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.