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 the molds to the bottom of the plate. After checking the seal between the molds and the well bottoms, collect the dissociated glioma spheres from a glioblastoma cell culture by centrifugation, and resuspend the pellet in one milliliter of cell dissociation enzyme.

After five minutes at room temperature, agitate the tube with gentle tapping and arrest the enzymatic reaction with four milliliters of complete medium. Centrifuge the cells again and resuspend the pellet in one milliliter of fresh complete medium before straining the cells through a 70-micrometer filter. Wash the strainer with an additional four milliliters of complete medium and split the suspension into two aliquots of 8 x 10⁴ cells per centrifuge tube.

Collect the cells by centrifugation, and resuspend one pellet in 80 microliters of freshly prepared polyethylene glycol maleimide, or PEG-Mal-RGD, and one pellet in 80 microliters of freshly prepared PEG-Mal-CYS solution per mold. Using a 200-microliter wide-orifice micropipette tip, add 40 microliters of hyaluronic acid solution into each rubber silicone mold, followed by 40 microliters of either the PEG-Mal-CYS or PEG-Mal-RGD cell solution, quickly pipetting up and down no more than 10 times per mold to mix.

Because of the rapid gelation time, once the two hydrogel components are mixed, it is important to use a wide-orifice pipette tip to mix the solutions together quickly yet thoroughly. This step requires practice.

Then, place the gel-encapsulated cells into a 37 degrees Celsius and 5% CO₂ cell culture incubator for 5 to 10 minutes. At the end of the incubation, add 2 to 2.5 milliliters of culture medium to each gel and use a sterile, two-microliter pipette tip and forceps to gently separate the gels from the sides of the molds. Then, use sterilized forceps to remove the molds from the plate wells and return the plate to the cell incubator.