Hello,
I've been making primary neuronal cultures for many years and have troubleshot all kinds of issues. However, I've recently encountered one that after weeks of troubleshooting I am still simply at a loss about how to solve.
I have been making p0 hippocampal neuron cultures for several months. After returning to lab from a short Christmas break, I found that my cultures were no longer surviving past DIV4-8. The neurons look fine after plating with the exception of a bit of debris. The cells grow seemingly normally in the serum media for 24 hours. Ater 24 hours in plating media, I switch 80% of the media to serum-free media and swap 50% of the media out every 3-4 days for fresh serum free media. The Neurons immediately produce additional cellular debris once the neurobasal media is added and start slowly dying. Each media swap seems to exacerbate the issue. Cultures that I track for longer than a week either show no signs of surviving cells, or the reduced number of surviving neurons all migrate together, produce bundles of neurites and no glia can be seen between the neuron bundles. These cultures are not usable for experiments.
My prepration includes:
- Acid-etching coverslips overnight followed by numerous washes with water and baking in an oven for sterility
- Overnight coating of the coverslips with PLL in Trizma followed by multiple washes with water.
- Hippocampi are dissected out, dissociated with papain for 20min at 37C in HBSS.
- Hippocampi are washed 2x with warmed HBSS before being triturated with flamed glass pipettes in 1mL plating media.
- Neurons are counted and then plated at the appropriate density in 24well plates in serum-containing plating media.
- After 24 hours in plating media, 80% of the media is swapped out for serum-free media.
- Every 4 days after plating, 50% of the media is swapped out.
The serum-containing plating media consists of:
MEM
50% Horse Serum
A hair extra glucose
Glutamine (1x)
B-27 (1x)
The growth media contains:
Neurobasal-A
Glutamine (1x)
B-27(1x)
Has anyone experienced something similar? How was it resolved? This issue is beyond my realm of experience and the experience of those in my lab.
I am probably wrong but how old is your glutamine? We have had issues with thawed glutamine at 4C for a few months. Is it passed its expiration date? Has it been siting at 4C for more than a month?
Hi Elizabeth Renee Belland , thanks for the input. Glutamine does spontaneously degrade to ammonia after a time in aqueous solution at 4C, so I think your instincts are right to check the glutamine. In my case I have tried new frozen stocks of glutamine from the manufacturer and tried substituting GlutaMax, which is a more stable dipeptide and unfortunately neither have improved my issue.
Out of ideas except new lot of Horse serum? B27 expired? New lot of PLL? Water source for washing off acid & PLL? I have years of experience with E15 cortical neurons so try: coat with HMW PDL in PBS instead. Plate in the neurobasal +B27 only. Good luck!
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