Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The aim of this procedure is to assay the metabolic profile of primary mouse crypt organoids in real time. This is accomplished by first collecting the mouse small intestine, followed by isolating the intestinal crypts Andre bending in matrigel. The second step is to culture the crypts until they form fully grown crypt organoids.

The next step is the preparation of a cartridge assay for metabolic studies. The final step is to load the cartridge and utility plate into the XF 24 extracellular flux analyzer and run extracellular flux analysis to measure the oxygen consumption rate and extracellular acidification rate of K crypt organoids. Ultimately, this procedure allows the study of the metabolic profile of primary mouse intestinal crypt organoids in real time.

The main advantage of this technique over existing methods like using cell culture for metabolics essays is that it allows us to assess intestinal crypto organ metabolism in real time. Organoid cultures mimic make the immune state more accurately, and this helps to generate more physiological relevant data. The technique can also be extended to analyzing other XI 3D organoid cultures.

This method can help answer key questions in the cancer field, such as characterizing metabolic changes in the small intestinal Crips during tumorgenesis, and in diabetes and metabolism research like comparing metabolism of normal and disease states. To begin this procedure, open up the abdomen of a euthanized mouse longitudinally. Fill the small intestine with ice cold calcium and magnesium free PBS and two x antibiotic antimycotic.

Then rapidly isolate the small intestine. Remove the mesenteric fat with a scalpel. Be careful not to perforate the tissue and keep the tissue moist with ice cold PBS during the entire procedure.

Open up the intestine and wash it thoroughly with ice cold PBS. After that, cut the small intestine into two sections. Flatten them out using a wet cotton tip.

Next, gently scrape off the VII using a preco slide. Wash the tissues vigorously in ice cold PBS several times. Then incubate them in 20 milliliters of one XPBS with three millimolar EDTA and 0.5 millimolar DTT for three minutes.

Afterwards, cut the tissues into two to four millimeter pieces on a preco slide. Using a razor blade, transfer the pieces into a 50 milliliter tube containing 20 milliliters of ice cold PBS. Then gently pipette up and down 10 times using a 10 milliliter sterile disposable pipette.

After the tissue fragments settle by gravity, remove the SUP natum with a pipette. Repeat the procedure three more times and make sure the supernatant is clear. Now, transfer the tissue pieces into a 50 milliliter tube containing 20 milliliters of PBS with two milli molar EDTA SW and incubate the mixture at four degrees Celsius for 30 minutes with gentle rocking.

After that, let the tissue settle and discard the supinate. Add 15 milliliters of ice coal PBS and pipes up and down five times. Let the tissue fragments settle and collect the supinate as fraction one.

Repeat the procedures to collect fraction two to fraction five and keep the fractions separate. Next, add 15 milliliters of ice called PBS and shake vigorously by hand for 15 seconds. Collect fraction six and repeat the procedures for fraction seven and fraction eight.

If the tissue pieces start to float, tap the tube to help them settle. Then inspect an aliquot of each fraction for crips under the microscope. This is an example of a bad fraction, and this is an example of a good fraction.

Pull the ones that contain Crips together. After that, pass the pulled fractions through a 70 micron nylon cell strainer and collect the Crips in a 50 milliliter tube. Subsequently, centrifuge the sample at 100 times G for five minutes, four degrees Celsius, discard the supinate and re suspend the PT in 10 milliliter of ice cold PBS with two x antibiotic antimycotic.

Then transfer the Crips to a 15 milliliter tube and repeat the wash one more time. Next, wash the crypts once with 10 milliliters of ice cold, A DF advanced D-M-E-M-F 12 containing two x antibiotic antimycotic. Subsequently wash the crypts once with 10 milliliters of a DF containing one x antibiotic antimycotic.

After that, count the crypts using a hemo cytometer. Adjust the crypt solution volume and transfer it to a 1.5 milliliter tube so that the final crypt concentration when resuspended is 100 to 200 crypts per 20 microliters gelatinous protein mixture. Next, centrifuge the crypt solution at 100 times G for five minutes at four degrees Celsius.

Remove the Senna and resuspend the crypts in the gelatinous protein mixture. Afterwards, plate 20 microliters of the crypt gelatinous protein mixture suspension per well in a 24 well utility plate. Keep the plate in a CO2 incubator at 37 degrees Celsius for 30 minutes until the suspension solidifies.

Then add 500 microliters of ice cold, complete culture media to each well and culture the Crips until they grow into the fully developed organoids In this procedure, hydrate the cartridge overnight in a non-carbon dioxide incubator at 37 degrees Celsius. Then remove the culture medium and wash the organoids twice with 500 microliters of DMEM. After five minutes, add 675 microliters of assay medium to each.

Well then check the crypt organoids microscopic morphology for incubating them in a non-carbon dioxide incubator at 37 degrees Celsius for one hour. Afterwards, prepare 10 micromolar injectable compounds in the assay medium. Set up the cartridge by loading 75 microliters of 10 micromolar injectable compounds into the ports of the cartridge sequentially.

Next, incubate the cartridge for 30 minutes to one hour in a non-carbon dioxide incubator at 37 degrees Celsius. Simultaneously, turn on the XF analyzer and create the assay protocol template. Place the cartridge and utility plate into the XF analyzer and run calibrate cartridge After that, load the cell culture plate into the instrument and run the assay.

Protocol shown here are the crypt organoids derived from an eight month old C 57 black six mouse in two days or 18 days.Culture. This figure shows the metabolic assay using organoids derived from the eight month old C 57 black six mice in which the oxygen consumption rate and the extracellular acidification rate are presented. Crypts are indicated in red, and the control is indicated in blue.

The reagents preloaded in the cartridge were injected sequentially as a oligo mycin, B-F-C-C-P, and C rotenone. This technique paved the way for researchers in the field of cancer and metabolism to explore metabolic changes in patient derived crypt organoids. After watching this video, you should have a good understanding of how to measure intestinal crypt, organoid metabolism in real time.

Thanks for watching and good luck with your experiments.