I am culturing rat dorsal root ganglion neurons on poly-L-lysine + laminin, supplemented with mitogenic inhibitors. In a few days, neurons arborize but the processes slowly turned thick and straight which attach loosely to the culture plate. I am wondering if it's coating problem (referring to similar observations in other neuronal cultures) or low plating density. Any input is appreciated. Thanks a lot.
Hi we use 50 ug/ml P-D-L and 4 or 2 ug/ml of laminin overnight and then 3 x wash by ddH2O. We also add NGF into culture medium to DRGs culture (25 ng/ml). Then it works good.
Thanks Marie. I am using 50ug/cm2 PLL and 2ug/cm2 laminin overnight. One H2O wash. I add 50ng/ml NGF. Seems my concentrations are significantly higher than yours. So did you ever see something like what's shown in my picture? Or could you share a picture of your culture with me so that I can have some sense what I should be expecting? Thank you very much.
I don't know exactly for which purpose you culture DRG neurons, but similarly to your observation, I have noted that a simple coating of PLL or LN promotes rapid neurite outgrowth but not their maintenance. However, when cultured onto monolayers of feeder cells (e.g. embryonic fibroblasts) they grow fine and remain attached to the substratum without forming big bundles much longer than on amorphous ECM molecules. I hope this may help.
We culture DRG and trigeminal ganglion cells from adult mice on glass cover slips with no coating, and the cells grow well. See: Belzer V, Shraer N, Hanani M. Phenotypic changes in satellite glial cells in cultured trigeminal ganglia. Neuron Glia Biology, 6, 237-243, 2010.
Very important in the case of using plastic: take special Nunclon dishes:
- Nunclon cell culture dishes, 6 cm² (Sigma, D8045)
- Nunclon Δ Multidishes 4 well round (Sigma, D6789-1CS)
Also important: do not use too much medium. It should just cover the DRG explant. Pipette the medium slowly to the side of the dish to prevent sheering pressure
I personally have better experience with PDL (0.5 mg/ml 3h-o/n) and Laminin (1 ug/ml o/n)
With 10 ng/ml NGF I can maintain DRG axons for at least 12 days. For that period of time, you will have to treat the cultures with mitomycin C in a daily on/off regime to prevent Schwann cells from contaminating your cultures.
I just saw that you do dissociated cutlures. For those I guess the same things apply, but I do not know for how long they will remain good.
You may want to try polyornithine rather than Poly-L-lysine as the initial coating. Wash the polyornithine off well and completely dry before adding the laminin (10ug/ml). What media are you using? We have had good results using F14 plus Sato salts plus Albumax.
With time cells start digesting poly-L-lysine, so I rather grow neurons on poly-D-lysine. If you are growing them on glass support, note that coating can have different efficiency in sticking depending on the type of glass. You could either pre-treat your glass with acids or increase incubation with pDl to overcome the problem. I found that for non optimal glass supports pDl on 37°C+resting at 4°C prior to use give good results.
Hello Katie, I did use Matrigel, it is excellent for primary culture of DRG neurons, and as recommendation don´t remove all the old medium when you add new.
Best.
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