Bioreactor Inoculation
A miniature (100-ml working volume) wicking bioreactor was sterilized by ethylene oxide gas. The scaffold was pre-wetted with 100 ml DMEM/F12 medium overnight. The next day, hiPSC-derived pancreatic cells were trypsinized, and resuspended in 50 ml stage 4 culture medium. Cells were inoculated into the bioreactor by adding the cells to the feed bottle and recirculating the medium. Within 12 hours, no free-floating cells were seen in the feed bottle. Cell culture media were changed every other day, following the differentiation protocol. Upon completion of the bioreactor experiment, cells were removed by placing the scaffold in 20 ml of Dulbecco’s modified Eagle’s medium containing 0.5% Trypsin with EDTA and sonicating for 3 hours at 40Hz using an ultrasonic bath. Cell density of the released cells in the medium was assessed by two methods, Vi-cell counter and hemocytometer.