Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

This method provide a new model for modeling human glioblastoma using organoid. And by generating this organoid model, we can use it to define what's the relationship between genotype and phenotype in human GBM, and also use it for personalized treatment regarding drug screen application, for example.

The challenges that we face at the moment is that the current existing models for glioblastoma, like genetically modified mice or patient-derived xenograft, they do not accurately replicate how the tumor genotype influences the molecular phenotype. For example, the connection between the elephant mutation and the features of the glioblastoma have not been shown in these models.

We already made use of this very new model. By analyzing with modelomic approach, we could establish a link between genotype and methylome-proteome, also metabolomic heterogeneity. Furthermore, we already did the one-drug screen using FDA-approved drugs. We could identify drugs which are effective for particular genotypes of human GBM, and this paved the way toward personalized GBM treatment in the future.

[Instructor] To begin, digest the cultured iPSCs with two passaging reagents for four minutes respectively. Centrifuge the cells at 120 x g for five minutes, and then gently pipette to resuspend the cells into a single-cell suspension. After counting the cells, seed 1 x 10 to the power of 4 single cells of iPSCs into each well of a bone marrow Matrigel-coated 48-well plate. Culture for 24 hours in human embryonic cell-qualified culture medium containing 10-micromolar rock inhibitor. Next, infect the cells with lentivirus containing luciferase-expressing plasmids and eight micrograms per milliliter polybrene. After six hours, replace the medium with fresh human embryonic cell-qualified culture medium. After 24 hours, add 150 micrograms per milliliter of luciferase substrate to the cell culture medium. Then using bioluminescence imaging equipment with a five-second exposure, check for bioluminescent imaging signal. Next, add two micrograms per milliliter of puromycin to the culture medium. Incubate for two days, and change the medium every other day for one week to expand the cells. When the cells reach 70% confluency, digest iPSCs with two passaging reagents for four minutes respectively. Centrifuge at 120 x g for five minutes, and gently pipette to resuspend the cells into a single-cell suspension. After counting the cells, seed single cells at a density of 50 cells per 10-centimeter dish for two dishes. Check the bioluminescent imaging signal. Under the microscope, use sterilized 10-microliter pipette tips to pick clones with strong bioluminescent imaging signals. After a kneeling guide RNA oligos, linearize plasmids PX330A1X2 and PX330S2 With the BbsI enzyme. Ligate one of the guide RNAs with either the PX330A1X2 or PX330S2 plasmid using T4 ligase at room temperature for four hours. Then digest the two resulting plasmids with the BsaI enzyme before purifying the products using gel extraction. Ligate the guide RNA scaffolds from the PPX330S2 gRNA plasmid to the linearized PX330A1X2 gRNA plasmid using T4 ligase at room temperature for four hours. Clone the puromycin-resistance gene from the PX459 plasmid into the resulting plasmids by digesting with EcoRI followed by T4 ligation at room temperature for four hours. Centrifuge the digested iPSCs at 120 x g for five minutes, and resuspend them in human embryonic cell-qualified culture medium. After counting the cells and dividing them into several tubes, centrifuge at 120 x g for five minutes. Mix 15 micrograms of the plasmids in 135 microliters of resuspension buffer. Then use 120 microliters of the mixture to resuspend the cell pellet. Electroporate the cells with two pulses at 1,200 volts for 20 milliseconds, and plate the resulting cells in one well of a six-well plate containing human embryonic cell-qualified culture medium with the reagent to facilitate single-cell survival. Add two micrograms per milliliter of puromycin to the culture medium every 12 hours for two days, and then expand the cells for one week before harvest. Now digest the iPSCs having the highest knockout efficiency with two passaging reagents for four minutes respectively. After spinning down the cells for five minutes at 120 x g, resuspend them in human embryonic cell-qualified culture medium. Seed approximately 100 cells in two 10-centimeter dishes containing prewarmed human embryonic cell-qualified culture medium supplemented with a reagent to facilitate single-cell survival. When the single-cell colonies are between three and five millimeters in diameter, use 10-microliter pipette tips under the microscope to pick them and expand them in two wells of 48-well plates. After extracting DNA from one well of the 48-well plate, perform a polymerase chain reaction. after performing the CRISPR-Cas9 knockdown of iPSCs, digest iPSCs with two passaging reagents for four minutes respectively. Centrifuge the cells at 120 x g for five minutes, and resuspend into a single-cell suspension using low bFGF hES media. Once the cells are counted, add an appropriate number of cells to the designated culture environment. Add 50-micromolar rock inhibitor and six nanograms per milliliter bFGF to the low bFGF hES. Seed 150 microliters of cell suspension containing 9,000 cells into each well of a 96-well ultra-low attachment plate. On day three, remove 80 microliters of the old culture medium from each well and add 150 microliters of fresh low bFGF hES medium. When the embryoid bodies begin to brighten and develop smooth edges, replace the old culture medium with 150 to 200 microliters of fresh neural induction medium. Observe the embryoid bodies under the tissue culture microscope and select the brighter ones around the edges. Using a cut 200-microliter wide pipette tip, transfer the selected embryoid bodies to an organoid embedding sheet. After removing the excess medium, drop 30 microliters of bone marrow matrix onto the organoids. Using a 10-microliter pipette tip, position the embryoid bodies in the center of the droplet. Wash off the bone marrow matrix droplets into a six-well plate containing three milliliters of NeuroDMEM-A medium and three micromolar CHIR99021. Place the six-well plate on a shaker set to rotate at 75 RPM in the incubator. Transfer the organoids at the desired developmental age to a 24-well plate containing one milliliter of NeuroDMEM+A medium, placing one organoid per well. Add 150 micrograms per milliliter of D-luciferin to each well before incubating on the shaker for 15 to 30 minutes. On day zero, after incubating the organoids with luciferin and checking the bioluminescence, apply dimethyl sulfoxide or a drug of interest to the organoid culture medium. Once the organoids are treated continuously for six to 10 days, add 100 micromolar bromodeoxyuridine to the medium and incubate the organoids in a carbon dioxide incubator for two hours. LEGOs derived from mutant iPSCs displayed increased expansion over three months compared to wild-type isogenic controls. LEGOs exhibited atypical nuclear features after one month of culture, suggesting potential malignant transformation. Effective drug treatment on LEGOs led to a marked reduction in bioluminescence imaging signal, whereas noneffective treatment showed no significant signal change.