Primary neuronal cell culture is an ideal model system to study dissociated neurons in vitro culture. The advantage of using this method is being able to maintain the cellular features of dissociated cells, such as mechanisms, functions, and morphology in vitro conditions for a few weeks as close as possible to in vivo condition. Put the round cover slips in a 24-well plate under the biosafety cabinet.
Add 90 microliters of poly-L-ornithine onto each cover slip. Close the cap and gently place the plate in the 37 degree incubator for two days. Prepare the culture medium and seeding medium and keep them at four degrees Celsius.
Prepare the working trypsin solution in DMEM/F12 and keep it at four degrees. Place the culture medium and seeding medium in the incubator at 37 degrees. Take the 24-well plate out of the incubator.
Wash the cover slips three times with double distilled water. Leave the cover slips for at least two hours to completely dry. Prepare three Petri dishes filled with cold PBS, three Petri dishes filled with cold HBSS, and five Petri dishes filled with cold dissection medium on ice.
Excise the uterine horns and wash them in cold PBS three times on ice. From here all steps should be carried out on ice to minimize tissue damage. After the last washing step, separate the pups from the uterus in HBSS and transfer them to the cold dissection medium.
In dissection medium, decapitate the pups with scissors. Open the calvarium from the foramen magnum to the inferior border of the orbital cavity. Using a pair of fine forceps, remove the skull base and peel the skull away from the brain.
Carefully remove the meninges of the cerebellum starting from the lateral surface of the middle cerebellar peduncle and pons. Cut both cerebellar peduncles and separate the cerebellum from the rest of the brain. Immediately place the collected cerebella in a sterile 15-mL conical tube filled with DMEM/F12 on ice.
Centrifuge the tube at 1, 000 g at four degrees for one minute in DMEM/F12. Repeat this three times, each time gently removing the supernatant with a pipette and re-suspending the pellet in fresh DMEM/F12. Add two milliliters of pre-warmed trypsin and gently pipette to mix them together.
Place the tube in a 37 degree water bath for 12 minutes. After incubation, bring the tube to the safety cabinet and add 10 mLs of DMEM/F12 to inactivate the trypsin. Centrifuge the mixture at 1, 200 g's for five minutes.
Discard the supernatant and resuspend in fresh medium followed by centrifugation three times. Pre-wet a sterile plastic pipette with DMEM/F2. Add 3.5 milliliters of DNAse working solution to the same tube.
Triturate the tissue with a pipette several times until the fluid attains a homogeneous milky color. Add 10 mL of cold DMEM/F12 to the mixture and proceed to the centrifuge. Centrifuge the sample at 1, 200 g at four degrees for five minutes.
Carefully remove the supernatant without disturbing the pellet. Remove the seeding medium from the incubator. Add 500 microliters of prewarmed seeding medium and mix it well using the pipette.
Count the cells using a hemocytometer. Dilute the cell suspension with seeding medium to a density of 500, 000 cells per milliliter. Add 90 microliters of the mixture to each well at the center of the coverslip.
Place the plate in the incubator at 37 degrees for three to four hours. After incubation, add 500 microliters of prewarmed culture medium into each well. Replace the plate into the incubator at 37 degrees.
After seven days, replace the old medium with fresh culture medium II.Prepare a separate 24-well plate with corresponding numeric organization and add 100 microliters of PFA 4%to each well. After the desired days in culture, gently remove the cover slips from the wells of the original culture plate and place them into the corresponding wells of the PFA plate described in the previous step. Add PFA to the wells to completely immerse the cover slips.
Keep the PFA plate at four degrees for 30 to 120 minutes. After incubation, restore the plate to room temperature. Wash the cover slips gently with PBS for five minutes three times.
Figure two in the text shows cerebellar cell culture starting at E18. Immunofluorescence for calbindin shows Purkinje neurons with axonal extensions by three days in vitro. By seven to 10 days in vitro, dendritic processes are evident.
At 21 days, complex dendritic branches are present. Figure three in the text shows cerebellar cell cultures starting at various embryonic days. immunofluorescence detection of calbindin shows Purkinje neurons with early axons only after 18 days in vitro.
Purkinje neuron cultures started at E14 and E15 will develop dendrites, but never as complex as those that develop when E18 is the starting point. Figure four in the text shows that different cell types develop from E18 cultures after 21 days in vitro. Calbindin green immunofluorescence shows Purkinje neurons.
Red immunofluorescence for parvalbumin in B and C show inhibitory interneurons. Red immunofluorescence for sodium voltage-gated channel in E and F shows granule neurons. The present protocol is cost-effective and easy to conduct.
At the end of this video, you will be able to collect and culture cerebellar neurons and fix them at desired DIVs.
