Looking for a successful protocol to maintain confluent primary cell cultures at basal, non-dividing conditions for some time to allow testing the effects of cytotoxic drugs. I've found several protocols from some 15 years ago. Does anyone have more up to date information?
My experience on adherent or suspension primary cell cultures is that you can extend their life by renewing their medium 1 or 2 times per week (not more), always keeping about 1/3 of the previous medium: cells are secreting autokrines factors like interleukines that have a positive effect on their survival. I also avoid as much as possible to shock them: pre-heated medium and pH equilibrated medium before any medium change, minimal time out of the incubator etc
Actually more interested in endothelial cells and tiisues such as lung, liver and kidney, as these are in hand and more relevant to the project right now.
My guess that if you are looking for long-term cultures of monolayers, you may try the continuous feeding and see the evolution of the cultures. Are you considering such methods for "aging" effects or for tightening cell-cell contacts?
Brain microvascular endothelial cells are cultured for 6-8 days in average, other like Caco-2 (human colon carcinoma cell lines) have been reported to grow up to 21 days in order to "mature" their tight junctions.
Abraham,
I'm rather looking to have the cells in a basal, non-dividing state so I can then test the effect of drugs on them. The compounds we are testing arrest cells in G2/M so I want the normal cells to be out of cycle
I would say you may rather give a try and see how your cells behave when kept for prolonged cultures, feeding them on a regular basis. If they have inhibition contact (and thus should switch to quiescent mode) then you should have a stable monolayer with some floating cells. You may run a time course experiment and check if you can a marker for cell division to try to determine when your cells reached the "out of cycle" stage.
I don't know about endothelial cells but I usually work with spleen ex vivo explants in order to test different drugs. Spleen and liver explants have adherent and non-adherent cells so there is an extra problem for maintaining alive long-term without missing any cell population by adding fresh media. They should be in good conditions for at least 7 days. Alamar blue could be useful to check whether they are still alive or not.
I would like to join Abraham's comment. We have experience with brain and other endothelial as well as epthelial cells and cell lines from intestine, kidney, lung and nasal mucosa. These cell types do grow in monolayers and exhibit contact inhibition when confluent. Endothelial cells are prone to angiogenesis and tube formation when kept in confluent cultures over 10 days, but epithelial cells do not have such a problem and can be kept for 3 weeks easily. Lowering the serum concentration (endothelial cells dye in serum free media) can help to maintain confluent monolayers for longer period in a quiescent state.
In general, there are several ways to stop cell growth: contact inhibition, low serum drug treatment etc.
Cells tend to stop, when cells are confluent on dishes/plate because of contact inhibition. You might want to reduce serum concentration or factors which promote cell growth after cells reach a confluent state.
There are several chemical compounds to stop cell growth. Mitomycin c is used to stop cell growth of feeder cells for ES cells. Instead of mitomycin C, X-ray radiation is also used, I thought. Ara-c is used to maintain post-mitotic neurons but in this case growing cells would die by Ara-c.
Thymidine can stop cell growth of some kinds of cells at G1 phase. But I am not sure how long you can hold cells keeping cells healthy, because thymidine is used for cell cycle analysis which does not require long term culture.
I second Kazuo Fushimi's suggestion that mitomycin-C treatment will be your best bet. The cells stop dividing and remain viable for several days (up to about 21 days). You can grow the cells to the desired confluence and arrest them.
For primary cells I suggest that you grow cells until they are over confluent then you remove serum or growth factors by substituting medium with basal medium with 0.5% serum or BSA (in case you don't normally use serum). Leave them for three day (72 hours) and they will be in G0. If you leave them for 24 hours they will be in G1. You have to leave them 48 to 72 hours in order to get them into G0. You can take some and ensure they are in G0 using Ki-67. Please check my previous publication Ghebeh H. et al. International Journal of Cancer 2007. The beauty of this method that this arrest is reversible so cells can go back to G1 when subcultured. I believe this method is the closest to the physiological conditions. Mitomycin C arrest is irreversible.
Thank you all so much so the comments and tips. We will try them out and update how it went.
Michael
My experience on adherent or suspension primary cell cultures is that you can extend their life by renewing their medium 1 or 2 times per week (not more), always keeping about 1/3 of the previous medium: cells are secreting autokrines factors like interleukines that have a positive effect on their survival. I also avoid as much as possible to shock them: pre-heated medium and pH equilibrated medium before any medium change, minimal time out of the incubator etc
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