This article describes a method for isolating and culturing hippocampal neurons from postnatal mouse pups. The process involves enzymatic digestion and mechanical dissociation to obtain a single-cell suspension, which is then cultured in a supportive environment.
Take a harvested mouse pup hippocampus in a buffer.
Cut the hippocampus into smaller fragments and transfer them to a conical tube.
Wash the tissue with buffer. Once the fragments settle, remove the buffer. Now, add fresh buffer and trypsin.
During incubation, trypsin, a proteolytic enzyme, digests the tissue's extracellular matrix, loosening the hippocampal neurons.
Wash the digested tissue with buffer.
Once the fragments settle, remove the buffer. Add fresh buffer and pipette the tissue repeatedly to mechanically dissociate it, obtaining a single-cell suspension.
Once the undigested tissue settles, transfer the cell suspension onto poly-L-lysine-coated coverslips with wax supports within a culture plate containing media.
The cells attach to the poly-L-lysine-coated surfaces through electrostatic interactions.
Now, place the neuron-adhered coverslips facing down into a dish containing cortical glial or non-neuronal cells in serum-free media.
In culture, wax supports prevent direct contact between neurons and glial cells, creating a microenvironment where glial-cell-secreted factors promote neuronal survival and maturation.
To begin, dissect six to eight hippocampi from postnatal, one-day-old pups with HBSS medium in a Petri dish. Chop the hippocampi with dissection scissors into smaller pieces, and transfer them from the dish to a 15-milliliter tube. Wash the hippocampi twice with 5 milliliters of HBSS, and leave them in 4.5 milliliters of HBSS after the wash.
Add 0.5 milliliters of 2.5% trypsin into 4.5 milliliters of HBSS, and incubate in a 37-degree Celsius water bath for 15 minutes, inverting the tube every 5 minutes. Well-digested hippocampi should become sticky and form a cluster. If needed, extend the digestion for 5 more minutes.
Wash hippocampi with HBSS three times for 5 minutes per wash. After the wash, add 2 milliliters of HBSS, and pipette the hippocampi up and down with a Pasteur pipette 15 times. Triturate the tissue with a fire-polished Pasteur pipette 10 times, and rest the tube for five minutes until all chunks set to the bottom. Repeat the trituration with the remaining chunks until most of them have disappeared.
Then, use a 1-milliliter pipette tip to gently transfer the supernatant containing the dissociated neurons to plating dishes. Add it directly to the pre-incubated plating medium and shake the plate gently. 2 to 4 hours after seeding, check the plating dishes with a light microscope. The majority of the neurons should have attached to the coverslip.
Using a fine-tipped forceps, flip the coverslips to the glial cell feeder dishes containing pre-conditioned neuronal culture medium with the wax dot side facing downwards. Neurons can grow in the glia cell feeder dishes for up to one month. Feed neurons every seven days with 1 milliliter of fresh neuronal culture medium.
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