I have recently started experimenting with establishing rat E18 hippocampal cultures. I have had mixed successes and failures and have learned a great deal along the way. One issue that I have noticed is that when I plate my neurons at densities higher than about 40K/12mm coverslip they are forming non-adherent clumps. I don't mind the sparsity with a lower seeding number, however I would like to culture them to DIV21 and they seem to become less healthy after about two weeks at this density. Does anyone have any advice regarding improving plating density to avoid clumping?
I don't feel that it is a substrate issue because I do get nice adherence, just a little too sparse for my needs. I am culturing in standard supplemented Neurobasal on slips coated with PDL/laminin.
Hi Thomas - I routinely plate P0 rat hippocampal neurons at both low (40k) and high (>100K) densities to at least DIV21. I feel clumping is generally a result of unhappy/ unhealthy neurons trying to find partners to "feed" off of, so perhaps the neurons are not finding enough goodies in your growth media to keep them happy. I use NbActiv4 from Brainbits, LLC, a serum-free complete media. I replace half the media about 3 days after plating, then once weekly thereafter. Also, though you don't suspect your substrate, I use only high molecular wt PDL (Sigma P7405) coating for my 12mm glass coverslips. You might consider trying this. Good luck, hope this helps!
I would make sure you are coating the plates with borate buffer and laminin, which help decrease clumping and keep the environment a basic pH.
Thank you for the comments, Amelia and Brenda. What I have found is that the media exchanges and plating number are the two variables that have the greatest effect on my cultures. I have been using coverslips from Neuvitro because I don't have a lot of time to play with preparing coverslips and they have been quite consistent. I also realized that I get better survival with supplementing 25uM glutamate when plating. I thought that the Glutamax was the same (face palm) reagent. I am seeing better results and have cultures going out to DIV14 without troubles. I no longer think that my manipulations require older cultures.