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+