Isolation and cultivation of adipose tissue-derived mesenchymal stromal cells

Approximately 125 g of processed adipose tissue is incubated for 90 minutes at 37°C in dissociation medium (1:1) consisting of 1% (v/v) bovine serum albumin and 1 mg/mL collagenase A in phosphate buffered saline (PBS). After two filtration steps, the filtrate is carefully brought on top of 15 mL of Histopaque®-1077. Upon centrifugation for 20 minutes at 1000 g (4°C), the top layer is removed and the AT-MSC are collected in 50 mL PBS/BSA (1%). This procedure is carried out separately on two pieces of adipose tissue. Typically 5 - 20 x 10E7 viable cells are obtained per 250 g of processed adipose tissue. The isolated AT-MSC are then (sub)cultured as a monolayer in AT-MSC growth medium for 2 weeks, consisting of Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) foetal bovine serum (FBS), 50 µg/mL streptomycin sulphate, 7.33 IU/mL benzyl penicillin and 2.5 µg/mL fungizone. Cell cultures are incubated at 37°C in a 5% (v/v) CO2 humidified atmosphere and passaged at subconfluency using TrypLE® express. Growth media is changed every 3 days.