ISSN 2415-3060 (print), ISSN 2522-4972 (online)
  • 26 of 42
JMBS 2020, 5(2): 185–191

Cytomorphological Disorders in the Cell Culture of Rabbit Dermal Papilla after Incubation with Dimethysulphoxide

Novikova O. Yu., Bondarenko T. P.

Most of modern mammalian cell cryopreservation protocols use dimethyl sulfoxide solutions as cryoprotectant. That is why we studied the effect of dimethyl sulfoxide on cell culture quality after interaction and cryopreservation. Dermal papilla vibrissa is a source of pluripotent cells neural crest derivatives. Because of pluripotency this type of cell is of interest for transplantation, which is associated with the need for their long-term cultivation and storage. In this work, we studied the pathological changes in the cells of the diploid culture of the dermal papilla of the rabbit vibrissa that occur at the interphase and persist for 2 passages after exposure with cryoprotective media based on different concentrations of dimethyl sulfoxide. Material and methods. The cell culture of dermal papilla was obtained by the method of explants from hair follicles of vibrissa of newborn rabbits. Passaging of received adhesive cultures was performed every 7-10 days. Contact with dimethyl sulfoxide was performed for 20 minutes, after which the cells were placed in standard culture medium for further growth. The calculation of interphase pathologies was carried out on fixed cytological preparations stained with hematoxylin-eosin. Results and discussion. The incubation in a cryoprotective medium containing 5 and 7.5% was safe from the point of view of inducing cytological disorders. Dimethyl sulfoxide in concentrations exceeding 10% led to a dose-dependent increase in cell pathologies. Among the pathologies, violations most often occurred in the association with defects in membranes and the cytoskeleton such as vacuolization of the cytoplasm, the formation of microvesicles on the membrane. There were also disorders affecting the genetic and protein-synthesizing apparatus: polyploid, multinucleated cells, cells with abnormal nuclei, with micronuclei, at various terminal stages of apoptosis. Based on a comparison of cultures at passages 1 and 2 after exposure to dimethyl sulfoxide, it was found that the detected violations were not eliminated during cultivation, but were stored and accumulated. Conclusion. The study showed that protein-peptide additives were capable of exerting a protective effect on the background of high concentrations of dimethyl sulfoxide, which contributed to the preservation of cell viability, but led to the accumulation of anomalies in the culture during further cultivation.

Keywords: dermal papilla, mitosis, pathology of division, dimethyl sulfoxide, cryopreservation

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  1. Yuan C, Gao J, Guo Bai J, Marshall L, Cai Z, Wang L, et al. Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes. PLoS One. 2014; 9(9): e107447. PMID: 25238609. PMCID: PMC4169574.
  2. Wang H, Scott RE. Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO). Cell Prolif. 1993; 26: 55–66. PMID: 8439589.
  3. Zemlianskykh NG. Effect of substances with cryoprotective properties on surface marker CD44 in human erythrocytes. Tsitol Genet. 2016; 50(3): 66¬ –79. PMID: 30480411.
  4. Yazici I, Molski M, Siemionow MZ. Cryopreservation in Plastic Surgery. Our Experience and Review of the Literature. Experimental Models and Research Designs. London. Springer; 2015. p. 255-64.
  5. Kajiura S, Mii S, Aki R, Hamada Yu, Arakawa N, Kawahara K, et al. Cryopreservation of the Hair Follicle Maintains Pluripotency of Nestin-Expressing Hair Follicle-Associated Pluripotent Stem Cells. Tissue Eng Part C Methods. 2015; 21(8): 825–31. PMID: 25743086. PMCID: PMC4523096.
  6. Cao W, Li L, Tran B, Kajiura S, Amoh Y, Liu F, et al. Extensive Hair Shaft Growth after Mouse Whisker Follicle Isolation, Cryopreservation and Transplantation in Nude Mice. PLoS One. 2015; 10(12): e0145997. PMID: 26716690. PMCID: PMC4696652.
  7. Kajiura S, Mii S, Aki R, Hamada Y, Arakawa N, Kawahara K, et al. Protocols for Cryopreservation of Intact Hair Follicle That Maintain Pluripotency of Nestin-Expressing Hair-Follicle-Associated Pluripotent (HAP) Stem Cells. Methods Mol Biol. 2016; 1453: 173–8. PMID: 27431257.
  8. Hill RP, Gledhill K, Gardner A, Higgins CA, Crawford H, Lawrence C, et al. Generation and characterization of multipotent stem cells from established dermal cultures. PLoS One. 2012; 7(11): e50742. PMID: 23226372. PMCID: PMC3511366.
  9. Rajaraman R, Guernsey DL, Rajaraman MM, Rajaraman SR. Stem cells, senescence, neosis and self-renewal in cancer. Cancer Cell Int. 2006; 6: 21–5. PMID: 17092342. PMCID: PMC1664585.
  10. Dludla PV, Jack B, Viraragavan A, Pheiffer C, Johnson R, Louw J, et al. A dose-dependent effect of dimethyl sulfoxide on lipid content, cell viability and oxidative stress in 3T3-L1 adipocyte. Toxicology Reports. 2018; 5: 1014–20. PMID: 30364542. PMCID: PMC6197677.
  11. Song YM, Song SO, Jung YK, Kang ES, Cha BS, Lee HC, et al. Dimethyl sulfoxide reduces hepatocellular lipid accumulation through autophagy induction. Autophagy. 2012; 8: 1085–97. PMID: 22722716. PMCID: PMC3429545.
  12. Notman R, Noro M, O'Malley B, Anwar J. Molecular basis for dimethylsulfoxide (DMSO) action on lipid membranes. J Am Chem Soc. 2006; 128(43): 13982–3. PMID: 17061853.
  13. Cheng CY, Song J, Pas J, Meijer LH, Han S. DMSO induces dehydration near lipid membrane surfaces. Biophys J. 2015; 109(2): 330–9. PMID: 26200868. PMCID: PMC4621616.
  14. Najafian L, Babji AS. A review of fish-derived antioxidant and antimicrobial peptides: their production, assessment, and applications. Peptides. 2012: 33(1): 178-85. PMID: 22138166.
  15. Ruan R, Zou L, Sun S, Liu J, Wen L, Gao D, et al. Cell blebbing upon addition of cryoprotectants: a self-protection mechanism. PLoS One. 2015; 10(4): e0125746. PMID: 25875076. PMCID: PMC4395349.
  16. De Abreu Costa L, Henrique Fernandes Ottoni M, Dos Santos MG, Meireles AB, Gomes de Almeida V, de Fátima Pereira W, et al. Dimethyl Sulfoxide (DMSO) Decreases Cell Proliferation and TNF-α, IFN-γ, and IL-2 Cytokines Production in Cultures of Peripheral Blood Lymphocytes. Molecules. 2017; 22(1): E1789. PMID: 29125561. PMCID: PMC6150313.
  17. Finkenstaedt-Quinn SA, Ge S, Haynes CL. Cytoskeleton dynamics in drug-treated platelets. Anal Bioanal Chem. 2015; 407(10): 2803–9. PMID: 25701419. PMCID: PMC4370791.
  18. Castedo M, Perfettini JL, Medema J P, Kroemer G. Cell death by mitotic catastrophe: a molecular definition. Oncogene. 2004; 23: 2825–37. PMID: 15077146.
  19. Na J, Baker D, Zhang J, Andrews PW, Barbaric I. Aneuploidy in pluripotent stem cells and implications for cancerous transformation. Protein Cell. 2014; 5: 569–79. PMID: 24899134. PMCID: PMC4130921.