Hyqtase

MSC were isolated from the femurs of Balb/c and C3H mice using an adaptation of the plastic adherence method described by Phinney et al.^{23 (link)} Briefly, following femoral aspiration, the resulting cell suspensions were plated at a density of 10⁵ cells cm⁻² in T25 flasks (Nunc, Denmark) in culture medium (Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG; Invitrogen), 10% (v/v) foetal calf serum (FCS; Lonza, UK), 2 mM l-glutamine and 100 U mL⁻¹ penicillin/streptomycin (Invitrogen, UK)). The cells were incubated in a humidified incubator at 37°C in an atmosphere of 5% (v/v) CO₂ in air for 24 h before the first medium change. Culture medium was changed every 3–4 days and cells were passaged using Hy-Q-Tase™ (Thermo-Fisher Scientific, UK). Cultures were maintained up to passage 15 (P15).

The BMSCs were cultured in basal medium at 37°C in a humidified atmosphere containing 5% CO₂ for 6 days, and the culture medium was changed every 2 days. When the cell monolayers reached 80–90% confluence, they were detached using HyQTase (Thermo Scientific) and dispersed into a single cell suspension by gentle pipetting. The cells were then counted and their vitality was assessed using trypan blue in a Beckman hemocytometer (Beckman Coulter, Inc., Brea, CA, USA).
Prior to cell seeding, the 18 kGy γ-irradiated, freeze-dried fetal bovine acellular dermal matrix swatches were cut into 0.8 cm × 0.8 cm pieces and subjected to ultraviolet radiation for 15–20 minutes in a laminar flow hood. The pieces were then placed into the wells of 4- or 24-well cell culture plates and soaked in 1 mL of basal medium for 1 hour. After aspirating the media, approximately 20,000 cells were seeded into the wells directly on to the fetal bovine acellular dermal matrix or into control wells without fetal bovine acellular dermal matrix. The plates were incubated at 37°C for 1 hour to allow the cells to attach to the fetal bovine acellular dermal matrix, and then 1.5 mL of fresh basal medium was added to each well. Finally, the plates were placed in a humidified incubator (Thermo Fisher Scientific) at 37°C with 5% CO₂. The media were replaced with 1.5 mL of fresh basal medium every other day.