I have started to grow INS-1 Cells (P#49) in RPMI-1640 supplemented with pen-strep, Na- pyruvate, 50uM 2 Mercaptoethanol, 10mM HEPES PH7.4, 1mM Glutamine. I cryopreserved these cells a few months ago, but now I just resuscitate them in a six well plate in order to attains a confluency earlier, rather than in 10 cm2 cell culture plate. But they seem to be dead on a daily basis. I just changed media after 24 hours of resuscitation, and then I kept the cells in the media for 48 hours. But it has a growth problem.
Any suggestions or comments about it will be highly appreciated, thanks.
First I must admit that I never cultured Ins- 1 cells for my experiment by I frequently culture many primary and cancer cell lines. I was curious about the issue of stalling growth of Ins 1 cells in your culture and did some research. It seems to me that the protocol and the media composition you are following lacks exogenously added glucose (10mM). The attached Eur J Endocrinol article has a very good description for the optimized media composition for culturing Ins 1 cells.
I found two more important threads where people reported problems in culturing these cells and other experts helped them to solve the issues.
http://www.scientistsolutions.com/t3797-culture+of+ins+1+cells.html
http://biology.stackexchange.com/questions/175/how-much-time-do-ins1-e-and-min6-cells-need-after-splitting
After considering those little changes if you still have problems I think transferring the culture surface to a suitable coated material could be helpful. pre-coated dishes with 5 μg/ml of either fibronectin, laminin or collagen 1 provides a better surface for Ins 1 cell attachment and proliferation. Attached J. Cell Commun. Signal. article will give you an idea for surface coating for Ins 1 cell culture.
You can also try increasing the seeding density, most often poor seeding density restricts better cell attachment and division.
I am sharing two articles as a combined pdf doc.
Hope that helps.
Thanks Sir for both of you, for your detailed Instructions/suggestions,
i would like to add that, the RPMI 1640 (Cat#R0883), It includes glucose with 2g/l. sorry Amritlal i forgot to add it.
The only possible reasons which left behind is, i think which you point out the culture surface, i resuscitate in six well plate considering the fact that it will take less time to attain confluency. And those six plates are from TPP, which i read that their culture vessels/products are not coated,
Adam i use to centrifuge the cells and get pellet of it, so DMSO containing media is removed completely.
I would like to add updated information, i just observe it today at 2nd half, it was bacterial contaminated,
so i just discard it, by pouring bleach in it.
Now i think i should choose 10Cm2 plate, which will obviously coated.
Any other comments/suggestion will be much appreciated.
Once again Thanks,
Mr Shabbir Khan, I recently started working on Ins 1 cell line. I am also facing the problem with cell confluency. I saw in ATCC that cells will take 3 days to attach fully. So I think after seeding them, we should not change the media for 3 days (rather to the conventional 24 hrs). Did you find any solution to the issue. Plz let me know.
We had the same situation when resuscitate INS-1 cells,it is a very sensitive and weak cell line,and we solved this problem by incubate the cell directly after thawed without any centrifugation,(there was DMSO in the stocking reagent,generally we have to remove DMSO before the incubation by centrifugation,but this step will hurt the cell),then incubate the cell over night and it will detach very well,then,you can change the media and continue the experiment.
One more thing is,add Glutamine 2-3g/L every two weeks into the media,because the degradation time of Glutamine is short,media without fresh Glutamine can not maintain INS-1 cell condition very well especially in cell resuscitation procedure.
Good Luck
Dear Riyazuddin Mohammed,
I proceeded Exp with Min-6 (beta cell) cells. I would like to add that, grow INS-1 cells without mercaptoethanol if you face its growth problem. Because it worked with me, at that time. Now i m doing exp with Min-6 cells.
Best wishes for your Research...
Thanks
Hi Shabbir. Now I'm working with INS-1 cell line and facing the same situation. Not only frozen cells but also cells under culture became very unhappy and keep detaching. Everyday there will be group of cells floating. I use the same recipe of growth medium as yours. I suspected it was due to bacteria contamination and thawed new cell, changed medium, add pen-strep. But these didn't work well. So your solution is to grow INS-1 cells without 2-mercaptoethanol? Thanks.
Hello Every one !!
I am planning my work on in vitro insulin secretion study. Can you please let me know the appropriate source of INS 1 0r MIN 6 cell line?
I have been using the INS-1 and MIN-6 cells from AddexBio for 2 years. Very good quality cells. You may want to get it from them .
Can you please tell me that till how many passages should we use INS 1 or MIN 6 cell line? Is it finite cell line? Does it decrease its characteristics of insulin secretion and glucose response after particular passage no?
Not sure about INS-1, but MIN-6 cells do seem to have life. As to how many passages can these lines be extended, I have no data to tell. What we did was to expand the cells at early passages and froze multiple vials down. When we need them, we thaw one out and continuously using them. If something happens, we use a new vial. Hope the information helps.
Thank you Jimmy Chow!! For guiding me regarding handling of cell line. But can you please let me know according to your experience, you have worked with which no. of earlier passage and it is working fine?
Not a problem, Madhavi. We purchased it at around passage 8 or 10. But we have been culturing them for 2 years, so many passages now. They are still good.
I am interseted in INS-1 cell line. Can any body tell me where I can buy it? Thanks
- Badges
- Science method