To begin, take the plate of electroporated fibroblasts and observe the formation of hiPSC colonies under a 10x to 20x objective phase contrast microscope. The hiPSC colonies with 300 micrometer diameter, distinct borders, and a high nuclei to body ratio are suitable for picking. Before picking, coat the wells of a 96 well plate suitable for hiPSC culture with 100 microliters of coating solution.
And incubate for one hour at 37 degrees Celsius. During incubation, mark the colonies of interest at the bottom of the cell culture dish with a pen. For the final preparation of the 96 well plate, remove the coating solution and add 100 microliters of pre-warmed hiPSC culture medium, containing 10 micromoles of ROCK inhibitor Y-27632 to each well.
Next, wash the cells with pre-warmed PBS and add fresh pre-warmed hiPSC culture medium containing 10 micromoles of ROCK inhibitor Y-27632 before picking to remove dead cells. Pick the hiPSC colonies using a gauge needle and divide the colonies into small pieces by drawing a grid into each colony. Afterward, use a phase contrast microscope to check that the colonies are successfully divided into pieces.
Finally, transfer the colonies with a 100 microliter pipette to the prepared 96 well plate, holding the pipette upright over the colony. Allow the cells to attach overnight at 37 degrees Celsius and 5%carbon dioxide without disturbance. The following day, replace the medium with 200 microliters of fresh hiPSC culture medium to remove the ROCK inhibitor Y-27632 and place the plates back in the incubator.
Monitor the 96 well plate and mark the wells with successfully picked clones. The marked clones can be expanded and frozen at this point. Phase contrast images have successfully reprogrammed hiPSC colonies, selected hiPSCs, spontaneously differentiated colonies, non-hiPSC colonies, and a too dense hiPSC culture are presented.
