The overall goal of this procedure is to isolate primary myofibroblasts from mouse or human colon tissue. This is accomplished by first harvesting the colon from the donor mouse and rinsing it with ice cold PBS. Next, the epithelial cells are denuded through a series of washes and enzymatic treatments.
Then the epithelial cell suspension is plated on tissue culture dishes and allowed to incubate. Finally, non-adherent cells are washed off to isolate the myofibroblasts. Ultimately, results can be obtained that show isolation of primary myofibroblast cells through immunohistochemical staining, Demonstrating the procedure will be sharing, yay, a technician from my laboratory.
All experiments were approved by the University of California Office of Animal Research Oversight using a mouse that was euthanized with inhaled isof fluorine, followed by cervical dislocation. Begin by making a ventral midline incision. Retract the bowel away and locate the colon, retract the bladder laterally, and identify the pubic bone using fine scissors.
Cut the bone to expose the rectum, dissect the dorsal mesentary off of the rectum and colon, and immobilize the colon from the anus to the cecum. Then transect it to disconnect the colon from the small bowel. Place the specimen in ice cold PBS.
After filling a 10 cc syringe with ice cold, PBS flush the lumen of the colon several times to clear its contents. Then use scissors to cut open the colon lengthwise and place it in a 50 milliliter conical tube containing 20 milliliters of ice cold. PBS continue to replace the PBS until the fluid is clear with no particulate matter.
A protocol to obtain human tissue from surgical patients, including obtaining written informed consent from patients prior to surgery was approved by the University of California office of the Human Research Protection Program. Obtain a half inch full thickness section of colon wall from a colon resection performed using standard procedures and immediately place it on ice cold PBS. Remove any colon mesentary and epi loic appendages.
Since the presence of fat will affect the digestion process, place the entire mouse colon in a 50 milliliter conical tube with 25 milliliters of five millimolar EDTA in HBSS and incubate it in a 37 degrees Celsius shaking air bath for 15 minutes. Carefully pour off the solution and repeat the watches a total of five times. To process the human tissue.
Use scissors to cut it into four equal sized pieces and place two pieces into each of two 50 milliliter tubes containing 25 milliliters of five millimolar EDTA in HBSS. Wash the tissue five times has demonstrated for the mouse samples. Next, briefly wash the mouse and human tissues in 20 milliliters of ice cold PBS two times.
Then transfer the tissues to new 50 milliliter tubes containing 20 milliliters of RPMI five and 10 units of disc paste, and 2000 units of collagenase for the mouse tissue, or 20 units of dise and 4, 000 units of collagenase D for the human tissue. Place the tube standing up in a 37 degree Celsius shaking air bath and shake at 250 RPM for up to 60 minutes or until the tissue begins to look stringy. Shake the tubes two to four times to break up the tissue.
Then pellet the samples at 200 Gs at four degrees Celsius for five minutes to isolate and culture myofibroblasts. After pouring off the supernatant from the digested tissue, resus, suspend each pellet in 10 milliliters of cold ammonium chloride potassium or a CK lysis buffer, then centrifuge at four degrees Celsius and 200 Gs for five minutes. After discarding the supernatant, use a pipette to resuspend each pellet in 10 milliliters of RPMI five.
Using a 10 milliliter pipette pass half of the cell suspensions through a 70 micrometer mesh strainer into 100 millimeter TC treated dishes. Add an additional 15 milliliters of RPMI five to each dish. Strain the remaining five milliliter suspensions into separate TC treated dishes.
Incubate the samples in a 10%carbon dioxide incubator at 37 degrees Celsius for three hours. Then wash the cultures twice with HBSS to remove non-adherent cells and add fresh RPMI five. Adherent cells are composed of epithelial cells, macrophages, and myofibroblasts.
One week post seeding only myofibroblasts divide while macrophages and epithelial cells sse. After the first passage to passage, the cells trypsin eyes them and split them two to one. Once isolated primary human myofibroblasts can be grown in cell culture and used for up to four passages.
As shown here. The cells are characterized as alpha smooth muscle actin and fermenting positive and desmin negative. Consistent with intestinal subepithelial myofibroblasts, they also have a characteristic stellate morphology.
Primary myofibroblasts can be used to confirm experimental findings identified in a cell line. For example, exposure of 18 co cells to the pro-inflammatory cytokine. TNF alpha led to a time dependent increase in EGF receptor expression.
This correlated with enhanced EGF induced COX two expression and P 42 44 MA kinase phosphorylation. To validate these experimental findings. Experiments were repeated using primary human myofibroblasts, isolated from surgically resected colons of patients with colorectal cancer.
These figures show that primary human myofibroblasts closely mimic the experimental findings initially identified in the 18 co cell line align. After watching this video, you should have a good understanding of how to isolate primary myofibroblasts from mouse or human colon tissue.
