An Indirect Neuron-Astrocyte Co-Culture Technique for Studying Neuron-Glia Interactions

Begin with a multi-well plate containing a permeable membrane insert coated with poly-D-lysine.

Add astrocyte growth medium to the well and seed astrocytes, a specialized glial cell, into the insert.

Incubate to allow cell adherence on poly-D-lysine via electrostatic interactions and form a monolayer.

Replace the astrocyte medium with a neuron culture medium.

Transfer this insert into a multi-well plate containing adhered primary mouse embryonic neurons over a polymer-coated coverslip.

Incubate to establish an indirect neuron-astrocyte co-culture, where the two cell types are physically separated yet share the same culture medium.

Astrocytes secrete various neurotrophic factors essential for neuron survival and growth.

These factors migrate through the permeable support and interact with primary neurons. This promotes their differentiation and forms neuronal projections.

These projections elongate and establish synaptic connections between neurons, indicating neuron-glial interactions.

Coat each insert with 10 micrograms per milliliter of poly-D-lysine and incubate for one hour. After one hour, wash the inserts twice with PBS.

In the meantime, aspirate the astrocyte medium and wash the culture once with 10 milliliters of PBS to ensure that residual serum is removed. Next, add 3 milliliters of 0.05% trypsin EDTA to the flasks, and incubate the cells for trypsinization for approximately 10 minutes. Afterward, gently resuspend the cells in 7 milliliters of astrocyte medium.

Transfer the cell suspension to a 15-milliliter tube, and centrifuge for five minutes at 216 times g. Then, carefully aspirate the supernatant and resuspend the cell pellet in 1 milliliter of astrocyte medium. Count the cells using a counting chamber. Subsequently, fill the 24-well plate with 500 microliters of astrocyte medium per well. Aspirate the PBS and transfer 25,000 cells in 500 microliters of astrocyte medium into each individual insert, and incubate the culture at 37 degrees Celsius.

To dissect the hippocampi, prepare three 10-centimeter dishes filled with preparation medium and one 2-milliliter tube with 1 milliliter of preparation medium. Dissect the hippocampi from the cortex moieties and collect them in a 2-milliliter tube filled with preparation medium.

It is critical for the hippocampi to be isolated without any adjacent tissue from other regions of the CNS. Therefore, after removing the hippocampus, the foreign contaminating tissues have to be additionally removed.

After dissection, transfer the tube to a sterile laminar flow bench. Remove the preparation medium carefully, and digest the hippocampal tissue with 1 milliliter of digestion solution containing papain. After 15 minutes of digestion, carefully withdraw the digestion solution by gentle suction with a pipette.

Due to the neuron's high sensitivity, it is critical to triturate the hippocampi carefully in order to avoid bubbles and to obtain the optimal trituration intensity.

Wash the hippocampi three times with neuron medium by adding and carefully aspirating 1 milliliter of fresh culture medium per wash cycle. After the final washing step, carefully triturate the tissue in 1 milliliter of neuron medium. Then, count the cells using a counting chamber. Plate 35,000 cells in 500 microliters of neuron medium per well of the 24-well plate, and incubate the neurons at 37 degrees Celsius for one hour.

Now, take the prepared inserts, which have been seeded with confluent astrocyte monolayers out of the incubator. Exchange the medium by aspirating the astrocyte medium and replacing it with 500 microliters of fresh neuron medium.

Then, place the inserts with astrocytes carefully into the wells that contain the neuron cultures using sterile forceps. Subsequently, place the resulting indirect neuron astrocyte co-culture back into the incubator until the experiments.