Isolation of Myofibroblasts from Mouse and Human Esophagus

The overall goal of this procedure is to isolate primary myofibroblasts from mouse and human esophagus. This is accomplished by first harvesting the entire esophagus from the donor neonate mouse with human tissue. The mucosa is separated from the muscularis propria.

The second step is to mince the tissue and wash several times in HBSS. Next, the tissue is digested with collagenase diss and further minced releasing the mesenchymal cells along with attached epithelium. The heterogeneous cell population is then cultured in myofibroblast media in which myofibroblasts adhere while epithelial cells do not survive and are washed away.

Ultimately, immunochemistry and flow cytometry are used to confirm that the cells in primary culture are myofibroblasts. Demonstrating the procedures will be myself, Matthew Gargas, a technician, and chow new, a postdoc from my laboratory, Tori. After euthanizing an eight to 12 day old mouse, pin down the dorsal side of the animal and wet the ventral surface with 70%ethanol.

Next, using forceps, grab and lift up the skin anterior to the urethral opening. Cut along the ventral midline from the urethral opening to the chin. Only cut the skin.

Then make incisions from the urethral opening to the knees, forming an upside down Y.Following the incisions to the knees, make incisions on both sides of the rib cage. After completing the incisions, carefully peel the skin away and lay it aside. Now lift the peritoneum and cut through it up to the diaphragm and ribcage.

Keep the scissors pointed upwards to avoid damage to the intrathoracic and abdominal contents. Then gently lift up the left lobe of the liver to expose the underlying stomach, the esophageal gastric junction and the abdominal portion of the esophagus using surgical scissors cut along the midline of the sternum and ribcage up to the cervical girdle.Again. In doing this, keep the scissors angled away from the underlying organs.

Then pull aside the ribcage to expose the contents of the thoracic cavity. Now gently remove the heart and both lobes of the lungs. Use cotton applicators to absorb the bleeding as needed.

The thoracic esophagus is a narrow, flexible tube posterior to the trachea and anterior to the thoracic vertebra. In neonates, find the esophageal gastric junction to identify the esophagus from the stomach. Bluntly, dissect the vasculature surrounding the esophagus, including fat and mesentery tissue, up to the esophageal origin at the cervical cavity.

Now resect the entire length of the esophagus and transfer it to chilled HBSS if desired. Also, remove a portion of the stomach for orientation purposes. Begin this protocol by mincing mucosal fragments obtained of mouse or human tissue in HBSS in a 1.7 milliliter micro fuge tube or a five milliliter tube respectively.

Now, when obtaining human esophageal stromal cells, it is important to separate the mucosa from underlying muscularis propria by sharp dissection. Now in the tube further mints the tissue into two to three millimeter pieces using fine scissors. After the esophageal mucosal fragment sediment slowly decant the HBSS being careful not to inadvertently discard the tissue.

You can also use a large tip pipette to remove additional HBSS. Then add fresh HBSS. Mix the tube with a gentle shake and allow the tissue to settle.

Once settled, decant the HBSS again and repeat this wash. Step eight times in all. After the eight washes, incubate the tissue with collagenase 11 and diss incubate murine tissue up to 25 minutes on a rocking shaker set at a slow speed and at room temperature.

Under the same conditions, incubate human tissue for six hours. After the digestion, mix the tissue even further using fine scissors, and then centrifuge the tissue at 200 G for 10 minutes. Discard the supernatant and decant the pelleted tissue to a clean 1.7 milliliter tube.

Then under the tissue culture hood, wash the cells five times in DMEM with 2%sorbitol to eliminate the non-viable cells and other debris. For each wash, allow the cells to settle by gravity and decant off the wash solution. After the washes, seed the cells to three wells of a six.

Well plate and culture them in vacuum filtered supplemented DMEM. Human tissue may require more wells or plates, depending on the size of the specimen culture, the cells, until the wells are 80%confluent. Then pass the adherence cells into T 25 flasks first, use 0.05%trypsin in EDTA to dislodge the cells.

Then add media to neutralize the reaction. Collect the dislodged cells into a centrifuge tube. Then spin the cells at 400 G to collect them under a microscope.

Observe the spindle shaped morphology of the adherent cells to distinguish the cultured stromal cells from muscle cells immuno stain them for Desmond. Using a standard protocol, briefly plate 15, 000 cells in four well chamber slides and grow in myofibroblast media for 24 hours. The next day, fix the cells and then before applying the primary antibody block the fixed cells with 5%goat serum in PBS, both primary and secondary antibodies should be diluted in 5%Goat serum as well.

Incubate with primary antibody at room temperature or overnight at four degrees Celsius. Then rinse the cells three times with PBS following the washes. Add the secondary antibodies and incubate the cells for an hour.

At room temperature, use three washes in PBS to remove the secondary antibodies. Then the stained cells can be analyzed following the washes. Apply DPI with mounting media and cover slip to establish the purity of the esophageal cells.

Check them for hematopoietic and endothelial cell surface markers. Using flow cytometry. Grow sufficient numbers in culture flasks until they are ready to collect.

Then detach the cultured mouse esophageal stromal cells using non enzymatic cell dissociation solution at 37 degrees Celsius for 10 minutes. Following their collection. Wash the cells with fax buffer twice and count the cells.

Now using a standard staining procedure stain with CD 45 to identify immune cells and stain with CD 31 to identify endothelial cells, then run the cells through the flow cytometer. Esophageal stromal cells were isolated using mechanical and enzymatic digestion and cultured in six well plates within hours of plating. The cells were in a mixed suspension loosely adhered to the plate.

Over the next 24 hours, spindle shaped cells sprouted from the cell suspension and adhered firmly to the plate bottom. These cells covered the entire well within five days and were passage successfully retaining their morphology for at least 15 passages seen here, the cells are at a low density. These are the spindle shaped cells near co fluency, immuno staining of primary cultures of esophageal stromal cells.

Grown in chamber slides demonstrated an abundant expression of myofibroblast cytoskeletal markers. Alpha, SMA and menton culture purity was evaluated by flow cytometry. Forward and side scatter were established for primary murn esophageal stromal cells, followed by gating and analysis of live cells for cell surface proteins.

Primary murn esophageal stromal cells lacked expression of hematopoietic CD 45 and lacked expression of endothelial CD 31 cell surface markers. After watching this video, you should have a good understanding of how to establish primary cultures of myofibroblasts from mouse and human esophagus.Yes.