Generation of three-dimensional pannus-like tissues in vitro from single cell suspensions of synovial fluid cells from arthritis patients

2004 ◽  
Vol 24 (2) ◽  
pp. 71-76 ◽  
Author(s):  
Samuel Solomon ◽  
Madhan Masilamani ◽  
Subhasis Mohanty ◽  
J�rg E. Schwab ◽  
Eva-Maria Boneberg ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Single Cell ◽  
Three Dimensional ◽  
Cell Suspensions ◽  
Synovial Fluid Cells

An in vitro model for the study of human parathyroid gland tissue: Single cell suspensions and monolayer cultures

1982 ◽  
Vol 181 (2) ◽  
pp. 147-154 ◽  
Author(s):  
P. K. Wagner ◽  
A. Knuth ◽  
U. Krause ◽  
H. Gabbert ◽  
Th. Schärfe ◽  
...  
Keyword(s):  
Single Cell ◽  
Parathyroid Gland ◽  
In Vitro Model ◽  
Cell Suspensions ◽  
Monolayer Cultures ◽  
Gland Tissue ◽  
Vitro Model

scholarly journals Self-organising human gonads generated by a Matrigel-based gradient system

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Elizabeth Oliver ◽  
João Pedro Alves-Lopes ◽  
Femke Harteveld ◽  
Rod T. Mitchell ◽  
Elisabet Åkesson ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Germ Cell ◽  
Somatic Cell ◽  
Three Dimensional ◽  
Cell Suspensions ◽  
Testicular Development ◽  
Female Gonad ◽  
Gradient System ◽  
Three Dimensional Culture

Abstract Background Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro. Here we extend the model to human first-trimester embryonic gonadal tissue. Results Testicular cell suspensions reorganised into testis-like organoids with distinct seminiferous-like cords situated within an interstitial environment after 7 days. In contrast, tissue reorganisation failed to occur when mesonephros, which promotes testicular development in vivo, was included in the tissue digest. Organoids generated from dissociated female gonad cell suspensions formed loosely organised cords after 7 days. In addition to displaying testis-specific architecture, testis-like organoids demonstrated evidence of somatic cell differentiation. Within the 3-LGS, we observed the onset of AMH expression in the cytoplasm of SOX9-positive Sertoli cells within reorganised testicular cords. Leydig cell differentiation and onset of steroidogenic capacity was also revealed in the 3-LGS through the expression of key steroidogenic enzymes StAR and CYP17A1 within the interstitial compartment. While the 3-LGS generates a somatic cell environment capable of supporting germ cell survival in ovarian organoids germ cell loss was observed in testicular organoids. Conclusion The 3-LGS can be used to generate organised whole gonadal organoids within 7 days. The 3-LGS brings a new opportunity to explore gonadal organogenesis and contributes to the development of more complex in vitro models in the field of developmental and regenerative medicine.

scholarly journals Optical Cellular Micromotion: A New Paradigm to Measure Tumour Cells Invasion in 3D Tumour Environments

2021 ◽  
Author(s):  
Zhaobin Guo ◽  
Chih-Tsung Yang ◽  
Chia-Chi Chien ◽  
Luke Selth ◽  
Pierre Bagnaninchi ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Invasion ◽  
Tumour Cell ◽  
Single Cells ◽  
Three Dimensional ◽  
Tumour Cells ◽  
Digital Holographic Microscopy ◽  
New Paradigm ◽  
Cell Invasiveness

Measuring tumour cell invasiveness through three-dimensional (3D) tissues, particularly at the single cell level, can provide important mechanistic understanding and assist in identifying therapeutic targets of tumour invasion. However, current experimental approaches, including standard in vitro invasion assays, have limited physiological relevance and offer insufficient insight about the vast heterogeneity in tumour cell migration through tissues. To address these issues, here we report on the concept of optical cellular micromotion, where digital holographic microscopy (DHM) is used to map the optical thickness fluctuations at sub-micron scale within single cells. These fluctuations are driven by the dynamic movement of subcellular structures including the cytoskeleton and inherently associated with the biological processes involved in cell invasion within tissues. We experimentally demonstrate that the optical cellular micromotion correlates with tumour cells motility and invasiveness both at the population and single cell levels. In addition, the optical cellular micromotion significantly reduced upon treatment with migrastatic drugs that inhibit tumour cell invasion. These results demonstrate that micromotion measurements can rapidly and non-invasively determine the invasive behaviour of single tumour cells within tissues, yielding a new and powerful tool to assess the efficacy of approaches targeting tumour cell invasiveness.

scholarly journals High-Content Image-Based Single-Cell Phenotypic Analysis for the Testicular Toxicity Prediction Induced by Bisphenol A and Its Analogs Bisphenol S, Bisphenol AF, and Tetrabromobisphenol A in a Three-Dimensional Testicular Cell Co-culture Model

2019 ◽  
Vol 173 (2) ◽  
pp. 313-335 ◽  
Author(s):  
Lei Yin ◽  
Jacob Steven Siracusa ◽  
Emily Measel ◽  
Xueling Guan ◽  
Clayton Edenfield ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Three Dimensional ◽  
Testicular Cell ◽  
Phenotypic Analysis ◽  
Bisphenol S ◽  
Cytoskeletal Structure ◽  
Culture Model ◽  
Bisphenol Af

Abstract Emerging data indicate that structural analogs of bisphenol A (BPA) such as bisphenol S (BPS), tetrabromobisphenol A (TBBPA), and bisphenol AF (BPAF) have been introduced into the market as substitutes for BPA. Our previous study compared in vitro testicular toxicity using murine C18-4 spermatogonial cells and found that BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS. Recently, we developed a novel in vitro three-dimensional (3D) testicular cell co-culture model, enabling the classification of reproductive toxic substances. In this study, we applied the testicular cell co-culture model and employed a high-content image (HCA)-based single-cell analysis to further compare the testicular toxicities of BPA and its analogs. We also developed a machine learning (ML)-based HCA pipeline to examine the complex phenotypic changes associated with testicular toxicities. We found dose- and time-dependent changes in a wide spectrum of adverse endpoints, including nuclear morphology, DNA synthesis, DNA damage, and cytoskeletal structure in a single-cell-based analysis. The co-cultured testicular cells were more sensitive than the C18 spermatogonial cells in response to BPA and its analogs. Unlike conventional population-averaged assays, single-cell-based assays not only showed the levels of the averaged population, but also revealed changes in the sub-population. Machine learning-based phenotypic analysis revealed that treatment of BPA and its analogs resulted in the loss of spatial cytoskeletal structure, and an accumulation of M phase cells in a dose- and time-dependent manner. Furthermore, treatment of BPAF-induced multinucleated cells, which were associated with altered DNA damage response and impaired cellular F-actin filaments. Overall, we demonstrated a new and effective means to evaluate multiple toxic endpoints in the testicular co-culture model through the combination of ML and high-content image-based single-cell analysis. This approach provided an in-depth analysis of the multi-dimensional HCA data and provided an unbiased quantitative analysis of the phenotypes of interest.

scholarly journals Isolation of the stromal-vascular fraction of mouse bone marrow markedly enhances the yield of clonogenic stromal progenitors

Blood ◽  
2012 ◽  
Vol 119 (11) ◽  
pp. e86-e95 ◽  
Author(s):  
Colby Suire ◽  
Nathalie Brouard ◽  
Karen Hirschi ◽  
Paul J. Simmons
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Stromal Vascular Fraction ◽  
Cell Suspensions ◽  
Mouse Bone Marrow ◽  
Multilineage Differentiation ◽  
Low Oxygen ◽  
Low Incidence

Abstract The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of mouse bone marrow stromal cells (BMSCs), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of stromal cell stem/progenitors, we hypothesized that a potential reason for the low yield of mouse BMSCs is the flushing of the marrow used to remove single-cell suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of BMSCs physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs, which yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1631.8 + 199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32 + 1.9) by at least 2 orders of magnitude (P < .001; N = 8) with an accompanying 113.95-fold enrichment of CFU-F frequency when plated at low oxygen (5%). Purified BMSC populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single-cell suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays.

scholarly journals Single-cell transcriptomics elucidates in vitro reprogramming of human intestinal epithelium cultured in a physiodynamic gut-on-a-chip

2021 ◽  
Author(s):  
Woojung Shin ◽  
Zhe Su ◽  
S. Stephen Yi ◽  
Hyun Jung Kim
Keyword(s):  
Single Cell ◽  
Intestinal Epithelium ◽  
Drug Transport ◽  
Three Dimensional ◽  
Cancer Cell Line ◽  
Static Condition ◽  
On Chip ◽  
Epithelial Layers

The microphysiological human gut-on-a-chip has demonstrated in vivo-relevant cellular fidelity of intestinal epithelium compared to its cultures in a static condition. Microfluidic control of morphogen gradients and mechanical cues robustly induced morphological histogenesis with villi-like three-dimensional (3D) microarchitecture, lineage-associated cytodifferentiation, and physiological functions of a human intestinal Caco-2 epithelium. However, transcriptomic dynamics that orchestrates morphological and functional reprogramming of the epithelium in a microphysiological culture remains elusive. Single-cell transcriptomic analysis revealed that a gut-on-a-chip culture that offers physiological motions and flow drives three distinctive subclusters that offer distinct gene expression and unique spatial representation in 3D epithelial layers. The pseudotemporal trajectory of individual cells visualized the evolutionary transition from ancestral genotypes in static cultures into more heterogeneous phenotypes in physiodynamic cultures on cell cycles, differentiation, and intestinal functions including digestion, absorption, drug transport, and metabolism of xenobiotics. Furthermore, the inversed transcriptomic signature of oncogenes and tumor-suppressor genes of Caco-2 cells verified that a gut-on-a-chip culture drives a postmitotic reprogramming of cancer-associated phenotypes. Thus, we discovered that a physiodynamic on-chip culture is necessary and sufficient for a cancer cell line to be reprogrammed to elicit in vivo-relevant heterogeneous cell populations with restored normal physiological signatures.

scholarly journals Osteoarthritic Synovial Fluid Modulates Cell Phenotype and Metabolic BehaviorIn Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Eduardo Branco de Sousa ◽  
Gilson Costa dos Santos Junior ◽  
Ramon Pinheiro Aguiar ◽  
Rafaela da Costa Sartore ◽  
Ana Carolina Leal de Oliveira ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Adipogenic Differentiation ◽  
Mesenchymal Cell ◽  
Cell Phenotype ◽  
Elisa Assay ◽  
Metabolomic Profile ◽  
Cell Lineages ◽  
H Nmr ◽  
Synovial Fluid Cells

Synovial fluid holds a population of mesenchymal stem cells (MSC) that could be used for clinical treatment. Our goal was to characterize the inflammatory and metabolomic profile of the synovial fluid from osteoarthritic patients and to identify its modulatory effect on synovial fluid cells. Synovial fluid was collected from non-OA and OA patients, which was centrifuged to isolate cells. Cells were cultured for 21 days, characterized with specific markers for MSC, and exposed to a specific cocktail to induce chondrogenic, osteogenic, and adipogenic differentiation. Then, we performed a MTT assay exposing SF cells from non-OA and OA patients to a medium containing non-OA and OA synovial fluid. Synovial fluid from non-OA and OA patients was submitted to ELISA to evaluate BMP-2, BMP-4, IL-6, IL-10, TNF-α, and TGF-β1 concentrations and to a metabolomic evaluation using1H-NMR. Synovial fluid cells presented spindle-shaped morphologyin vitro. Samples from OA patients formed a higher number of colonies than the ones from non-OA patients. After 21 days, the colony-forming cells from OA patients differentiated into the three mesenchymal cell lineages, under the appropriated induction protocols. Synovial fluid cells increased its metabolic activity after being exposed to the OA synovial fluid. ELISA assay showed that OA synovial fluid samples presented higher concentration of IL-10 and TGF-β1 than the non-OA, while the NMR showed that OA synovial fluid presents higher concentrations of glucose and glycerol. In conclusion, SFC activity is modulated by OA synovial fluid, which presents higher concentration of IL-10, TGF-β, glycerol, and glucose.

scholarly journals Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marion Mandon ◽  
Sylvie Huet ◽  
Estelle Dubreil ◽  
Valérie Fessard ◽  
Ludovic Le Hégarat
Keyword(s):  
Single Cell ◽  
Gel Electrophoresis ◽  
Three Dimensional ◽  
Liver Model ◽  
Single Cell Gel Electrophoresis

scholarly journals A ROCK Inhibitor Promotes Graft Survival during Transplantation of iPS-Cell-Derived Retinal Cells

2021 ◽  
Vol 22 (6) ◽  
pp. 3237
Author(s):  
Masaaki Ishida ◽  
Sunao Sugita ◽  
Kenichi Makabe ◽  
Shota Fujii ◽  
Yoko Futatsugi ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Single Cell ◽  
Cell Transplantation ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cell Suspensions ◽  
Rpe Cells ◽  
Rpe Transplantation

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.

Sign in / Sign up

Export Citation Format

Share Document