Stem Cell Culture Support—Troubleshooting

Cells cultured in other feeder-free media systems, such as mTeSR™ Medium with BD Matrigel™ Basement Membrane Matrix, or Gibco™ StemPro™ hESC SFM with Gibco™ Geltrex™ Matrix, can be successfully cultured in Essential 8 Medium and on VTN-N. In addition, PSCs grown on feeders with Gibco™ KnockOut™ Serum Replacement have also been shown to be successfully cultured in Essential 8 Medium on VTN-N. However, when changing media systems, cells must be passaged either manually, or with EDTA prior to culturing in Essential 8 Medium on VTN-N.

Yes. PSCs cryopreserved from cultures of mTeSR™ Medium and BD Matrigel™ Basement Membrane Matrix may be thawed into Gibco™ Essential 8™ Medium and plated on VTN-N. Certain lines may benefit from thawing into the medium and substrate they were growing in at the time of cryopreservation. Then at the next passage, use EDTA to passage the cells into Gibco™ Essential 8™ Medium and VTN-N.

For optimal health of the cultures, cells should be passaged upon reaching ~85% confluency. We have observed improved cell health upon single-cell passaging for cells passaged between 40-85% confluency. If the cells are overly confluent, then inclusion of ROCK inhibitor/RevitaCell Supplement is recommended at the time of passaging. In general, we recommend carrying cells at a range of split ratios to prevent routine passaging at high confluencies as this can result in poor cell survival.

In such circumstances, we recommend either full media change on Sunday or overlaying the culture with additional media on Sunday.

Yes. The main reason that the workflow states this is to accommodate ROCK inhibitor or RevitaCell Supplement that have been added during passaging. In such circumstances where ROCK inhibitor or RevitaCell Supplement are added, you should feed your cells within 18-24 hours post-passaging. However, if ROCK inhibitor or RevitaCell Supplement are not included at the time of passaging, then you may skip the day post-passaging.

Depending on your cell type, you should expect to see some cytotoxicity 24–48 hours post-transduction, which can affect >50% of your cells. This is an indication of high uptake of the virus and is caused by the expression of exogenous genes. We recommend that you continue culturing your cells and proceed with the protocol. Please note that the new vector backbone included in the Invitrogen™ CytoTune™ 2.0 Kit has been shown to cause less cytotoxicity compared to the original kit. In addition because only three vectors are required (compared to four in the original kit), use of the 2.0 kit typically results in reduced cytotoxicity.

The Invitrogen™ CytoTune™-iPS Sendai 2.0 Reprogramming Kit contains a temperature sensitive mutant of c-Myc and KOS that facilitates the clearance of these vectors. To clear c-Myc and KOS, incubate the iPSCs at 38–39°C for 5 days. One caveat is that given the sensitive nature of iPSCs, only perform this temperature shift if Sendai virus is in your iPSC lines after more than 10 passages, and you have performed RT-PCR to show that the Klf4 vector is absent from your cells (this vector does not have temperature-sensitive mutations). Then you can perform temperature shift to remove the c-Myc and KOS vectors.

Valproic acid is typically thought to enhance reprogramming with integrating viral systems such as lentivirus. It is unlikely to enhance CytoTune™ reagent reprogramming since Sendai virus is a non-integrating RNA virus. 

Please note that the Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) has been discontinued.

Here are some reasons why your cell culture could have failed:

• Did not store cells correctly: These cells should be stored in liquid nitrogen.
• Medium used was not the recoomended one: The complete medium of Gibco™ H9-derived NSCs (Cat. No. N7800) and Gibco™ StemPro™ NSCs (Cat. No. A15654 or A15655) have different formulations. Extra components (heparin and ascorbic acid) need to be added into the medium to culture StemPro™ NSCs.
• Did not thaw cells correctly: Do not thaw the cells for longer than 2 mins at 37°C. After cells have thawed, transfer to 50-mL tube first, then add pre-warmed complete medium drop-wise (approximately 1 drop per second) while swirling the tube.
• Did not count cell viability after thawing cells and did not seed them appropriately: You need to count cell viability with trypan blue. At least 1 x 10E6 viable cells/mL are provided in each vial. For H9-derived NSCs, recommended seeding density is > 1 x 10E5 viable cells/cm². For StemPro™ NSCs, recommended seeding density (suspension) is >7 x 10E4 viable cells/mL.
• Plate is not coated or has been coated incorrectly: For H9-derived NSCs and adherent culture, you need to properly coat the tissue culture plate with Gibco™ Geltrex™ Matrix, fibronectin, or poly-L-ornithine/laminin, following instructions for coating. For StemPro™ NSCs, we recommend that you grow them in suspension culture, as adherent culture would trigger differentiation.

NSCs do not stain with neuron-specific markers. If staining was observed, this could have resulted from antibody non-specific staining. We recommend checking antibody specificity and the staining protocol: 

• Titrate primary antibody to find the optimal antibody working concentration. 
• Change the permeabilization protocol, because a high concentration of permeabilization reagent(s) could result in high background.
• Use enough blocking solution such as 5–10% serum to block non-specific staining.
• Include isotype control.

These cells are very fragile. We recommend following the procedure in the manual and using the correct medium. Fast thawing is the key for healthy culture. There are several critical points to consider:

• Pre-rinse all materials with medium before use. Do not use PBS, DPBS, or HBSS to rinse because they do not contain proteins.
• Thaw cells quickly and do not expose cells to air. 
• Transfer cells to a pre-rinsed tube first, then slowly add medium in a drop-wise manner. Do not add the full amount of medium to the cells at once because this may result in osmotic shock and decreased cell viability.
• Use pre-warmed complete growth medium and correct seeding density. 
• Matrix coating is required for some cell cultures.
• For primary neuron cells, do not centrifuge the cells as they are extremely fragile upon recovery from cryopreservation.

There are several possibilities causing the failure of neural induction:

• High quality of hPSC cells is critical to the success of neural induction. Remove differentiated and partially differentiated hPSC cells before neural induction.
• Before plating hPSCs for induction, cell counting is recommended because too low or too high cell confluency will reduce induction efficiency. The recommended plating density for induction is 2–2.5 x 10E4cells/cm².
• Cell clumps (and not a single cell suspension) should be plated for induction.
• To increase induction efficiency, overnight treatment with 10 µM ROCK Inhibitor Y27632 at the time hPSC splitting can be used to prevent extensive cell death.

• Check if the correct B-27™ Supplement was used to culture the cells.
• Check expiration date of B-27™ Supplement to see if it has expired.
• Check if the B-27™-supplemented medium used was fresh. The supplemented medium is stable for only 2 weeks at 4°C. 
• Check if the B-27™ Supplement was exposed to excessive heat. Thawed B-27™ Supplement should not be exposed to room temperature (or higher temperatures) for more than 30 mins. 
• Check if the B-27™ supplement was thawed and refrozen multiple times. Thawed B-27™ Supplement at 4°C should be used within 1 week.
• Check the appearance of the B-27™ Supplement. It should be a transparent yellow liquid. Green color appearance indicates that the supplement is not good.