Here, we have demonstrated the protocol for obtaining and preserving the good and viable cells for uses of therapeutic steps, like the infusion of pluripotent mesenchymal cells. The main advantage of this, that using this technique, we can obtain the viable cell volume versus time gained in the isolation step of the cells from the collected tissue. This technique can be extended toward all disease or conditions where heterogenic medicine and cell therapy are applied.
This method can also be applied to cytogenomic stability testing in areas such as drug testing, where long-term cell cultures may be encouraged. Whenever manipulating the live cells, be calm and confident. Take extreme care with sepsis and be constantly alert with a scientist's eyes to observe any adversity and make good decisions.
Begin by weighing the bag and gauging the temperature with a digital non-contact infrared clinical thermometer. Leave the bag for five minutes inside the laminar flow chamber to precipitate the greasier layers and separate the tissue containing the cells. Inject 100 milliliters of DPBS with calcium into the transfer bag and mix it with the hands.
Let it stand for five minutes. Then use a 60 milliliter syringe attached to the bag adapter to discard most of the basal liquid that precipitates. Repeat this process two times.
Add 100 milliliters of the digestion solution to the bag, and leave it at 37 degrees Celsius for 30 minutes under slow stirring. Then transfer all the bag's contents to four 50 milliliter conical tubes and centrifuge them at 400G for 10 minutes at 22 degrees Celsius. Discard the supernatant and add 5 milliliters of DMEM low glucose supplemented with 20%fetal bovine serum to the cell pellet.
Mix a fresh solution of 10 microliters of trypan blue at 0.05%in distilled water with 10 microliters of cellular suspension for five minutes. Use an inverted light microscope at 20X magnification and count the viable cells in a Neubauer cell counting chamber. Suspend the cell pellet in a cryo-protective medium at a concentration of 1 million cells per milliliter.
Then place 1 milliliter of this mixture in cryo vials. Use a freezing container with a cooling rate of 1 degree Celsius per minute and store at 80 degrees Celsius for one year. After a year, store it in standard cassette boxes immersed in liquid nitrogen vapor phase.
Data from different steps of the procedure from the nine patients are presented in this table. According to the initial cellular yield, a variable volume of cells for each sample was determined. 1 milliliter of samples with higher cellular yield, 1.1 milliliters for samples with intermediate cellular yield and 2 milliliters per samples with lower cellular yield.
The cellular yield before passage one broadly varied even when the same confluence before trypsinization was observed. Therefore, here the cells may have grown in layers. The representative image shows the plastic-adherent mesenchymal ADSCs at the first passage after isolation at light microscopy.
The cells show adhesion to the plastic-and fibroblast-like morphology. The viable cells counted in the Neubauer chamber in trypan blue assay are shown here. The flow cytometry data from six patients are presented in this table.
From these CD45 negative cells, the percentage of ADSCs with different combinations of stem cell markers was determined. The representative images show the subpopulation of cells positive for stem cell-associated markers in SVF of case nine after eight months of cryo-preservation. Here, R1 is the total cellular region analyzed in the forward scatter versus side scatter plot.
And R2 is the CD45 negative region. CD73, CD90, and CD105 populations are positive in this region. ADSC differentiation in chondrocytes, osteocytes, and adipocytes are shown here.
While attempting this procedure, remember to stir slowly, preferably with the hands, while leaving at 37 Celsius degrees for 30 minutes to observe the adhesions and blistering. Keep the work room temperature around 25 Celsius degrees, and avoid any thermal shock at much larger temperatures. The technique that we have shown here can be used to successfully isolate the pluripotent mesenchymal cells from other tissues.
