The goal of this method is to isolate stem cells from the mouse tracheal submucosal gland ducts to study, repair and regeneration. First, the trachea is dissected from a euthanized mouse. The trachea is cleaned, cut and digested.
Then the epithelium is mechanically stripped and further digested to generate a single cell suspension. The cells are then stained for facts sorted and finally processed for use in in vivo and in vitro assays. Ultimately, this method can help identify key pathways regulating airway, epithelial stem cell, self-renewal and differentiation.
These techniques are important because they provide us with models of in vitro and ex vivo airway, epithelial repair and regeneration. This allows us to study normal airway repair as well as diseases of the airway, such as chronic obstructive pulmonary disease, cystic fibrosis, and asthma. We first had the idea for this method when we were not able to find specific surface markers to isolate the basal from the duct stem cells, and so we decided to strip the surface epithelium with the basal cells away and leave behind the duct cells Demonstrating the procedures of tracheal dissection.
Airway epithelial stripping, a creation of a single cell suspension and fact sorting will be armored hega. A postdoctoral scholar in my lab demonstrating the procedures of processing sorted cells in the in vitro and ex vivo models is yes, orga our lab manager. Begin this procedure by cutting open the abdominal wall of a euthanized mouse.
Move the intestines to the side to expose the abdominal aorta and then dissect it. Open the chest by cutting through the diaphragm and interior chest wall on both sides to expose the heart and lungs. Continue cutting up into the neck, removing the fat and salivary glands to expose the trachea using fine scissors.
Remove the thymus and sever the lung from the connective tissue below and behind it. Then insert the forceps between the trachea and the esophagus to separate them. Cut the trachea at its uppermost part above its connection with the larynx.
Next, hold the trachea with the forceps and dissect it from its attachment. Continuing downward to remove it with the heart and lung and block, cut away the heart and lungs near the hilum to include most of the right and left main bronchi. Place the trachea in a small Petri dish containing collecting medium on ice under the dissecting microscope.
Use very fine point number five, forceps and venous tube and scissors to remove any tissue attached to the trachea. This includes lymph nodes, recurrent laryngeal nerve fat, the thyroid gland, and remaining pulmonary vessels. The upper part of the trachea needs special attention to remove the large pieces of laryngeal cartilage without compromising the biggest bunch of submucosal glands that is lodged between the CRICO cartilage and the first tracheal cartilaginous ring C one.
Next, cut the trachea into the upper part and lower part between C four and C five, and then cut. Open both parts through the lumen of the trachea. Transfer the lower part of the trachea to a one milliliter micro centrifuge tubes and the upper part of the trachea to a 15 milliliter conical tube for enzyme digestion and processing into single cell suspensions.
In the next part of the procedure, the lower and upper portions of the trachea are processed to generate single cell suspensions. While these should be processed concurrently here, the two methods will be demonstrated sequentially. To process the lower part of the trachea, add it to one milliliter of 16 unit dis space in a micro centrifuge tube, incubate at room temperature for 30 minutes.
Then transfer the trachea to a new tube containing 0.5 milligrams per milliliter, DNA one in DMM for an additional 20 minutes at room temperature following the digest, use fine forceps to transfer the tissue to a sterile Petri dish containing fresh collecting medium. Then under the dissecting microscope, strip the surface epithelium by pulling gently on the epithelial sheets to separate them from the underlying basement membrane. Next, use a pipette to transfer the medium containing the stripped epithelium to a 50 milliliter conical tube and centrifuge at 1000 G at four degrees Celsius.
Following the spin, remove the SNAT and add 0.1%tripsin EDTA, incubate the tube in a 37 degree Celsius shaking incubator for 30 minutes after the incubation, using a 1000 microliter tip pipette up and down repeatedly to break up clumps of cells and ensure the cells form a single cell suspension. Place the upper trachea in a conical tube containing one milliliter of 0.15%Pronase, then incubate at four degrees Celsius for four hours. Transfer the lower part of the trachea to a one milliliter micro centrifuge tube containing one milliliter of medium containing 16 units of disc base.
Following the incubation, gently vortex the tube and then place the trachea into fresh collecting medium. Under the dissecting microscope, confirm that there is complete detachment of the surface epithelium. Use fine scissors to mince the trachea and open up the submucosal gland compartments.
Incubate for one more hour in 0.15%pronase at room temperature with slow shaking. Spin down the tracheal tissue after removing the pronase. ADD 0.1%tripsin EDTA and incubate at 37 degrees Celsius for 30 minutes with shaking.
Next, filter the big chunks with a 70 micron strainer. Then wash the strainer with medium. Pass the digested cells clumps and any remaining chunks of glands through 20 gauge, 23 gauge and 26 gauge needles using a 10 milliliter syringe.
Repeat the packaging several times with each needle size before stepping down. Now most cells should be in a single cell suspension. Filter the suspension through a 40 micron strainer, then wash the strainer with medium and spin down the cells.
Then reconstitute the cells in the appropriate volume of medium, count the cells and proceed immediately to fact staining and sorting. Take enough cells for preparing the unstained and single stained compensation tubes. Then stain the rest of the cells with primary antibodies against trop tube and I TGA six for 15 minutes at room temperature After the incubation wash with PBS are collecting medium, remove the supernatant very carefully.
Break the cell pellet stain with the appropriate secondary antibody. Incubate in the dark at room temperature for 10 minutes. Then wash and remove the sane and then break the pellet.
The cells can now be sorted by fluorescence activated cell sorting. Now that the cells have been sorted, they can be used to investigate the mechanisms of repair and re regeneration to generate in vitro sphere forming cultures. Mix 100 microliters of growth factor reduced matri gel with 100 microliters of complete medium containing up to 50, 000 sorted cells.
Pipette this mixture into the upper chamber of a 24 well trans well plate into the lower chamber pipette 400 microliters of complete medium incubate at 37 degrees Celsius. Change the medium in the lower chamber every other day and add extra 100 microliters of matri gel every week or earlier. If it becomes thin.
Spheres typically form after seven days and can be grown for several weeks or serially passaged. For the in vivo model, reconstitute the sorted cell pellet in complete medium mixed one-to-one with matri gel at a concentration of 5, 000 to 10, 000 cells per 50 microliters. After anesthetizing the recipient C 57 black six mouse sterilize and shave, its back then in the upper back.
Make a central longitudinal skin incision. Use the blunt side of the scissor blade to widen pouches between the skin and back of the chest wall on both sides. Push the mouse for limb up and backward to move and identify the medial border of the scapula.
Then using a 300 microliter insulin syringe, inject 50 microliters of the cell suspension into the fat pad just medial to the scapula and lateral to the vertebrae. A bleb should form repeat the injection on the other side, then clip or stitch the skin incision after three weeks. Open the skin of the euthanized mouse as before and dissect the whole fat pad from both sides with a safety margin.
To avoid missing any tissue structure that might've formed and extended around. Fix the dissected fat pad in PFA and embed it in paraffin to avoid bypassing or overlooking structures during block cutting. Trim 40 microns once, then cut a four micron slice.
Place it on a glass slide, then examine it under the microscope to determine whether epithelial structures are present. If there is nothing seen, then trim once more and repeat this step. Stripping of the mouse trachea as described in this video, will result in a denuded trachea that is visible by light microscopy as shown in here denuded tracheas.
After 30 minutes of dys pasted, digestion are shown on the left and denuded tracheas. After four hours of pronase digestion are shown on the right. After epithelial stripping, submucosal glands are released from the tissues and resemble bunches of grapes.
Removal of surface epithelial cells is shown with black arrows. Removal of cells in the submucosal gland, ducts is shown with green arrows. Flow cytometry of the single cell suspensions should show forward scatter and side scatter plots as shown here.
Good separation of duct cells from the rest. If the tracheal cells should be seen with the Trop two antibody in flow cytometry for fluorescent activated cell sorting, after about one week in culture, spheres should be visible in the matri gel and our luminal when derived from basal cells and din appearance when derived from duct cells. The efficiency of this process is the order of one to 2%and single cells will still be present in the matrigel and likely represent cells from the submucosal gland ducts that do not possess the capacity for self-renew.
Basal spheres are larger and luminal in appearance. Injection of duct stem cells into the mouse fat pad results in the formation of submucosal gland, like structures that are spherical and possess a central lumen. These are seen in the blocks even without h and e staining.
Many cross-sections need to be made through the fat pad into obtain a section that contains these epithelial structures. After watching this video, you should have a good understanding of how to isolate a stop a stem cell population from subiaco gland duct cells, and once mastered, this technique can be completed in 10 hours. Following this procedure, the culture conditions can be changed in order to answer several different questions in relation to sphere formation and regeneration boost growth factors.
