I went through a basic protocol provided by Brewer et al., 1994
1. The media was stored according to instruction (-4 to -20 for B27, -20 for trypsin and 2-8C for neurobasal and 15C for Glutamax)
2. The hippocampus of mature rat (6W) was extracted under the sterile condition
3. The extracted hippocampus was stored in HANK and Pen/Step for 2 min
4. The hippocampus was homogenized for 15min using trypsin-EDTA 0.05 (using micropipette with blue and yellow tips). i had extrac care about buble formation
5. The trypsin was inactivated and centrifuged, the pellet was re-suspended in 500ul hank+pen
6. The cells was counted (2 * 10E6) and inoculated into the culture media (5ml neurobasal+ 2% b27+glutamax 1%) in a T25 flask (adherent + filter cap)
7. The cells were incubated at 37C and 5% Co2
* No antibiotic in culture medium / even addition of antibiotic dosen't make change. Furthermore, i didnt have contamination
* No serum in the medium
* No bFGF/ even addition of bFGF dosen't make change
Still i have some growth that i don't know what they are. I think they are not typical neural cells. In my though, the problem is that the cells are die within few days.
what went wrong?
Hi
I didnt have any experience with stem cells but I think serum is necessary for any cell culture because cells need it for growth
@Alireza
Hi
Due to undefined composition of the serum (FBS) researcher are using serum-free media to decrease experimental variability.
@Nicolas
Much appreciate for the detail suggestions.
- I am attempting to culture monolayer of differentiated neurons.
- I used treated T25 flask with vent cap (https://goo.gl/nGY2S3). The manufacturer didn't disclose what reagent they have used to treat the surface, this would be source of the problem. Base on your suggestion i am going to coat the flasks with PLL.
- Im curious if i can use FGF instead of serum. FGF has increases the cell number equal to serum (Kitchens et al. 1994) and also may reduces oxidative stress in the cell culture medium(Galderisi et al., 2013)
@Alireza
In my opinion, as long as media composition is important for you, utilization of serum would lead to experimental variability.
@Nicolas,
Much appreciate for your advice.
I have been cultured a fresh cells in a PLL coated T25 flask as you instructed. The shelf life of the cells significantly increased in comparison with previous cultures. But i still have challenge with short life of the cells.
According to your pic, cell shape is circle and it looks like they did not attach to bottom and died. In my opinion, cell number is too low and T25 flask is too big. Use the 8-10 cover slips and 100pi petri dish (which is treated by after alcohol and baking) before spreading neuronal cell. You can easily deal with neuron on coverslip with 12 well plate to perform other experiments. I used to use 8-10 cover slips and 100 pi petri dish with 1 pair of hippocampus of embryonic stage.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining