The overall goal of this procedure is to isolate adult stem cells from musculoskeletal soft tissues such as skeletal muscle. This is accomplished by first taking a biopsy of the soft tissue, debriding it and chopping it finely. The second step of the procedure is to enzymatically digest the tissue.
The third step of the procedure is to isolate the stem cells through repeated pre plating steps. The final step of the procedure is the identification of the isolated stem cells. Ultimately, results can be obtained that show the cells morphological and cellular behaviors using immunofluorescence, microscopy and or flow cytometry.
Hi, I'm y Lee from the Lab of Molecular Pathology in the Department of Orthopedic Surgery at the University of Pittsburgh. Today I will show you how to isolate a adult stem cell from the soft tissues of Mus skeletal system such as skeletal muscle. Hello, I'm Dr.Johnny Ward.
I'm a professor at the University of Pittsburgh School of Medicine within the Department of Orthopedic Surgery. I've been working, you know, closely with Dr.Young Lee and this procedures to insulate, you know, different type of stem cell from skeletal muscle and alter tissues such as standard. We use the procedures in our laboratory to isolate stem cell for basically research studies on the translational medicine applications.
So let's get studied. Let the tissues from which stem cells can be isolated include tendons and skeletal muscles harvested from the tibia or soleus muscles of the hind limb of wild type mice. After removing remnants of skin and bone, place tissues in a dish containing cold HBSS supplemented with 5%FBS.
Next, place the tissue separately on collagen coated dishes containing cold HBSS. We'll use skeletal muscle tissues to demonstrate the basic procedures for tissue dissociation and digestion. Use micro scissors and or scalpel blades to mince the skeletal tissues into a coarse slurry pH enzymatic digestion of the tissues.
Start by transferring the MIT tissue slurries into separate 15 milliliter tubes in centrifuge at approximately 3, 500 RPM at four degrees Celsius for five minutes. Remove the SANE wash by Resus suspending an HBSS and repeat the centrifugation. After removing the supernat, digest the slurries by adding 10 milliliters of prewarm.0.2%collagenase.
Type 11 incubate for 60 minutes at 37 degrees Celsius, shaking the tubes by hand every 10 minutes when the 60 minute incubation is done. Centrifuge and then resuspend the slurries in 10 milliliters of DYS space solution. Incubate for 45 minutes at 37 degrees Celsius, shaking by hand every 10 minutes after the 45 minute incubation centrifuge and resus suspend the slurries in 10 milliliters of 0.2%trips in HBSS solution.
We usually incubate for 15 to 20 minutes at 37 degrees Celsius while inverting the tubes every 10 minutes, the incubation is complete. When enough single cells are released from the tissues as illustrated here centrifuge the resulting cells and resus suspend the cell pellet in five milliliters of proliferation. Medium PM dissociate the cell suspension by passing the extract through a series of needles two times through an 18 gauge needle, then once through a 23 gauge needle and finally once through a 27 gauge needle.
Lastly, pass the cell extract through a 70 micron cell strainer to begin the prepl technique. Centrifuge the cell extract that was just prepared and resuspend the pellets in proliferation. Medium plate five milliliters of the cell mixture onto a collagen coated T 25 flask and market as PP one incubated 37 degrees Celsius in a humidified 5%carbon dioxide incubator for two hours.
After two hours. Transfer the non-adherence cells to a new collagen coated T 25 flask and market as PP two. Add five milliliters of PM into the previous T 25 flask labeled PP one.
Return both flasks to the incubator for 24 hours. After 24 hours, transfer non-adherent cells from PP two to a new collagen ENC coated T 25 flask and mark it as PP three. Add five milliliters of proliferation medium into the flask labeled PP two.
Return the flasks to the incubator. Repeat the procedure every 24 hours until population PP six. The stem cell population is created.
The isolated PP six cells are very few in number and need to be maintained in FBS rich proliferation medium. That has changed daily. Although most of the cells in the PP six culture will die during the following one to two weeks of culturing.
A large healthy PP six population is usually created after an additional one to two weeks of expansion. Maintain all other groups of suspended cells as well, allowing them to proliferate and examining them regularly using an inverted microscope. The early adhering cells that attach to the PP one and PP two flasks are mostly fibrotic cells.
Cells that adhere to collagen coated flasks after a longer time period within 48 to 96 hours. PP three and PP four are mostly myoblasts. While muscle satellite cells are often seen mostly in PP five, the later prepl cells 96 hours or later PP six are regarded as adult stem cells and appear small, round and translucent.
At the beginning of their isolation, they can be further characterized by immunohistochemistry for their expression of stem cell markers such as stem cell, antigen one and CDC 34. We've just show you how to isolate stem cell from a second mass of the mouse. When do this procedure, it's important to remember to maintain steroid during all the procedures.
Once the stem cell has been isolated, make sure to culture those cells as low density to maintain the stem cell characteristics of the cells. So that's it. Thank you for watching and good luck with your experience And I wish you good luck to try to use this in your experiment.
Thank you.
