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Differentiation of respiratory epithelium in a 3-dimensional co-culture with fibroblasts embedded in fibrin gel.

ABSTRACT: Tracheal tissue engineering is a promising option for the treatment of tracheal defects. In a previous study we proved the suitability of fibrin gel as a scaffold for tracheal tissue engineering. This study investigates whether the differentiation of respiratory epithelium can be increased by culturing epithelial cells in a three dimensional system containing fibroblasts embedded into fibrin gel.Respiratory epithelial cells were isolated from porcine trachea, seeded onto a fibrin gel and kept in air-liquid-interface culture for 33 days. Morphology as well as pan-cytokeratin, MUC5AC and claudin-1 expression of cells cultured on pure fibrin gel were compared to culture on gels containing fibroblasts.After two weeks, cells seeded on pure fibrin gel were multilayered, showed hyperproliferation and dedifferentiation. Co-cultured cells built up a pseudostratified epithelium. The differentiation and organization of epithelial structure improved with respect to time. After four weeks, morphology of the co-cultured respiratory epithelium resembled native tracheal epithelium. Immunohistochemistry showed that respiratory epithelium co-cultured with fibroblasts had an increasing similarity of pan-cytokeratin expression compared to native trachea. Cells cultured without fibroblasts differed in pan-cytokeratin expression from native trachea and did not show any improvement of differentiation. Immunohistochemical staining of MUC5AC and claudin-1 proved seeded cells being respiratory epithelial cells.This study indicates that adding fibroblasts to fibrin gel positively influences the differentiation of respiratory epithelium.

SUBMITTER: Albers S

PROVIDER: S-EPMC4772366 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Cardiac fibroblasts support endothelial cell proliferation and sprout formation but not the development of multicellular sprouts in a fibrin gel co-culture model.

Project description:A primary impediment to cardiac tissue engineering lies in the inability to adequately vascularize the constructs to optimize survival upon implantation. During normal angiogenesis, endothelial cells (ECs) require a support cell to form mature patent lumens and it has been demonstrated that pericytes, vascular smooth muscle cells and mesenchymal stem cells (MSCs) are all able to support the formation of mature vessels. In the heart, cardiac fibroblasts (CFs) provide important electrical and mechanical functions, but to date have not been sufficiently studied for their role in angiogenesis. To study CFs role in angiogenesis, we co-cultured different concentrations of various cell types in fibrin hemispheres with appropriate combinations of their specific media, to determine the optimal conditions for EC growth and sprout formation through DNA analysis, flow cytometry and immunohistology. ECs proliferated best when co-cultured with CFs and analysis of immunohistological images demonstrated that ECs formed the longest and most numerous sprouts with CFs as compared to MSCs. However, ECs were able to produce more multicellular sprouts when in culture with the MSCs. Moreover, these effects were dependent on the ratio of support cell to EC in co-culture. Overall, CFs provide a good support system for EC proliferation and sprout formation; however, MSCs allow for more multicellular sprouts, which is more indicative of the in vivo process.

| S-EPMC3969781 | biostudies-other

Injectable and biofunctionalized fibrin hydrogels co-embedded with stem cells induce hair follicle genesis.

Project description:Fibrin-based hydrogels have been widely used in various tissue engineering because of their biocompatibility, biodegradability, tunable mechanical characteristics and nanofibrous structural properties. However, their ability to support stem cells for hair follicle neogenesis is unclear. In this study, we investigated the effect of fibrin hydrogels in supporting skin-derived precursors (SKPs) in hair follicle neogenesis. Our results showed that SKPs in fibrin hydrogels with high cell viability and proliferation, the stemness of SKPs could be maintained, and the expression of hair induction signature genes such as akp2 and nestin was enhanced. Moreover, hair follicle reconstruction experiments showed de novo hair genesis in mice and the hairs persisted for a long time without teratoma formation. More importantly, the blood vessels and sebaceous glands were also regenerated. Our study demonstrated that fibrin hydrogels are promising in hair follicle regeneration and have potential application in clinical settings for alopecia and wound healing.

| S-EPMC9847531 | biostudies-literature

Structural hierarchy governs fibrin gel mechanics.

Project description:Fibrin gels are responsible for the mechanical strength of blood clots, which are among the most resilient protein materials in nature. Here we investigate the physical origin of this mechanical behavior by performing rheology measurements on reconstituted fibrin gels. We find that increasing levels of shear strain induce a succession of distinct elastic responses that reflect stretching processes on different length scales. We present a theoretical model that explains these observations in terms of the unique hierarchical architecture of the fibers. The fibers are bundles of semiflexible protofibrils that are loosely connected by flexible linker chains. This architecture makes the fibers 100-fold more flexible to bending than anticipated based on their large diameter. Moreover, in contrast with other biopolymers, fibrin fibers intrinsically stiffen when stretched. The resulting hierarchy of elastic regimes explains the incredible resilience of fibrin clots against large deformations.

| S-EPMC2872216 | biostudies-literature

Effects of HGF neutralizing antibody on three-dimensional co-culture of periodontitis-associated fibroblasts (PAF)

Project description:We compared gene expression profiles of three-dimensional co-culture of periodontitis-associated fibroblasts embedded in collagen gels treated with control IgG or HGF neutralizing antibody.

2022-01-01 | GSE138926 | GEO

Selective Expansion of Skeletal Muscle Stem Cells from Bulk Muscle Cells in Soft Three-Dimensional Fibrin Gel.

Project description:Muscle stem cells (MuSCs) exhibit robust myogenic potential in vivo, thus providing a promising curative treatment for muscle disorders. Ex vivo expansion of adult MuSCs is highly desired to achieve a therapeutic cell dose because of their scarcity in limited muscle biopsies. Sorting of pure MuSCs is generally required for all the current culture systems. Here we developed a soft three-dimensional (3D) salmon fibrin gel culture system that can selectively expand mouse MuSCs from bulk skeletal muscle preparations without cell sorting and faithfully maintain their regenerative capacity in culture. Our study established a novel platform for convenient ex vivo expansion of MuSCs, thus greatly advancing stem cell-based therapies for various muscle disorders. Stem Cells Translational Medicine 2017;6:1412-1423.

| S-EPMC5442710 | biostudies-literature

Biphenotypic Differentiation of Pancreatic Cancer in 3-Dimensional Culture.

Project description:OBJECTIVE:Pancreatic ductal adenocarcinoma (PDAC) is the third most common cause of cancer death in the United States. Improved characterized models of PDAC are needed for drug screening. METHODS:We grew 4 established pancreatic cancer cell lines in hanging drop cultures to produce spheroids. We also grew organoids from explanted xenografted PDAC and surgically resected primary PDAC. We performed transmission and scanning electron microscopy and compared findings with those of the normal pancreatic duct. We also performed single-cell cloning to determine the potential options for differentiation. RESULTS:Spheroids contained tight junctions and desmosomes but lacked zymogen granules, as expected. The former features were present in normal pancreatic duct but absent from PDAC cell lines grown in standard 2-dimensional culture. Spheroids functionally excluded macromolecules in whole mounts. Cells on the surface of PDAC spheroids were carpeted by microvilli except for rare cells with prominent stereocilia. Carpets of microvilli were also seen in low passage organoids produced from xenografts and surgically resected human PDAC, in addition to normal human pancreatic duct. We performed single-cell cloning and resulting spheroids produced both cell phenotypes at the same approximate ratios as those from bulk cultures. CONCLUSIONS:Pancreatic cancer spheroids/organoids are capable of biphenotypic differentiation.

| S-EPMC6791773 | biostudies-literature

Embryonic Stem Cell Engineering with a Glycomimetic FGF2/BMP4 Co-Receptor Drives Mesodermal Differentiation in a Three-Dimensional Culture.

Project description:Cell surface glycans, such as heparan sulfate (HS), are increasingly identified as co-regulators of growth factor signaling in early embryonic development; therefore, chemical tailoring of HS activity within the cellular glycocalyx of stem cells offers an opportunity to control their differentiation. The growth factors FGF2 and BMP4 are involved in mediating the exit of murine embryonic stem cells (mESCs) from their pluripotent state and their differentiation toward mesodermal cell types, respectively. Here, we report a method for remodeling the glycocalyx of mutant Ext1-/- mESCs with defective biosynthesis of HS to drive their mesodermal differentiation in an embryoid body culture. Lipid-functionalized synthetic HS-mimetic glycopolymers with affinity for both FGF2 and BMP4 were introduced into the plasma membrane of Ext1-/- mESCs, where they acted as functional co-receptors of these growth factors and facilitated signal transduction through associated MAPK and Smad signaling pathways. We demonstrate that these materials can be employed to remodel Ext1-/- mESCs within three-dimensional embryoid body structures, providing enhanced association of BMP4 at the cell surface and driving mesodermal differentiation. As a more complete understanding of the function of HS in regulating development continues to emerge, this simple glycocalyx engineering method is poised to enable precise control over growth factor signaling activity and outcomes of differentiation in stem cells.

| S-EPMC6252183 | biostudies-literature

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture.

Project description:The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase (p < 0.05) in secretion of ?-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation.

| S-EPMC5578114 | biostudies-literature

FITC-Dextran Release from Cell-Embedded Fibrin Hydrogels.

Project description:Fibrin hydrogel is a central biological material in tissue engineering and drug delivery applications. As such, fibrin is typically combined with cells and biomolecules targeted to the regenerated tissue. Previous studies have analyzed the release of different molecules from fibrin hydrogels; however, the effect of embedded cells on the release profile has yet to be quantitatively explored. This study focused on the release of Fluorescein isothiocyanate (FITC)-dextran (FD) 250 kDa from fibrin hydrogels, populated with different concentrations of fibroblast or endothelial cells, during a 48-h observation period. The addition of cells to fibrin gels decreased the overall release by a small percentage (by 7-15% for fibroblasts and 6-8% for endothelial cells) relative to acellular gels. The release profile was shown to be modulated by various cellular activities, including gel degradation and physical obstruction to diffusion. Cell-generated forces and matrix deformation (i.e., densification and fiber alignment) were not found to significantly influence the release profiles. This knowledge is expected to improve fibrin integration in tissue engineering and drug delivery applications by enabling predictions and ways to modulate the release profiles of various biomolecules.

| S-EPMC7926394 | biostudies-literature

Aligned human microvessels formed in 3D fibrin gel by constraint of gel contraction.

Project description:This study aimed to form microvessels in fibrin gels, which is of interest both for studying the fundamental cell-matrix interactions as well as for tissue engineering purposes, and to align the microvessels, which would provide natural inlet and outlet sides for perfusion. The data reported here demonstrate the formation of highly interconnected microvessels in fibrin gel under defined medium conditions and the ability to align them using two methods, both of which involved anchoring the gel at both ends to constrain the cell-induced compaction. The first method used only defined medium and resulted in moderate alignment. The second method used defined and serum-containing media sequentially to achieve high levels of microvessel alignment.

| S-EPMC3851923 | biostudies-literature

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