ReNcell VM cell culture problems - why are they dying after I split them?

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Reshma Bhagat

Hi Sophia, I have used ReNcells in the lab. Before I can answer your query could you please answer following:

1) What media are you using?

2) Are you differentiating them?

3) Have tried laminin coating instead of matrigel?

Thanks and Regards

Sophia Chan

Hi Reshma Bhagat thanks for getting back to me.

1) I'm using 50% DMEM/F12, 50% Neurobasal supplemented with Glutamine, N2 and NEAA, bFGF, EGF

2) I haven't been able to passage them beyond the first passage so I haven't tried to differentiate them yet

3) No I have not but I can give it a go.

Hope that has been useful information!

Hi Sophia, thank you for your response

I feel that you should first try changing the coating to laminin- it works best for us.

You can try 100% Neurobasal supplemented with Glutamine, BSA, N2, bFGF, EGF, and NSF-1(from lonza). We have used this media for expanding ReN-Cells to several passages.

You can also try 100% ReNSc Media (provided by millipore) supplemented with bFGF and EGF.

For both media we have used laminin coating only.

Hope this helps you. All the best for you experiments.

Reshma Bhagat.

Hi Reshma,

Okay! I'll look into those changes. Thank you very much!

Happy New Year!

Happy new year :)

Sorry I was just wondering what your protocol is for coating the plates with laminin?

Hi, We have used working stock of laminin as 1mg/ml and dilute it to working concentration of 5ug/ml in sterile 1X PBS.

We put laminin on culture-wares so that it covers properly and leave at 37 degrees for 5h followed by one wash with 1X PBS. You need not dry the culture-wares, just seed your cells.

Also we never coat laminin and store the culture-wares they are used fresh.

Thanks and Regards.

Thank you so much for getting back to me. For coating the laminin, I saw that you can coat for 2h as well: does the timing make a huge difference?

Thanks!

Yes it does. You can try 2h also. See if it works for you. I have never used 2h coated culture wares.