Recombinant human bfgf

Human placental tissues (n = 4) from first trimester gestation (≤12 weeks of gestation) were collected from healthy consented patients during elective abortions at the UC Davis Medical Center, with approval from the Institutional Review Board. CV-MSCs were isolated from chorionic villus tissue using an explant culture method, previously established in our lab^{48 (link)}. Chorionic villus tissue was washed in phosphate buffered saline (PBS, Hyclone), containing 100 U/ml penicillin and 100 µg/ml streptomycin (1% pen-strep, Thermo Fisher Scientific), and dissected into smaller pieces. Tissues were evenly spread across tissue culture-treated flasks and cultured in D5 media containing DMEM high glucose (Hyclone), 5% fetal bovine serum (FBS, Hyclone), 20 ng/mL recombinant human bFGF (R&D systems), 20 ng/mL recombinant human EGF (R&D systems), and incubated at 37°C. Cells were allowed to migrate from the tissue and grow to 80–90% confluency, before the first passage. The media was changed every 3–4 days. CV-MSCs were used between P3 to P5 for all experiments. HCAECs were purchased from ATCC. BM-MSCs were obtained from Dr. Arnold I. Caplan (Case Western Reserve University). AT-MSCs were obtained from Dr. David E. Sahar (University of California, Davis).

RGD-coupled alginate (NovaMatrix FMC Biopolymer, Norway) was used as the scaffold material. Alginate was purified and partially oxidized (2%) to increase its degradability according to published methods in the literature.^{30 (link),31} The prepared alginate was concentrated, freeze-dried under reduced pressure, and mixed with a cocktail containing 10 µM forskolin (FSK) (Sigma), 10 µM MeBIO (Santa Cruz Biotechnology), and 10 ng/ml recombinant human bFGF (R&D Systems).
To study the effects of elasticity of the alginate hydrogel on myogenic differentiation capacity of encapsulated GMSCs the elastic modulus (E) of alginate hydrogels in presence of different concentrations of CaCl₂ (10, 25, 50, 75, and 100 mM) was measured using an Instron mechanical testing machine (Norwood, PA) at a compression rate of 0.5 mm.min⁻¹ according to the methods already in literature.^{18 (link)}To characterize the release profile of all the components of the cocktail, the hydrogel microspheres were loaded at the abovementioned concentrations and at each selected time interval (1, 3, 5, 7, 10, 12, and 14 days), the amounts of medium were collected and analyzed for released of FSK and MeBIO, using a UV spectrophotometer at 320 nm (Beckman, Brea, CA). Moreover, the release profile of bFGF was characterized using human b-FGF Immunoassay kit (BioSource International Inc. Camerillo, CA).