Has anyone dealt with technical issues with both primary rat neuron/glia co cultures and human iPSC neuron astrocyte co cultures (both on PDL), where the cells seem to ball up near the cell body and cause long filament string like processes to one another? We think it could be due to the coating or a cell signaling process? See image comparison of healthy vs. "spindled" like cells. Apologies for the bad quality image due to uploading on here.
Any technical advice welcome. Thank you!
I see this when my glia cultures are proliferating too fast. I do co-cultures with human neurons (iPS derived) and mouse glia. If this occurs I add a low concentration of ARAC to stop glia cell division. This usually helps. Note: ARAC can become toxic so try to limit the amount of time you have it in your culture. Good luck!
Hello,
I routinely do primary cultures of hippocampal and cortical neurons. Sometimes, I I saw such similar neuron clumps in the dish. Here are two important things you might want to consider to solve this problem. 1) Number of cells and (2) PDL coating. You could think of reducing the number of cells to seed. And, also change your PDL stock solutions. In my case, PDL coating was the reason. I have changed the stock solution and also increased the working concentration of PDL for coating the dishes.
One more thing, do you observe neuron clumping only on glass coverslips or also on plastic coated dishes?
Dear Colleen,
Appears like the differentiated cells are establishing communication via signaling processes. The process in the medium seem to be smooth to me.
Hope it helps.
Best,
AMB
Dear Colleen,
I agree with Satish above. This behavior seems to be usually due to a defective coating. Unable to properly attach to the offered substrate, the neurites have a preference to grow along other neurites resulting in these sort of fasciculated structures. Also cell bodies associate with other cell bodies that are better anchored to the substrate than themselves forming cell body islets. Defective coating could arise from a bad stock of coating agent, badly cleaned coverslips, insufficient incubation of the coating or precense of UV light during the incubation time.
Cheers,
Dear Colleen,
I agree that it looks like a coating problem, and that you are seeing fasiculated neurites, as suggested by Sebastian. In addition, it may be possible that you are getting incomplete dissociation of your tissue, so that you are seeding very small clumps of cells rather than individual cells. Particularly when dissociating primary tissue, each prep can differ depending on how long the dissection takes and how long the cells sit before plating. When you count your cells in the hemocytometer, are you seeing individual cells, and then you see the aggregates form after plating? If that is the case, change your coating protocol, or even get new Poly-D-Lysine.
I always freeze stock aliquots of 1 mg/ml PDL (dissolved in water), and the lab has agreed on an aliquot size that will coat a certain number of plates or flasks and can be used up in a week. By making the stock solution in water, it can be diluted to a range of concentrations, and by using different buffers depending on whether we are using it to bind ECM proteins to the dish, or on its own. Avoid freeze/thaw.
Good Luck!
Jill
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods