences 1 (December 24, 2021): 10–15. http://dx.doi.org/10.25259/srjhs_5_2021.

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Objectives: Three-dimensional (3D) printing has gained significance for human health-care applications in recent years. Some of these applications include obtaining models which mimic anatomical parts. One other parallel development in the biological research area is the development of 3D cell cultures. Such cultures are now becoming the material of choice for in vitro experiments, fast replacing the traditional adherent/monolayer 2D culture approaches. We present here, a method to obtain 3D prints of 3D aggregates of three human cancer cell lines. Such 3D prints can be useful models to understand solid tumor morphologies and also as effective teaching models. Materials and Methods: Photomicrographs of the 3D aggregates of the human cancer cell lines SiHa, MCF-7, and A549 (human cervical cancer, breast cancer, and non-small cell lung cancer cell lines, respectively) were obtained using inverted phase contrast microscopy. Conversion of normal jpeg images into 3D files was performed using the lithophane method and CAD files obtained. The CAD files thus generated were used to print the objects using the Stratasys Polyjet J750 3D Printer. Results: We could obtain 3D prints of SiHa, MCF-7, and A549 (human cervical cancer, breast cancer, and non-small cell lung cancer cell lines, respectively) 3D aggregates/spheroids. Conclusion: It is hoped that this approach will be useful for studying solid tumor morphologies in finer details. Furthermore, other benefits of such 3D prints would be in them being excellent models for teaching purposes.

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Kupchik, H. Z., E. A. Collins, M. J. O'Brien, and R. P. McCaffrey. "Chemotherapy screening assay using 3-dimensional cell culture." Cancer Letters 51, no. 1 (May 1990): 11–16. http://dx.doi.org/10.1016/0304-3835(90)90224-l.

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Lee, J., M. Park, J. Byeon, N. Gu, I. Cho, and S. Cha. "Angiogenic effects of 3 dimensional cell culture system." Cytotherapy 19, no. 5 (May 2017): S234—S235. http://dx.doi.org/10.1016/j.jcyt.2017.02.344.

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Chitturi Suryaprakash, Ravi Teja, Omar Kujan, Kate Shearston, and Camile S. Farah. "Three-Dimensional Cell Culture Models to Investigate Oral Carcinogenesis: A Scoping Review." International Journal of Molecular Sciences 21, no. 24 (December 14, 2020): 9520. http://dx.doi.org/10.3390/ijms21249520.

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Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiologically representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qualitative analysis including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiological relevance of these models continues to improve.

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Ceresa, Claudia C., Alan J. Knox, and Simon R. Johnson. "Use of a three-dimensional cell culture model to study airway smooth muscle-mast cell interactions in airway remodeling." American Journal of Physiology-Lung Cellular and Molecular Physiology 296, no. 6 (June 2009): L1059—L1066. http://dx.doi.org/10.1152/ajplung.90445.2008.

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Increased airway smooth muscle (ASM) mass and infiltration by mast cells are key features of airway remodeling in asthma. We describe a model to investigate the relationship between ASM, the extracellular matrix, mast cells, and airway remodeling. ASM cells were cultured in a three-dimensional (3-D) collagen I gel (3-D culture) alone or with mast cells. Immunocytochemistry and Western blotting of ASM in 3-D cultures revealed a spindle-shaped morphology and significantly lower α-smooth muscle actin and vimentin expression than in ASM cultured in monolayers on collagen type I or plastic (2-D culture). In 3-D cultures, basal ASM proliferation, examined by Ki67 immunocytochemistry, was reduced to 33 ± 7% ( P < 0.05) of that in 2-D cultures. The presence of mast cells in cocultures increased ASM proliferation by 1.8-fold ( P < 0.05). Gelatin zymography revealed more active matrix metalloproteinase (MMP)-2 in 3-D than in 2-D culture supernatants over 7 days. Functional MMP activity was examined by gel contraction. The spontaneous gel contraction over 7 days was significantly inhibited by the MMP inhibitor ilomastat. Mast cell coculture enhanced ASM gel contraction by 22 ± 16% (not significant). Our model shows that ASM has different morphology, with lower contractile protein expression and basal proliferation in 3-D culture. Compared with standard techniques, ASM synthetic function, as shown by MMP production and activity, is sustained over longer periods. The presence of mast cells in the 3-D model enhanced ASM proliferation and MMP production. Airway remodeling in asthma may be more accurately modeled by our system than by standard culture systems.

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Kim, Minseok S., Ju Hun Yeon, and Je-Kyun Park. "A microfluidic platform for 3-dimensional cell culture and cell-based assays." Biomedical Microdevices 9, no. 1 (November 11, 2006): 25–34. http://dx.doi.org/10.1007/s10544-006-9016-4.

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Jenkins, James, Ruslan I. Dmitriev, Karl Morten, Kieran W. McDermott, and Dmitri B. Papkovsky. "Oxygen-sensing scaffolds for 3-dimensional cell and tissue culture." Acta Biomaterialia 16 (April 2015): 126–35. http://dx.doi.org/10.1016/j.actbio.2015.01.032.

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Wu, Min-Hsien, Yu-Han Chang, Yen-Ting Liu, Yan-Ming Chen, Shih-Siou Wang, Hsin-Yao Wang, Chao-Sung Lai, and Tung-Ming Pan. "Development of high throughput microfluidic cell culture chip for perfusion 3-dimensional cell culture-based chemosensitivity assay." Sensors and Actuators B: Chemical 155, no. 1 (July 2011): 397–407. http://dx.doi.org/10.1016/j.snb.2010.11.027.

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Ferraz, M. A. M. M., H. H. W. Henning, K. M. A. Van Dorenmalen, P. L. A. M. Vos, T. A. E. Stout, P. F. Costa, J. Malda, and B. M. Gadella. "52 USE OF TRANSWELL CELL CULTURE AND 3-DIMENSIONAL PRINTING TECHNOLOGY TO DEVELOP AN IN VITRO BOVINE OVIDUCT." Reproduction, Fertility and Development 28, no. 2 (2016): 156. http://dx.doi.org/10.1071/rdv28n2ab52.

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Oviduct epithelial cells (OECs) generate the microenvironment for mammalian fertilization. When cultured in vitro OECs rapidly lose their differentiated cell properties (e.g. secretory activity and cilia), while suspended cells have a limited lifespan. These limitations, likely due to the lack of folded tubular geometry of the oviduct, prompted us to combine transwell cell culture and 3-D printing technologies to mimic the in vivo OEC niche in order to better study the unique role of the oviduct and its microenvironment during the processes of fertilization and early embryonic development. U-shape inserts were 3-D printed using a multi-arm acrylate-based resin (PIC100) on an Envisiontec Perfactory P3 stereolithographer. Post-printing treatments of custom-made tubular transwell inserts were first tested in order to determine any possible negative impact of the plastics on cell growth. Inserts were either untreated, post-cured with 1000 flashes/side (Otoflash, 66 W), post-cured and Soxhlet-extracted overnight in isopropanol, or post-cured and Soxhlet-extracted over the weekend in water at 37°C. The post-cured and Soxhlet-extracted overnight in isopropanol inserts were selected as best pretreatment for culturing OECs. These inserts were mounted with track-etched PET membranes (12 µm thick, 0.4 µm pore diameter) to create a U-shape geometry that allows perfusion. Bovine OECs were obtained by squeezing the whole oviduct collected from slaughterhouse cows (on luteal phase) and cultured as monolayers for 7 days (n = 2 cows). These de-differentiated OECs were seeded on the membranes, grown to confluence (7 days), and cultured (1) at an air-liquid interface for 6 and 14 days (air-liquid culture) or (2) under perfusion (6 mL h–1) for 6 days (perfusion culture). OECs were also cultured on coverslips as monolayers (2-D culture) for 6 and 14 days. After this period, the OECs were fixed and immune labelled to determine their polarized state. Polarization of OECs (laminin and primary cilia detection) was observed on Day 6 for perfusion culture, on Day 14 for air-liquid culture, and was not detected in 2-D culture. The presence of secondary cilia (acetylated α-tubulin) was observed in 6% of the cells cultured under perfusion at Day 6; secondary cilia was not present in air-liquid or 2-D cultures during the period analysed. In conclusion, post-curing and Soxhlet extraction of leachable compounds is crucial to avoid toxic effects on cell growth. The U-shape custom-designed inserts are able to create a tube-like surface in which bovine oviducal cells can be cultured to confluency and thereafter repolarize (presence of primary cilia and detection of laminin); this polarization occurs faster when the U-shape culture is under perfusion. Further studies will examine the ability of the cells to differentiate further (development of secondary cilia and secretory ability) and support in vitro fertilization. To this end, 3-D designs will be tested to determine their use for live cell imaging and for collecting secreted fluids.

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Jeong, Yun Yeong, Mi Sun Kim, Ko Eun Lee, Ok Hyung Nam, Ji-Hyun Jang, Sung-Chul Choi, and Hyo-Seol Lee. "Comparison of 2- and 3-Dimensional Cultured Periodontal Ligament Stem Cells; a Pilot Study." Applied Sciences 11, no. 3 (January 25, 2021): 1083. http://dx.doi.org/10.3390/app11031083.

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This study compared the characteristics of periodontal ligament stem cells (PDLSCs) cultured using 3-dimensional (3D) versus conventional 2-dimensional (2D) methods. PDLSCs were cultured in either a 3D culture with a non-adhesive culture plate (Stemfit 3D®) or a conventional 2D culture using a 6-well plate. Morphology, viability, proliferation ability, and osteogenic differentiation were analyzed to characterize the differences induced in identical PDLSCs by 3D and 2D culture environments. In addition, gene expression was analyzed using RNA sequencing to further characterize the functional differences. The diameter and the viability of the 3D-cultured PDLSCs decreased over time, but the shape of the spheroid was maintained for 20 days. Although osteogenic differentiation occurred in both the 2D- and 3D-cultured PDLSCs, compared to the control group it was 20.8 and 1.6 higher in the 3D- and 2D-cultured cells, respectively. RNA sequencing revealed that PDLSCs cultured using 2D and 3D methods have different gene expression profiles. The viability of the 3D-cultured cells was decreased, but they showed superior osteogenic differentiation compared to 2D-cultured cells. Within the limitations of this study, the results demonstrate that the structure and function of PDLSCs are influenced by the cell culture method.

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Aljitawi, Omar S., Dandan Li, Da Zhang, Jonathan Mahnken, Suman Kambhampati, Peter Van Veldhuizen, and Rama Garimella. "A 3 Dimensional in Vitro Model to Test HL-60 Cell Line Sensitivity to Chemotherapy." Blood 118, no. 21 (November 18, 2011): 4882. http://dx.doi.org/10.1182/blood.v118.21.4882.4882.

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Abstract Abstract 4882 Introduction: Current in vitro drug testing models are based on 2-dimensional (2D) cell culture systems and therefore do not always predict in vivo responses. This lack of predictability of the 2D assays is believed to be related to the 3-dimensional (3D) microenvironment present in tissues or tumors. This 3D microenvironment, were cell-cell and cell-extracellular matrix (ECM) interactions occur, is fundamental for cell biologic activities. This is especially true for acute myeloid leukemia, were current 2-D cell culture models do not always predict clinical responses. This discrepancy in leukemia cell responses to chemotherapy in vivo, in comparison to in vitro, is at least partly related to leukemia cells interaction with the bone marrow microenvironment and their ability to establish niches. These niches offer partial protection from the effects of cytotoxic chemotherapy, otherwise termed cell adhesion-mediated drug resistance. In these experiments, we investigate the apoptotic effects of cytotoxic chemotherapy on HL-60 cell line cultured in a designed 3D AML cell culture model. In this 3D microenvironment, HL-60 cells were co-cultured with ex vivo expanded bone marrow mesenchyaml stem cells in a 3D synthetic scaffold. Aim: To examine the apoptotic effect of cytotoxic chemotherapy on HL-60 co-cultured with human bone marrow mesenchymal stem cells (huBM-MSCs) in 3D conditions. Methods: After several passages, expanded huBM-MSCs were seeded into PGA/PLLA 90/10 copolymer discs, 5-mm in diameter and 2-mm in thickness and allowed to attach to scaffold fibers and to expand over 2 weeks. Then, HL-60 were added and allowed to grow in the 3D culture system for another 10 days. HL-60 cells in 3D culture system were then exposed to doxorubicin given in two concentrations (25 and 50 μM) and incubated for 24 hours. HL-60 were then retrieved applying a combination of mechanical forces and using cell dissociation solution. FITC Annexin V Apoptosis Detection Kit was used to determine apoptosis. Apoptosis was confirmed by TUNEL assay. Proliferation of HL-60 cells in the 3D scaffold was assessed using Ki-67 stain of scaffold's cryosections. All tests were done in triplicates, and untreated HL-60 served as controls for treatment. Comparison was made with HL-60 cells alone and with HL-60 cells growing on a hu-BM-MSC monolayer. SAS version 9.2 (SAS Institute, Inc., 2002–2008) was used for statistical analysis Results: Virtually, all HL-60 cells treated with 25 or 50 μM underwent late apoptosis. Around.03% of HL-60 cells survived 25 μM concentration, none, however, survived 50 μM concentration. In 2D, most of HL-60 cells underwent necrosis, and to lesser extent late apoptosis. In sharp contrast, 17.8% of HL-60 cells survived 25μM concentration, nevertheless, only.27% of HL-60 cells treated with 50 μM concentration survived. The differences in apoptosis patterns between the three groups was statistically significant (P<.0001). Conclusion: compared to traditional cell culture conditions, the designed 3D culture conditions protected a higher percentage of HL-60 cells from undergoing apoptosis and necrosis. Disclosures: No relevant conflicts of interest to declare.

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Oh, Eun-Taex, Ha Gyeong Kim, Min-Ho Choi, Jae-Seon Lee, Sang Jeong Kim, Jong-Young Kwak, and Heon Joo Park. "Multi-Layer Nanofibrous PCL Scaffold-Based Colon Cancer Cell Cultures to Mimic Hypoxic Tumor Microenvironment for Bioassay." Cancers 13, no. 14 (July 15, 2021): 3550. http://dx.doi.org/10.3390/cancers13143550.

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Three-dimensional (3D) cancer cell culture systems have been developed to aid the study of molecular mechanisms in cancer development, identify therapeutic targets, and test drug candidates. In this study, we developed a strategy for mimicking the hypoxic tumor microenvironment in a 3D cancer cell culture system using multi-layer, nanofibrous poly(ε-caprolactone) (PCL) scaffold (pNFS)-based cancer cell cultures. We found that human colon cancer cells infiltrated pNFS within 3 days and could be cultured three-dimensionally within the NFS. When incubated in four stacks of 30 µm-thick pNFS for 3 days, colon cancer cells in layer three showed partially reduced entry into the S phase, whereas those in layer four, located farthest from the media, showed a marked reduction in S-phase entry. As a consequence, cells in layer four exhibited hypoxia-induced disorganization of F-actin on day 3, and those in layers three and four showed an increase in the expression of the hypoxia-induced transcription factor HIF-1α and its target genes, Glut1, CA9, VEGF, and LDHA. Consistent with these results, doxorubicin- and ionizing radiation-induced cell death was reduced in colon cancer cells cultured in layers three and four. These results suggest that pNFS-based multi-layer colon cancer cell cultures mimic the hypoxic tumor microenvironment and are useful for bioassays.

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Kim, Choong, Kang Sun Lee, Jae Hoon Bang, Young Eyn Kim, Min-Cheol Kim, Kwang Wook Oh, Soo Hyun Lee, and Ji Yoon Kang. "3-Dimensional cell culture for on-chip differentiation of stem cells in embryoid body." Lab on a Chip 11, no. 5 (2011): 874. http://dx.doi.org/10.1039/c0lc00516a.

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Liu, Haijun, Xiaoniu Dai, Yusi Cheng, Shencun Fang, Yingming Zhang, Xingang Wang, Wei Zhang, Hong Liao, Honghong Yao, and Jie Chao. "MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts." American Journal of Physiology-Lung Cellular and Molecular Physiology 310, no. 2 (January 15, 2016): L121—L132. http://dx.doi.org/10.1152/ajplung.00278.2015.

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Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis.

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MATSUURA, Tomokazu. "Bioreactors for 3-dimensional high-density culture of human cells." Human Cell 19, no. 1 (February 2006): 11–16. http://dx.doi.org/10.1111/j.1749-0774.2005.00002.x.

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Jongpaiboonkit, Leenaporn, William J. King, Gary E. Lyons, Amy L. Paguirigan, Jay W. Warrick, David J. Beebe, and William L. Murphy. "An adaptable hydrogel array format for 3-dimensional cell culture and analysis." Biomaterials 29, no. 23 (August 2008): 3346–56. http://dx.doi.org/10.1016/j.biomaterials.2008.04.040.

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Suderman, Michael T., Kevin B. Temeyer, Kristie G. Schlechte, and Adalberto A. Pérez de León. "Three-Dimensional Culture of Rhipicephalus (Boophilus) microplus BmVIII-SCC Cells on Multiple Synthetic Scaffold Systems and in Rotating Bioreactors." Insects 12, no. 8 (August 19, 2021): 747. http://dx.doi.org/10.3390/insects12080747.

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Tick cell culture facilitates research on the biology of ticks and their role as vectors of pathogens that affect humans, domestic animals, and wildlife. Because two-dimensional cell culture doesn’t promote the development of multicellular tissue-like composites, we hypothesized that culturing tick cells in a three-dimensional (3-D) configuration would form spheroids or tissue-like organoids. In this study, the cell line BmVIII-SCC obtained from the cattle fever tick, Rhipicephalus (Boophilus) microplus (Canestrini, 1888), was cultured in different synthetic scaffold systems. Growth of the tick cells on macrogelatinous beads in rotating continuous culture system bioreactors enabled cellular attachment, organization, and development into spheroid-like aggregates, with evidence of tight cellular junctions between adjacent cells and secretion of an extracellular matrix. At least three cell morphologies were identified within the aggregates: fibroblast-like cells, small endothelial-like cells, and larger cells exhibiting multiple cytoplasmic endosomes and granular vesicles. These observations suggest that BmVIII-SCC cells adapted to 3-D culture retain pluripotency. Additional studies involving genomic analyses are needed to determine if BmVIII-SCC cells in 3-D culture mimic tick organs. Applications of 3-D culture to cattle fever tick research are discussed.

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Mueller-Klieser, Wolfgang. "Three-dimensional cell cultures: from molecular mechanisms to clinical applications." American Journal of Physiology-Cell Physiology 273, no. 4 (October 1, 1997): C1109—C1123. http://dx.doi.org/10.1152/ajpcell.1997.273.4.c1109.

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This article reviews actual advances in the development and application of three-dimensional (3-D) cell culture systems. Recent therapeutically oriented studies include characterization of multicellular-mediated drug resistance, novel ways of quantifying hypoxia, and new approaches to more efficient immunotherapy. Recent progress toward understanding the development of necrosis in tumor spheroids has been made using novel spheroid models. 3-D cultures have been used for studies on molecular mechanisms involved in invasion and metastasis, with a major focus on the role of E-cadherin. Similarly, tumor angiogenesis and the significance of vascular endothelial growth factor have been investigated in a variety of 3-D culture systems. There are many ongoing developments in tissue modeling or remodeling that promise significant progress toward the development of bioartificial liver support and artificial blood. Perhaps one of the most interesting areas of basic research with 3-D cultures is the characterization of embryoid bodies obtained from stable embryonic stem cells. These models have greatly increased the understanding of embryonic development, in particular through the notable exceptional advances in cardiogenesis.

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Aljitawi, Omar S., Yinghua Xiao, Karthik Ramachandran, Brea C. Lipe, Rama Garimella, Amrita Krishnan, Lisa Stehno-Bittel, and Linheng Li. "A 3-Dimensional Co-Culture Model to Investigate Adhesion-Mediated Drug Resistance in Multiple Myeloma." Blood 120, no. 21 (November 16, 2012): 1826. http://dx.doi.org/10.1182/blood.v120.21.1826.1826.

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Abstract Abstract 1826 Background: In multiple myeloma (MM), evidence suggests that the adhesion of malignant plasma cells to bone marrow microenvironment elements is central to MM growth and drug resistance, otherwise referred to as adhesion–mediated drug resistance. As other investigators have shown, the MM-stroma interaction is in part mediated by adhesion molecules like N-cadherin. Goal: To develop a 3-dimensional MM in vitro co-culture model where MM cells interact with stromal elements mimicking the bone marrow microenvironment and use the model to investigate N-cadherin mediated drug resistance. Methods: We co-cultured U266 cells, a MM cell line, with mesenchymal stromal cells (MSCs) isolated from Wharton's jelly (WJ) in a synthetic copolymer (polyglycolic acid/ poly L-lactic acid 90/10 copolymer) (3-dimensional culture conditions). For controls, we cultured U266 cells over a monolayer of WJMCS (2-dimensional conditions) or U266 cells in suspension. Cell proliferation was measured by Ki-67 immunohistochemistry (IHC) stain and lambda light chain expression was measure by enzyme-linked immunosorbent assay (ELISA) and IHC stains. Bortezomib in 1, 5, and 10 nM concentrations was used to treat U266 cells for 24 hours under the three culture conditions. Cytotoxicity was measured by modified Alamar Blue assay. Results: U266 cells in our 3-D model adhered to stromal cells and formed clusters close to matrix material. U266 cells proliferated as evidenced by positive Ki-67 stain, and expressed lambda light chain by IHC and ELISA. Lambda light expression measured by ELISA peaked at 1 week of co-culture in the 3-D model. Though N-cadherin was expressed in both stromal cells and U266 MM cells in 3-D, it was expressed only in stromal cells in 2-D culture conditions. Finally, pre-incubating U266 cells with N-cadherin blocking antibody resulted in less population of the 3-D model by U266 MM cells. Treatment of U266 with bortezomib resulted in less cell cytotoxicity in 3-D and 2-D co-culture conditions compared to U266 cells treated in suspension. Conclusions: Using a 3-D in vitro co-culture model, we demonstrated that only 3-D co-culture conditions resulted in N-cadherin expression in U266 MM cell line. We also demonstrated that U266 population of the 3-D model was successfully blocked using an N-cadherin blocking antibody. We propose N-cadherin mediated adhesion as a mechanism to explain reduced cytotoxicity to bortezomib in 3-D. We propose this model to be used to investigate adhesion-mediated drug resistance. Disclosures: No relevant conflicts of interest to declare.

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Kreeger, J. M., J. T. Payne, J. L. Tomlinson, and J. L. Cook. "Effects of EGF, FGF, and PDGF on Canine Chondrocytes in Three-Dimensional Culture." Veterinary and Comparative Orthopaedics and Traumatology 10, no. 04 (1997): 210–13. http://dx.doi.org/10.1055/s-0038-1632598.

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SummaryThree-dimensional (3-D) culture of chondrocytes has been shown to promote cell proliferation, differentiation, and production of matrix while providing for cell morphology and matrix characteristics that resemble normal articular cartilage (1-3). In a recently published study, enhanced glycosaminoglycan (GAG) production was demonstrated in canine chondrocytes in 3-D culture on cancellous bone substrate (3). The effects of growth factors known to be present in cryopreserved bone were suggested to be responsible. Various growth factors including epidermal growth factor (EGF), fibroblast growth factor (FGF), and plateletderived growth factor (PDGF) have been shown to enhance chondrocyte DNA production, mitotic activity, and matrix production (5-10). The objective of the study reported here was to evaluate the effects of one concentration of EGF, FGF, and PDGF on canine chondrocyte proliferation and production of normal matrix constituents in 3-D culture.Canine articular chondrocytes were cultured in three-dimensional medium for 25 days. EGF, FGF, and PDGF were added to the culture medium. Chondrocytes in 3-D culture maintained viability and differentiation. Cell morphology and matrix production resembled that of intact hyaline cartilage. Significant differences with respect to cell counts, glycosaminoglycan concentration, or collagen type II immuno-reactivity as the result of added growth factors were not found.

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Saotome, Toshiki, Naoki Shimada, Kumiko Matsuno, Koichiro Nakamura, and Yasuhiko Tabata. "Gelatin hydrogel nonwoven fabrics of a cell culture scaffold to formulate 3-dimensional cell constructs." Regenerative Therapy 18 (December 2021): 418–29. http://dx.doi.org/10.1016/j.reth.2021.09.008.

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KIM, KI-UP, and So-My Koo. "Abstract B36: Synthesizing new proteins from 3-dimensional spheroids, compare to 2-dimensional cell line culture." Clinical Cancer Research 20, no. 2 Supplement (January 15, 2014): B36. http://dx.doi.org/10.1158/1078-0432.14aacriaslc-b36.

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Ballestreri, Érica, Carlos Alexandre Fedrigo, Carlos Alexandre Fedrigo, Gabriel Ratund, Gabriel Ratund, Felipe Umpierre Conter, Felipe Umpierre Conter, Ivana Grivicich, and Ivana Grivicich. "Development of three-dimensional cell culture spheroid model for non-small-cell lung carcinoma cell line nci-h460 exposed to cisplatin / Desenvolvimento de modelo de cultura celular tridimensional para a linhagem celular de carcinoma de pulmão de não-pequenas células nci-h460 expostas á cisplatina." Brazilian Journal of Development 7, no. 12 (December 29, 2021): 11140–115148. http://dx.doi.org/10.34117/bjdv7n12-331.

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The three-dimensional culture models have been developed to promote better simulation of in vivo conditions of cellular interactions and phenotypic expressions. Since each cell line behaves differently and not all aggregate three-dimensionally, the objectives of this study were to standardize the conditions for cultivation spheroids produced from the human non-smal-cell lung cancer cell line NCI-H460, and evaluate the effects of cisplatin in this model. We investigated the best cell density for the formation of spheroids and their growth time in culture conditions. For this, spheroids were treated with different concentrations of cisplatin. The analysis was performed by measuring the diameter of the spheroids before treatment and measured 48 h after treatment and then every 3 days. The standard cell density conditions obtained was 10 x 104 cells. Spheroids were collected from the 7th day and isolated in a non-adherent matrix well (covered with 2% agar and culture medium), remaining viable until the 17th day. From the 8th day of isolated culture, the cisplatin promoted a reduction in the growth of spheroids dependent on the concentration used. Thus, it was concluded that it was possible to standardize the effective methodology for the formation and maintenance of spheroids from this cell line, as well as cisplatin inhibited the growth of the spheroids, suggesting its cytotoxic effect also cultures in three-dimensional model.

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Rosellini, Alfredo, Giulia Freer, Paola Quaranta, Veronica Dovere, Melissa Menichini, Fabrizio Maggi, Paola Mazzetti, and Mauro Pistello. "Enhanced in vitro virus expression using 3-dimensional cell culture spheroids for infection." Journal of Virological Methods 265 (March 2019): 99–104. http://dx.doi.org/10.1016/j.jviromet.2018.12.017.

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Sudar, D., D. Callahan, B. Parvin, D. Knowles, C. Ortiz de Solorzano, and M. H. Barcellos Hoff. "Design of a Microscopy System for Quantitative Spatial and Temporal Analysis of Multicellular Interactions." Microscopy and Microanalysis 7, S2 (August 2001): 32–33. http://dx.doi.org/10.1017/s1431927600026234.

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The challenge of the post-genomic era is functional genomics, i.e. understanding how the genome is expressed to produce myriad cell phenotypes. A phenotype is the result of selective expression of the genome in response to the microenvironment. to use genomic information to understand the biology of complex organisms, the biological responses and signaling pathways in cells need to be studied in context, i.e. within the proper tissue structure. Nonetheless, most current biology is conducted using cells cultured in monolayers on traditional tissue culture plastic. These non-physiological models impede the ability to predict in vivo responses from model systems. The same cells cultured in 2-dimensions (i.e. monolayers) vs. 3-dimensions (e.g. multicellular tumor spheroids) differ in their responses to external stimuli such as ionizing radiation, viral infection, cytotoxic drugs, and chemotherapeutic agents. Our laboratory has led the way in promoting and developing 3-dimensional cell culture models that more accurately reflect in vivo biology, beginning with the establishment 15 years ago of physiologically functional reconstituted mammary acini in culture.Quantitation of spatial and temporal concurrent behavior of multiple markers in these 3-dimensional cell cultures is hampered by the currently routine mode of sequential image acquisition, measurement and analysis of specific targets. This precludes the detailed analysis of multi-dimensional, time sequence responses and fails to relate features in novel and meaningful ways that will further our understanding of basic biology. Thus new methodology was needed for high-throughput, dynamic evaluations of large numbers of live multicellular specimens. Rather than using confocal microscopy methods, which interfere with live cell systems due to photo-damage, optical sectioning of the 3-dimensional structures is achieved with structured light illumination using the Wilson grating in an implementation described by Lanni.

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Kunz-Schughart, Leoni A., Sabine Wenninger, Thomas Neumeier, Paula Seidl та Ruth Knuechel. "Three-dimensional tissue structure affects sensitivity of fibroblasts to TGF-β1". American Journal of Physiology-Cell Physiology 284, № 1 (1 січня 2003): C209—C219. http://dx.doi.org/10.1152/ajpcell.00557.2001.

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Transforming growth factor-β (TGF-β) is known to induce α-smooth muscle actin (α-SMA) in fibroblasts and is supposed to play a role in myofibroblast differentiation and tumor desmoplasia. Our objective was to elucidate the impact of TGF-β1 on α-SMA expression in fibroblasts in a three-dimensional (3-D) vs. two-dimensional (2-D) environment. In monolayer culture, all fibroblast cultures responded in a similar fashion to TGF-β1 with regard to α-SMA expression. In fibroblast spheroids, α-SMA expression was reduced and induction by TGF-β1 was highly variable. This difference correlated with a differential regulation in the TGF-β receptor (TGFβR) expression, in particular with a reduction in TGF-βRII in part of the fibroblast types. Our data indicate that 1) sensitivity to TGF-β1-induced α-SMA expression in a 3-D environment is fibroblast-type specific, 2) fibroblast type-independent regulatory mechanisms, such as a general reduction/loss in TGF-βRIII, contribute to an altered TGFβR expression profile in spheroid compared with monolayer culture, and 3) fibroblast type-specific alterations in TGFβR types I and II determine the sensitivity to TGF-β1-induced α-SMA expression in the 3-D setting. We suggest that fibroblasts that can be induced by TGF-β1 to produce α-SMA in spheroid culture reflect a “premyofibroblastic” phenotype.

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Johnson, Chassidy, Kiah Sanders, and Hung Fan. "Jaagsiekte Sheep Retrovirus Transformation in Madin-Darby Canine Kidney Epithelial Cell Three-Dimensional Culture." Journal of Virology 84, no. 10 (March 10, 2010): 5379–90. http://dx.doi.org/10.1128/jvi.02323-09.

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ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep that shares similarities with human bronchioloalveolar carcinoma (BAC). JSRV is unique because the envelope gene (env) is the oncogene, as it can transform cells in culture and induce tumors in animals. The phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR and H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) pathways have been shown to be critical for Env transformation. However, the question still remains of how disruption of these pathways relates to tumor formation. To address this, JSRV Env transformation was studied in the context of epithelial structure, using the polarized Madin-Darby canine kidney (MDCK) epithelial cell three-dimensional (3-D) culture system. The results indicated that JSRV Env-transformed MDCK cells were larger and had full or multiple lumens, in contrast to the single lumens observed in controls. The altered phenotype was largely mediated by an increase in proliferation, in addition to overcoming the proliferative suppression signal. JSRV Env was not found to disrupt polarity or tight junctions or to inhibit lumen apoptosis. The PI3K-Akt-mTOR pathway was important for Env transformation in MDCK cells, although the mechanisms of action differed in 3-D and monolayer cultures. PI3K-dependent signaling to mTOR occurred in monolayers, while PI3K-independent signaling to mTOR occurred in 3-D culture. In contrast, the H/N-Ras-MEK-MAPK pathway was found to be inhibitory to transformation in both normal and transformed MDCK cells in 3-D culture. However, in monolayer culture, inhibition of MEK reverted the transformed phenotype, suggesting a different mechanism(s) of action in monolayer versus 3-D culture.

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Boyer, Jean Zheng, Gail D. Lewis Phillips, Hiro Nitta, Karl Garsha, Brittany Admire, Robert Kraft, Eslie Dennis, Elizabeth Vela, and Penny Towne. "Activity of trastuzumab emtansine (T-DM1) in 3D cell culture." Breast Cancer Research and Treatment 188, no. 1 (June 5, 2021): 65–75. http://dx.doi.org/10.1007/s10549-021-06272-x.

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Abstract Background Cell spheroids and aggregates generated from three-dimensional (3D) cell culture methods are similar to in vivo tumors in terms of tissue morphology, biology, and gene expression, unlike cells grown in 2D cell cultures. Breast cancer heterogeneity is one of the main drug resistant mechanisms and needs to be overcome in order to increase the efficacy of drug activity in its treatments. Methods We performed a unique 3D cell culture and drug efficacy study with trastuzumab emtansine (Kadcyla®, T-DM1) across five breast cancer cell lines (BT-474, SK-BR-3, MDA-MB-361, MDA-MB-175, and MCF-7) that were previously investigated in 2D cell culture. We performed HER2 IHC staining, cell viability experiments, Gene-protein-assay (GPA), and T-DM1 internalization studies. Results We obtained significantly different results including higher IC50 for some of the cell lines. Our GPA showed some significant heterogeneous HER2 gene and protein expression in 3D cultured spheroids or aggregates. The fluorescent images also showed that a longer incubation time is needed for T-DM1 to be internalized effectively into 3D cultured spheroids or aggregates. Conclusion Our study demonstrated that the difference of T-DM1 drug activity in 3D spheroids or aggregates might be due to tumor heterogeneity and less efficient internalization of T-DM1 that is not seen using 2D cell culture models. Drug studies using 3D cell culture are expected to provide biologically relevant models for determining drug activity in tumor tissue in future drug response and resistance research.

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Huang, Song-Bin, Min-Hsien Wu, Shih-Siou Wang, and Gwo-Bin Lee. "Microfluidic cell culture chip with multiplexed medium delivery and efficient cell/scaffold loading mechanisms for high-throughput perfusion 3-dimensional cell culture-based assays." Biomedical Microdevices 13, no. 3 (January 14, 2011): 415–30. http://dx.doi.org/10.1007/s10544-011-9510-1.

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Toda, Shuji, Shigehisa Aoki, Kazuyoshi Uchihashi, Aki Matsunobu, Mihoko Yamamoto, Akifumi Ootani, Fumio Yamasaki, Eisuke Koike, and Hajime Sugihara. "Culture Models for Studying Thyroid Biology and Disorders." ISRN Endocrinology 2011 (July 12, 2011): 1–9. http://dx.doi.org/10.5402/2011/275782.

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The thyroid is composed of thyroid follicles supported by extracellular matrix, capillary network, and stromal cell types such as fibroblasts. The follicles consist of thyrocytes and C cells. In this microenvironment, thyrocytes are highly integrated in their specific structural and functional polarization, but monolayer and floating cultures cannot allow thyrocytes to organize the follicles with such polarity. In contrast, three-dimensional (3-D) collagen gel culture enables thyrocytes to form 3-D follicles with normal polarity. However, these systems never reconstruct the follicles consisting of both thyrocytes and C cells. Thyroid tissue-organotypic culture retains 3-D follicles with both thyrocytes and C cells. To create more appropriate experimental models, we here characterize four culture systems above and then introduce the models for studying thyroid biology and disorders. Finally, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types.

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Baudino, Troy A., Alex McFadden, Charity Fix, Joshua Hastings, Robert Price, and Thomas K. Borg. "Cell Patterning: Interaction of Cardiac Myocytes and Fibroblasts in Three-Dimensional Culture." Microscopy and Microanalysis 14, no. 2 (March 3, 2008): 117–25. http://dx.doi.org/10.1017/s1431927608080021.

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Patterning of cells is critical to the formation and function of the normal organ, and it appears to be dependent upon internal and external signals. Additionally, the formation of most tissues requires the interaction of several cell types. Indeed, both extracellular matrix (ECM) components and cellular components are necessary for three-dimensional (3-D) tissue formationin vitro. Using 3-D cultures we demonstrate that ECM arranged in an aligned fashion is necessary for the rod-shaped phenotype of the myocyte, and once this pattern is established, the myocytes were responsible for the alignment of any subsequent cell layers. This is analogous to thein vivopattern that is observed, where there appears to be minimal ECM signaling, rather formation of multicellular patterns is dependent upon cell–cell interactions. Our 3-D culture of myocytes and fibroblasts is significant in that it modelsin vivoorganization of cardiac tissue and can be used to investigate interactions between fibroblasts and myocytes. Furthermore, we used rotational cultures to examine cellular interactions. Using these systems, we demonstrate that specific connexins and cadherins are critical for cell–cell interactions. The data presented here document the feasibility of using these systems to investigate cellular interactions during normal growth and injury.

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Elkington, Paul. "Investigating multi-drug resistant tuberculosis in the 3-dimensional bioelectrospray cell culture model – MRC." Impact 2017, no. 4 (May 8, 2017): 20–22. http://dx.doi.org/10.21820/23987073.2017.4.20.

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Nascimento, Leonardo do, Natália Fontana Nicoletti, Manuela Peletti-Figueiró, Daniel Marinowic, and Asdrubal Falavigna. "Hyaluronic Acid In Vitro Response: Viability and Proliferation Profile of Human Chondrocytes in 3D-Based Culture." CARTILAGE 13, no. 2_suppl (November 15, 2021): 1077S—1087S. http://dx.doi.org/10.1177/19476035211057244.

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Objectives This study aimed to evaluate the efficacy of hyaluronic acid in the viability and proliferation profile of human femoral-tibial joint cartilage affected by osteoarthritis using in vitro models of chondrocytes in a 2-dimensional (2D)- and 3-dimensional (3D)-based culture model by spheroids. Design In vitro study of knee cartilage affected by osteoarthritis that required surgical treatment. Samples were cultured and exposed to hyaluronic acid (100 and 500 μM; intervention group) or vehicle solution. In monolayer or 2D culture, proliferation and cell viability were measured, and nuclear morphometry was analyzed by 4′,6′-diamino-2-fenil-indol (DAPI) staining. The 3D-based culture established from the culture of articular cartilage of patients submitted to total knee arthroplasty evaluated the diameter, viability, and fusion ability of the chondrospheres created. Results Samples from 3 patients resulted in viable cultures, with chondrocyte cells exhibiting a potential for cell proliferation and viability to establish a culture. Hyaluronic acid (100 and 500 μM) improved chondrocyte viability and proliferation up to 72 hours in contact when compared with the control group, and no nuclear irregularities in morphology cell characteristics were observed by DAPI. In the 3D evaluation, hyaluronic acid (500 μM) improved the cellular feedback mechanisms, increasing the survival and maintenance of the chondrospheres after 7 days of analysis, showing the intrinsic capacity of chondrospheres grouped in the attempt to rearrange and reestablish new articular tissue. Conclusions The 2D- and 3D-based culture models with hyaluronic acid improved chondrocyte viability and proliferation and demonstrated the ability of freshly formed chondrospheres to undergo fusion when placed together in the presence of hyaluronic acid.

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Tobin, Brian W., Sandra K. Leeper-Woodford, Brian B. Hashemi, Scott M. Smith та Clarence F. Sams. "Altered TNF-α, glucose, insulin, and amino acids in islets of Langerhans cultured in a microgravity model system". American Journal of Physiology-Endocrinology and Metabolism 280, № 1 (1 січня 2001): E92—E102. http://dx.doi.org/10.1152/ajpendo.2001.280.1.e92.

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The present studies were designed to determine effects of a microgravity model system upon lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNF-α) activity and indexes of insulin and fuel homeostasis of pancreatic islets of Langerhans. Islets (1,726 ± 117, 150 islet equivalent units) from Wistar-Furth rats were treated as 1) high aspect ratio vessel (HARV) cell culture, 2) HARV plus LPS, 3) static culture, and 4) static culture plus LPS. TNF-α (L929 cytotoxicity assay) was significantly increased in LPS-induced HARV and static cultures; yet the increase was more pronounced in the static culture group ( P < 0.05). A decrease in insulin concentration was demonstrated in the LPS-stimulated HARV culture ( P< 0.05). We observed a greater glucose concentration and increased disappearance of arginine in islets cultured in HARVs. Although nitrogenous compound analysis indicated a ubiquitous reliance on glutamine in all experimental groups, arginine was converted to ornithine at a twofold greater rate in the islets cultured in the HARV microgravity model system ( P < 0.05). These studies demonstrate alterations in LPS-induced TNF-α production of pancreatic islets of Langerhans, favoring a lesser TNF activity in the HARV. These alterations in fuel homeostasis may be promulgated by gravity-averaged cell culture methods or by three-dimensional cell assembly.

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von Bomhard, Achim, Joseph Faust, Alexander F. Elsaesser, Silke Schwarz, Katharina Pippich, and Nicole Rotter. "Impact of expansion and redifferentiation under hypothermia on chondrogenic capacity of cultured human septal chondrocytes." Journal of Tissue Engineering 8 (January 1, 2017): 204173141773265. http://dx.doi.org/10.1177/2041731417732655.

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A critical limitation in the cultivation of cartilage for tissue engineering is the dedifferentiation in chondrocytes, mainly during in vitro amplification. Despite many previous studies investigating the influence of various conditions, no data exist concerning the effects of hypothermia. Our aim has been to influence chondrocyte dedifferentiation in vitro by hypothermic conditions. Chondrocytes were isolated from cartilage biopsies and seeded in monolayer and in three-dimensional pellet-cultures. Each cell culture was either performed at 32.2°C or 37°C during amplification. Additionally, the influence of the redifferentiation of chondrocytes in three-dimensional cell culture was examined at 32.2°C and 37°C after amplification at 32.2°C or 37°C. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to measure cell proliferation in monolayer, whereas the polymerase chain reaction and immunohistochemical and histological staining were used in three-dimensional pellet-cultures. Real-time polymerase chain reaction was employed to measure the relative expression of the target genes collagen II, collagen I, aggrecan and versican. Ratios were estimated between collagen II/collagen I and aggrecan/versican to evaluate differentiation. A higher value of these ratios indicated an advantageous status of differentiation. In monolayer, hypothermia at 32.2°C slowed down the proliferation rate of chondrocytes significantly, being up to two times lower at 32.2°C compared with culture at 37°C. Simultaneously, hypothermia in monolayer decelerated dedifferentiation. The ratio of aggrecan/versican was significantly higher at 32.2°C compared with that at 37°C. In three-dimensional pellet-culture, the chondrocytes redifferentiated at 32.2°C and at 37°C, and this process is more distinct at 37°C than at 32.2°C. Similar results were obtained for the ratios of collagen II/collagen I and aggrecan/versican and were supported by immunochemical and histological staining. Thus, hypothermic conditions for chondrocytes are mainly advantageous in monolayer culture. In three-dimensional pellet-culture, redifferentiation predominates at 37°C compared with at 32.2°C. In particular, the results from the monolayer cultures show potential in the avoidance of dedifferentiation.

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Alföldi, Balog, Faragó, Halmai, Kotogány, Neuperger, Nagy, Fehér, Szebeni, and Puskás. "Single Cell Mass Cytometry of Non-Small Cell Lung Cancer Cells Reveals Complexity of In vivo And Three-Dimensional Models over the Petri-dish." Cells 8, no. 9 (September 16, 2019): 1093. http://dx.doi.org/10.3390/cells8091093.

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Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing methods: two-dimensional (2D), carrier-free, or bead-based 3D culturing with in vivo xenografts. Proliferation, viability, and cell cycle phase distribution has been investigated. Gene expression analysis enabled the selection of markers that were overexpressed: TMEM45A, SLC16A3, CD66, SLC2A1, CA9, CD24, or repressed: EGFR either in vivo or in long-term 3D cultures. Additionally, TRA-1-60, pan-keratins, CD326, Galectin-3, and CD274, markers with known clinical significance have been investigated at single cell resolution. The described twelve markers convincingly highlighted a unique pattern reflecting intra-tumor heterogeneity of 3D samples and in vivo A549 lung cancer cells. In 3D systems CA9, CD24, and EGFR showed higher expression than in vivo. Multidimensional single cell proteome profiling revealed that 3D cultures represent a transition from 2D to in vivo conditions by intermediate marker expression of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, Gal-3, CD66, GLUT1, and CD274. Therefore, 3D cultures of NSCLC cells bearing more putative cancer targets should be used in drug screening as the preferred technique rather than the Petri-dish.

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Bogunovic, Natalija, Jorn P. Meekel, Jisca Majolée, Marije Hekhuis, Jakob Pyszkowski, Stefan Jockenhövel, Magnus Kruse, et al. "Patient-Specific 3-Dimensional Model of Smooth Muscle Cell and Extracellular Matrix Dysfunction for the Study of Aortic Aneurysms." Journal of Endovascular Therapy 28, no. 4 (April 26, 2021): 604–13. http://dx.doi.org/10.1177/15266028211009272.

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Introduction: Abdominal aortic aneurysms (AAAs) are associated with overall high mortality in case of rupture. Since the pathophysiology is unclear, no adequate pharmacological therapy exists. Smooth muscle cells (SMCs) dysfunction and extracellular matrix (ECM) degradation have been proposed as underlying causes. We investigated SMC spatial organization and SMC-ECM interactions in our novel 3-dimensional (3D) vascular model. We validated our model for future use by comparing it to existing 2-dimensional (2D) cell culture. Our model can be used for translational studies of SMC and their role in AAA pathophysiology. Materials and Methods: SMC isolated from the medial layer of were the aortic wall of controls and AAA patients seeded on electrospun poly-lactide- co-glycolide scaffolds and cultured for 5 weeks, after which endothelial cells (EC) are added. Cell morphology, orientation, mechanical properties and ECM production were quantified for validation and comparison between controls and patients. Results: We show that cultured SMC proliferate into multiple layers after 5 weeks in culture and produce ECM proteins, mimicking their behavior in the medial aortic layer. EC attach to multilayered SMC, mimicking layer interactions. The novel SMC model exhibits viscoelastic properties comparable to biological vessels; cytoskeletal organization increases during the 5 weeks in culture; increased cytoskeletal alignment and decreased ECM production indicate different organization of AAA patients’ cells compared with control. Conclusion: We present a valuable preclinical model of AAA constructed with patient specific cells with applications in both translational research and therapeutic developments. We observed SMC spatial reorganization in a time course of 5 weeks in our robust, patient-specific model of SMC–EC organization and ECM production.

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Ibold, Yvonne, Simone Frauenschuh, Christian Kaps, Michael Sittinger, Jochen Ringe, and Peter M. Goetz. "Development of a High-Throughput Screening Assay Based on the 3-Dimensional Pannus Model for Rheumatoid Arthritis." Journal of Biomolecular Screening 12, no. 7 (October 2007): 956–65. http://dx.doi.org/10.1177/1087057107307147.

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The 3-dimensional (3-D) pannus model for rheumatoid arthritis (RA) is based on the interactive co-culture of cartilage and synovial fibroblasts (SFs). Besides the investigation of the pathogenesis of RA, it can be used to analyze the active profiles of antirheumatic pharmaceuticals and other bioactive substances under in vitro conditions. For a potential application in the industrial drug-screening process as a transitional step between 2-dimensional (2-D) cell-based assays and in vivo animal studies, the pannus model was developed into an in vitro high-throughput screening (HTS) assay. Using the CyBi™-Disk workstation for parallel liquid handling, the main cell culture steps of cell seeding and cultivation were automated. Chondrocytes were isolated from articular cartilage and seeded directly into 96-well microplates in high-density pellets to ensure formation of cartilage-specific extracellular matrix (ECM). Cell seeding was performed automatically and manually to compare both processes regarding accuracy, reproducibility, consistency, and handling time. For automated cultivation of the chondrocyte pellet cultures, a sequential program was developed using the CyBio Control software to minimize shear forces and handling time. After 14 days of cultivation, the pannus model was completed by coating the cartilage pellets with a layer of human SFs. The effects due to automation in comparison to manual handling were analyzed by optical analysis of the pellets, histological and immunohistochemical staining, and real-time PCR. Automation of this in vitro model was successfully achieved and resulted in an improved quality of the generated pannus cultures by enhancing the formation of cartilage-specific ECM. In addition, automated cell seeding and media exchange increased the efficiency due to a reduction of labor intensity and handling time. ( Journal of Biomolecular Screening 2007:956-965)

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Irawan, Vincent, Akon Higuchi, and Toshiyuki Ikoma. "Physical cues of biomaterials guide stem cell fate of differentiation: The effect of elasticity of cell culture biomaterials." Open Physics 16, no. 1 (December 31, 2018): 943–55. http://dx.doi.org/10.1515/phys-2018-0116.

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Abstract It is widely accepted that stem cells directly sense the elasticity of two-dimensional (2-D) substrates and differentiate into a distinct cell type dependent on the substrate elasticity (direct-sensing differentiation: soft and hard substrates promote differentiation into soft and hard tissue lineage, respectively). Biologically, native extracellular matrices (ECMs) are constantly remodeled through out the life of individuals, which inadvertently introduce changes of mechanical properties. Therefore, direct-sensing differentiation might not fully take into account the responses of stem cells in the actual ECMs microenvironment. Recent investigations in three-dimensional (3-D) cell culture environment suggested the inconsistency of direct-sensing differentiation. Stem cells specifically differentiate not only by sensing the elasticity of materials but also by considering the cellular traction exerted to reorganize the matrices and the matrices deformation. This paper aims to expand further how the cells incorporate the elasticity cues and traction-mediated deformation in final differentiation fates of stem cells. To achieve the aim, we introduced an empirical model based on the investigations in 3-D cell culture environment. The empirical model would serve as a useful framework for future studies intended to investigate the relationship of traction-mediated deformation and commitment of stem cells for variety of tissue lineage in 2-D or 3-D cell culture environment.

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Itoh, Y., J. I. Sasaki, M. Hashimoto, C. Katata, M. Hayashi, and S. Imazato. "Pulp Regeneration by 3-dimensional Dental Pulp Stem Cell Constructs." Journal of Dental Research 97, no. 10 (April 27, 2018): 1137–43. http://dx.doi.org/10.1177/0022034518772260.

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Dental pulp regeneration therapy for the pulpless tooth has attracted recent attention, and clinical trial studies are underway with the tissue engineering approach. However, there remain many concerns, including the extended period for regenerating the dental pulp. In addition, the use of scaffolds increases the risk of inflammation and infection. To establish a basic technology for novel dental pulp regenerative therapy that allows transplant of pulp-like tissue, we attempted to fabricate scaffold-free 3-dimensional (3D) cell constructs composed of dental pulp stem cells (DPSCs). Furthermore, we assessed viability of these 3D DPSC constructs for dental pulp regeneration through in vitro and in vivo studies. For the in vitro study, we obtained 3D DPSC constructs by shaping sheet-like aggregates of DPSCs with a thermoresponsive hydrogel. DPSCs within constructs remained viable even after prolonged culture; furthermore, 3D DPSC constructs possessed a self-organization ability necessary to serve as a transplant tissue. For the in vivo study, we filled the human tooth root canal with DPSC constructs and implanted it subcutaneously into immunodeficient mice. We found that pulp-like tissues with rich blood vessels were formed within the human root canal 6 wk after implantation. Histologic analyses revealed that transplanted DPSCs differentiated into odontoblast-like mineralizing cells at sites in contact with dentin; furthermore, human CD31–positive endothelial cells were found at the center of regenerated tissue. Thus, the self-organizing ability of 3D DPSC constructs was active within the pulpless root canal in vivo. In addition, blood vessel–rich pulp-like tissues can be formed with DPSCs without requiring scaffolds or growth factors. The technology established in this study allows us to prepare DPSC constructs with variable sizes and shapes; therefore, transplantation of DPSC constructs shows promise for regeneration of pulpal tissue in the pulpless tooth.

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Poon, Christine, Mei Zhang, Andrew Ruys, Angela Hong, Christelle Catuogno, and Philip Boughton. "A Novel Dynamic 3-Dimensional Construct for Respiratory Tissue Engineering." Journal of Biomimetics, Biomaterials and Tissue Engineering 14 (July 2012): 31–42. http://dx.doi.org/10.4028/www.scientific.net/jbbte.14.31.

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Tissue engineering of airway tissues poses many complex challenges. As tissue form is determined by function and vice versa, it is necessary to consider mechanical and physiological constraints in conjunction with standard biologic and biochemical factors when culturing tissues in vitro. This study involved the development and validation of a novel 3-dimensional (3-D) construct with the capacity to periodically expose a cell scaffold to air and medium at application of physiologic strain rates. The ultimate objective was to mimic respiratory conditions experienced by airway tissues during breathing whilst ensuring compatibility with proven cell culture techniques. The Biaxx design consists of an elastomeric porous synthetic scaffold integrated with a unique biopolymer coupling unit which engages with an IAXSYS bioreactor actuator. Uniform biaxial strain was imparted by the coupling unit whilst simultaneously creating a periodic air-liquid interface. Biaxx scaffolds with and without a coating of particulate 45S5 bioglass were employed in an assay to assess cell attachment and proliferation whilst subject to periodic strain. Physiologic lung tissue strain of 5-15% was achieved for over 200,000 cycles at 0.2Hz. Preliminary biological studies with H460 human lung carcinoma cells confirmed cell attachment, growth and proliferation on this promising construct.

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Zhang, Lanlan, Hong Song, and Xiaojun Zhao. "Optimum Combination of Insulin-Transferrin-Selenium and Fetal Bovine Serum for Culture of Rabbit Articular Chondrocytes in Three-Dimensional Alginate Scaffolds." International Journal of Cell Biology 2009 (2009): 1–6. http://dx.doi.org/10.1155/2009/747016.

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Fetal bovine serum (FBS) has been reported to affect chondrocyte biosynthesis in monolayer culture. Insulin-Transferrin-Selenium (ITS) was investigated as a partial replacement for FBS during in vitro culture of rabbit articular chondrocytes in three-dimensional alginate scaffold. Chondrocyte-seeded alginate hydrogels were cultured in Dulbecco's modified Eagle's medium plus 10% FBS, 1% ITS plus 2% FBS, 1% ITS plus 4% FBS, or 1% ITS plus 8% FBS. At designed time point, the Chondrocyte-seeded alginate hydrogels were harvested and evaluated with histological staining, immunohistochemistry, and quantitative gene expression analysis. Viable cell density and cell division were also evaluated. Chondrocytes biosynthesis and cell division in 1% ITS with 2% FBS medium were similar to that in medium added with 10% FBS. For a total culture of 3 weeks, phenotypic gene expression in chondrocyte-seeded hydrogels was maintained at high levels in medium with 1% ITS plus 2% FBS, while it was decreased to varying degrees in the other groups. In conclusion, with 1% ITS, medium with 2% FBS could promote chondrocyte biosynthesis and cell division, and prevented cell dedifferentiation in three-dimensional alginate scaffolds.

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44

Chudakova, D. A., E. Yu Skorova, and I. V. Reshetov. "Practical Applications of 3D Cell Culture Biotechnologies for Translational Oncology and Personalized Therapy." Biotekhnologiya 36, no. 3 (2020): 3–15. http://dx.doi.org/10.21519/0234-2758-2020-36-3-3-15.

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The creation of in vitro three-dimensional cellular model systems (in vitro 3D cultures) is a fast-growing leading-edge segment of the biotechnological industry. We have examined in this work the key 80 articles published after 2008, and focused on applications of in vitro 3D culture in translational oncology. We described a broad range of 3D culture systems, including models with and without extracellular matrix (ECM). 3D culture models based on decellularized ECM were discussed in more detail. The role of ECM in pathogeneis of malignant neoplasms, in particular, in the phenomenon of the tumor resistance to chemotherapy, was evaluated. 2D and 3D culture systems were compared, and natural and synthetic ECM were described, as well as the model creation based on 3D bioprinting. Particular attention was paid to in vitro models of various cancers, including those at the metastatic stage, based on 3D cell cultures, which maximally mimic the in vivo tumor behavior. The prospects of the practical application of 3D cell culture models in preclinical drag screening and in personalized therapy were discussed. We also presented our data on in vitro 2D and 3D culturing of human cells on various substrates. 3D cellular models, 3D bioprinting, biotechnology, extracellular matrix, cancer, translational medicine, personalized medicine, drag development, in vitro, ex vivo, oncology The authors are grateful to Dr. E. Shabalina for providing part of the experimental data and to OKA-Biotech Company for the samples of recombinant Funding-The work was supported by a Grant from the Russian Science Foundation (no. 18-15-00391). doi: 10.21519/0234-2758-2020-36-3-3-15

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Caicedo-Carvajal, Carlos, Qing Liu, Andre Goy, Andrew L. Pecora, Anthony R. Mato, Tatyana Feldman, and K. Stephen Suh. "A Novel 3D Co-Culture System for Isolation and Amplification of Primary Liquid Cancer Cells." Blood 116, no. 21 (November 19, 2010): 426. http://dx.doi.org/10.1182/blood.v116.21.426.426.

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Abstract Abstract 426 One of the important challenges in screening anti-cancer drugs is the lack of available “primary cultures systems” that is easy to use to screen new compounds or their combinations. The low yield of primary cancer cell cultures is mainly due to suboptimum environment in vitro and inefficient 2-dimensional cell culture conditions. To create an optimum in vitro environment, lymphoma cell lines were grown in 3-dimension model by using a scaffold and the stromal cells derived from neonatal foreskin was used as the feeder component. This 3-dimensional (3D) stromal co-culture generates an in-vitro model that may mimic the conditions/microenvironment of blood cancer cells interacting with stromal compartments. A specific 3D tissue culture scaffold 3D Insert-PS™ (300 μ m in fiber diameter and 400μ m in pore size) significantly enhances the cell proliferation and maintenance of liquid cancer cells in comparison to 2D stromal co-culture control. The combination of the neonatal stroma cells, a novel 3D scaffold, the constant gyration and a frequent nutrient stimulation allows the lymphoma cells to proliferate 10-fold faster than the cells grown in 2D under the same condition. Starting from the 2nd day of 3D cell culture, these lymphoma cells grew to form layers of aggregated clusters and caused disappearance of single cells morphology and phenotype that is typical of cells growing in suspension. The cell aggregates are continuously produced from the 3D scaffold, subsequently dislodge from the scaffold and then remain viable at the bottom of the dish below the scaffold. When the cell clusters are harvested and cultured in 3D condition, the contamination of fibroblasts is over 1,000 fold less than the cell clusters that are generated from 2D environment. In addition, the clusters of cancer cells generated from 3D co-culture using 3D scaffolds contained the fibroblasts contamination that is less than 0.00001% of the total cell count, suggesting that this novel 3D environment can be implicated for the isolation of primary lymphoma/cancer cells from patient's blood or tissue specimen. To investigate this feasibility, <1% lymphoma cells were premixed with 100 fold excess of neonatal stroma cells, and the mixture was grown using our 3D scaffolds. In 7 days, the 3D culture system was able to amplify lymphoma cells over 100 fold or over 10,000 % of the starting cell number. This preliminary data indicate that this 3D scaffold and co-culturing environment can be customized to amplify primary cancer cells from blood or tumor tissues and subsequently used for personalized drug screening procedures. Disclosures: Goy: Allos Therapeutics, Inc.: Consultancy, Honoraria.

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HAMMOND, T. G., E. BENES, K. C. O’REILLY, D. A. WOLF, R. M. LINNEHAN, A. TAHER, J. H. KAYSEN, P. L. ALLEN, and T. J. GOODWIN. "Mechanical culture conditions effect gene expression: gravity-induced changes on the space shuttle." Physiological Genomics 3, no. 3 (September 8, 2000): 163–73. http://dx.doi.org/10.1152/physiolgenomics.2000.3.3.163.

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Hammond, T. G., E. Benes, K. C. O’Reilly, D. A. Wolf, R. M. Linnehan, J. H. Kaysen, P. L. Allen, and T. J. Goodwin. Mechanical culture conditions effect gene expression: gravity-induced changes on the space shuttle. Physiol Genomics 3: 163–173, 2000.—Three-dimensional suspension culture is a gravity-limited phenomenon. The balancing forces necessary to keep the aggregates in suspension increase directly with aggregate size. This leads to a self-propagating cycle of cell damage by balancing forces. Cell culture in microgravity avoids this trade-off. We determined which genes mediate three-dimensional culture of cell and tissue aggregates in the low-shear stress, low-turbulent environment of actual microgravity. Primary cultures of human renal cortical cells were flown on the space shuttle. Cells grown in microgravity and ground-based controls were grown for 6 days and fixed. RNA was extracted, and automated gene array analysis of the expression of 10,000 genes was performed. A select group of genes were regulated in microgravity. These 1,632 genes were independent of known shear stress response element-dependent genes and heat shock proteins. Specific transcription factors underwent large changes in microgravity including the Wilms’ tumor zinc finger protein, and the vitamin D receptor. A specific group of genes, under the control of defined transcription factors, mediate three-dimensional suspension culture under microgravity conditions.

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Winters, B. S., B. K. Mohan Raj, E. E. Robinson, R. A. Foty, and S. A. Corbett. "Three-dimensional Culture Regulates Raf-1 Expression to Modulate Fibronectin Matrix Assembly." Molecular Biology of the Cell 17, no. 8 (August 2006): 3386–96. http://dx.doi.org/10.1091/mbc.e05-09-0849.

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Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell–FN interactions in 3D.

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Clayton, Natasha P., Alanna Burwell, Heather Jensen, Barbara F. Williams, Quashana D. Brown, Pamela Ovwigho, Sreenivasa Ramaiahgari, Tonia Hermon, and Darlene Dixon. "Preparation of Three-dimensional (3-D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation." Toxicologic Pathology 46, no. 6 (August 2018): 653–59. http://dx.doi.org/10.1177/0192623318789069.

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The use of three-dimensional (3-D) in vitro culture systems (spheroids, organoids) in biomolecular and drug discovery research has become increasingly popular. The popularity is due, in part, to a diminished reliance on animal bioassays and a desire to develop physiologically relevant cell culture systems that simulate the in vivo tissue microenvironment. Most evaluations of 3-D cultures are by confocal microscopy and high-content imaging; however, these technologies do not allow for detailed cellular morphologic assessments or permit basic hematoxylin and eosin histologic evaluations. There are few studies that have reported detailed processes for preparing 3-D cultures for paraffin embedding and subsequent use for histochemical or immunohistochemical staining. In an attempt to do so, we have developed a protocol to paraffin-embed human liver spheroids that can be sectioned with a microtome and mounted onto glass slides for routine histochemical and immunohistochemical staining and light microscopic evaluations.

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Qwarnströ, E. E., S. A. MacFarlane, and R. C. Page. "Effects of interleukin-1 on fibroblast extracellular matrix, using a 3-dimensional culture system." Journal of Cellular Physiology 139, no. 3 (June 1989): 501–8. http://dx.doi.org/10.1002/jcp.1041390308.

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Suenaga, Hideyuki, Katsuko S. Furukawa, Tsuyoshi Takato, Takashi Ushida, and Tetsuya Tateishi. "Cell Condensation and 3-Dimensional Dynamic Environment in a Rotation Culture Upregulates Osteogenic Differentiation of Mesenchymal Stromal Cells." Asian Journal of Oral and Maxillofacial Surgery 20, no. 4 (December 2008): 177–83. http://dx.doi.org/10.1016/s0915-6992(08)80022-2.

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