The overall goal of this procedure is to invitro characterize CD146 positive resident Mesenchymal Stromal Cell or MSCs isolated from the Rat Lung using a combination of enzymatic digestion, density gradient separation, plastic adherence and bead sorting. This method can help answer key questions in the pulmonary disease field about how lung MSCs are infected in disease and how they contribute to immune modulation and tissue repair. The main advantages of this technique are that it is fast, reproducible, and easily applicable to different species of experimental animals or tissues.
Demonstrating the first steps of the procedure will be Arul Vadivel, a research associate from my laboratory. To remove the lung from the thorax hold the trachea with small forceps and use surgical scissors to separate the trachea on the rostral side. Then while gently pulling the lung package out of the thorax cut away any connective tissue on the dorsal side along the rib cage.
Cut along the diaphragm to sever the lungs from the aorta and esophagus, and use gauze to gently remove any remaining blood from the lung tissue. Using scissors remove the trachea and bronchi and carefully place the lung lobes in a 50 milliliter tube containing 35 milliliters of cold CPDA anticoagulant and PBS to remove the blood and debris. After five minutes, transfer the lungs to a new 50 milliliter tube containing 35 milliliters of sterile, room temperature PBS.
And invert gently to remove the citrate. After five minutes rinse the lungs in a new 50 milliliter tube containing 35 milliliters of sterile room temperature Dulbecco's PBS supplemented with sodium pyruvate and glucose and invert gently. Then transfer the lungs into another 50 milliliter tube and use a scalpel to finely mince the tissue against the wall of the tube.
It is very important to cut the tissue into very small pieces to ensure a maximum cell yield. And to work quickly to ensure a robust cell viability. When all of the tissue has been minced add freshly prepared enzyme to the tube and tightly cap the lid.
Then incubate the tissue pieces at 38 degrees Celsius with gentle agitation for the appropriate digestion period. At the end of the incubation stop the digestion with 200 microliters of sterile filtered EDTA. Add 20 milliliters of PBS supplemented with FBS to the tissue slurry and pass the entire suspension through a 100 micron cell strainer into a new 50 milliliter tube.
Then rinse the digestion tube and cell strainer with 10 milliliters of FBS/PBS and pull the wash with a filtered sample. After spinning down the cells two times, resuspend the second pellet in four millimeters of room temperature FBS/PBS. And slowly layer the cells over three milliliters of density gradient medium at a greater than 45 degree angle.
By patting at an angle greater than 45 degrees between the cell suspension and the density gradient medium prevents sheer forces from mixing the cells with the gradient medium allowing the layers to be created, if mixing occurs there will be no density gradient. Separate the cells by centrifugation and collect the interphase. Then wash the lung MSCs in 10 milliliters of sterile PBS and resuspend the pellet in prewarmed complete lung MSC culture medium for plating.
To isolate the CD146 positive cell population first coat magnetic beads with 10 micrograms of biotinylated secondary antibody per 100 microliters of magnetic beads, in a two milliliter sterile round bottom cryo vial under sterile conditions. Incubate the beads and antibody on a rotating sample mixer for 30 minutes at room temperature and 30 RPM. Then resuspend the antibody conjugated beads in 1.5 milliliters of BSA supplemented PBS and transfer the beads to a three milliliter round bottom flow cytometry tube.
Rinse the cryo vial with an additional 1.5 milliliters of BSA/PBS and pull the wash in with the beads. Place the tube on an appropriate magnet for two minutes until all of the beads are attached to the rear wall of the tube and the solution becomes clear. Then remove the supernatant and wash the beads two more times with three milliliters of BSA/PBS.
Rinse the MSCs three times with unsupplemented PBS. After the last wash treat the cells with a gentle non trypsin solution for 10 minutes at 37 degrees Celsius. When all the cells have detached add fresh lung MSC culture medium and spin down the cells.
Resuspend the pellet in PBS plus BSA and count the cells. Then transfer the cells to a three milliliter tube containing CD146 primary antibody, cap the tube, and incubate the cells on a rotating sample mixer at 15 RPM and four degrees Celsius. At the end of the incubation wash the cells two times in three milliliters of PBS plus BSA resuspending the second pellet in three millimeters of the secondary antibody conjugated beads.
After 30 minutes on the rotating sample mixer at four degrees Celsius, place the tube on the magnet and use a sterile pasteur pipette to slowly and carefully remove the negative cells. Next with the tube removed from the magnet resuspend the cells in three milliliters of BSA supplemented PBS and repeat the selection process four more times. After the last negative cell removal wash the CD146 positive lung MSCs in prewarmed culture medium.
Finally, resuspend the bead conjugated cells in fresh lung Mesenchymal Stromal Cell culture medium and place 5, 000 cells per square centimeter in an appropriately sized culture flask. Plastic adherence followed by three to five days of culture enriches the density gradients separation interphase population for CD90.CD73. and CD 146 positive lung MSCs.
Moreover, these cells are capable of a colony forming unit efficiency of about 30%Much higher than that generally reported for bone marrow or other MSC subsets that differentiates along the three classic MSC lineages. Positive selection at the CD146 positive subpopulation ostensibly results in a cell population that is highly enriched in lung MSCs as demonstrated by a higher percentage of MSC associated surface marker expressing cells. These cells also demonstrate a considerably higher colony forming potential and a stronger differentiation response, particularly in the Chondrogenic lineage cells compared to the total Mesenchymal cell population obtained before CD146 selection.
Of note, these lung MSCs form very few true adipocytes exhibiting many more spindle shaped cells filled with small lipid vesicles possibly reflecting the lipofibroblast lineage that is crucial for both lung development and alveolar type two cell support. Once mastered this technique can yield CD146 positive lung MSCs within six to 10 days although the yield will depend on the age and the species of the animal. While attempting this procedure it's important to work fast to preserve the viability of the cells.
Following this procedure, other methods, like mix lymphocyte reaction or wound healing SAs can be performed to answer additional questions about the efficiency of MSCs at inhibiting T Cell proliferation and supporting wound healing. After watching this video, you should have a good understanding of how to isolate CD146 positive lung MSCs using enzymatic digestion, density gradient separation, plastic adherence, and bead sorting.
