“…The previously described iPSC line 006‐BIOTR‐0001, obtained from fibroblasts and reprogrammed with Sendai virus kit (Thermo Fisher, Waltham, MA; clone 1, Mayo Clinic) was used for all experiments . Additional lines used to validate reproducibility of the fibrinogen coating included the previously described IMR‐90‐4, obtained from a immortalized human fibroblast cell line and reprogrammed using lentiviral vectors (clone 4; WiCell; Madison, WI), and a new line, 018‐BIOTR‐0089 (clone 18), produced by reprogramming of human peripheral blood lymphocytes using episomal DNA transfection.…”
Section: Methodsmentioning
confidence: 99%
“…Fibrin gels were made as previously described using a clinical grade kit (Evicel; Ethicon, Somerville, NJ) . Briefly, a mixture of 30 mg/ml fibrinogen and 50 U/ml thrombin (final concentrations) was mixed in a well of a 12‐well plate (total 100 μl), and a custom polycarbonate mold with parafilm lining was used to flatten the gel within the well and minimize the minicus effect.…”
Section: Methodsmentioning
confidence: 99%
“…Briefly, a mixture of 30 mg/ml fibrinogen and 50 U/ml thrombin (final concentrations) was mixed in a well of a 12‐well plate (total 100 μl), and a custom polycarbonate mold with parafilm lining was used to flatten the gel within the well and minimize the minicus effect. The 30 mg/ml fibrinogen concentration was chosen because it is comparable to fibrin glue concentrations and our previous work demonstrating iPSC‐RPE culture . The 50 U/ml thrombin concentration was chosen to provide some time for mixing and applying the mold prior to gelation.…”
Section: Methodsmentioning
confidence: 99%
“…Previously, our group had reported using fibrin hydrogels as a scaffold for transplantation of induced pluripotent stem cell derived retinal pigment epithelium (iPSC‐RPE) . The results of this work showed that the partially differentiated iPSC‐RPE can directly adhere to the fibrin gel, form a monolayer and express phenotype specific marker proteins . Similarly, work from Willerths' group has demonstrated culture of iPSC‐derived neural aggregates on fibrin hydrogels can be used to generate a mixed population of dorsal and ventral spinal neurons .…”
Section: Introductionmentioning
confidence: 97%
“…Previously, our group had reported using fibrin hydrogels as a scaffold for transplantation of induced pluripotent stem cell derived retinal pigment epithelium (iPSC‐RPE) . The results of this work showed that the partially differentiated iPSC‐RPE can directly adhere to the fibrin gel, form a monolayer and express phenotype specific marker proteins .…”
Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)‐derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.g., laminin) for expansion in adherent cell culture for use in cell therapy. To address this, we investigated the potential of culturing iPSCs on fibrin hydrogels for three‐dimensional applications and further examined the use of fibrinogen, the soluble precursor protein, as a coating substrate for traditional adherent cell culture. iPSCs successfully adhered to and proliferated on fibrin hydrogels. The two‐dimensional culture with fibrinogen allows for immediate adaption of culture models to a nonxenogeneic model. Similarly, multiple commercially available iPSC lines adhered to and proliferated on fibrinogen coated surfaces. iPSCs cultured on fibrinogen expressed similar levels of the pluripotent stem cell markers SSea4 (98.7% ± 1.8%), Oct3/4 (97.3% ± 3.8%), TRA1‐60 (92.2% ± 5.3%), and NANOG (96.0% ± 3.9%) compared with iPSCs on Geltrex. Using a trilineage differentiation assay, we found no difference in the ability of iPSCs grown on fibrinogen or Geltrex to differentiate to endoderm, mesoderm, or ectoderm. Finally, we demonstrated the ability to differentiate iPSCs to endothelial cells using only fibrinogen coated plates. On the basis of these data, we conclude that human fibrinogen provides a readily available and inexpensive alternative to laminin‐based products for the growth, expansion, and differentiation of iPSCs for use in research and clinical cell therapy applications.
stem cells translational medicine
2019;8:512–521
“…The previously described iPSC line 006‐BIOTR‐0001, obtained from fibroblasts and reprogrammed with Sendai virus kit (Thermo Fisher, Waltham, MA; clone 1, Mayo Clinic) was used for all experiments . Additional lines used to validate reproducibility of the fibrinogen coating included the previously described IMR‐90‐4, obtained from a immortalized human fibroblast cell line and reprogrammed using lentiviral vectors (clone 4; WiCell; Madison, WI), and a new line, 018‐BIOTR‐0089 (clone 18), produced by reprogramming of human peripheral blood lymphocytes using episomal DNA transfection.…”
Section: Methodsmentioning
confidence: 99%
“…Fibrin gels were made as previously described using a clinical grade kit (Evicel; Ethicon, Somerville, NJ) . Briefly, a mixture of 30 mg/ml fibrinogen and 50 U/ml thrombin (final concentrations) was mixed in a well of a 12‐well plate (total 100 μl), and a custom polycarbonate mold with parafilm lining was used to flatten the gel within the well and minimize the minicus effect.…”
Section: Methodsmentioning
confidence: 99%
“…Briefly, a mixture of 30 mg/ml fibrinogen and 50 U/ml thrombin (final concentrations) was mixed in a well of a 12‐well plate (total 100 μl), and a custom polycarbonate mold with parafilm lining was used to flatten the gel within the well and minimize the minicus effect. The 30 mg/ml fibrinogen concentration was chosen because it is comparable to fibrin glue concentrations and our previous work demonstrating iPSC‐RPE culture . The 50 U/ml thrombin concentration was chosen to provide some time for mixing and applying the mold prior to gelation.…”
Section: Methodsmentioning
confidence: 99%
“…Previously, our group had reported using fibrin hydrogels as a scaffold for transplantation of induced pluripotent stem cell derived retinal pigment epithelium (iPSC‐RPE) . The results of this work showed that the partially differentiated iPSC‐RPE can directly adhere to the fibrin gel, form a monolayer and express phenotype specific marker proteins . Similarly, work from Willerths' group has demonstrated culture of iPSC‐derived neural aggregates on fibrin hydrogels can be used to generate a mixed population of dorsal and ventral spinal neurons .…”
Section: Introductionmentioning
confidence: 97%
“…Previously, our group had reported using fibrin hydrogels as a scaffold for transplantation of induced pluripotent stem cell derived retinal pigment epithelium (iPSC‐RPE) . The results of this work showed that the partially differentiated iPSC‐RPE can directly adhere to the fibrin gel, form a monolayer and express phenotype specific marker proteins .…”
Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)‐derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.g., laminin) for expansion in adherent cell culture for use in cell therapy. To address this, we investigated the potential of culturing iPSCs on fibrin hydrogels for three‐dimensional applications and further examined the use of fibrinogen, the soluble precursor protein, as a coating substrate for traditional adherent cell culture. iPSCs successfully adhered to and proliferated on fibrin hydrogels. The two‐dimensional culture with fibrinogen allows for immediate adaption of culture models to a nonxenogeneic model. Similarly, multiple commercially available iPSC lines adhered to and proliferated on fibrinogen coated surfaces. iPSCs cultured on fibrinogen expressed similar levels of the pluripotent stem cell markers SSea4 (98.7% ± 1.8%), Oct3/4 (97.3% ± 3.8%), TRA1‐60 (92.2% ± 5.3%), and NANOG (96.0% ± 3.9%) compared with iPSCs on Geltrex. Using a trilineage differentiation assay, we found no difference in the ability of iPSCs grown on fibrinogen or Geltrex to differentiate to endoderm, mesoderm, or ectoderm. Finally, we demonstrated the ability to differentiate iPSCs to endothelial cells using only fibrinogen coated plates. On the basis of these data, we conclude that human fibrinogen provides a readily available and inexpensive alternative to laminin‐based products for the growth, expansion, and differentiation of iPSCs for use in research and clinical cell therapy applications.
stem cells translational medicine
2019;8:512–521
Fibrin, the prominent extracellular matrix in early wound tissue, is discussed to influence immune cells and healing. The nature of fibrinogen/fibrin to form fibrillary networks is frequently exploited to engineer microenvironments for cellular analysis. This study focuses on revealing the correlation of fibril formation kinetic and the resulting network microstructure of engineered 3D fibrin networks. Different concentrations of fibrinogen (1–3 mg mL−1), thrombin (0.01–0.15 U mL−1), sodium chloride (40–120 mm), and calcium chloride (1–10 mm) are applied to assess the impact on the fibril growth kinetics by turbidity analysis and on the resulting fibril and pore diameter by laser scanning microscopy. The results highlight a direct influence of the sodium chloride concentration on fibrillation kinetics and reveal a strong correlation between fibrillation kinetics and network microstructure. With the assumption of a first‐order growth kinetic, an increase of the growth constant k (0.015–0.04 min−1) is found to correlate to a decrease in fibril diameter (1–0.65 µm) and pore diameter (11–5 µm). The new findings enable an easy prediction of 3D fibrin network microstructure by the fibril formation kinetic and contribute to an improved engineering of defined scaffolds for tissue engineering and cell culture applications.
Fibrin is a degradable biopolymer with an excellent clinical safety profile. Use of higher mechanical strength fibrin hydrogels is limited by the rapid rate of fibrin polymerization. We recently demonstrated the use of higher mechanical strength (fibrinogen concentrations >30 mg/ml) fibrin scaffolds for surgical implantation of cells. The rapid polymerization of fibrin at fibrinogen concentrations impaired our ability to scale production of these fibrin scaffolds. We serendipitously discovered that the azo dye Trypan blue (TB) slowed fibrin gelation kinetics allowing for more uniform mixing of fibrinogen and thrombin at high concentrations. A screen of closely related compounds identified similar activity for Evans blue (EB), an isomer of TB. Both TB and EB exhibited a concentration dependent increase in clot time, though EB had a larger effect. While gelation time was increased by TB or EB, overall polymerization time was unaffected. Scanning electron microscopy showed similar surface topography, but transmission electron microscopy showed a higher cross‐linking density for gels formed with TB or EB versus controls. Based on these data we conclude that addition of TB or EB during thrombin mediated fibrin polymerization slows the initial gelation time permitting generation of larger more uniform fibrin hydrogels with high‐mechanical strength.
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