Cultivation and cryopreservation of rat stem cells and their interaction with lyophilised acellular matrix
DOI:
https://doi.org/10.61751/bmbr.2706-6290.2023.2.51Keywords:
in vitro culture, cryopreservation, umbilical cord, muscles, dermis, lyophilised matrix, pericardiumAbstract
With the rapid development of regenerative medicine in the 21st century, the study of the therapeutic potential
of stem cells in both preclinical research and clinical trials has become particularly relevant. Preclinical studies on
animals allow for a detailed understanding of the mechanisms of action of allogzeneic cell preparations, exploring their
regenerative activity, pharmacodynamics, and potential side effects. The purpose of the study was to select optimal
conditions for obtaining, cultivating, and cryopreserving mesenchymal stem cells from rats and analyse their interaction
with the lyophilised acellular matrix. The enzymatic method was applied to obtain primary cell cultures from the umbilical
cord, dermis, and muscles of Rattus norvegicus fetuses. Cell cultures were cultivated in vitro, and cell line proliferation rates
were analysed using an inverted microscope. In addition, cryopreservation was performed to store cellular materials. The
interaction of mesenchymal stem cells with an acellular matrix and cryopreservation of the obtained cells was at the 4 and
5th passages. It was shown that the optimal nutrient medium for cultivating the obtained lines of mesenchymal stem cells
from the umbilical cord and dermis of rat fetuses is DMEM/F12 Advanced. It was established that the method of thawing
the cell suspension by 10-fold dilution of dimethyl sulfoxide is more effective than the alternative method of immediate removal of cryoprotectant by centrifugation. The lyophilised acellular dermal matrix was found to have a cytotoxic effect
on all cultured rat cells, while the pericardial matrix showed a positive effect on the growth of the investigated cell lines.
Thus, the optimal nutrient medium and conditions for freezing/thawing of rat stem cells were selected, and the effect of
lyophilised acellular matrix, planned for therapeutic use, on the obtained cell lines was determined
Received: 30.03.2023 | Revised: 30.05.2023 | Accepted: 26.06.2023
References
Budash HV, Bilko NM. Embryonic and induced pluripotent stem cells and their differentiation in the cardiomyocyte direction in the presence of dimethyl sulfoxide. Cytol. Genet. 2019;53:34–41. DOI: 10.3103/S0095452719010055
Ray SK, Mukherjee S. Clinical practice of umbilical cord blood stem cells in transplantation and regenerative medicine – prodigious promise for imminent times. Recent Pat Biotechnol. 2022;16(1):16–34. DOI: 10.2174/1872208315666211026103227
Redko O, Dovhalyuk A, Dovbush A, Nebesna Z, Yakubyshyna L, Krynytska I. Liver injury associated with acute respiratory distress syndrome and the prospects of mesenchymal stem cells therapy for liver failure. Cell Organ Transpl. 2021;9(2):136–42. DOI: 10.22494/cot.v9i2.130
Börger V, Bremer M, Ferrer-Tur R, Gockeln L, Stambouli O, Becic A, Giebel B. Mesenchymal stem/stromal cell-derived extracellular vesicles and their potential as novel immunomodulatory therapeutic agents. Int J Mol Sci. 2017;18(7):1450. DOI: 10.3390/ijms18071450
Lopes-Pacheco M, Robba C, Rocco PRM, Pelosi P. Current understanding of the therapeutic benefits of mesenchymal stem cells in acute respiratory distress syndrome. Cell Biol Toxicol. 2020;36:83–2. DOI: 10.1007/s10565-019-09493-5
Chaleshtori SS, Dezfouli MRM, Fakhr MJ. Mesenchymal stem/stromal cells: The therapeutic effects in animal models of acute pulmonary diseases. Respir Res. 2020;21:110. DOI: 10.1186/s12931-020-01373-5
Cao J, Hou S, Ding H, Liu Z, Song M, Qin X, et al. In vivo Tracking of systemically administered allogeneic bone marrow mesenchymal stem cells in normal rats through bioluminescence imaging. 2016;3970942. DOI: 10.1155/2016/3970942
Miao J, Ren Z, Zhong Z, Yan L, Xia X, Wang J, Yang J. Mesenchymal stem cells: Potential therapeutic prospect of paracrine pathways in neonatal infection. J Interferon Cytokine Res. 2021 Oct;41(10):365–74. DOI: 10.1089/jir.2021.0094
Fu X, Liu G, Halim A, Ju Y, Luo Q, Song G. Mesenchymal stem cell migration and tissue repair. Cells. 2019;8(8):784. DOI: 10.3390/cells8080784
Keshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine. Stem Cell Res Ther. 2018;9(1):63. DOI: 10.1186/s13287-018-0791-7
Park KS, Bandeira E, Shelke GV, Lässer C, Lötvall J. Enhancement of therapeutic potential of mesenchymal stem cell-derived extracellular vesicles. Stem Cell Res Ther. 2019;10(1):288. DOI: 10.1186/s13287-019-1398-3
Alzahrani FA, Saadeldin IM, Ahmad A, Kumar D, Azhar EI, Siddiqui AJ, et al. The potential use of mesenchymal stem cells and their derived exosomes as immunomodulatory agents for COVID-19 patients. Stem Cells Int. 2020;8835986. DOI: 10.1155/2020/8835986
Bhattacharya SA, Prajapati BG. A review on cryoprotectant and its modern implication in cryonics. Asian J Pharm. 2016;10(3):1–6. Available from: https://www.researchgate.net/publication/305323767_A_Review_on_Cryoprotectant_and_its_Modern_Implication_in_Cryonics
Marquez-Curtis LA, Janowska-Wieczorek A, McGann LE, Elliott JAW. Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects. Cryobiology. 2015 Oct;71(2):181–97. DOI: 10.1016/j.cryobiol.2015.07.003
Awan M, Buriak I, Fleck R, Fuller B, Goltsev A, Kerby J, et al. Dimethyl sulfoxide: A central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regen Med. 2020;15(3):1463–91. DOI: 10.2217/rme-2019-0145
Fedoniuk LY, Kulyanda IS, Dovgalyuk AI, Lomakina YV, Kramar SB, Kulianda OO, Valko OO. Morphological characteristics of acellular dermal matrix manufacturing. Wiad Lek. 2021;74(3 p.I):418–22. DOI: 10.36740/WLek202103107
Krynytska IY, Dovhalyuk AI. Research on the regenerative potential of cellular therapy agents in acute respiratory distress syndrome. In: Ministry of Health [Internet]. 2023 [cited 2023 June 5]. Available from: https://www.tdmu.edu.ua/naukovo-doslidni-roboty-shho-finansuyutsya-ministerstvom-ohorony-zdorov-ya/
European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS No. 123). Available from: https://www.coe.int/en/web/conventions/full-list?module=treaty-detail&treatynum=123
Law of Ukraine No. 3447-IV “On Protection of Animals from Cruelty” (21.02.2006). Available from: https://zakon.rada.gov.ua/laws/show/3447-15#n45
Dzhyvak VG, Dovhalyuk AI, Paliy IR, Klishch IM. Research of stimulating efficiency of blood allogeneic plasma fractions on cell proliferation in vitro conditions. J Educ Health Sport. 2020;10(8):559–69. DOI: 10.12775/JEHS.2020.10.08.068
Heger JI, Froehlich K, Pastuschek J, Schmidt A, Baer C, Mrowka R, et al. Human serum alters cell culture behavior and improves spheroid formation in comparison to fetal bovine serum. Exp Cell Res. 2018 Apr 1;365(1):57–65. DOI: 10.1016/j.yexcr.2018.02.017
Kim JH, Jo CH, Kim HR, Hwang YI. Comparison of immunological characteristics of mesenchymal stem cells from the periodontal ligament, umbilical cord, and adipose tissue. Stem Cells Int. 2018 Apr 1;2018:8429042. DOI: 10.1155/2018/8429042
Ecke A, Lutter AH, Scholka J, Hansch A, Becker R, Anderer U. Tissue specific differentiation of human chondrocytes depends on cell microenvironment and serum selection. Cells. 2019 Aug;8(8):934. DOI: 10.3390/cells8080934
McGarvey SS, Ferreyros M, Kogut I, Bilousova G. Differentiating induced pluripotent stem cells toward mesenchymal stem/stromal cells. Methods Mol Biol. 2022;2549:153–67. DOI: 10.1007/7651_2021_383
Kohno Y, Lin T, Pajarinen J, Romero-Lopez M, Maruyama M, Huang JF, et al. Osteogenic ability of rat bone marrow concentrate is at least as efficacious as mesenchymal stem cells in vitro. J Biomed Mater Res B Appl Biomater. 2019;107(8):2500–6. DOI: 10.1002/jbm.b.34340
Mitchell A, Rivas KA, Smith R III, Watts AE. Cryopreservation of equine mesenchymal stem cells in 95% autologous serum and 5% DMSO does not alter post-thaw growth or morphology in vitro compared to fetal bovine serum or allogeneic serum at 20% or 95% and DMSO at 10% or 5%. Stem Cell Res Ther. 2015;6:231. DOI: 10.1186/s13287-015-0230-y
Lässer C, Jang SC, Lötvall J. Subpopulations of extracellular vesicles and their therapeutic potential. Mol Aspects Med. 2018 Apr;60:1–14. DOI: 10.1016/j.mam.2018.02.002
Mathew DD, Sharma AK, Kumar N, Kumar V, Maiti SK, Remya V, et al. Safety study of xenogenic mesenchymal stem cells seeded rat dermal matrix in healing of skin defects. Trends Biomater Artif Organs. 2021 Jul;35(2):148–60. Available from: https://www.researchgate.net/publication/343725075_Safety_study_of_xenogenic_mesenchymal_stem_cells_seeded_rat_dermal_matrix_in_healing_of_skin_defects
Nassiri Mansour R, Hasanzadeh E, Abasi M, Gholipourmalekabadi M, Mellati A, Enderami SE. The effect of fetal bovine acellular dermal matrix seeded with Wharton's Jelly mesenchymal stem cells for healing full-thickness skin wounds. Genes. 2023 Apr;14(4):909. DOI: 10.3390/genes14040909
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