Recently in the lab that I currently work in, we have had trouble growing our cell culture. We use a 3D matrigel insert cell culture method to culture mouse alveolar lung epithelium (specifically SPC+ AECIIs co-cultured with PDGFRa+ fibroblasts). We have also recently begun culturing human HTII-280+ AECIIs. However, the growth of organoid spheres that we had been able to achieve in the past has not been seen over the last few weeks. At day 14 of our culture the spheres are quite small and take up very little space of the insert compared to normal. I am speculating that maybe there is something wrong with the FBS, EGF, or BPE that we use, as our protocol for medium preparation and lung dissection/dissociation has not changed. Is there an easy way to determine if this is the issue? Or is there some other troubleshooting method we could try?
May want to try all new stock of FBS, EGF, BPE .
Check the expiration date for all the reagents you are using.
Can incubate FBS alone or with GFs to make sure no microbial contamination.
If you are not getting what you usually do anymore, in deed the reagents may be the cause. If expiration dates are not involved, are you using the same type of reagents (company, lot number, etc.). Check lot numbers, different batches of reagents may have different effects, the extent mat be variable according to many factors. That's specially true with FBS, which its compositon is very heterogenous and variation between batches can be great according to the element in the serum that is giving you the desired effect. Some collegues try different lots of FBS and then buy big amounts of the batch that produces the desired effect for future use.
The role of the specific reagents varies. If you have a slight difference with a batch that is essential in your culture, the effect will be bigger eventhough other reagents are doing their role fine. The opposite is also true.
Good luck.
Looks to me it is the tissue, as previously you were growing good.
Mannan
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