Fresh Primary Tumor Tissue Processing to Establish Tumor Organoids and Primary Fibroblasts

Begin by placing the tissue in a sterile tissue culture plate and removing excess media. Measure the tissue with a sterile metal ruler and photograph it. Add three milliliters of advanced DMEM/F-12 media to the cut tissue and pipette the tissue up and down to wash it.

Then wash the tissue with three milliliters of PBS. Aspirate the medium from the tissue culture plate and cut it into small pieces using sterile blades and two milliliters of digestion media. Then collect the tissue into a 50-milliliter tube containing five milliliters total of the digestion medium.

Incubate the tube at 37 degrees Celsius for one hour. Periodically mix the suspension by pipetting up and down. Inactivate the trypsin by adding five milliliters of advanced DMEM/F-12 to the tube.

Then filter the sample through a 70-micrometer pore strainer to remove any large debris. Now pipette two milliliters of DMEM containing 10%FBS to the top of the filter, and use a pipette to collect the debris and tissue remnants for fibroblast culturing. Then plate the debris for fibroblast culture.

Centrifuge the filtrate at room temperature at 200 to 300 G for five minutes, and then aspirate the supernatant. Add five milliliters of advanced DMF 12 to the pellet and centrifuge the suspension again at 300 G for five minutes. For red blood cell lysis, resuspend the pellet in four milliliters of ammonium chloride potassium lysis buffer, and transfer it to a 15-milliliter tube.

Incubate the cells at room temperature for two minutes. After centrifuging the mixture at the same conditions as demonstrated previously, aspirate the supernatant. To the pellet, add five milliliters of advanced DMEM/F-12, and centrifuge again at four degrees Celsius.

After aspirating the supernatant, add one milliliter of commercially available cell dissociation reagent to the pellet and incubate it for two to three minutes at room temperature. After incubation, centrifuge and aspirate the supernatant once again. Then resuspend the pellet in five milliliters of advanced DMEM/F-12.

Dissociate cell clumps by pipetting the suspension multiple times with a P1000 pipette. Centrifuge the suspension and remove the supernatant. Next, obtain pre-chilled P1000 and P200 pipette tips, and use the cold tips fixed onto the P1000 pipette to resuspend the cell pellet in the membrane matrix, before placing the Falcon on ice to prevent solidification.

Obtain the preheated plate from the incubator. Using the P200 pipette set at 48 microliters and using cold tips, create basement membrane matrix domes in the preheated plate. Then incubate it to solidify the basement membrane mix.

Once the matrix is solidified, add two to two and a half milliliters of advanced DMEM/F-12, supplemented with the growth factors and inhibitors. Tumor cells were isolated and tumor organoids were established from fresh tissue over a 15-day period. Occasionally, large cell debris volumes prevent accurate visualization of developing tumor organoids.

Developing organoids were observed to phenotypically vary from isolated, rounded to spheroid or aggregate-like cultures, depending on the tumor origin. Organoid cultures may be contaminated by fibroblasts, a common PI product of primary tumor cell culture. After approximately seven days of culture, the fibroblasts migrate from the basement membrane matrix domes and adhere to the cell plates, which may compromise optimal organoid growth.

Fibroblast cultures are established from the tissue debris recovered from the filter. The cells migrate out of the tissue debris and adhere to the culture plates.