The present PhD thesis is dedicated to the study of cultivation of mesenchymal sem cells from Wharton jelly (WJ-MSC) under physiologic oxygen concentrations.
In present work, two systems for composing the gas mixtures with low oxygen concentrations, based either on nitrogen or argon, were developed and tested for cultivation of WJ-MSC.
The obtaining of WJ-MSC primary cultures by explant method in gas mixtures with 3% oxygen showed, that the number and size of WJ-MSC colonies were higher under conditions of mild hypoxia, comparing to standard CO2-incubator conditions. On average, the number of WJ-MSC in conditions of mild hypoxia was 1,27 fold higher. The study of metabolic activity of primary WJ-MSC cultures showed the high level of heterogeneity between populations, obtained from different donors. On average, the optical density in MTT-test was 1,35 fold higher under 3% oxygen, comparing to standard CO2-incubator conditions.
The study of WJ-MSC proliferative activity in gas mixtures, containing 3% oxygen, based either on nitrogen or argon, showed, that after 7 days of cultivation, at passages 1 to 5, the numbers of cells in WJ-MSC cultures maintained in 3% oxygen gas mixtures were larger, than in control group from standard CO2-incubator conditions, even after slight decrease in proliferative activity after passage 3. No changes in MSC surface marker proteins were detected.
We also developed the method of multiplication of WJ-MSC, spontaneously detached from substrate, presumably, before or during cell division. The study revealed that the proliferative activity in "side lines", obtained from spontaneously detached cells (SDC) at passage 0 and 1, was nearly similar to one of cultures, passed with trypsin-EDTA solution. The proliferation level in cultures, obtained from SDC collected on passage 2, appeared to be significantly lower, than that in the trypsin-EDTA passed line. We also found, that WJ-MSC cultures of "main" and "side" lines from 0 and 1 passage, contained similar numbers of cells with senescent phenotype at passage 3 and 4, while their numbers in "side line" from passage 2 was significantly higher. The study showed, that under 3% oxygen, the number of MSC-WJ in cultures, obtained from SDC, was higher comparing to the standard CO2 incubator conditions.
The analysis of WJ-MSC morphology revealed, that the degree of heterogeneity in nuclear-cytoplasmic ratio and “width\length ratio” in cultures, changed during cultivation period. For all groups, the highest level of homogeneity was observed at passage 2. Comparing the effects of gas mixtures, the highest level of homogeneity was observed in nitrogen-based gas mixtures. We also showed the heterogeneity in the activity of senescence-associated -β-galactosidase at passage 3 in all conditions. The lowest level of SA-β-gal activity was detected in nitrogen-based gas mixture. We also showed that the number of SA-β-gal was on average, greater, than the number of cells with flattened shape.
In this study, the novel optimization for methods of non-viral WJ-MSC transfection was developed, by conducting all the stages of transfection procedure under physiological oxygen tensions. The transfection with nano-sized polyplexes containing plasmid DNA with marker gene (enhanced green fluorescent protein - eGFP), in gas mixtures with 3% oxygen, showed, that the percent of eGFP-positive cells was significantly higher under mild hypoxia, than in control group, maintained in standard CO2-incubator conditions. In general, the number of eGFP-positive cells was 2,58-time higher in nitrogen-based mixture, and 1,37 in argon-based mixture, comparing to CO2-incubator conditions.
The present work showed the positive effect of mild hypoxic conditions on WJ-MSC cultures and is the first to reveal the difference in biological effects of gas mixtures, with nitrogen and argon as "base" component, on WJ-MSC.
