A Hyaluronic Acid-Based Hydrogel for 3-Dimensional Culture of Glioblastoma Cells

Begin with gliomaspheres, cell aggregates derived from a brain tumor or glioblastoma.

Add dissociation enzymes. Incubate to separate the glioblastoma cells and obtain a single-cell suspension.

Next, introduce a serum-enriched culture medium to stop the enzyme activity.

Centrifuge and remove the supernatant, then resuspend the cells.

Filter the suspension, then centrifuge and remove the supernatant.

Resuspend the cells in hydrogel precursors that contain small peptides.

Load this mixture into wells with a pre-assembled silicone mold containing hyaluronic acid.

Mix the solution. Incubate to allow the interaction between the hyaluronic acid and hydrogel precursors, forming a hydrogel that facilitates cell entrapment.

Overlay the hydrogel with culture media to sustain cell survival.

After disassembling the mold, incubate the hydrogel.

Within the hydrogel, the adhesion proteins on cells interact with the precursor peptides and hyaluronic acid.

This causes cells to form small clusters and develop a three-dimensional culture.

To begin, place clean, dry silicone rubber molds into each well of a non tissue culture-treated 12-well plate, and use the clean blunt end of a pipette tip to adhere