The overall aim of this procedure is to establish primary neuron colon tumor organoids. This is accomplished by first collecting mouse colon tumor tissue. The second step is to dissociate adjacent normal colon epithelium.
Next, the colon tumor cells are digested into single cells. The final step is to embed colon tumor cells into matrigel and selectively culture them with limited nutrient conditions. Ultimately, light microscopy is used to show the time course of a colon tumor organoid formation.
The main advantage of this technique over existing methods such as transformed colon cancer cell lines, is that organoid cultures closely mimic the in vivo state and generate more physiologically relevant data. After euthanizing the mouse with carbon dioxide and collecting the colon, flush the colon with PBS. Use scissors to open the colon longitudinally, then place it under a stereo microscope and use scissors to remove regions containing tumors.
Collect and wash the tumor tissue with PBS. After washing, transfer the intestinal fragments to EDTA chelation, buffer, and incubate on ice for 60 minutes after chelation, most of the normal intestinal epithelial cells will be detached while tumor cells will remain attached to the mechy. Aspirate the chelation buffer containing normal epithelial cells and wash the remaining tumor fragments.
One more time. With five milliliters of cold chelation buffer. After aspirating the chelation buffer, wash the tumor fragments with five milliliters of cold one XPBS.
After aspirating the one XPBS add digestion buffer to the tissue fragments and incubate for two hours. At 37 degrees Celsius, allow the tumor fragments to settle under normal gravity for one minute and then collect the S supernatant in a 15 milliliter centrifuge tube. Pellet the single cell tumor suspension supinate by centrifuging at 200 times G for three minutes.
Then wash once with five milliliters of PBS and centrifuge. Again, gain resuspend the tumor pellet with 500 microliters of PBS. Then count isolated single tumor cells using a hemo cytometer.
Next, reell the tumor cells by centrifuging at 200 times G for three minutes and resend in five milligrams per milliliter matrigel on ice. Then plate 15, 000 cells in a 50 microliter volume of matrigel per well in a 24 well plate. After allowing the matrigel cell mixture to polymerize for 15 minutes at 37 degrees Celsius, add 500 microliters of basal culture medium containing 50 nanograms per milliliter urine EGF to each well change basal culture medium containing EGF every two days and massage organoids one to five once a week to massage.
After replacing the culture medium with fresh basal medium, use a P 1000 pipette with a cut pipette tip to mechanically disrupt the organoids and matrigel. Transfer the organoid suspension into a 15 milliliter falcon tube. Use a fire polished pasta pipette to further disrupt the organoids.
Wash dissociated organoids with five milliliters of basal culture, medium and centrifuge 200 times G for two minutes. Then discard the supinate, resuspend the pellet with matri gel and plate as previously described to freeze organoids dissociate wash and centrifuge. As before, discard the supinate and resus.
Suspend the pellet in DMEM containing 20%fetal bovine serum and 10%dimethyl sulf oxide. Next, transfer the cell suspension into 1.5 milliliter cryo tubes. Transfer the tubes to a Nalgene Mr.Frosty freezing container and store in a minus 80 degrees Celsius freezer to achieve a cooling rate of minus one degrees Celsius per minute.
After overnight incubation transfer the cells into liquid nitrogen. The cells can be stored in this manner for over two years. For RNA extraction, the cells are collected.
As for passaging, the RNA is isolated using the pico pure RNA isolation kit. According to the manufacturer's instructions for protein extraction, the cell pellet is collected in the usual manner and then lies in 100 microliters of radio immunoprecipitation assay buffer for immunohistochemistry. After aspirating the cell culture medium, use a cut P 1000 pipette to transfer tumor organoids into a cryo mold.
Place the mold on dry ice and add tissue freezing medium to the mold cut frozen sections at seven microns and stain According to previously published protocols, this image shows a representative organoid formation derived from a colon tumor taken from a three month old, a PC min mouse, a short while after plating. This image shows the same organoid one day after plating. Here, the organoid has been in culture for three days.
While this image shows the organoid six days after plating. Here two organoids that have been in culture for 14 days are shown. Note that at this stage, each organoid consists of a cluster of cells.
This histogram shows the successful rate of organoid formation under two different collagenase digestion conditions. These images show organoids derived from a colon tumor taken from a three month old, a PC min mouse with immunofluorescent staining for beta-catenin. DPI was used to stain nuclei.
After watching this video, you should have a good understanding of how to establish primary urine colon tumor OID by enzyme digestion.
