Hi all. I've been working with iPS feeder free culture for almost 4 months now. But I am always facing problem after the third passage. It seems like the cells got washed out after media change after the third passage. Currently I am using Geltrex as my coating agent. I tried to do as gentle as I can during the media change. Unfortunately, the same problem occurs after the third passage. Can anyone suggest any solution for this matter? Thank you in advance.
Despite the issue only arising at passage 3, I suspect you are doing something wrong with the Geltrex. Please describe how you are using the Geltrex and how you are doing the passaging.
Ron Hrstka Hi, thank you for the feedback. For geltrex i use 1:60 in DMEM/F-12 media for coating. Incubate minimum 2 hours (usually overnight).
As for the passage, wash with DPBS, dissociate with 0.5x Tryple-SELECT for 3 minutes, wash with DPBS, add media, scrap off and do the cell count and seed. The media change after 24 hours (supplemented with FGF2)
I hope you can give some suggestion.
I have no idea about geltrex, but we use feeder free iPS culture using laminin 511 coating. http://www.matrixome.co.jp/en/products/imatrix-511silk . cell washing out is never an issue. 40-50 passages works fine. we use PBS for imatrix preparation. Type of tissue culture dishes may play important role too.
A dilution ratio is meaningless unless you know the protein concentration of the specific lot of geltrex you have received. In most cases, I use 1 mg of geltrex per 100mm plate or per 3 60mm plates or per single 48, 24, 12, or 6 well tissue cluster. Geltrex works fine when used properly and, in comparison to the mention of Lam521, I have taken clones out past 100 passages and they still worked fine for both cardiac and neural differentiaions.
I never have had a use for Tryple. Since you are doing cell counts, does that mean that you are taking them to single cells? I hope not.
Ron Hrstka do you mean that I need to passage using ratio instead of cell count? Can you suggest the best ratio for 6 wells plate?
Mazharul Islam thank you for you suggestion. We will try to use laminin after this.
- Badges
- Science topic
- Similar topics
- Philosophy
- Moral Philosophy
- Free Will