Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Finding treatments for glioblastoma is challenging because of the cellular diversity that exists in tumors and the interactions between diverse microenvironments. Organoids can help recreate some of that spatial diversity, and we can use this to better understand the biology of the tumors. While traditional cell culture methods tend to produce homogeneous tumor cell populations, organoids can better mimic the molecular and cellular diversity of glioblastoma and better model intratumoral interactions.

Organoids can be used to investigate drug efficacy and mechanisms of resistance. Resistance to clinical therapies may only appear in organoids and not in the traditional sphere culture, reflecting the clinical reality of this disease. Organoids can help demonstrate the regional effects of chemotherapies.

They can be used to discover new niche specific therapies, and they can also act as a predictive tool for personalized medicine in the future. Some materials used to create organoids are temperature sensitive, and neglecting to keep these materials at their proper temperature can lead to difficulties creating or maintaining organoids. Start preparing the organoid molds under a culture hood by taking the wax paper off the parafilm sheet and placing it between two sterile 96-well polymerase chain reaction or PCR plates.

Ensure that the inside of the parafilm is clean. Apply even pressure to the top plate to form small two-millimeter-deep dimples in the parafilm without creating holes. Separate the plates.

The dimpled parafilm will stick to the top plate. Then place the top plate on dry ice for 30 seconds. Use sterile forceps to pull the parafilm off the top plate in a quick motion.

Frozen parafilm is easier to remove. Place the completed parafilm mold in a covered, sterile 10-centimeter cell culture dish. To prepare the single-cell suspension from glioblastoma or GBM adherent culture, remove the used media from the plate.

Then add two milliliters of cell detachment solution at room temperature to the plate, and incubate the plate at 37 degrees for three minutes. After confirming cell detachment from the plate under the microscope, add eight milliliters of Neurobasal Medium complete, or NBMc medium, to neutralize the cell detachment solution. Strain the suspension through a single 70-micron cell strainer.

Spin down the cells at 120 times G for five minutes. Then remove the supernatant and resuspend the cells in one milliliter of NBMc. Count the cells from the single-cell suspension using a cell impermeant stain.

To make organoids from a single-cell suspension, add an appropriate amount of laminin-rich extracellular matrix, or lrECM, into a small centrifuge tube in an ice bucket or cold block. Prepare a cell mixture containing 20, 000 cells per organoid. Mix the lrECM cell suspension mixture thoroughly to avoid settling the cells, which would result in non-uniform organoid formation.

Next, carefully pipette 20 microliters of the mixture onto a parafilm mold to form a pearl-like droplet. Ensure to cool the pipette tip every two to three organoids to prevent lrECM polymerization. Once the desired number of organoids are pipetted onto the parafilm mold in a 10-centimeter culture plate, incubate the organoids at 37 degrees Celsius for one to two hours in a cell culture incubator.

After the organoids are solidified, flush the organoids gently off the parafilm mold with NBMc medium using a P1000 tip and collect them into a new 10-centimeter culture plate containing 20 milliliters of NBMc. Place the culture plate in an incubator without shaking for four days. After four days, replace the media in the plate.

To do this, place a piece of dark paper beneath the cell culture plate. Tilt the plate and wait for 20 seconds. As the organoids completely settle at the bottom of the dish, slowly remove the media with a large-opening glass pipette.

After adding new media, place the plate in the incubator on an orbital shaker at 80 RPM, and exchange media every two to three days thereafter. The growth of the organoid was observed using light microscopy. In the center view, early organoid growth showed migration and single-cell invasion through lrECM.

The mature organoids at seven weeks were relative to the size of a dime. The immunohistochemical staining of the GBM organoids with phospho-histone H3 revealed highly proliferative cells with a brown or purple appearance in the organoid perimeter compared to the core. The cell viability data for organoids in 0.1%DMSO demonstrated intraorganoid and interorganoid consistency.

Pay attention to organoid biomass and media acidification. If the organoids are going through media too quickly, be sure to exchange the media more frequently or split the organoids between additional plates. Mature organoids can be used for experiments with therapies or embedded and sectioned for use with immunohistochemistry or immunofluorescence.

The dissociated organoids can be used for FACS analysis. These organoids were recently used in three-dimensional functional screening to discover targets essential in spatially distinct cancer stem cell niches. We're also using these models to prioritize different drugs and therapies prior to in vivo testing.