Any useful tips for transitioning/thawing cell culture in DEF-CS?
Any useful tips for transitioning/thawing cell culture in DEF-CS?
| Make sure to seed the cells dense, especially the first couple of passages (it did look a little bit sparse in the images as compared to our images even though 100k/cm2 was seeded. It might be that the cells do not attach so well, could be worth an even higher start density). Do allow the cells to grow quite dense the first passage, so that the cells are not passaged to early, it might be good to allow 4-6 days between first and second passage. Double the volume of coating, dilute COAT-1 1:10 in D-PBS, and coat for at least 1h at 37C Double the volume of GF-1, using 6 µl/ml medium. Try to passage the cells as small clusters, for example by adding TrypLE Select only for one minute, when the cells start to round up. Aspirate the enzyme, add some medium for passage (for example 4 ml/T25 flask), scrape the cells of surface by using a cell scraper. Transfer small clusters of cells to a new flask. This could be tested for the first and second passage, then go over to single cell passage again. If the cell line seem to consume medium quite fast (i.e. medium turns yellow overnight), increase the medium volume used. Some cell lines seem to have a higher metabolic rate, and could be sensitive to lower pH. Do not use GF-2, to see if that might help. Most cell lines do need GF-2. Stem cell products |