hi all!!
i am trying to culture adult human brain cortical cells from autopsy brain tissue. but my culture do not adhere or grow. kindly help regarding the same .
The brain sample were taken within 12 hours of post mortem time. this the procedure i followed: firstly the plates were coated with poly d lysine . the plating media used was dmem f12+10 Fbs + NEAA+ bFGF pen strep+ gentamicin. i used a 2 min treatment with 0.1 % trypsin to dissociate the cell. and further triturate using a 1ml pipette tip.
any suggestions regarding the same will be appreciated
thanks in advance
Dear Parmita,
Although 12 hs postmortem is probably the fresher that this type of samples can be obtained, the time elapsed may be still a bit too long to obtain a good amount of neurons viable for culturing, specially if refrigeration was not applied quickly after the patient's passing.
Assuming your samples derived from adult donors, you may need to modify the protocol for dissociation. It is possible that the 2 minutes incubation with trypsin is too short to dissociate the ECM surrounding the neurons and you just end up tearing them during the mechanical dissociation.
For this, I would recommend to cut the tissue in smaller fragments and increase the incubation time in trypsin to at least 10 minutes. Additionally, I strongly recommend to use gentler dissociation systems like papain (30 minutes @ 37C), essential to increase neuron yield from adult tissue [http://www.worthington-biochem.com/pap/cat.html].
Next step would be to check on your culture media, perhaps using osmotically corrected medias like Neurobasal A instead of F12.
The attached paper from Gregory J Brewer may give you additional tips.
Good luck,
Dear sebastian.. thankyou for ur suggestion. I am planning for mixed culture of neuron and astrocytes. I will implement ur suggestions and check if there is some betterment in culture yeild
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