The overall goal of this method is to isolate viable mucosal myofibroblasts and fibroblasts from frozen gastrointestinal tissue samples, originally obtained from biopsies or surgical specimens. The isolation of myofibroblasts and fibroblasts from properly frozen gastrointestinal tissue allows processing of fresh samples to be delayed. This facilitates the use of samples from different sources worldwide.
If the tissue was preserved in freeze medium, place the cryovial in a 37 degree Celsius water bath until the tissue and medium are thawed. For four to five two square millimeter tissue fragments, add 10 milliliters of HBSS without calcium and magnesium, tightly close the lid, and wash the tissue by gently inverting the tube one time. Once the tissue has settled by gravity, carefully discard the supernatant, and wash the tissues again.
Then, add 10 millilites of collagenase solution to the tube, and transfer the tissue suspension into a sterile DNase/RNase free tube with built-in rotors. Tightly close the rotor tube and attach it upside down onto the sleeve of a dissociator. Then, run the preset H-tumor zero one program.
At the termination of the program, incubate the tissue slurry for 45 minutes at 37 degrees Celsius, under continuous 140 RPM rotation on a shaker. Then, return the tube to the dissociator, and run the H-tumor zero two program. After the second dissociation, add 50 microliters of DNA stock solution to the cell solution for 30 minutes at 37 degrees Celsius, under continuous 60 RPM rotation on the shaker.
At the end of the second incubation, run the H-tumor zero three program. Now, filter the suspension through a sterile 70 micron cell strainer, into a 50 milliliter conical tube for centrifugation. Discard the supernatant, and wash the cell pellet in 25 milliliters of HBSS without calcium and magnesium two times, counting the cells after the first wash.
For myofibroblast primary culture generation, resuspend the pellet in up to 4 x 10 to the 6th cells per three milliliters of medium, and seed them in 6 well cell culture-treated plates. Grow the cells at 37 degrees Celsius, and five percent carbon dioxide, changing the medium every two to three days until the formation of an 80 percent confluent myofibroblast monolayer. The cells are then ready to be passaged in T25 culture flasks.
To analyze or sort the isolated myofibroblasts by flow cytometry, plate up to 2 x 10 to the 6th cells per two milliliters of fibroblast isolation medium, in a 24 well low binding plate for overnight incubation at 37 degrees Celsius and five percent carbon dioxide. Using this enzymatic digestion protocol, myofibroblasts and fibroblasts got CD-90 positive mucosal stromal cell populations, can be consistently isolated and grown from gastrointestinal surgical specimens and biopsies. While there is a slight decrease in the viability of the mucosal single cell preparation after freezing the surgical specimens, there is an increase in the efficiency of the myofibroblast and fibroblast culture generation due to a concomitant decrease in the bacterial and fungal concentration.
After two passages, the myofibroblasts and fibroblasts are positive for CD90, the mesenchymal marker lineage marker, Vimentin, and smooth muscle actin, a marker of differentiated mesenchymal cells, as determined by both confocal microscopy and flow cytometric analysis. Once mastered, if performed properly, this technique can be completed in approximately two hours. While attempting this procedure, it is important to remember to allow the necessary time for the tissue to be fully enzymatically digested.
Following this procedure, other methods, like flow cytometry, immunocytochemistry, and various functional assays may be performed. They may be used to determine the contribution of myofibroblasts and fibroblasts to gut mucosal homeostasis as well as to the pathology of acute and chronic inflammatory gastrointestinal disease. After watching this video, you should have a good understanding of how to isolate myofibroblasts and fibroblasts from frozen gastrointestinal tissue.
Don't forget, unfixed human tissue can be contaminated by pathogenic microorganisms. Universal precautions while working with human biological samples should always be taken while performing this procedure.
