This method can help answer key questions about glioblastoma tumors and how the extracellular matrix can facilitate treatment resistance. This technique enables modular construction of adaptable 3D cell culture platforms which can be used to culture patient-derived glioblastoma cells in an environment that better approximates native brain. 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 gliomaspheres from a glioblastoma cell culture by centrifugation and re-suspend 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 re-suspend 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 eight times 10 to the fourth cells per centrifuge tube. Collect the cells by centrifugation and re-suspend 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 five to 10 minutes. At the end of the incubation, add two to two and a half 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.
For single cell extraction, at the appropriate experimental endpoint, remove the supernatant from the wells and transfer the gel culture from each well into individual 1.5 milliliter microcentrifuge tubes. Incubate the gels with 500 microliters of cell dissociation enzyme for five minutes at 37 degrees celsius with occasional flicking and transfer the mixtures into individual 50 milliliter centrifuge tubes containing five milliliters of complete medium per tube on ice. Next, use a 10 milliliter syringe equipped with a 20-gauge needle to gently aspirate each cell suspension through the needle tip eight times and filter the mixtures through a 70 micrometer cell strainer into new 50 milliliter centrifuge tubes.
When dissociating cells from the hydrogel, make sure to depress the syringe plunger slowly to avoid sheering which can compromise cell membrane integrity. Wash the strainers with five milliliters of fresh medium and collect the cells by centrifugation. Then, re-suspend the pellets in the appropriate flow cytometry buffer for staining and sorting according to standard protocols.
To cryopreserve the hydrogels for sectioning, remove the supernatant from the plate wells and incubate the gel cultures with two milliliters of 4%paraformaldehyde at four degrees celsius overnight. The next morning, replace the fixative with two milliliters of 5%sucrose in PBS for a one hour incubation at room temperature. At the end of the incubation, replace the 5%sucrose with two milliliters of 20%sucrose in PBS for two 30 minute incubations at room temperature.
After the second 30 minute incubation, refresh the 20%sucrose one more time for a four degrees celsius incubation overnight. The next morning, gently replace the sucrose in PBS solution with 20%sucrose in optimal cutting temperature or OCT medium taking care to cover each gel entirely and place the plate at four degrees celsius for an additional three hours. At the end of the incubation, use a large flat spatula to carefully transfer each gel into the center of an embedding mold and fill the embedding molds with pure OCT to just below the top edges of the molds.
Then, freeze the hydrogel samples in cooled 2-methylbutane and section the hydrogel samples on a cryostat according to standard protocols. Seating 80 microliter hydrogels to a four times 10 to the fourth cells per gel density, consistently results in proliferation rates that are comparable to, or better than, gliomasphere cultures. For example, four days after encapsulation, glioblastoma cells in RGD containing hydrogels exhibit an invasive phenotype while cells cultured in hydrogel using PEG-Mal-CYS demonstrate a spherical morphology.
These robust cultures allow the evaluation of the effects of reagents like soluble cyclo RGD which competitively disrupt interactions of cells with RGD incorporated into hydrogels. Using bioluminescence imaging, relative numbers of viable cells can be observed in hydrogel cultures, for example, during the course of a 12 day erlotinib treatment. Western blot analysis of cleaved poly ADP polymerase indicates that the relative degree of apoptosis in treated CD44 knockdown cells is higher than that observed in wildtype glioblastoma cells cultured in 0.5%hyaluronic acid hydrogels.
In addition, cryosections of hydrogel based 3D cultures preserve extracellular matrix deposited by the cultured cells. For example, allowing analysis of the deposition patterns of type four collagen shifts upon erlotinib treatment. When attempting this procedure, it is important to evaluate the degree of thiolation of each batch of hyaluronic acid.
It is also important to make up fresh precursor solutions for each time you do a cell encapsulation experiment. Development of this technique has paved the way for cancer researchers to evaluate the efficacy of new and existing treatments and to explore the mechanisms of treatment resistance and brain cancer progression, all using an ex vivo model.
