Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research

The protocols for the culture of canine hepatic and intestinal organoids are important for standardizing this novel technology. This technique allows the reliable and reproducible establishment of the canine organoid culture including the organoid isolation, maintenance, harvesting, and subsequent biobanking of samples. Canine organoids can provide an excellent model for numerous applications spanning from biology, infectious disease development, drug testing and discovery, and also transmission medicine.

To begin, prepare five 15 milliliter centrifuge tubes with five milliliters of 1X complete chelating solution, one tube with three milliliters of 1X complete chelating solution, one empty tube, and one tube with five milliliters of 1X complete chelating solution, and two milliliters of fetal bovine serum. On the day of isolation, place a sterile Petri dish, scalpel, ice bucket, and cold advanced DMEM nutrient mixture F12 in the biosafety cabinet. Place the required number of 24-well cell culture plate in the carbon dioxide incubator to pre-warm.

Place solubilized extracellular membrane matrix on ice for thawing. Pre-chill the centrifuge to four degrees Celsius. Move complete media with growth factors from the freezer to a 37 degree Celsius water bath avoiding direct light exposure.

Shake tissue samples in the transport tube for 30 seconds. Remove excessive supernatant without discarding any tissue by slow pipetting near the fluid surface until 0.5 milliliters is left in the tube. Transfer tissue and remaining supernatant to a sterile Petri dish.

Using a disposable scalpel, cut and mince the tissue into smaller pieces resembling a mash consistency for approximately five minutes. Pipette the minced tissue with liquid from the Petri dish to the first complete chelating solution tube, then add two milliliters of advanced DMEM nutrient mixture F12 to the Petri dish. Flush the remaining tissue and transfer to the first complete chelating solution tube.

Vortex the 1X complete chelating solution tube approximately five times for five seconds. Allow biopsies to settle at the bottom of the tube for one minute and remove the supernatant until five milliliters is left. Transfer the 1X complete chelating solution from the new tube to the sample tube.

Repeat the previous step for the following two tubes. And on the final two washes, remove the supernatant down to three milliliters remaining in the tube. Transfer the biopsies and 1X complete chelating solution from the sample tube to one well of a six-well plate.

Add three milliliters of 1X complete chelating solution to the sample tube. Gently swirl to collect any remaining tissue and transfer to the same well of the plate. Add 150 microliters of 0.5 molar ethylene diamine tetraacetic acid in a well and place the six-well plate on a 20 degree 24 RPM rocker at four degrees Celsius.

Incubate the liver samples for 10 minutes and the intestinal samples for one hour on the moving rocker. Transport the six-well plate back to the biosafety cabinet and transfer the minced tissue and liquid to a tube containing 1X complete chelating solution with fetal bovine serum. Allow the tissues to settle and transport the supernatant with 0.2 milliliters of the upper portion of the tissue to an empty tube.

Spin down the tube containing the sample at 700 XG for five minutes at four degrees Celsius. Stem cells are now pelleted along with the minced tissue. Remove and discard the supernatant carefully to not disturb the pellet.

Resuspend the pellet in advanced DMEM F12. Spin the tube again and aspirate the supernatant without disturbing the pellet. Add the calculated volume of solubilized extracellular membrane matrix to the sample tube.

Be careful when removing media so as not to lose the cell pellet. When adding an extracellular membrane matrix, pipette slowly to not create excessive bubbles in the organoid suspension. Keep the sample with the solubilized extracellular membrane matrix on ice and seed the suspension in the middle of the wells so that the solubilized extracellular membrane matrix forms a dome-like structure.

Transport the plate to an incubator and allow the solubilized extracellular membrane matrix to solidify for 30 minutes. Mix ROCK inhibitor and GSK3 beta in complete media with growth factors and add 500 microliters of this solution to every well. Place the plate in the incubator.

The canine organoid protocol typically generates approximately 50, 000 to 150, 000 intestinal or hepatic cells per well. After stem cell isolation, hepatic canine organoids start their lifecycle as expanding spheroids, and after seven days, they turn into budding and differentiating organoids. The volume, surface area, 2D ellipse area, and circumference of the spheroids were calculated.

2D ellipse area and circumference were used to assess the organoid culture’s health. Assessment of the expression of the markers in a semi-quantitative manner showed that the spheroids preferentially expressed the cholangiocyte marker KRT7 ranging from one to 26%The stem cell marker LGR5 expression ranged between 0.17 to 0.78%while hepatocyte markers were expressed to a lower extent at 0.05 to 0.34%for Forkhead box 1A and 0.03 to 0.28%for cytochrome-P450. A 12-minute incubation with trypsin-like protease did not inhibit organoid growth.

However, the growth of organoids was negatively impacted using a 24-minute incubation. The survivability of canine hepatic organoids in an unfavorable environment was analyzed to determine the volume of organoid media and basement matrix needed for hepatic organoids’successful growth and survival. The survival rate of organoids ranged from 12.9 days to 18 days.

Basic cell culture techniques and aseptic approaches are the essential prerequisites for proper organoid isolation and maintenance.