The Influence of DMSO and a Fraction (below 5 Kda) from Cord Blood on MSC Culture Growth

Gulevsky A. K., Lavrik A. A., Trifonova A. V.

CLINICAL AND EXPERIMENTAL MEDICINE

Scentific article

The study aim was to estimate the influence of a fraction (below 5 kDa) from cord blood on MSC cul- ture growth after equilibration with dimethyl sulfoxide (DMSO). Materials and Methods. MSC were obtained from rat bone marrow and used at the 3rd – 4th passages. Equilibra- tion of cell suspension was carried out at 20-22oC for 30 minutes in medium with 95 % FBS (fetal bovine serum) and 5 % DMSO. Cell concentration was 106 cells/mL. DMSO was removed by centrifugation. MSC were cultivated in growth medium DMEM/F12 (1:1) with 10 % FBS. Seeding concentration was 104 cells/ cm2. The fraction (below 5 kDa) was extracted from cattle whole cord blood by ultrafiltration (CBF). It was added to MSC growth medium in the concentrations of 28 µg/mL to 400 µg/mL. Growth properties were estimated in terms of PI (Proliferation Index) and MI (Mitotic Index). The number of pathological divisions and cell morphologic characteristics were also estimated. Results and Discussion. The growth properties of MSC culture deteriorated after equilibration with 5 % DMSO. PI was 70 % of the pre-equilibration value. Possibly, it was associated with cytoplasmatic membrane alterations, which damaged cell adhesion. This resulted in a later start of cell divisions as compared with the control. Therefore, it may be a cause of growth property deterioration of MSC culture after contact with DMSO. Interactions of DMSO with membrane proteins and lipids impaired oxidative phosphorylation and, as a consequence, cell energy. If CBF was added in the concentration of 28 µg/mL or 56 µg/mL, the PI was significantly lower as compared with native culture. The addition of CBF in the concentration range from 112 µg/mL to 400 µg/mL resulted in normaliza- tion of MSC culture growth. However, a significant difference from the native PI was found for the variant with 400 µg/mL CBF. As one can see, the cell damage after equilibration with DMSO was reversible, and cells may recover. At the 3rd day MI in the variant after equilibration with DMSO didn’t differ from native or CBF variants. The per- centage of pathological divisions of native MSC culture was about 40 %. No significant difference in the number of pathological divisions between all variants was found. Morphological differences were found in the variants with DMSO and CBF as compared with the control. They were manifested as cytoplasmic and nuclear vacuolations, changes of cell and nucleus shapes and nucleus-cytoplasm ratio. The cell morphology normalized after 24-h cul- tivation. CBF didn’t influence recovery rate of cell morphological abnormalities and the number of pathological divisions. Previously, it was shown that CBF affected cell cultures as a growth-stimulating factor. It increased cell culture growth via accelerating cell adhesion and activating mitosis. We also showed a direct stimulating effect of CBF on cell energy metabolism. So, both of the mechanisms could ameliorate cell damage after DMSO contact and con- tribute to recovery of cell culture growth. Conclusions. MSC culture growth indices significantly decreased after contact with 5 % DMSO. Cell morpholog- ical abnormalities caused by DMSO were the most manifested within the first 24 h of cultivation. The fraction (below 5 kDa) from cord blood at the concentration of 400 µg/mL normalized the MSC culture growth indices affected by contact with DMSO and did not influence cell morphological abnormalities.

equilibration, cultivation, DMSO, fraction (below 5 kDa) from cord blood, MSC

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