So our research focuses on improving generation of patient derived organoids for glioblastoma patients. And our key questions here revolve around whether this approach can efficiently produce more organoids, reduce manufacturing time, and at the same time maintain the characteristics of the original tumor. And we would use that for an effective drug and immunotherapy screening in glioblastoma patients.
The recent developments in the glioblastoma field include advancement in understanding the genetic and molecular aspects leading to the recognition of new targets. Additionally, there's a great growing emphasis on using 3D organoids, to better replicate the complexity of tumors and allow drug testing. Currently, technologies like manual dissection, enzymatic dissociation, organ on a chip, and bioprinting are being widely used to prepare organoids.
On the other hand, mechanical chopping provides metabolically active brain slices in various studies, but lacks the metabolic studies for organoid preparation. Our research has introduced an innovative method for generating patient-derived 3D organoids using an automated chopping machine. We demonstrated a nearly 70%reduction in manufacturing time and a significantly higher organoids count compared to the manual dissection.
Offering a more efficient method for drug and immunotherapy screening in glioblastoma patients. The advancement of our study significantly accelerates the organoid production process, and at the same time is able to maintain the main characteristics of the original tumors. And by reducing the time as well as the resources required for organoid generation, our approach enhances the feasibility of large scale drug and immunotherapy screening for glioblastoma patients.
To begin, transfer the tumor tissue immersed in 25 milliliters of hibernate A into a sterilized glass Petri dish under a laminar flow cabinet. Using a microscope carefully eliminate all the necrotic tissue, then use a scalpel and tissue forceps to dissect the blood vessels out. Cut the tumor tissue into one to two cubic centimeter pieces.
Transfer them into a plastic Petri dish containing three milliliters of H-GPSA medium, and place the dish on ice. To manually process the tumor, transfer the tumor piece to a fresh glass Petri dish. Using two scalpels, dissect the segment into 0.5 milliliter sections under a microscope.
To mechanically process the tumor, first, position the blade properly, then adjust the slice thickness to 0.45 to 0.50 millimeters, and fix the table release knob to start mode. Cut the Angros block into a two-centimeter long cylinder and glue it onto the circular plastic dish of the chopper. Using a scalpel, create a pit in the Angros cylinder and place the tumor tissue in the pit.
Then position the plastic disc onto the mounting disc of the cutting table, and press the reset button to start the chopping process. After the first round, rotate the mounting disc by 90 degrees and repeat again to cut the tissue into rectangular pieces. Remove the plastic disc, and then using a tissue spatula rotate only the tumor tissue by 90 degrees.
Return the plastic disc to the cutting table and press reset to continue cutting. Then using a five-milliliter pipette, transfer the processed material along with the medium to the petri dish. Place the dish immediately on ice.
To begin, obtain the tumor tissue from the patients and process it with a chopper. Tilt the Petri dish containing chopped tumor upwards to 45 degrees, and carefully remove 2.5 milliliters of the H-GPSA medium. To wash the tumor pieces, add two milliliters of RBC lysis buffer, and incubate in an orbital shaker at a slow speed.
Then, discard the lysis buffer completely. Next, add four milliliters of patient-derived organoid medium to the tissue pieces, and transfer the tissue chunks to an ultra low attachment six-well plate. Incubate the plate in an orbital shaker for two to four weeks.
Every two days, change half of the spent medium with a prewarmed fresh patient derived organoid medium. To prevent tissue hypoxia, transfer the patient derived organoids to a sterile glass petri dish and observe the tissue morphology under a microscope. Then using a scalpel, cut the adhesive tissue.
Patient derived organoids obtained using the chopper method, reached the desired rounded shape within one week as compared to the manually chopped ones. And the number of organoids formed was significantly higher using this method.
