In our lab, we focus on the rare type of brain tumors which is called diffuse low-grade gliomas. These gliomas will affect often young adults, and they are characterized by a mutation in the gene called IDH-1. The main advance in the field of gliomas are, first, the use of 3D models, like tumoroid or organoid, second, the use of imaging directly in the brain of mice transplanted with gliomas, and third, the use of special transcriptomic and single-cell RNA.
The main challenge we face when we work on IDH-1 mutant gliomas is the low proliferation of these cells and their difficulty to be maintained in culture, and so that we have derived and define a protocol to keep the cells alive, and for some of them, we have been able to derive a cell line, but not for all patients. By studying IDH-1 mutant cells in vitro, but also in vivo, we have found that these cells are quite plastic, and they can adopt two cells'fate, either being astrocyte-like or oval-like, and we have also found that nudge is playing a major role in this plasticity. With this new explant protocol, we have been able to maintain the IDH-1 mutant cells in vitro for weeks, but even more, for months, and we have shown that there are different type of tumoral cells, as we found in patient.
We have also found that there are some tumoral environment cells, like microglial cells, which make it possible to study interaction between tumor cells and their environment, so these models offer a more accurate representation of the biological diversity found in the tumors. To begin, prepare the workspace and arrange all the sterilized tools. Transfer the tumor resection onto ice to preserve tissue viability for culture.
Examine the tumor resection for size and heterogeneity. Select portions displaying a gray color with a soft or jelly-like texture, as these areas are rich in tumor cells. Transfer the selected tumor samples into a 1.5 milliliter microtube or cryovial and add one milliliter of media.
Using sterile scissors, chop the samples into small pieces measuring one to two cubic millimeters. Next, with sterile scissors, trim the end of a 1, 000 microliter pipette tip to create a wide opening approximately three to four millimeters in diameter. Pipette 100 to 200 microliters of the minced tissue fragments with a cut tip into wells of a plate.
Gently rehomogenize the suspension to ensure uniform distribution, as the smaller fragments tend to settle rapidly. Place the plate in an incubator set at 37 degrees Celsius with 5%carbon dioxide and 100%humidity. Replace the media daily if the tumor's metabolic activity causes rapid yellowing or if the fragment density requires it.
To begin, obtain the minced tumor tissue suspension. For the freezing medium preparation, add two milliliters of DMSO to eight milliliters of cell culture medium. Combine one volume of the minced tumor suspension with an equal volume of the prepared 20%DMSO freezing medium.
Carefully pipette the mixture up and down to ensure thorough blending. Rapidly transfer the cryovials into a cryogenic container to facilitate gradual temperature reduction, then incubate overnight at minus 80 degrees Celsius before transferring to liquid nitrogen for long-term storage. For thawing, remove the cryovials from liquid nitrogen and immediately place them in a 37 degrees Celsius water bath.
Warm them until approximately 80%of the cells have thawed. To remove the DMSO, quickly transfer the suspension into a 15 milliliter centrifuge tube containing five milliliters of prewarmed 1X PBS and centrifuge the tube at 300 G for five minutes. Remove the supernatant and repeat the PBS wash, followed by centrifugation.
Aspirate the supernatant, ensuring the pellet remains undisturbed. Resuspend the pellet in an appropriate amount of medium and transfer it into a precoated PDL Laminin 24-well plate. Incubate the plate at 37 degrees Celsius.
Cells exhibiting elongated, bipolar, or multipolar morphologies were observed growing from tumor explants attached to the well surface after a minimum culture duration of four weeks. Tumor-derived cells exhibited comparable morphology between fresh and cryo-preserved tumors across three cases, as shown by elongated and bipolar appearances forming intricate networks. Radial migration of the tumor-derived cell along the fiber was clearly observed.
Unsuccessful explant cultures showed non-adherent fragments and numerous gitter cells, characterized by autofluorescence and lipid content.
