The overall goal of this procedure is to isolate and purify subsets of human blood vessel derived multipotent precursor cells from a single skeletal muscle biopsy first, dissociate the muscle biopsy and digest with a combination of collagenase type one, two, and four. Then label the single cell suspension with fluorescence conjugated antibodies that recognize a combination of select cell surface markers. Next, perform fluorescence activated cell sorting by flow cytometry.
See and expand the sorted cell subtypes in specific culture conditions at optimal densities. Ultimately, the post sort analysis of purified cells, as well as subsequent flow cytometry and R-T-P-C-R analysis of cultured cell subsets is used to show the purity of these homogeneous BVSE subsets. Yeah, this method, you know, can answer really, you know, what is the origin of those meso camel stem cells?
You know, because a lot of people has found different type of stem cells in different tissues. So I think this can answer the origin of those stem cells, as well as the role that perivascular and any other blood vessel derived cells play in tissue regeneration and repair. Visual demonstration of this method is critical as the muscle biopsy.
Dissociation steps can be difficult to learn. Technical details and optimization of collagenase digestion are very critical to the yield and the purification of target cells. Transport the human skeletal muscle biopsy on ice.
In DM M supplemented with 5%FBS and 1%penicillin streptomycin under sterile conditions. Wash the specimen twice with PBS containing 2%antibiotic antifungal then in dms, supplemented with 2%aa. Remove the attached adipose and connective tissue.
Also remove the large blood vessels under a dissection microscope. Now cut the muscle specimen into small pieces. Transfer the samples to 20 milliliters of preservation medium for storage at four degrees Celsius for up to five days.
On the day of cell isolation. Remove the muscle pieces and wash them twice in PBS containing 2%pen strip. Now to make the digestion solution add 100 milligrams per milliliter, each of type one, type two, and type four collagenase into pm.
Finally, chop and mechanically mince the muscle pieces with a small amount of pm. Pass the solution through a 10 milliliter serological pipette monitoring for absence of clotting. Remove the residual adipose and connective tissue during this process.
Then transfer half the adult sample to 20 milliliters or all of the fetal sample to 10 milliliters of the digestion solution. Digest a sample for 50 to 60 minutes at 37 degrees Celsius on an orbital shaker at 70 to 80 RPM. Observe the digestion status every 15 to 20 minutes.
Optimize cell yield and surface antigen preservation by adjusting the digestion time and or agitation speed accordingly, based on the amount of tissue, when the turbidity almost clears vigorously pipette the digestive tissue three to five times. Add an equal amount of PM to stop the reaction and then centrifuge at 400 Gs.For four minutes, carefully remove the supra natant to wash the pellet resuspend in 10 milliliters of pm. Filter through a 100 micron cell strainer centrifuge at 400 Gs for four minutes, and carefully remove the SUP natant.
Next resus, suspend the pellets in erythrocyte lysis buffer and filter through a 70 micron cell strainer to obtain a single cell suspension. After incubating for 10 minutes at room temperature, filter the suspension through a 70 micrometer cell strainer. If any precipitation is observed, pellet the cells by centrifugation resus.
Suspend in 0.5 milliliters of PBS and count the number of cells for staining. Dilute the single cell suspension to less than 5 million cells per milliliter with PBS aliquot, 50 to a hundred microliters of the cell suspension and split into 11 tubes for the unstained Control, the negative controls and the single color positive controls. If necessary, block the single cell suspension for 10 minutes at four degrees Celsius in mouse serum.
Add the antibody solutions according to experimental design and incubate for 20 minutes at four degrees Celsius. Pellet the cells by centrifugation and wash each sample pellet once in diluted PM at a final concentration of 5 million cells per milliliter for dead cell exclusion at a one to 100 dilution of seven a a d and incubate for 15 minutes at room temperature for the negative control and the single color positive controls. Resuspend the cell pellets in 0.5 milliliters of diluted pm.
Transfer all of the cell suspensions to round bottom polystyrene flow cytometry tubes. Now prepare cell collection tubes with 500 microliters of the appropriate coer medium. Transport the cell suspensions on ice to the cell.
Sort run the cell suspensions on the cell sorter in the order of the unstained control negative controls single color positive controls and the main cell suspension while adjusting laser intensity, channel compensation and cell population gating stringently to maximize cell purity. Collect the desired cell populations in the appropriate collection tubes. Store collected cells at four degrees Celsius.
If not seeding immediately. Seed freshly sorted. Myogenic endothelial cells in MPM onto plates that were pre-coded with Type one collagen for subsequent packaging of ECS seed.
3, 500 to 4, 000 cells per square centimeters in MPM onto plates or flasks pre-coated with type one collagen seed. Freshly sorted parasites in e gm two onto plates freshly coated with 0.2%gelatin for the first and second passages. Split the cells at a one to three ratio in PC medium onto regular polystyrene culture.
Plates from P three onward. See the cells at 6, 500 to 7, 000 cells per square centimeter in PC medium onto regular polystyrene culture plates. Flasks see the freshly sorted advent tissue cells in AC medium onto regular polystyrene culture plates for subsequent packaging of ACS seed cells at 6, 000 to 7, 000 cells per square centimeters in AC medium onto regular polystyrene culture plates.
Flasks facts parameters are first corrected based on the data obtained from the unstained control, negative controls and single color positive controls after exclusion of dead cells, the fluorescent labeled cell suspension is subjected to a series of negative and positive cell surface marker selections. First CD 45 positive cells are gated out. Then CD 56 positive and CD 56 negative cells are separated from the CD 45 negative fraction.
The CD 56 positive fraction is further subjected to CD 34, CD 1 44 selection where only dual positive cells are marked as myogenic endothelial cells and subsequently collected. Similarly, the CD 56 negative fraction is further subjected to CD 34, CD 1 46 selection where only CD 34 negative CD 1 46 positive cells are marked as parasites and subsequently collected the CD 34 positive CD 1 46 negative fraction is subjected to an additional negative CD 31 selection to exclude endothelial cells, and only the CD 34 positive CD 31 negative subset is marked as adv cells for collection. Here, CD 1 44 can be used to substitute CD 31 for EC exclusion.
These H-P-V-S-C subsets were purified from muscle biopsies of a single donor. Unlike the typical heterogeneous muscle stem cells, cultures of human blood vessel derived stem cell subsets remain homogeneous even after long-term expansion. As you know, I think it is very important for auto researcher to use the same technique to isolate, you know, those blood vessel derived stem cell that we have described in our labs.
So we believe that this technique in a side by side manner will allow people to reproduce our result and to really compare the result from one team to the other. Don't forget that the working with fresh human tissue can be extremely hazardous. Precautions such as wearing nitrile gloves and eye protection should be taken while performing this procedure.
