- Volume 31 Issue 3
DOI QR Code
The importance of post-thaw subculture for standardizing cellular activity of fresh or cryopreserved mouse embryonic stem cells
- Ko, Dong Woo ;
- Yoon, Jung Ki ;
- Ahn, Jong il ;
- Lee, Myungook ;
- Yang, Woo Sub ;
- Ahn, Ji Yeon ;
- Lim, Jeong Mook
- Received : 2017.04.17
- Accepted : 2017.08.07
- Published : 2018.03.01
Objective: Remarkable difference in cellular activity was found between early and late subpassaged embryonic stem cell (ESCs) lines, which can be created by subtle changes in cell manipulation protocol. This study subsequently examined whether post-thaw subculture of early subpassaged ESC lines could further affect the activity of the ESCs. Methods: Fresh (as a control treatment) or cryopreserved F1 hybrid (B6CBAF1) early ESC lines (C57BL/6xCBA) of the 4 (P4) or the 19 passage (P19) were subcultured once, twice or six times under the same condition. The post-thaw survival of the ESCs was monitored after the post-treatment subculture and the ability of cell proliferation, reactive oxygen species (ROS) generation, apoptosis and mitochondrial ATP synthesis was subsequently examined. Results: Regardless of the subculture number, P19 ESCs showed better (p<0.05) doubling time and less ATP production than P4 ESCs and such difference was not influenced by fresh or cryopreservation. The difference between P4 and P19 ESC lines became decreased as the post-treatment subculture was increased and the six times subculture eliminated such difference. Similarly, transient but prominent difference in ROS production and apoptotic cell number was detected between P4 and P19 ESCs only at the 1st subculture after treatment, but no statistical differences between two ESC lines was detected in other observations. Conclusion: The results of this study suggest that post-thaw subculture of ESCs under the same environment is recommended for standardizing their cellular activity. The activity of cell proliferation ability and ATP synthesis can be used as parameters for quality control of ESCs.
Apoptosis;ATP;Cryopreservation;Embryonic Stem Cells;Mouse;Reactive Oxygen Species
- Bardelli S, Moccetti M. Stem Cell banking and its impact on cardiac regenerative medicine. Adv Exp Med Biol 2016;951:163-78.
- Hilkens P, Driesen RB, Wolfs E, et al. Cryopreservation and banking of dental stem cells. Adv Exp Med Biol 2016;951:199-235.
- Woods EJ, Perry BC, Hockema JJ, et al. Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use. Cryobiology 2009;59:150-7.
- Perry BC, Zhou D, Wu X, et al. Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use. Tissue Eng Part C Methods 2008;14:149-56.
- Stacey G. Banking stem cells for research and clinical applications. Prog Brain Res 2012;200:41-58.
- Sun C, Yue J, He N, et al. Fundamental principles of stem cell banking. Adv Exp Med Biol 2016;951:31-45.
- Murphy A, McKenna D, McCullough J. Cord blood banking and quality issues. Transfusion 2016;56:645-52. https://doi.org/10.1111/trf.13388
- Vaidya A, Singhania S. Quality control measures in cord blood banking in India - critical appraisal and recommendations. J Stem Cells 2013;8:105-13.
- Diaferia GR, Cardano M, Cattaneo M, et al. The science of stem cell biobanking: investing in the future. J Cell Physiol 2012;227:14-9. https://doi.org/10.1002/jcp.22732
- Kim GA, Lee ST, Ahn JY, Park JH, Lim JM. Improved viability of freeze-thawed embryonic stem cells after exposure to glutathione. Fertil Steril 2010;94:2409-12. https://doi.org/10.1016/j.fertnstert.2010.01.073
- International Stem Cell Banking I. Consensus guidance for banking and supply of human embryonic stem cell lines for research purposes. Stem Cell Rev 2009;5:301-14. https://doi.org/10.1007/s12015-009-9085-x
- Ilic D, Ogilvie C. Human embryonic stem cells-what have we done? What are we doing? Where are we going? Stem Cells 2017;35:17-25. https://doi.org/10.1002/stem.2450
- Kadota S, Aiba K, Nakatsuji N. Embryonic stem cell research. Nihon Rinsho 2011;69:2109-13.
- Nakamura Y. Bio-resource of human and animal-derived cell materials. Exp Anim 2010;59:1-7. https://doi.org/10.1538/expanim.59.1
- Healy LE, Ludwig TE, Choo A. International banking: checks, deposits, and withdrawals. Cell Stem Cell 2008;2:305-6.
- Nottola SA, Albani E, Coticchio G, et al. Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes. J Assist Reprod Genet 2016;33:1559-70. https://doi.org/10.1007/s10815-016-0798-x
- Morgenstern DA, Ahsan G, Brocklesby M, et al. Post-thaw viability of cryopreserved peripheral blood stem cells (PBSC) does not guarantee functional activity: important implications for quality assurance of stem cell transplant programmes. Br J Haematol 2016;174:942-51. https://doi.org/10.1111/bjh.14160
- Smagur A, Mitrus I, Giebel S, et al. Impact of different dimethyl sulphoxide concentrations on cell recovery, viability and clonogenic potential of cryopreserved peripheral blood hematopoietic stem and progenitor cells. Vox Sang 2013;104:240-7. https://doi.org/10.1111/j.1423-0410.2012.01657.x
- Woods EJ, Thirumala S, Badhe-Buchanan SS, Clarke D, Mathew AJ. Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use. Cytotherapy 2016;18:697-711. https://doi.org/10.1016/j.jcyt.2016.03.295
- Pal R, Totey S, Mamidi MK, Bhat VS, Totey S. Propensity of human embryonic stem cell lines during early stage of lineage specification controls their terminal differentiation into mature cell types. Exp Biol Med (Maywood) 2009;234:1230-43. https://doi.org/10.3181/0901-RM-38
- Park YB, Kim YY, Oh SK, et al. Alterations of proliferative and differentiation potentials of human embryonic stem cells during long-term culture. Exp Mol Med 2008;40:98-108. https://doi.org/10.3858/emm.2008.40.1.98
- Enver T, Soneji S, Joshi C, et al. Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells. Hum Mol Genet 2005;14:3129-40. https://doi.org/10.1093/hmg/ddi345
- Maitra A, Arking DE, Shivapurkar N, et al. Genomic alterations in cultured human embryonic stem cells. Nat Genet 2005;37:1099-103. https://doi.org/10.1038/ng1631
- Huang HL, Hsing HW, Lai TC, et al. Trypsin-induced proteome alteration during cell subculture in mammalian cells. J Biomed Sci 2010;17:36.
- Li Y, Ma T. Bioprocessing of cryopreservation for large-scale banking of human pluripotent stem cells. Biores Open Access 2012;1:205-14. https://doi.org/10.1089/biores.2012.0224
- Miki T, Wong W, Zhou E, et al. Biological impact of xeno-free chemically defined cryopreservation medium on amniotic epithelial cells. Stem Cell Res Ther 2016;7:8.
- Sart S, Ma T, Li Y. Cryopreservation of pluripotent stem cell aggregates in defined protein-free formulation. Biotechnol Prog 2013;29:143-53. https://doi.org/10.1002/btpr.1653
- Kim GA, Kim HY, Kim JW, et al. Ultrastructural deformity of ovarian follicles induced by different cryopreservation protocols. Fertil Steril 2010;94:1548-50.e1. https://doi.org/10.1016/j.fertnstert.2009.12.029
- Wagh V, Meganathan K, Jagtap S, et al. Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing. Stem Cell Rev 2011;7:506-17. https://doi.org/10.1007/s12015-011-9230-1
- Li XY, Jia Q, Di KQ, et al. Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation. Cell Tissue Res 2007;327:607-14. https://doi.org/10.1007/s00441-006-0354-6
Supported by : Ministry for Food, Agriculture, Forestry and Fisheries, National Research Foundation of Korea (NRF)