1 To begin, coat the 6-well plate2 with two milliliters of 0.2%gelatin solution per well. 3 Incubate the plate at 37 degrees Celsius 4 and 5%carbon dioxide for 30 minutes. 5 Next, drop-wise add iMEFs into a 15 milliliter tube 6 containing 10 milliliters 7 of pre-warmed human fibroblast medium, 8 and mix by gently inverting the tube.
9 Centrifuge the cell suspension at 309 G for three minutes. 10 And remove the supernatant before re-suspending the pellet 11 in 12 milliliters of human fibroblast medium. 12 Place two milliliters of the cell suspension 13 into each well of the gelatin-coated six well plate.
14 And incubate overnight at 37 degrees Celsius 15 and 5%carbon dioxide. 16 Discard the medium from the lentivirus-infected 17 and non-infected PDAC, and wash the cells twice with PBS. 18 Then add 500 microliters of trypsin to dissociate the cells.
19 And incubate at 37 degrees Celsius 20 with 5%carbon dioxide and 5%oxygen 21 for 15 minutes. 22 Centrifuge the cell suspension at 300 G for five minutes. 23 And re-suspend the pellet in one milliliter of PDAC medium.
24 Wash the iMEF plate with PDAC medium. 25 Then add 50, 000 lentivirus-infected PDAC cells 26 per well in five wells of iMEF plate, and incubate overnight 27 as demonstrated previously. 28 After preparing the reprogramming medium, 29 add 100 microliters of basic fibroblasts growth factor 30 to 100 milliliters of reprogramming medium.
31 The next day, wash the cells growing on iMEF feeders twice 32 with pre-warmed reprogramming media. 33 Then add two milliliters of media to each well, 34 and incubate overnight at 37 degrees Celsius 35 with 5%carbon dioxide and 3%oxygen. 36 After passing the IPSC pool five times, 37 observe the robust colonies 38 maintaining ESC-like morphology.
39 The PDAC, BxPc3, 40 H6c7, and human fibroblast 41 showed different days of reprogramming 42 to form a mature ESC-like morphology. 43 Clonal IPSC lines were established 44 from each reprogramming of PDAC, 45 BxPc3, H6c7, 46 and human fibroblast cells. 47 All established IPSC lines stained positive 48 for TRA-160, confirming their reprogramming to pluripotency.
