Hello guys!
So, Its been 4 years that I do primary cortical neuronal culture as the same way and they are cultived for 12 days at plates covered by Poly D lisine and with the medium: Neurobasal + B27 + Glutamax + Gentamicin. In the past month, some of my neurons didnt developed! Actually, the cells stayed at progenitors cells (the same as they looked like at day 1). The cells are not dead, as I noticed at microscope and by medium color changes, but they dont develop to neurons. Any ideas of why this could be happening? The incubator is the same as usual, and I share with another researcher that cultivates astrocytes and they are just fine!
Dear Amanda,
Here few things to try in your next culture:
> Avoid Gentamicin >> check what happens
> Try replacing Neurobasal by DMEM in your culture media (with and without Gentamicin
> Check for mycoplasma contamination either in culture or you animals (more if you breed them yourself).
Dirty troubleshooting :
> What age are the embryos pups you use? Assuming you use a Banker's style protocol, If using E16-E19 embryos, try leaving them longer (4-6hs some use overnight) in media with serum to allow more astrocytes to attach (dirtier culture). This is so after change to neuronal media, you will have many astrocytes to condition de media. If under this condition you see your cells differentiating better than in your regular cultures, then you will know that something in your original media formulation isn't working properly, and your neurons are not getting all the goodies they need. You can also add Gentamicin in this condition to test whether it could be the cause of your problems. I have never used any antibiotic for primary neuronal cultures outside for selection and for short periods of time.
Happy to clarify is something of the above doesn't make sense to you.
Best and good luck
Hi Amanda,
- Sudden “no maturation” after years usually means a reagent/lot issue — recheck lots of PDL, B27 (try SM1 from STEMCELL Technologies - they screen lots for performance), and add a fresh laminin overlay; rerun with a known-good batch.
- Ensure seeding density is high enough and there’s no serum carryover; add trophic support (e.g., BDNF/NT-3) if neurites stall.
- Rule out silent stressors: mycoplasma, CO₂/pH drift, osmolarity, or harsh media changes in the first 48 h.
- For more reliable adhesion and maturation, DendroTEK dPGA | Protease-Resistant Culture Matrix under a laminin overlay is a stable alternative to PDL/PLO.
Best of luck, Jason Hamlin
Source: I work at DendroTEK Biosciences, and I used to work at STEMCELL Technologies.
www.dendrotek.ca
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