What are some things that can go wrong with a primary neuronal cell culture? Pro

Question

What are some things that can go wrong with a primary neuronal cell culture?

Protocol:

1. Treat dishes with fibronectin or collagen, allow to dry, and sterilize for 10 min under UV

2.Dissect tissue and put it in a 15ml vial with 1ml of HBSS (we dissected the cerebellum from fetal mouse and cerebellum from a newborn mouse)

3.Wash tissue twice with HBSS (WE FORGOT TO DO THIS)

4. Add 2 ml of Cell dissociation media and incubate for 15 min at 37 degrees celcius

5. Pipette up and down. If there's still chunks incubate at 37 degrees celcius an extra 5 min

6. Add at least 2 ml of Complete Growth Media

7. Plate cells

Explanation / Answer

The possible problems associated with this protocol are as discussed below:

Step 2: Tissue dissection should be highly careful. The meninges should be carefully and slowly removed and maximum tissue should be excised at a single event to avoid any mechanical damage to neurons.

Step 3: The tissue should be washed atleast once with HBSS. However, not washing the tissue with HBSS will not impair neuronal growth but can cause contamination of other cells.

Step 4: The volume of cell dissociation media seems to be less. Secondarily, simple sedentary incubation will not yield sufficient cells. Incubation should be carried out on a shaker with temperature controller. The media should be changed every 2-3 minutes and the cells dissociated in it should be collected by centrifugation. This will help in enrichment of the culture.

Step 5: Pipetting can rupture the cells due to mechanical shear. This step should be very slow and smooth.

Step 6: Direct plating of cells is not recommended. Only a counted number of cells i.e. 10,000 cells/cm square should be plated for good culture growth.

Thus, these troubleshoots should be taken care of while performing primary neuronal culture.

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