Frequently Asked Questions on the Maintenance of hPSCs
How do I choose the optimal TeSR™ media for my research applications?
Each media within theTeSR™ family is produced using high-quality raw materials that are rigorously pre-screened to ensure the highest levels of quality and minimal batch-to-batch variability. This ensures experimental reproducibility time after time. Each media is based on published formulations.1-3 Use our Choose Your TeSR Maintenance Medium infographic to make an informed decision on the right media for the expansion of hPSCs in culture or downstream applications.
Can I transition cells cultured in other feeder-free media to mTeSR™1?
Human ES and iPS cells cultured in other TeSR™ media, or other feeder-free media, can be conveniently transferred to mTeSR™1. Cells should transition smoothly into mTeSR™1 with minimal differences in morphology, pluripotency, and growth rate. Please see our technical manual for the protocols.
Why should TeSR™ supplement be thawed at room temperature or in the refrigerator instead of at 37°C?
We do recommend warming or thawing TeSR™ media or supplements in the fridge overnight, or at room temperature instead of at 37°C. There is a large temperature discrepancy from storage at -20°C to thawing in a 37°C water bath. Components may precipitate with such a large temperature change, and more importantly, this may affect the performance of the components. Once the supplement is thawed, use the supplement immediately or aliquot and store at -20°C for up to 3 months. After thawing the aliquoted supplement, use immediately or store at 2 - 8°C for up to two weeks, and do not refreeze.
My TeSR™ medium supplement appears slightly turbid after thawing. Can I still use it?
If this is noted, ensure that the supplement is at room temperature. If turbidity persists, place the bottle in a 37°C water bath for approximately 5 minutes, swirling occasionally until the supplement becomes clear. Supplement must be free of turbidity before adding to the basal medium. If the turbidity does not clear, please contact Scientific Support.
Is it necessary to add antibiotics to the media?
No, it is not necessary to add antibiotics to the media as aseptic technique should be sufficient to maintain sterile cultures.
Why is it necessary to use non-tissue culture-treated cultureware for Vitronectin XF™(Catalog #07180)?
We recommend the use of Vitronectin XF™ with non tissue culture-treated (TC) cultureware. If used with TC treated plates, there may be an issue with substrate attachment TC treated plates are recommended when coating plates with Corning® Matrigel® (Corning® Catalog #354277) or CellAdhere™ Laminin-521 (Catalog #77003).
Passaging and Maintenance
Which is better, non-enzymatic dissociation or enzymatic dissociation?
For optimal cell survival and attachment, it is important to select an appropriate passaging reagent that is compatible with the matrix that is used. ReLeSR™ and Gentle Cell Dissociation Reagent are compatible with both Vitronectin XF™ and Matrigel®. However, Dispase is not recommended for the passaging cultures maintained on Vitronectin XF™ substrate nor in cell culture in TeSR™-E8™.
Should I passage my hPSCs as aggregates or as single cells?
Passaging hPSCs as aggregates have been shown to allow the long-term expansion of many different cell lines while maintaining a normal karyotype. It is possible to passage human ES and iPS cells as single cells; however, it has been demonstrated that this practice can place unwanted selective pressure on cell populations and could lead to genetic aberrations in the culture.4,5 Therefore, if you perform single-cell passaging of human ES or iPS cells in any culture medium, check the karyotype frequently to ensure that the culture has retained a normal karyotype.
How does one determine the optimal number of aggregates to plate?
To determine what is the best aggregate size (i.e. the one that gives optimal colonies with the least amount of differentiation), we recommend performing the aggregate count method at passage and plate a range of aggregate numbers (starting with 300 aggregates per well and plating some wells with less than 300 and some with more). Determine which aggregate number gives you the optimal colony morphology for the passaging schedule you are working with (e.g. 7 days).
When is ROCK inhibitor required?
The use of Y-27632 (Catalog #72302), also known as ROCK (Rho-associated, coiled-coil containing protein kinase) inhibitor has been shown to enhance survival of human embryonic stem (ES) cells as single cells by preventing dissociation-induced apoptosis.6 Y-27632 can be used when passaging cells as single cells (not aggregates), when cryopreserving cells, and in specific differentiation protocols, such as neural progenitor generation. When passaging hPSCs as aggregates, there is no requirement to add Y-27632 to the medium.7 It may also result in a decrease in the quality of cell morphology or have other unintended effects on the culture.8
When is spontaneous differentiation a problem?
Limited amount (5 - 10%) of spontaneous differentiation is not an issue in culture, as long as the areas of differentiation are removed on the day of passaging. hPSCs are prone to differentiation when grown in vitro. This is a characteristic of hPSCs: they have the ability to differentiate into cells of the three different germ layers. The pluripotency factors in TeSR™ media will help prevent the cells from differentiating, but a low level (< 10%) of spontaneous differentiation in culture is normal and healthy. Many labs use different techniques (e.g. expression markers, colony morphology, karyotyping, functional pluripotency tests, differentiation to three germ layers) to ensure their hPSCs are maintained in an undifferentiated state.
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- Ludwig TE et al. (2006) Feeder-independent culture of human embryonic stem cells. Nat Methods 3(8): 637–46.
- Ludwig TE et al. (2006) Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24(2): 185–7.
- Chen G et al. (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8(5): 424–9.
- Draper JS et al. (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 22(1): 53–4.
- Buzzard JJ et al. (2004) Karyotype of human ES cells during extended culture. Nat Biotechnol 22(4):381–2.
- Watanabe et al. (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25(6): 681-6.
- Beers et al. (2012) Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat Protoc 7(11): 2029–40.
- Närvä et al. (2017) A Strong Contractile Actin Fence and Large Adhesions Direct Human Pluripotent Colony Morphology and Adhesion. Stem Cell Reports 9 (1): 67-76.
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Frequently Asked Questions on the Maintenance of hPSCs