Cell Culture
hiPSC-derived pancreatic cells were generated following an established
stepwise protocol. Briefly, hiPSCs (F3.5.2, Chang 2015) were cultured in
mTesr-1 (STEMCELL Technologies) in Matrigel-coated 6-well plates to
reach 80% confluence. When confluent, cells were incubated with
Accutase (STEMCELL Technologies) for 5 minutes to detach and pipetted to
form single cells. Single cells were centrifuged at 300 x g for 5
minutes; the supernatant was removed, and cells were loaded into
custom-made polydimethylsiloxane microarrays prepared by soft
lithography (Tomov 2015) with 500-µm wells to form embryoid bodies (EB).
After four days, EBs were transferred to Matrigel-coated petri dishes
and differentiation initiated. Approximately 30 EBs were plated in each
well of a 6-well plate. From Day 1 to Day 12 of differentiation, Kroon’s
protocol (Kroon 2008) was followed to generate endocrine precursors. On
day 12, Millman’s protocol (Millman 2016) was initiated to further
differentiate the cells to mature beta cells.
A frozen vial of primary pancreatic cells was purchased from Celprogen
and cultured on pre-coated flasks (Celprogen) according to
manufacturer’s protocol.
Seeding NKX6-1+/PDX-1+