Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells

Take a microplate with an adherent culture of glial cells derived from a rat brain. The culture contains astrocytes, microglia, and oligodendrocytes.

Remove the medium, incubate with an enzyme solution to detach the cells, and then transfer them into a tube.

Centrifuge and remove the supernatant with cellular debris. Resuspend the cells in a warm medium.

Triturate using a syringe to break up cell clumps and filter using a strainer to obtain a single-cell suspension.

Add a photocrosslinkable biopolymer, crosslinking agents, and extracellular matrix components.

Pour the mixture into a mould attached to a coverslip. Expose the mixture to high-intensity light to induce crosslinking, forming a gel and encapsulating the glial cells.

Remove the mould and place the coverslips in a microplate. Add a warm medium and incubate the gel under physiological conditions.

The cells proliferate within the supporting extracellular matrix and create a cellular network, establishing a three-dimensional culture of glial cells.

24 hours before cell encapsulation, aspirate the medium from two-week primary cultures, and add 1 milliliter of fresh medium to each well. On the day of cell encapsulation, prepare