Human Pluripotent Stem Cells (iPSC) Generation, Culture, and …

1If FW of material is not 320.26, dilute appropriately to achieve a 10 mM solution.

2Work quickly; if the Matrigelis allowed to warm at all, it will become gel and will not be appropriate for plating. Matrigel cannot be thawed and refrozen.

3Warm complete medium required for that day at room temperature until it is no longer cool to the touch. Do not warm the medium at 37 C.

4Vials stored in liquid nitrogen may accidentally explode when warmed. Wear ultra-low temperature cryo gloves and also wear eye protection.

5The feeder should be used within a week.

6Wear ultra-low temperature cryo gloves and eye protection when taking the cells from nitrogen tank.

7Number of wells receiving cells is based on the thaw recommendation found in the certificate of analysis if you bought it from the company. If you thaw the frozen vial prepared previously in laboratory follow the labs instruction. For example: When the thaw recommendation is to thaw 1 vial into 1 well, resuspend the pellet in 3 mL of stem cell culture medium.

8To reduce the contamination potential, do not reinsert a used pipette into sterile medium for any reason. If feeding more than one plate, use a different pipette for each plate to reduce risk of contamination.

9Observe the pluripotent stem cells using a microscope. If they require passaging, follow the passaging protocol below.

10At least one well of cells should be left and used as a backup to protect against problems with the split.

11Make sure that the cells remain adhered to the plate. To avoid the iPSC colonies peeling off, do not dispense the medium in a continuous stream.

12iPS cells will grow at a different rate, and the split ratio will need to be adjusted every single time the iPSC cells are passaged. The split ratio is variable and generally is between 1:2 and 1:4. Always, as a general rule, observe the iPSC colonies from the last split ratio and adjust the ratio according to the appearance of the iPSC colonies. If the cells look healthy and colonies have enough space, split the iPSC using the same ratio, if they are dense and crowding, increase the ratio, and if the cells are sparse, decrease the ratio. iPS cells will need to be split every 47 days based on the morphology of the colonies.

13After splitting the iPSC, while cells are attaching, open and close the incubator carefully. This will prevent disturbing the even distribution of cells to the surface of the well.

14iPSC can be passaged with the same method using dispase enzyme solution (1 mg/mL). To use dispase at step 7, add 1 mL room temperature dispase instead of adding 1 mL room temperature collagenase solution to each well to be passaged. Incubate for 35 min at 37 C and continue with the following steps after step 7.

15The isopropanol must be replaced every five uses.

16Try not to break iPSC colonies up into small clumps. If iPSCs are frozen in large aggregates, they will recover from the thaw more efficiently.

17Once cells are in contact with DMSO, they should be aliquoted quickly and initiate freezing within 23 min.

18Wrap the extra plates in Parafilm and store in refrigerator at 28 C and use the plates within 7 days after preparation. If any portion of the well dries out, do not use the well.

19Vials stored in liquid nitrogen may accidentally burst when warmed due to influx of liquid nitrogen into the vial (rare). Wear ultra-low temperature cryo gloves and eye protection.

20number of wells receiving cells is based on the thaw recommendation found in the certificate of analysis if you bought the iPSC line from company. If you thaw the frozen vial of iPSC prepared previously in the laboratory, follow the lab instructions. For example: When the thaw recommendation is to thaw 1 vial into 1 well, resuspend the pellet in 2.5 mL of MTeSR medium.

21To reduce the contamination potential, do not reinsert a used pipette into sterile medium. If feeding more than one plate, use a different pipette for each plate.

22Observe the pluripotent stem cells using a microscope. Follow the passaging protocol below when they require passaging.

23Make sure that the cells remain adhered to the plate. Do not dispense the medium in a continuous stream in one spot to avoid of detaching iPSC from the wells.

24The split ratio is variable, and is generally between 1:2 and 1:4. Always as a general rule, observe the last split ratio and adjust the ratio according to the appearance of the iPSC colonies in the wells. If the colonies on Matrigel have enough space, split using the same ratio, if they are dense and crowding, increase the ratio, and if the cells are sparse, decrease the ratio. Cells will need to be split every 57 days based on the iPSC cell colonies appearance.

25To prevent disturbing the even distribution of cells to the surface of the well, try to limit opening and closing the incubator for few first hours after passaging the iPSC, while cells are attaching. Feed the iPSC daily until ready to passage or freeze.

26Try not to break the iPSC to small clumps. Cells will recover from the thaw more efficiently if frozen in large aggregates.

27Wrap the extra plates in Parafilm and store in refrigerator at 28 C. The plates should be used within a week after preparation. Do not allow the wells to dry. If any portion of the well dries out, do not use the well. Prior to use, pre-warm the plate to room temperature for at least 1 h.

28Warm complete medium required for that day at room temperature until it is no longer cool to the touch. Do not warm the medium at 37 C.

29With certain cell lines, this may take longer than 5 min.

30At least one well of cells should be left and used as a backup to protect against problems with the split.

31Work quickly to remove cells after adding Essential 8 Medium to the well. Do not passage more than one to three wells at a time.

32The split ratio is variable, though generally between 1:2 and 1:4. A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the iPSC colonies. If the cells look healthy and colonies have enough space, split using the same ratio, if they are dense and crowding, increase the ratio, and if the cells are sparse, decrease the ratio.

33While cells are attaching, try to limit opening and closing the incubator doors, and if you need to access the incubator, open and close the doors carefully. This will prevent disturbing the even distribution of cells to the surface of the well.

34Try not to break clumps into little collections of cells. Cells will recover from the thaw more efficiently if frozen in large aggregates.

35Proper maintenance of human iPSC in culture is critical for efficiency of endoderm induction. If the efficiency gradually decreases over several differentiations for a specific cell line, check the maintenance methods and reagents.

36Prepare a 6-well plate with human ECM protein 2448 h before transferring DE cells onto the ECM coated plate. Wrap the extra plates in Parafilm and store in a refrigerator at 28 C and use the plates within 710 days after preparation. If any portion of the well dries out, do not use the well.

37To assess the appropriate anterior foregut endoderm induction, stain a couple of wells without switching to lung progenitor differentiation medium for SOX2 and FOXA2.

38It is important to use low passage HEK293T cells for the production of viruses. To make sure the cells are always in the fastest growth phase, never let the cells grow to 100 % confluence.

39Prepare eight plates of HEK293T, each plate for making one type of virus.

40For each transcription factor to be tested, incubate a Dox-induced (1 L Dox for 48 h) and non-induced well with both the primary and secondary antibodies. The remaining Dox-induced well should be used as a negative control by incubating with the secondary antibody only.

41Add 10 mL of human iPSC culture medium to one plate, which will serve as a negative control.

42to ensure completion of the reprogramming process, it is necessary to remove Dox from the induction medium once colony with good morphology are observed. We recommend removing Dox from the medium at day 25. Removal of Dox ensures that the iPS cell colonies picked and passaged around day 30 are reliant on endogenous expression of pluripotency genes and are not the result of sustained induction of ectopic transcription factor expression by Dox.

43Some transduced fibroblasts in a 10 cm dish may reprogram later. To get the maximum reprogramming efficiency, trypsinize the rest of the cells into a 10 cm plate. Transfer the cells from each of the 10 cm plates into a new MEF cell plate and change medium every 48 h with hESC culture medium. Wait for another 12 weeks for iPSC colonies to develop.

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Human Pluripotent Stem Cells (iPSC) Generation, Culture, and ...