This method is significant because patient-derived organoids provide a rapid and powerful tool to study urological cancers as they frequently mimic the genetic and phenotypic heterogeneity of this multispectrum disease. Organoids can be isolated from a limited amount of patient biopsy material and can be rapidly expanded for downstream analysis. The organoids generated using this protocol can be used both as models to help us understand the cellular and molecular basis of urological cancers and as precision medicine tools to let us predict which treatments and individual patients cancer might respond to.
To begin, collect a fresh macroscopically viable tumor specimen from surgery and ensure that the sample is submerged in transport medium in a sterile 50 milliliter conical tube or urine specimen jar during transit. Record specimen details including tissue weight, sample description, and details regarding any blood and urine samples on the patient specimen processing sheet. Carefully replace the transport medium with 10 milliliters of basal media and allow the tumor tissue to settle by gravity.
Next, using forceps, remove the tumor tissue and place it in a sterile 90 millimeter Petri dish. Record the weight of the tissue in grams or milligrams on the clinical specimen processing sheet and dissection grid. Then using sterile forceps in a disposable scalpel blade mounted to a scalpel handle, remove the non-cancerous tissue and macroscopically visible necrotic regions.
Wash the tumor pieces once or twice with cold DPBS, then collect and transfer them to a new sterile 90 millimeter Petri dish. Take a photograph, draw a tissue diagram, and plan the tissue dissection on a clinical processing grid. For histopathological analysis, place approximately 50 milligrams of the tumor tissue into a labeled disposable plastic histology cassette.
Then submerge the histology cassette into a container with 5X to 10X volume of 10%neutral buffered formalin and incubate overnight. The following day, replace the formalin with 70%ethanol for storage at four degrees Celsius until the tissue can be processed. For molecular analysis, snap freeze at least one tumor piece in an RNase, DNase free 1.5 milliliter cryovial using liquid nitrogen and store at minus 80 degrees Celsius.
Next, dispense five milliliters of organoid medium in the 90 millimeter Petri dish containing the remaining tumor pieces and mechanically mince the tissue as fine as possible. using a sterile number 10 scalpel blade. Transfer the finely minced tissue to a 50 milliliter conical tube and add four milliliters of organoid medium, one milliliter of 10X collagenase/hyaluronidase and 0.1 milligram per milliliter DNase I to avoid cell clumping.
Incubate the minced tumor tissue in enzyme solution for one to two hours on an orbital shaker or rotator in an incubator to dissociate fragments into a cell suspension and break down collagens. Then stop the digestion by adding twice the volume of basal medium to the sample. Centrifuge the sample, aspirate, and discard the supernatant.
Next, to lyse contaminating red blood cells, resuspend the pellet in five milliliters of ammonium chloride potassium buffer and incubate the tube at room temperature for three minutes or until the complete lysis of the red blood cells is seen. Then add 20 milliliters of basal medium into the tube. Centrifuge again and aspirate the supernatant.
At this step, place a 10 milliliter aliquot of 2X and 1X organoid medium in a 37 degree Celsius water bath to warm. Filter the sample first through a pre-wet reversible 100 micron strainer into a new 50 milliliter tube to remove large insoluble material. Then filter the elute through a pre-wet reversible 37 micron strainer to collect single cells in small clusters for single cell and immune cell isolation.
Next, reverse the 37 micron strainer and add 10 milliliters of basal media to collect small and moderate-sized clusters. Top up each of these new 50 milliliter tubes with DPBS to 40 milliliters, then centrifuge the suspension and discard the supernatant. Next, add 10 milliliters of basal media to the tube containing the single cells and count the cells using trypan blue exclusion dye in an automated cell counter.
Determine the cell number and viability of cells. Centrifuge the remaining sample and replace the medium with cell freezing solution or basal media containing 10%fetal bovine serum 1%penicillin and streptomycin and 10%DMSO, then place the samples in 1.5 milliliter cryovials, store them in a self-freezing container, and immediately transfer the containers to a minus 80 degree Celsius freezer. The next day, transfer the cryovials into cryogenic liquid or air phase storage for long-term storage.
Next, resuspend the collected small and moderate-sized clusters with 500 microliters of pre-warmed 2X organoid medium. Then with ice cold P-1000 sterile filter pipette tips, add BME to the cells and mix gently. Quickly and carefully pipette 100 microliters of the reconstituted cells BME mixture to wells of an ultra low attachment, flat-bottom 96-well plate, and place the plate in an incubator for 20 to 30 minutes to solidify.
After incubation add organoid medium on top of the BME cell suspension in a one to two ratio depending on the empirically assessed volume, and place the plate in an incubator for 20 minutes to equilibrate. After incubation, remove the plate from the incubator and assemble it on a specimen holder on the stage of a microscope to assess organoids visually under phase contrast or bright field settings. Top up the medium every two to three days using 50 microliters of pre-warmed organoid medium to replenish depleted growth factors and overall volume.
Acquire the time-lapse series images on days 1, 2, and 3, and 5, 7, and 10 before passaging. Two hour digestion of bladder cancer tissue with commercially available collagenase/hyaluronidase and DNase I leads to sufficient digestion of 0.5 to 1 cubic millimeter pieces of more complex cystectomy biopsy tissue, including the neoplastic cells within the lamina propria and muscular layers of the bladder. Larger post-digestion fragments are suitable for culture and standard tissue culture plates of any size to isolate novel two dimensional cultures.
Organoids generated by this procedure undergo dynamic self-assembly, including phases of aggregation, compaction, and final formation of tight cellular structures. Histologically, these structures recapitulate the original patient tumor. Organoids generated in this procedure are suitable for many purposes, including further histopathological characterization, biobanking, and drug efficacy testing.
Following generation of organoids, additional methods can be used to profile these tumors, including the role of anti-cancer therapies, metabolic profiling, or transcription profiling. Within this 3D framework, these additional methodologies provide powerful insight into bladder cancer biology. This method was an important foundation for studies investigating immunooncology and cellular metabolism.
