Bacterial collagenase type 2

Cell counting and cell viability were performed to determine the cell growth and viability of ADSCs encapsulated in Col-Tgels by using a cell counter and a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Rockville, MD), as described18 (link). Briefly, cells were released from Col-Tgel-Cell constructs using 20 units of bacterial collagenase type II (Worthington, Lakewood, NJ) prior to counting by a cell counting machine (Beckman Coulter, Brea, CA). For the CCK-8 assay, 500 μL of the CCK working solution (CCK-8 stock solution in growth medium, 1:40) was directly applied to the Col-Tgel-Cell construct for 4 h. Subsequently, 100 μL of the supernatant was transferred into 96-well plates and the absorbance was measured in a multiplate reader at 450 nm (Molecular Devices, Sunnyvale, CA).

ADSCs were isolated from Fisher 344 retired breeders (18–20 months old) as described56 (link). Briefly, adipose tissues were dissected from the abdomen and digested with bacterial collagenase type II (Worthington, Lakewood, NJ). The digested cells were filtered and washed twice with the medium consisting of high glucose Dulbecco’s modified Eagle medium (DMEM, Mediatech, VA) supplemented with 2% (v/v) penicillin-streptomycin (PS, Mediatech, VA). Then, two millions of cells were plated on individual 60 mm Petri dishes containing DMEM growth medium supplemented with 10% (v/v) fetal bovine serum II (FBS, Thermo Scientific, MD) and 1% (v/v) PS in a humidified atmosphere of 95% O₂/5% CO₂ at 37 °C. The medium was changed every 2–3 days. When cells reached 70% confluence, they were sub-cultured using 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) solution.