Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

Our protocol for isolating intestinal mesenchyme results in a high yield of telocytes. This method offers an initial platform to study cell-cell interaction involving telocytes in homeostasis and disease. Telocytes are unique cells sensitive to available tissue dissociation protocols.

Therefore, the main advantage of this technique is the isolation of mesenchyme, including enriched viable telocytes. Mesenchyme, including telocyte, can serve as a source for signaling molecules and growth factors required for organized growth ex vivo. To begin, wash the intestine, separated from a euthanized mouse, in a Petri dish containing cold, sterile PBS.

Place the intestine in a Petri dish. And using ball-tip scissors, open the intestinal tube longitudinally, and wash out the feces. Transfer the intestine into a new dish containing fresh, cold PBS.

After washing the intestine one more time, cut the small intestine into one-centimeter-long segments, and transfer them into a 15-milliliter conical tube filled with eight milliliters of PBS. Shake the tube manually at one or two cycles per second for one minute. Pour the solution into a Petri dish.

And using forceps, transfer the segment into a 50-milliliter conical tube filled with 20 milliliters of Solution A.Place the tubes in an orbital shaker incubator at 37 degrees Celsius for 20 minutes. After incubation, shake the tube vigorously by hand at four or five cycles per second for one minute to dissociate the epithelium. Repeat this step once more.

After cutting and washing the fragments as demonstrated previously, transfer them into a new 50-milliliter tube filled with 10 milliliters of sterile PBS, and invert the tube at one or two cycles per second for one minute. Pour the solution in a Petri dish. Transfer the segments into a new 15-milliliter tube filled with 10 milliliters of sterile PBS, and tilt up and down gently at one or two cycles per second for two minutes.

Under a biosafety cabinet, use forceps to place the segments on a sterile laboratory wipe to dry them. Once dried, cut the segments further into pieces. Transfer the cut pieces using forceps into a six-well plate filled with four milliliters of pre-warmed digestion solution per well.

Incubate the plate at 37 degrees Celsius for 50 minutes, and gently shake the plate manually every 20 minutes. Then, transfer the pieces using a Pasteur pipette into a 15-milliliter conical tube filled with four milliliters of DMEM. Shake the tube manually at four or five cycles per second for one minute to get a single cell suspension.

Filter the suspension through a 100-micrometer strainer into a 50-milliliter conical tube. Centrifuge the filtrate at 700 g for five minutes at four degrees Celsius. Discard the supernatant by aspiration, and resuspend the cell pellet in five milliliters of 2%FBS/PBS.

After centrifuging the suspension one more time at 700 g for five minutes, discard the supernatant, and resuspend the cell pellet in 12 milliliters of culture medium. Finally, seed the cell suspension in six-well plates. Following mesenchyme dissociation, telocytes lose their cellular characteristics, showing round cellular morphology, and reflect less number of GFP-positive cells on day one compared to the following days.

A few days later, telocytes exhibit a small, stretched cell morphology with short cellular processes. After seven to 10 days of seeding, telocytes regain their cellular characteristics, showing large, stretched cell morphology with long cytoplasmic processes. Flow cytometry reveals cell composition.

Overall, 69%of the isolated cells were viable based on DAPI staining. And of these, 60.9%represented epithelial contamination and immune and endothelial cells. The telocyte fraction scattered above 100k and 70k FSC and SSC, respectively, and represented almost 10%of the gated mesenchyme.

FACS analysis revealed that the subset of telocytes can be defined by positive staining to CD201 and GP38. Immunostaining of one-day cultured mesenchyme using these markers showed expression of these molecular markers, despite cells not exhibiting their cellular characteristics. This procedure results in a viable single-cell suspension of stromal cells, which can be used not only for 2D culture but for any other application, such as 3D co-culture with organoids or bioprinting.