Primary cortical and hippocampal neuron culture

Neuroscience, as an important discipline, studying the functions and structures of neurons is of great significance for understanding the working mechanism of the human brain and treating neurodegenerative diseases.

Primary cortical and hippocampal neuron culture experiments are a common experimental method in neuroscience research. By culturing neurons, we can study their behavior and functions under different physiological and pathological states. This time, we will introduce in detail the whole process of primary cortical and hippocampal neuron culture experiments, from the acquisition, culture, observation and other aspects of neurons, to provide practical experimental guidance and reference for neuroscientific researchers.

1. Preparation before culture:

• High-pressure sterilization: instrument package, pipette tip box (1mL, 200ul, 10ul), gauze, ultra-pure water. Sterilize in advance with high pressure, and try to use the sterilized items within 1 week.

• Cell plate coating: Coat the cell plate with 0.1mg/mL poly-L-lysine (Catalog number: sigma: P1024) 1-2 days in advance, rinse the cell plate with ultra-pure water 3 times the next day, and dry in the incubator for standby use.

• Cell culture medium preparation:

DMEM with FBS:

DMEM 40mL + F-12 5mL + FBS 5mL + double antibody 500ul

Neurobasal: Neurobasal 48.5mL + B27 1mL + glutamine 250ul (0.5mM)

2. Animals: Purchase pregnant mice (16-18 days pregnant) or 1-3 day old newborn mice.

3. Ultraviolet disinfection workstation, previously sterilized instrument package, pipette tip box, gauze, ultra-pure water, etc. Turn on the ultraviolet in the workstation and cell room at the same time for 30 minutes, then ventilate for 10 minutes.

4. Take the fetal mice: Disinfect the workstation with alcohol, anesthetize the pregnant mouse with gas, cut the lower abdomen of the pregnant mouse in a “Y” shape, take out the intact uterus, avoid uterine rupture. Put the intact uterus into a clean container (10 cm cell dish), disinfect the outside of the container with alcohol, and then transfer it to the cell room.

5. Take the fetal mouse brain: Take out all the fetal mouse brains, put them into a 10 cm dish filled with DMEM, and place the 10 cm dish on an ice box (for newborn mice, soak in alcohol and then take out the brain directly).

6. Under the microscope, separate and extract the hippocampus or cortical tissue: Take a fetal mouse brain and place it in a 35mm small dish containing DMEM. Place the fetal mouse brain with the dorsal side facing up, use tweezers to separate the left and right sides along the midline，fix the fetal mouse brain with tweezers, use another pair of tweezers to pull the cortex outward, expose the hippocampus, strip the vascular membrane (try to strip it clean to effectively reduce the interference of fibroblasts), and separate the hippocampal segment. Place the separated hippocampal segment in a 35mm small dish, the small dish contains DMEM, and place the small dish on an ice box.

7. Digest the hippocampal tissue with trypsin. Transfer the small dish containing all the hippocampal tissue to the super clean workstation for operation, place it on an ice box for operation, lay the small dish on its side, use a 1mL pipette to suck clean the DMEM in the dish, then use ophthalmic scissors to cut the hippocampus into pieces, about 1mm3 in size, add an appropriate amount of trypsin (generally, for the fetal mouse hippocampus taken out from one pregnant mouse, use 2mL of trypsin), properly shake and disperse the hippocampal tissue pieces, and put them in the 37°C incubator as soon as possible, digest for 15 minutes. If there is more tissue, appropriately extend the digestion time.

8. Terminate the digestion. Take the small dish back to the ice box on the workstation, add 2mL DMEM to terminate the digestion, and transfer all the liquid in the dish to a 15mL centrifuge tube.

9. Centrifugation. Use a 1mL pipette to mix and blow the hippocampal tissue suspension evenly, centrifuge at 1500rpm for 3 minutes, and use another 15mL centrifuge tube to hold 4mL of waste liquid for balancing.

10. Prepare the cell suspension: Take out the centrifuge tube after centrifugation, discard the supernatant, leave the precipitate, add 1mL DMEM, blow evenly with a 1mL pipette tip, blow about 10-15 times, let it stand for 1 minute, then take the supernatant to a new 15mL centrifuge tube, repeat this step 2 times, and obtain 3-4mL of cell suspension.

11. Plate seeding: Prepare the appropriate plating cell suspension with DMEM containing FBS, seed the plates. The plating density for the 6-well plate is 7.5 X 10^5 cells/mL, and for the 12-well plate, it is 4 X 10^5 cells/mL. Place them in a 37℃ incubator for culture.

12. Change the medium 4-6 hours later, replace all the DMEM containing FBS with Neurobasal.

13. Cell medium change. Generally change the medium every 2 days, it is recommended to treat with Ara-C on the 2nd day. After 24 hours, change the medium fully with Neurobasal, and perform OGD or other subsequent treatments on the 7th-8th day.

14. Neuronal purity can be verified with MAP2, as shown in the following figure:

Brain tissue dissociation is not only manually enzymatic, but also RWD single cell suspension dissociator fully automatic dissociation of neural tissue helps users with neural research. It can gently, quickly, and efficiently prepare mouse brain tissue into a single-cell suspension while retaining important surface antigen epitopes of the cells. The obtained single-cell suspension can continue to be used for primary cell culture or cell sorting and other downstream experimental applications.