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6MEDICAL SCIENCES
OPTIMIZATION OF THE PROTOCOL FOR THE ISOLATION OF MMSC FROM LIPOASPIRATE
Makeyev O.,
doctor of medical sciences, professor, Ural State Medical University, Yekaterinburg, Russia, Institute of
Medical Cell Technology, Yekaterinburg, Russia
Korotkov A.,
candidate of biologssycal sciences, associate professor, Ural State Medical University, Yekaterinburg,
Russia, Institute of Medical Cell Technology, Yekaterinburg, Russia
Kostukova S.,
candidate of medical sciences, associate professor, Ural State Medical University, Yekaterinburg, Russia,
Institute of Medical Cell Technology, Yekaterinburg, Russia
Schumann Eu.,
senior lecturer, Ural State Medical University, Yekaterinburg, Russia
Desyatova M.
Senior Lecturer, Ural State Medical University, Yekaterinburg, Russia
https://doi.org/10.5281/zenodo.7377243
Abstract
Recent years have been marked by the widespread introduction of cell therapy into clinical practice. Multipotent cells, namely multipotent mesenzymal stromal cells (MMSC), have the greatest therapeutic value. However, their production is associated with significant technical difficulties. We propose a simplified protocol for the isolation of MMSC from lipoaspirate.
Keywords: Multipotent cells, a simplified protocol for the isolation
Recent studies have shown that MMSCs are able to transdifferentiate into tissue cells of various germ layers [1, 2]. It was shown that the regenerative potential of mesenchymal multipotent stromal cells derived from adipose tissue (aMMSC) is higher than that extracted from bone marrow [3], as well as their proliferative potential and transdifferentiation ability. Isolation of aMMSC is less traumatic, and the content per gram of tissue is 40 times higher [4].
However, their production is associated with significant technical difficulties
Classic MMSC extraction technology [5] includes 16 stages, including the processing of tissue with enzymes. The latter is difficult to control due to the fact that the activity of enzymes varies widely from lot to lot, and the composition and amount of connective tissue varies from patient to patient. As a result, the classical technology is labor-intensive (it takes 8-10 hours of continuous operation), and the results of its application are difficult to predict. However, it is known that the greatest amount of MMSC is in the perivascular space [5]. Given this and liposuction technique, one should expect that MMSC will be located in the "salt" part of the lipoaspirate.
Materials and methods
Multipotent mesenchymal stromal cells were obtained from lipoaspirate from six clinically healthy women aged 34-41 years with their informed consent.
Vital morphology of cell cultures and their photodocumentation was performed using an Olympus CKX 41 inverted microscope.
The viability of the obtained cells was evaluated by trypan blue staining.
In order to increase cell mass, MMSC was cultured using DMEM culture medium (Sigma-Aldrich,)
supplemented with 10% fetal bovine serum (Hy-clone). For differentiation in the chondrogenic direction, a combination of insulin, transforming growth factor and ascorbate was introduced into the culture medium [5]. Upon reaching the monolayer, cells from the surface of the culture vial were removed with a trypsin-Versen solution at concentrations of 0.25% and 0.2%, respectively.
Fluorescence microscopy (qualitative analysis) and flow cytofluorimetry (quantitative analysis) were used to evaluate the expression of specific positive and negative markers. Requirements for the expression of receptors on the surface of the studied cells: for CD73, CD90, CD105 - at least 95% of the cells in the sample, for CD31, CD1, and CD14 - no more than 2% of the cells.
The cryopreservation of the obtained MMSC cell mass was performed by the stepwise method: gradual cooling to -84 ° C in a Mr. container Frosty (Nanc), followed by immersion in liquid nitrogen. DMSO (MP Biomedicals) at a concentration of 10% was used as a cry-oprotectant.
The data obtained were processed by nonparamet-ric statistics. The statistical difference between the groups is established using the Kruskal-Wallis method with further processing by the method of multiple comparisons according to Dunn. The significance of differences compared to baseline is determined using the Wilcoxon test for related samples.
Results and Discussion
In the course of a study to optimize the release of MMSC from adipose tissue, the following technology was obtained:
1. Aspirate the "salt" part of the lipoaspirate.
2. Centrifuge at 400g for 10 minutes at room temperature
3. Resuspend the pellet in hypotonic PBS for 5 minutes at room temperature
4. Centrifuge at 400g for 10 minutes at room temperature
5. Resuspend the pellet in DMEM supplemented with 40% FBS, 100 units / ml penicillin and 100 units / ml streptomycin and transfer to vials and place in a CO2 incubator at 37 ° C and a CO2 content of 5%
6. A day later, cells and tissue fragments that did not adhere to the surface of the vial were washed three times with standard PBS, replacing the medium with standard (in our case, DMEM / F12 with 10% FBS, 10 ng / ml FGF, ABAM).
Identification of MMSC derived from human adipose tissue
Cells obtained from human anterior abdominal wall fat showed high adhesion on culture plastics. Even at the isolation stage, the adhesion property was used as a sort of sorting method: only those cells that were able to fix on plastic during the first days of cultivation were preserved in the culture, the rest were removed during the first or subsequent passages.
Adhesive cells had a spindle-shaped or process (fi-broblast-like) shape. During proliferation, the cells showed the ability to locomotion on the cultural surface, gradually spreading to the maximum possible area with the loss of contact with each other.
As the culture approached the state of the mono-layer, cell proliferation slowed noticeably, and then stopped. Thus, the culture demonstrated the ability to contact inhibition, which refers to one of the manifestations of feelings of their own pool. This characteristic is typical of all cells that completely or partially differ in the properties of "stemming" and / or that realize restoration of the cell population.
The removal of cell culture from the surface by trypsinization was accompanied by a change in the shape of the cells from the process to spherical, which is associated with cell adhesion. Trypsin, destroying surface adhesive proteins, deprived the cell of the basis of fixation on the surface, after which the cells acquired a spherical shape and were washed off from the surface of the culture plastic.
After neutralizing trypsin and transferring the removed culture to a new surface, after 30-40 minutes the
cells were firmly fixed on the plastic, ceased to give in to the usual mechanical washing off and gradually acquired a process form.
Thus, we can conclude that the fixation of cells on the culture plastic and their process shape indicates the ability to adhere - one of the main distinguishing features of MMSC, and the trypsinization effect - to the adhesion mechanism realized by trypsin-labile surface proteins.
The second necessary identification criterion for MMSC is the expression of a set of specific surface markers. In accordance with the accepted criteria, MMSCs should express CD73, CD90, CD105 markers and differ in the minimal expression of CD34, CD45, CD14, CD19 (and some other negative markers).
The method of fluorescence microscopy of cells isolated from fat of the anterior abdominal wall, demonstrated the expression of the necessary set of positive markers (CD73, CD90, CD105). Despite the fact that the technique is not quantitative, it leaves the possibility only for a qualitative determination, however, the fluorescence intensity suggested a sufficiently strong expression of the studied markers.
According to the definition of CD14, CD34 and CD45, fluorescence microscopy did not actually allow us to identify the fluorescence of colors corresponding to the fluorochromes used, which indicates an extremely low level of expression of negative markers.
Quantitative analysis of markers using flow cyto-fluorimetry showed unidirectional results with fluorescence microscopy. The average expression level of the CD73, CD90, and CD105 markers was higher than 90% (92.35 to 95.19) (CytoFLEX ™).
It seems that the MMSC culture under study contains a small fraction of cells that do not express positive markers. However, the proportion of these cells does not quantitatively exceed 4-7%, which suggests that most of the cells in the studied samples belong to MMSC and the culture parameters as a whole correspond to the MMSC properties.
In favor of the aforesaid, the expression of negative markers CD34, CD45, and CD14 is indicated, the analysis of which revealed no differences from the indicators of negative control (less than 1%). This indicator fully complies with the requirements for pure MMSC cultures (expression of negative markers no more than 2%) (Figure 1).
100 80 -
(0
c 60H ®
w 4020-
CD90 95.19 ± 11.46
100 80 -
10° 10' 10" 103 10" Fluorescence intensity
CD14
0.46 ± 0.31
i | 60- | 1 J
CD105 94.69 ±17.19
80 -
10° 10' 102 1 03 1 04 Fluorescence intensity
CD34
0.51 ± 0.73
80
10° 10' 10* 103 10* Fluorescence intensity
CD45
0.35 ± 0.21
W 40-1 40 - L
20-1 20l
L
10» 10' 102 1 03 1 0' Fluorescence intensity
10° 101 102 10s 104 Fluorescence intensity
10° 101 10s 103 10< Fluorescence intensity
Figure 1 - Quantitative analysis of CD markers
A standard analysis of signals received from intact cells by laser beam scattering showed that the sample contained at least 95% of intact cells, which is sufficient to verify the results as reliable.
The resulting technology is significantly different from the classical one in the following ways:
1. The end result is well predicted
2. Less labor intensive
3. The duration of the main part of the MMSC allocation process is no more than 1 hour.
4. Not inferior to the classical technology in the number of viable cells obtained with the MMSC phe-notype (2 - 4 x 105 cells from 100 ml of lipoaspirate).
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