The Effect of Allogenic Cell Cultures on Type I Diabetic Rats

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURE

Abstract

Introduction

Materials and research methods

Research results

Conclusion

Bibliographical references

LIST OF TABLES

Table 1 Results of a cytogenetic analysis of the rat red bone marrow stem cells culture at passages I-VI

Table 2 Results of a cytogenetic analysis of the rat adipose tissue stem cells culture at passages I-VI

Table 3 Results of a cytogenetic analysis of the rat pancreas stem cells culture at passages I-VI

Table 4 Morphometric indicators of Langerhans islets in rats of different groups on the 50th day after modeling of alloxan diabetes mellitus

LIST OF FIGURE

Fig. 1. Micrographics of rat bone marrow stem cell cultures (passage 0)

Fig. 2 . Micrographics of rat adipose tissue stem cell culture

Fig. 3. Micrographics of the stem cell culture of rat adipose tissue

Fig. 4. Micrographics of pancreas stem cell culture, (passage 0)

Fig. 5. Micrographics of pancreas stem cell culture I and IV passages

Fig. 6. CD48 expression in rat stem cell cultures, passage IV

Fig. 7. Micrographics of metaphase plates of rat chromosomes (passage I)

Fig. 8. Micrographics of cells with changes in the nucleus (passage VI)

Fig. 9. Rat pancreas with experimental alloxan diabetes

Fig. 10. Identification in the pancreas of Hoechst-labeled stem cells derived from bone marrow

Fig. 11. The level of glucose in the blood of rats with diabetes mellitus against the background of the introduction of stem cells of different origin cultures

Fig. 12. The pancreas of rat with experimental alloxan diabetes mellitus after administration the red bone marrow stem cell culture

Fig. 13. The pancreas of rat with experimental alloxan diabetes mellitus after administration the adipose tissue stem cell culture

ABSTRACT

Despite the shortcomings of the modern methods of treating animals with type I diabetes using insulin therapy, as well as the fact that pancreatic β-cell death is one of the important elements in the diabetes pathogenesis, new approaches to the treatment of this disease using cell technologies are being studied. Recent studies of β-cells in the in vitro system have shown that they have a fairly high regenerative capacity, but in the in vivo system with diabetes, these cells almost do not recover. The development of methods that can activate β-cell regeneration is an important area of the scientific research. To date, mainly red bone marrow is used as the source of stem cells for research, because this is the only tissue of the adult body that normally contains immature, undifferentiated and low-differentiated cells. However, adipose tissue is being increasingly used as an alternative source for stem cells, from which they can be isolated in significantly larger quantities using less invasive methods compared to using red bone marrow. It is worth noting that there are still a lot of unclear issues in the study of the pancreas regeneration ways, therefore, in the treatment of patients with diabetes, the direction of the use of cell culture obtained from the pancreas is especially relevant. Given the above, the aim of our study was to study the effect of cell cultures obtained from adipose tissue, bone marrow, and pancreas on the course of experimentally formed insulin-dependent type I diabetes in rats with the aim of developing scientifically based and effective cell therapy methods in veterinary medicine.

To solve this problem, we studied changes in the pancreas of rats with the introduction of alloxan, confirming histologically the presence of pathological processes characteristic of type I diabetes. We performed a cytogenetic analysis of rat cells in culture obtained from red bone marrow, adipose tissue and pancreas during subcultivation in the in vitro system and gave their phenotypic characterization. We studied the histological changes in the pancreas of rats with the introduction of cell cultures obtained from different sources against experimental diabetes and studied the changes in the blood glucose of experimental animals with the transplantation of cellular material.

According to the results of the studies, it was found that cell cultures obtained from bone marrow, adipose tissue and pancreas differ not only morphologically, but also in the expression of the studied CD markers. During cultivation, the studied cultures reveal genetic changes in the form of aneuploidy and polyploidy, however, the variability of their karyotype during passage does not go beyond the spontaneous level characteristic of mammals. The introduction of stem cells of pancreas, adipose tissue and bone marrow cultures with experimentally formed type I diabetes significantly contributes to a decrease in the level of glucose in the blood of animal recipients. With the transplantation of stem cells of the pancreas culture, an increase in the total volume of Langerhans islets was noted, primarily due to the islets neogenesis, and acceleration of cell regeneration in previously established islet tissue was also observed. The introduction of stem cells of the bone marrow culture leads to an increase in the total volume of Langerhans islets to a greater extent due to the regeneration by enhancing the proliferative activity of islet cells. The transplantation of adipose tissue stem cells stimulated the Langerhans islets neogenesis in the pancreas. Adipose tissue stem cell efficiency was the lowest.

INTRODUCTION

At the moment the upward trend in animal diabetes incidence is observed in the world [18, 24, 39, 43]. However, the etiology and pathogenesis of type I diabetes in animals is still understudied, and there are no effective treatment methods.

Despite the shortcomings of the modern methods of treating animals with type 1 diabetes using insulin therapy, as well as the fact that pancreatic β-cell death is an important element of the diabetes pathogenesis [8, 22], new approaches to the treatment of this disease using cell technologies are being studied [2, 27, 34]. The development of methods that can activate β-cell regeneration is an important area of the scientific research [4, 32, 42, 45].

Recent studies of β-cells in the in vitro system have shown that they have a rather high regenerative capacity [14, 15], but in the in vivo system with diabetes, these cells almost do not recover 31.

The prospects for the successful use of the cell-regenerative therapy in veterinary medicine depend largely on the results of the detailed study of the animal stem cells properties and their use for therapeutic purposes.

To date, mainly red bone marrow is used as the source of stem cells for research, because this is the only tissue of the adult body that normally contains immature, undifferentiated and low-differentiated cells. Cell culture obtained from red bone marrow is heterogeneous in its composition and contains hematopoietic stem cells, endothelial progenitor cells [23, 36], mesenchymal stem cells [26, 41], pluripotent 30 and multipotent 3 adult stem cells , very small embryonic-like cells 35.

However, adipose tissue is being increasingly used as an alternative source for stem cells, from which they can be isolated in significantly larger quantities using less invasive methods compared to using red bone marrow. Adipose tissue contains adipocytes, as well as the cells that make up the stromal vascular fraction (SVF): preadipocytes, endothelial and smooth muscle cells of blood vessels, perivascular fibroblasts, and supporting fibrous collagen stroma 9. The primary adipose tissue culture is also heterogeneous in composition 49 and contains mesenchymal stem cells 46, hematopoietic stem cells, and endothelial progenitor cells.

Since there are still a lot of unclear issues in the study of the pancreas regeneration ways, therefore, in the treatment of patients with diabetes, the direction of the use of cell culture obtained from the pancreas is especially relevant. Most scientists are inclined to think that the main regenerative function in the pancreas is performed by the ductal epithelium, the cells of which are believed to be “optional stem cells” [7, 12]. However, some researchers point to the presence of multipotent progenitor cells in the pancreas [21, 47]. Other scientists prove the ability of β-cells to divide 32. Given the above, the pancreas cell culture should also be considered heterogeneous in its cellular composition.

The important areas of the stem cell research are the study of biological activity, cytogenetic stability during cultivation in the in vitro system in order to prevent their neoplastic transformation in the in vivo system, as well as the reaction of the animal recipients to the introduced cellular material during clinical use 5.

So, the study of the animal stem cells properties and their further use in experimental insulin-dependent diabetes are very relevant questions, and their solution will contribute to the development of scientifically based and effective methods of cell therapy in veterinary medicine.

MATERIALS AND RESEARCH METHODS

All manipulations with animals were conducted in compliance with the Law of Ukraine "On the Protection of Animals from Cruelty" (No. 3447 - IV, dated 02.21.2006) (Law of Ukraine 2006).

Cell culture of pancreas, bone marrow (obtained from bone marrow of the tubular bones and pancreas of rat pups at the age of 12 days) and adipose tissue (obtained from rats aged 4-5 months) were used in the study. The resulting cell mass was cultured in standard medium: 80 % - Dulbecco's modified Eagle medium; 20 % fetal bovine serum; 10 μl/cm3 – antibiotic-antimycotic "Sigma", USA; in a CO2 incubator at 37 °C and 5 % concentration of CO2 33, in 90–100 % confluence.

Cells were detached according to standard methods (0.25 % trypsin/EDTA solution) 33. Further passage was carried out at a 1:3 dilution. Microscopic analysis and culture evaluation were carried out using an Axiovert 40 (Carl Zeiss) inverted microscope.

Phenotype changes were monitored by detecting CD markers (CD10, CD38, CD34, CD45, CD48, CD54, CD56, CD66e, CD96, CD227, CD326b, CD pan-keratin). Preparation of specimen was carried out according to standard methods) 33. Analysis of the results was carried out according to the number of cells with expression (green luminescence of the cells) and evaluated using the classical H-Score method: S=1×A+2×B+3×C, where S is the “H-Score” indicator, the value of which is ranging from 0 (protein is not expressed) to 300 (strong expression in 100 % of cells); A – cells with weak expression; B – percentage of cells with moderate protein expression; C – percentage of cells with strong expression. The degree of expression was determined as negative if the number of points was in the range from 0 to 50; low – from 51 to 100; moderate – from 101 to 200; high – 201 and higher [38, 48]. Studies were performed using a Leica DMR fluorescence microscope (Germany).

Cytogenetic analysis was performed on 30 metaphase plates of cell cultures from each passage. To obtain chromosome preparations, a modification of the standard cytogenetic method was used 33. The resulting preparations were stained using a stain pack (Leucodif 200), according to the manufacturer's instructions. Analysis of metaphase plates was carried out using a Leica DMR microscope (Germany), magnification ×400, ×1000. In the slides prepared by the above method the quantitative chromosome abnormalities – aneuploidy (A), polyploidy (PP) – were identified, and the number of binuclear cells (BN), cells with micronuclei (MY), mitotic index (percentage of cells in the division stage of the total number of analyzed cells (MI) 2, apoptotic index (percentage of cells with signs of apoptosis of the total number of analyzed cells (AI) were calculated. BN, MN and AI were calculated per 500 cells (%).

The experimental model of diabetes was reproduced by a single subcutaneous injection of alloxan monohydrate (Sigma, USA) at a dose of 150 mg/kg in the form of a 5 % solution in citrate buffer, pH 4.5 after a preliminary 24-hour food deprivation (with free access to water). To reduce the death of animals as a result of hypoglycemic shock after the injection of alloxan, they received a 5 % glucose solution instead of water within 24 hours after the diabetes induction.

To study the effectiveness of cultures, 2 million cells were transplanted into recipient animals with alloxan diabetes (in a volume of 50 μl) under a pancreatic capsule.

Tissue sampling for histological studies was performed on the 50th day of the experiment (the control group and the experimental group – 30th day after cell transplantation). After samples collection, they were fixed in a 10 % solution of neutral formalin for 24 hours, then the tissues were dehydrated and embedded in paraffin according to standard methods 33. Sections with a thickness of 5 μk were made using a rotary microtome NM 320 E (MICROM, Germany) and a section transfer system (STS, MICROM, Germany). To study the microstructure of tissues, sections were stained with hematoxylin and eosin, after which the preparations were subjected to light microscopy. Evaluation and analysis of the preparations was carried out using a Leica DMR microscope (Germany). The state of the pancreas in animals was investigated against the background of diabetes (the 50th day of the experiment) and on the 30th day after the cell cultures transplantation. Morphometric studies of the insular apparatus condition were performed on stained preparations. The total number of islets per 10 mm2 in the section was calculated, and the number of cell nuclei in the islets was additionally determined. The studies were carried out on 3 non-serial sections made at the 0.5 mm distance from each other (the distance exceeds the size of one islet).

RESEARCH RESULTS

Stage I of the study included work with cell cultures of red bone marrow, pancreas and adipose tissue obtained from rats.

During cultivation in the in vitro system, the cells in the culture change, this can cause differences in the phenotype. First of all, it depends on the characteristics of the tissue or organ that were used to obtain the primary culture. Each individual tissue contains a spectrum of identifying markers that distinguish it from others. Therefore, first of all, the changes that occur in the early stages of the cultivation process are due to retrograde shifts in state of differentiation. Phenotypic changes that occur during these processes can be detected visually (morphological evaluation) and by immunophenotypic analysis of the differentiation markers expression.

Phenotyping of cultures was carried out to create an experimental system for assessing the optimal source of cellular material for the treatment of diabetes.

Red bone marrow stem cell culture. The primary culture of rat red bone marrow stem cells was characterized by morphological heterogeneity (Fig. 1).

Within a few days after planting, a significant number of attached rounded cells that did not divide could be observed in combination with fibroblast-like cells (Fig. 1), which subsequently covered most of the area of culture plastic. Primary bone marrow culture reached 90–100 % confluence on average in 8 days.

In the process of subcultivation, 70–80 % confluence was achieved in 3 days. In passage I, culture heterogeneity was noted. The stem cell culture of rat red bone marrow contained a small number of polygonal cells surrounded by fibroblast-like cells (Fig. 1 c).

The morphological heterogeneity of red bone marrow culture stem cells at passages 0 and I can be explained by the fact that the hematopoietic cells contained in bone marrow are able to survive and self-sustain for a long time without a significant increase in their number 28. Therefore, against the background of a rapid increase in fibroblast-like cells, the percentage of polygonal cells decreases significantly, which we noted at the following passages.

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Fig. 1. Micrographics of rat bone marrow stem cell cultures (passage 0): a) the 3rd day of cultivation; b) the 7th day of cultivation; c) I passage; d) IV passage. Native preparation. Magnification in Fig. a - × 100; b, c, d - × 50. (Author’s own work).

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Adipose tissue stem cell culture. The primary stem cell culture of rat adipose tissue was characterized by morphological heterogeneity (Fig. 2 a, b), resulted from the presence of cells that are part of the stromal-vascular fraction.

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Fig. 2 Micrographics of rat adipose tissue stem cell culture. Native preparations: a) the 5th day of cultivation; b) the 14th day of cultivation. M agnification: ×100 . (Author’s own work).

Within a few days after planting, the presence of a significant number of weakly adhesive round cells was observed, which were removed during the passage. Starting from the 5th day of culturing cells obtained from adipose tissue, the culture became homogeneous (Fig. 2 a). The primary culture reached 90–100 % confluence on average in 14 days (Fig. 2 b). In the process of subcultivation, 70–80 % confluence was achieved in 4 days.

At passage I, the heterogeneity of the stem cell culture of rat adipose tissue was noted. Its basis was a small number of polygonal cells surrounded by fibroblast-like cells (Fig. 2 a).

With each passage, the number of polygonal cells decreased. At passage IV, the most homogeneous composition of the culture was noted. The morphology of the culture obtained from rat adipose tissue at passage IV was characterized mainly by a fibroblast-like structure (Fig. 3 b).

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Fig. 3. Micrographics of the stem cell culture of rat adipose tissue. Native preparations: a) I passage; b) IV passage. Magnification : ×50. (Author’s own work).

As can be seen from the above data, the stem cells of the adipose tissue culture were morphologically characterized by heterogeneity at the beginning of cultivation. Most scientists explain this phenomenon by the fact that the stromal-vascular fraction of adipose tissue acts as a source of proliferating cells, is quite scattered and consists of preadipocytes, endothelial and smooth muscle cells of blood vessels, perivascular fibroblasts and supporting fibrous collagen stroma 11. In the process of passage, the adipose tissue stem cell culture became more homogeneous due to fibroblast-like cells, which had high proliferative activity.

Pancreas stem cell culture. The primary stem cells culture of rat pancreas was characterized by morphological heterogeneity (Fig. 4).

On the 5th day of cultivation, we noted the formation of colonies, which were visually different. So, the culture consisted of colonies containing fibroblast-like cells, and individual colonies of polygonal cells.

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Fig. 4. Micrographics of pancreas stem cell culture, (passage 0): a) the 3rd day of cultivation; b) the 10th day of cultivation. Native preparations. Magnification in Fig. a - ×320; b - ×50. (Author’s own work).

Starting from the 2nd day of cultivation, we observed proliferation of cells of predominantly fibroblast-like morphology, which originated from the explant (Fig. 4 a.).

Starting from the 10th day of cultivation, fusion of cell colonies of different morphology was noted (Fig. 4 b). It should be noted that cells of fibroblast-like morphology were characterized by a higher division rate compared to polygonal cells. At the time 90 % confluence was achieved, the number of fibroblast-like cells was approximately 80 % of the cell mass of the primary pancreas culture.

The primary pancreas cell culture reached 90–100 % confluence on average in 14 days. In the process of subcultivation, 70–80 % confluence was achieved in 4 days.

At passage I, the culture of the rat pancreas continued to be heterogeneous; it consisted of individual polygonal cells surrounded by fibroblast-like cells (Fig. 5 a). Starting from passage II, the culture became homogeneous due to fibroblast-like cells (Fig. 5 b).

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Fig. 5. Micrographics of pancreas stem cell culture a) passage I; b) passage IV. Native preparations. Magnification: ×50 . (Author’s own work).

The morphology of the pancreas culture stem cells was the most heterogeneous among the studied cultures. So, in the primary culture, the formation of colonies of different morphology was noted, which merged during cultivation. The heterogeneity of the primary pancreas culture is confirmed by data from other authors, who, in turn, claim that exocrine cells make up 95 % of the monolayer, while 5 % include endocrine, ductal, and vascular endothelial cells 1.

Some scientists point to the preservation of the polygonal shape of β-cells during cultivation in the in vitro system [1, 44], while acinar cells have a fibroblast-like shape 25. It should be noted that the cells of the polygonal shape preserved longer than the culture of red bone marrow and the culture of adipose tissue, which indicates not only the adhesive properties of these cells, but also their ability to divide. However, cells of a fibroblast-like form divide faster, which leads to their gradual predominance in culture and its visual homogeneity.

Immunophenotyping of the population of stem cells obtained from rat red bone marrow revealed changes in the expression of the studied CD markers during cultivation.

So, in the rat red bone marrow stem cells culture, the expression of CD10 and CD54 was not observed throughout the entire study period. There was a decrease in the level of CD34 expression from moderate to low. The CD38 expression decreased sharply from a moderate level (passage I) to its absence (passage IV).

The degree of CD45 and CD326 expression gradually decreased with passages from low to "no expression". While the levels of CD48, CD66e and CD95 grew from low to moderate. Expression of CD56 and CD227 ranged low throughout the study period. During the study, a significant decrease in the degree of pan-keratin manifestation from a high to a moderate level was noted.

Immunophenotyping of the stem cell culture derived from adipose tissue made it possible to trace the changes occurring in it during cultivation. So, during all passages CD10, CD54 and CD56 had a negative degree of expression. Expression of CD34, CD38, CD45, and CD48 decreased from low to negative levels (Fig. 6). When studying the CD227 and CD326 expression, an inverse relationship was noted, since the level of expression increased with each passage. Pan-keratin expression was recorded at a low level throughout the study. Manifestation of CD66e was characterized by a high level at the beginning of cultivation and a low level at the end of the study.

The level of CD95 expression in the culture of stem cells of rat adipose tissue increased from low to moderate levels with each passage.

Immunophenotyping of the culture of cells obtained from rat pancreas also revealed changes in the expression of the studied CD markers during cultivation.

So, the expression of CD38 changed from a low level at passage I to a high level at passage IV. A low level of CD227 expression was observed throughout the entire cultivation period. Changes in expression from its absence to a moderate level were observed in CD66e and CD326. The degree of CD95 expression was moderate throughout the duration of the study. It should be noted that in the pancreas cell population there was no expression of CD10, CD34; CD45, CD48, CD54, CD56 and pan-keratin.

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Fig. 6. CD48 expression in rat stem cell cultures, passage IV: a) red bone marrow; b) adipose tissue. Fluorescence microscopy. Magnification ×1000 . (Author’s own work) .

Analyzing the data obtained during the immunophenotyping of stem cells in different cultures, we can conclude that they are heterogeneous in their morphology and additionally change from passage to passage. It should be noted that the panel of CD markers we selected is characteristic of weakly differentiated (progenitor, stem) cells.

Thus, cells that express markers of hematopoietic (CD34, CD45, CD48), epithelial (CD66e, CD326, pan-keratin) cells were detected in bone marrow culture. The expression of CD56 and CD66e, which manifest on mesenchymal stem cells, was also noted. From the data obtained, it can be concluded that rat red bone marrow cell culture contains hematopoietic cells (both stem and non-stem cells), mesenchymal stem cells, and possibly epithelial cells. Since the indicators of CD markers characteristic of epithelial cells are very different in their expression, it can be assumed that the role of these differentiation clusters in red bone marrow and its cultures is understudied.

The expression of markers that are manifested on hematopoietic, epithelial, mesenchymal cells, as well as preadipocytes was also noted on adipose tissue culture cells. It should be noted that the expression of markers characteristic of hematopoietic cells decreased from passage to passage, which indicates a decrease in the percentage of these cells in culture.

In the pancreas cell culture, cells with a minimal spectrum of used CD markers expression were observed. The pancreas culture was negative for markers characterizing hematopoietic cells. However, the expression of epithelial markers was well defined. Analyzing the above data, we can assume that the culture originated in the bulk by the cells of small ducts, which underwent dedifferentiation during cultivation.

Despite the inconsistency of data on the genetic stability of the cell during cultivation, the next step in the study was the cytogenetic analysis of the rat red bone marrow, adipose tissue, and pancreas cells at early passages. The potential use of stem cells for therapeutic use provides for their long-term cultivation in vitro. Research on genetic stability is an inherent problem of passage, since most cultures consist of a heterogeneous mass of cells in different states of differentiation and dedifferentiation.

Red bone marrow stem cell culture. Based on the analysis of the karyotype of the rat red bone marrow stem cell culture, it was found that they are characterized by quantitative chromosomal abnormalities (aneuploidy and polyploidy). The results of studies of a cytogenetic analysis of the rat red bone marrow stem cell culture are shown in Table 1. The appearance of aneuploid cells (Fig. 7 b) was observed from passage I to VI in an amount of 8.9±1.3 % (passage I) to 17.8±1.3 % (passage VI).

Table 1 Results of a cytogenetic analysis of the rat red bone marrow stem cells culture at passages I-VI (M±m, n=3)

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Note. *** p <0.001; ** p <0.01; * p <0.05, compared with the control (the control was the indicators obtained at passage I)

Fig. 7. Micrographics of metaphase plates of rat chromosomes (passage I), Leucodif-200 stain: a) normal karyotype, n=42; b) aneuploidy, n=38; c) polyploidy, n=84. Magnification ×1000 . (Author’s own work) .

Cytogenetic variation (aneuploidy) was mainly caused by the appearance of hypoploid cells, the karyotype of which is 2n=39, 2n=30 chromosomes. The difference in the average values for this trait in the populations of cells in passages III, V, VI was significant, compared with passage I. It should be noted that the highest level of aneuploidy (18.9±1.3 %) was observed in passage III, after which a decrease in the number of aneuploid cells (passage IV) and a gradual increase in their number with the following passages were noted.

A multiple increase in the number of chromosomes (polyploidy) in the cell population also manifested itself from passages I through VI (Fig. 7 c). From passage I to III, a stable number of cells with polyploidy was noted – 1.1±1.2 %. Starting from passage IV, there was a tendency to increase in the frequency of such a genomic mutation in stem cell culture to 4.4±1.3 %. However, our result was lower than the spontaneous chromosome variation characteristic of mammals (6–15 %) 50.

A micronucleus test was also performed to evaluate cytogenetic changes in the stem cell culture of rat red bone marrow. In the process of research, micronuclei were found in all passages. In addition, with each subsequent passage, a significant increase in their number was observed. However, the percentage of cells with micronuclei was within the normal range for mammals (1.6–5.6 %) 50.

During the studies, a significant increase in the number of binuclear cells in the red bone marrow culture from passage I to VI was noted. However, their number did not exceed the indices of spontaneous mutation of blood lymphocytes characteristic of mammals (5.4 %) 50. The increase in the number of binuclear cells in the culture with an increase in the duration of cultivation can be explained by the cell cycle lengthening with the cell culture aging, in particular cytokinesis.

In the process of research, a decrease in the mitotic index from passage I (4.1±0.2 %) to passage IV (2.7±0.3 %) and its gradual increase at passages V (3.3±0.1 %) and VI (3.5±0.1 %) were observed. This indicator did not go beyond the norm characteristic of mammals, which is 2.9–4.1 % 19.

In addition, an insignificant percentage of cells in the state of apoptosis was noted at each passage, the number of which gradually increased by passage IV (0.7±0.2 %). At V–VI passages, a decrease in the number of cells in the state of apoptosis to 0.5 % was observed. The level of apoptotic cells was within normal limits.

Adipose tissue stem cell culture. The analysis of the karyotype of the rat adipose tissue stem cells during their cultivation showed that they are also characterized by quantitative abnormalities (aneuploidy and polyploidy). The results of a cytogenetic analysis of the rat adipose tissue culture stem cells are shown in Table 2.

The presence of aneuploid cells was observed from passage I to VI in an amount of 4.4±1.3 % to 12.2±1.3 %. Cytogenetic variability (aneuploidy) was mainly observed in cells with a karyotype of 2n=38 chromosomes. The percentage of cells with aneuploidy increased slightly from passage I (4.4±1.3 %) to V (6.7±0 %). It is worth noting that at the passage VI there was a sharp significant increase in the number of cells with aneuploidy to 12.2±1.3 %. A multiple increase in the number of chromosomes (polyploidy) was manifested in the cell population only at passages I (5.6±1.3 %) and II (3.3±2.0 %). At the next passage, no polyploidy was noted.

During the study of the adipose tissue stem cell culture at the first passages we noted a large number of binuclear cells (2.9±0.2 % – passage I; 2.5±0.3 % – passage II) and a high mitotic index (4.3 % – passage I; 4.1 % – passage II). This suggests that the polyploidy detected in the first passages of cultivation is due to the high rate of division, which led to the discovery during analysis of a large number of cells in the process of cytokinesis. However, our results were lower than the indices of spontaneous chromosomal variability of blood leukocytes of mammals (6–15 %) 19.

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