Recombinant human basic fibroblast growth factor

Primary ECs isolated from aortas of human (two 21 year old Caucasian males, lot# 2139 and 1487; a 15 year old male, lot# 2102; and a 60 year old male, lot# 2366; Cell Application cat# 304-05a), mouse (two biological replicates of C57BL/6 males pooled from multiple mice, Cell Biologics cat# C57-6052, lot# A092913T2MP and B092913T2MP), and cow (two biological replicates, Cell Applications cat# B304-05, lot# 1165 and 1190) were thawed into T75 cell culture flasks and then further grown in T225 flasks at 37 °C and 5% CO₂ in Endothelial Cell Growth Media MV2 (PromoCell) supplemented with 5% fetal calf serum (PromoCell), 5-ng/ml recombinant human epidermal growth factor (PromoCell), 0.5-ng/ml recombinant human vascular endothelial growth factor 165 (PromoCell), 10-ng/ml recombinant human basic fibroblast growth factor (PromoCell), 20-ng/ml long R3 insulin-like growth factor-1 (PromoCell), 1-μg/ml ascorbic acid (PromoCell), and 0.2-μg/ml hydrocortisone (PromoCell). All experiments were carried out before passage 9. Telomerase-immortalized aortic ECs (TeloHAECs, ATCC CRL-4052) were cultured between passages 3 and 40 in the same way as described for the primary aortic ECs.

Primary human cardiac myocytes (HCM, lot 436Z024.4, catalog number PC-C-12810, two independent vials) isolated from the ventricles of the adult heart (33-year-old Caucasian female) were obtained from PromoCell GmbH (Heidelberg, Germany) and cultured at 37°C in a low-serum Myocyte Growth Medium (MGM) containing 5% fetal calf serum (FCS), recombinant human epidermal growth factor (0.5 ng/ml), recombinant human basic fibroblast growth factor (2 ng/ml), recombinant human insulin (5 μg/ml) (PromoCell GmbH, Heidelberg, Germany), 100 U/ml penicillin, 0.1 mg/ml streptomycin and 0.25 μg/ml amphotericin B (Corning, Tewksbury, MA, USA) in a cell culture incubator in the presence of 5% CO₂. HCM cells were passaged using Trypsin/EDTA ratio of 0.04%/0.03% designed for gentle detachment of adherent primary human cells according to the manufacturer’s instructions (PromoCell GmbH, Heidelberg, Germany). Cells were seeded at the concentration of 10000 cells per cm² of a 25 cm² culture flask or a 6-well plate, cultured overnight, pre-treated with 8 ng/ml remifentanil hydrochloride (Ultiva, lot U22B, dissolved in sterile 0.9% NaCl) (Aspen Pharma Ireland Limited, Dublin, Ireland) for 1 h and then cultured for 48 h in the presence of 200 μM cobalt chloride (Merck KGaA, Darmstadt, Germany).

Hair shaft elongation assay was performed according to previously described^{23 (link)–25 (link)}. For organ cultures of mouse vibrissa follicles, individual hair follicles were dissected from the upper lip pad of 5-week-old female C57BL/6 mice. Vibrissa follicles were placed individually in 24-well plates containing Follicle Dermal Papilla Cell Basal Medium (PromoCell GmbH) supplemented with 4 µl/ml bovine pituitary extract, 0.04 ml/ml fetal calf serum, 1 ng/ml basic recombinant human fibroblast growth factor, 5 µg/ml recombinant human insulin (PromoCell) with 1 x penicillin- streptomycin solution (Gibco; Invitrogen, Carlsbad, CA, USA, #15140-122) and each chemical was added to the culture medium. Protocols approved by the Institutional Animal Care and Use Committee (Konkuk University, Republic of Korea). No. KU16199