The overall goal of this procedure is to generate human enters from intestinal tissues. This is accomplished by first dissecting the intestinal mucosa from the underlying submucosal tissue. The second step is to incubate the mucosa with EDTA to promote the dissociation of the epithelium from the connective tissue.
Next, the intestinal crypts are separated from the dissociated epithelium. Finally, the intestinal crypts are cultured in basement membrane matrix. Ultimately, immunohistochemistry techniques are used to show the epithelial organization as well as specific epithelial markers of the cultured OIDs.
The implications of this method is the fact that we can now generate OIDs from patient-specific biopsies, which means that we now can use that tissue in the lab to look at intestinal diseases and diagnoses, which before now we've never been able to do. After preparing the buffers and reagents, according to the text protocol, thaw the basement membrane on ice and pre incubate a 24 well plate in a carbon dioxide incubator at 37 degrees Celsius when the tissue has been received. Use KO's PBS without calcium and magnesium or DPBS to wash the tissue until the DPBS is clear.
Place the tissue in a silicone coated glass Petri dish filled with ice cold DPBS with the mucosal side facing up and with minuchin pins secure the tissue by stretching and pinning it. Next, under a dissecting microscope, use micro dissecting scissors and fine point curved forceps to remove the underlying mucosa from the submucosa and connective tissue. Then with the mucosal side facing up, stretch and pin the dissected mucosa flat on the silicone coated glass Petri dish.
Using curved forceps, gently scrape the surface of the mucosa. This step is necessary to improve the quality of the preparation. Cover the mucosa with freshly prepared two millimolar EDTA chelation buffer.
Then after washing the tissue on ice, three to four times according to the text protocol under a dissecting microscope, use curved forceps to gently scrape the mucosa to release intestinal crypts with a pipette. Gently remove the crypt suspension from the Petri dish and transfer it into a 50 milliliter conical tube. Then check the tissue to make sure that almost all the crypts have been removed from the mucosa.
Filter the crypt suspension through a 150 micrometer filter two times and use a microscope to check the flow through After washing the crypt suspension and transferring the number of crypts needed to a five milliliter round bottom tube, according to the text protocol, centrifuge the crypt fraction for 10 minutes at 150 Gs and four degrees Celsius. Then remove the supernatant using pre chilled pipette tips and basement membrane matrix Resuspend. The crypt pellet.
Apply 50 microliters of crypt suspension in basement membrane matrix per well to the prewarm plate by slowly ejecting the basement membrane matrix in the center of the, well. Incubate the plate in a 37 degree Celsius 5%carbon dioxide incubator for 30 minutes to allow a complete polymerization of the basement membrane matrix. Then overlay the basement membrane matrix with 500 microliters of complete human mini gut medium, supplemented with 2.5 micromolar each of CHIR 9 0 2 2 1.
And Thien incubate the tissue changing the medium after two days, then every other day thereafter. This figure shows a typical example of freshly isolated crypts from whole tissue. The number of crypts isolated from a biopsy is smaller than in whole tissue.
Two biopsy bites are usually performed on a single pass. Each biopsy bite results in a 10 square millimeter surface with an average of 50 to 100 crips per biopsy. After culture and basement membrane matrix for five to six days, the Crips round up to form entero spheres for small intestine and colono spheres for colon and are passaged After seven days.
The enters or OIDs established from biopsies shown here undergo the same development at a lower density at seeding. Therefore, the packaging is usually done after 10 to 12 days in culture. As shown here, both OIDs and OIDs present a luminal side and are lined with an epithelium.
Proliferative cells can be observed within the OIDs and are located within the bud tips. Confocal imaging of OIDs stained with e catrin here in green shows the epithelial cells. This figure shows enters growing from a patient with cystic fibrosis and a tufting enteropathy due to a congenital mutation in the epithelial cell adhesion molecule gene.
Following this procedure or the method like real time PCR or Western blood can be used to answer additional question regarding a gene or prob of interest. After watching this video, you should have a good understanding how to consistently isolate human Crips to be able to efficiently generate enters in your lab to answer your patient specific questions.
