1. Which enzymes do you recommend for passaging PSCs growing on laminin?
Generally you can use your enzyme of choice. We recommend TrypLE select, but 0.05% Trypsin-EDTA, or 0.5-1.0 mM EDTA also works. The incubation time depends on the solution used to dissociate the cells and the specific cell line. Stem cells are sensitive and too long exposure to dissociation enzymes or too much mechanical force applied may result in lower cell viability.
2. Does the LN521™ only work with certain hPSC media?
The robust support of pluripotent stem cells by LN521 you to work with your medium and enzyme of choice. As a result, your protocols can easily be made totally defined and xeno-free (if applicable). We have successfully tested many different commercial media, such as NutriStem™, mTeSR™1 & TeSR™2 and Essential 8™. If you are using AF Nutristem XF for feeder containing culture, remember to switch to NutriStem XF/FF when using LN521 as coating material.
3. Can I passage my stem cells as clumps using the stem cell matrix?
Yes, LN521 supports both single cell and clump passaging. However, we do recommend single cell passaging since it is a much easier and more reliable method that allows standardized cultures. Each cell will have equal contact with the coating and the medium resulting in an homogenous environment. LN521 is the natural niche protein for the cells and enables cell-cell contact since it promotes high cell migration. The survival rate after single cells seeding is high. When using LN521 no treatment with apoptosis inhibitors, such as Rho-kinase (ROCK) inhibitor or blebbistatin, is needed to prevent anoikis. Hence, the conventional method where colony state is maintained to prevent apoptosis due to failure to adhere at re-seeding is unnecessary. Single cell passaging also decreases the risk of spontaneous differentiation.
4. Which range of seeding density do you recommend for human pluripotent stem cells?
Optimal seeding densities will vary from one cell line to another and should be determined empirically for your system. LN-521™ has been shown to support cell survival of as low as 5,000 cells/cm2 (Rodin et al., 2014,www-ncbi-nlm-nih-gov.proxy.kib.ki.se/pubmed /24463987). However, we generally recommend seeding your cells at a concentration of 30,000-50,000 cell/cm2 or split your cells with a ratio of 1:10 to 1:30. When changing to LN521 from another feeder-free matrix (i.e. Matrigel) we recommend that you seed the cells at a higher cell density (50,000–100,000 cells/cm2) for the first passage.
5. Can I make clonal cultures on LN521?
Clonal cultures can efficiently be done with CLONEstem-To-Go™, which is a product based on the LN-521™ matrix in combination with other natural stem cell niche molecules. CLONEstem-To-Go™ allows efficient clonal derivation (about 20% success rate), clonal survival and long-term self-renewal of hESC under defined and xeno-free conditions. Furthermore, CLONEstem-To-Go™ even allows hESC derivation from a single blastomere without the need to destroy the embryo. The science behind this is further described in an article in Nature Communications by Rodin and colleagues (Rodin et al., 2014).
6. Why do I see that cells stick to the outer rim of the wells in 96-well plates?
This is probably caused by the high surface tension created in the smaller well formats.
7. Do I need to pre-warm all solutions before usage and why?
The solutions should be pre-warmed since a large variation in temperature is stressful for the cells.
8. Can I perform non-enzymatic passage of my PSCs on LN-521?
You can choose to do non-enzymatic dissociation, such as EDTA, of pluripotent stem cells cultured on LN521. The concentration and incubation time is cell line dependent. Make sure the incubation time is sufficient as too much mechanical force may result in low cell viability. Mechanical force should be minimized not to cause signiﬁcant physical damage to the cells.
9. Can I use BioLamina’s human recombinant laminins for my animal ES and IPS cells?
Yes, most likely. Laminins, as well as many other basal membrane proteins, are highly conserved proteins. Customers working with mouse and monkey stem cells successfully use our human recombinant laminins.
10. When culturing my mESC feeder-free on LN511 and LN521, do I have to add LIF to prevent the cells from differentiating?
No, LN511 and LN521 supports self-renewal of mouse ES cells in the absence of feeder cells, LIF, or other differentiation inhibitors, even at low cell density. LN511 and LN521 successfully support both naive and primed stem cells since it has been shown to activate the PI3/Akt pathway while the MAPK/ERK pathway is unaffected (Rodin et al., 2014).