Improved viability of freeze-thawed embryonic stem cells after exposure to glutathione
2010; Elsevier BV; Volume: 94; Issue: 6 Linguagem: Inglês
10.1016/j.fertnstert.2010.01.073
ISSN1556-5653
AutoresGil Ah Kim, Seung Tae Lee, Ji Yeon Ahn, Jong Heum Park, Jeong Mook Lim,
Tópico(s)Renal and related cancers
ResumoAdding a potent antioxidant, glutathione (GSH), to a cryoprotective solution consisting of dimethyl sulfoxide and ethylene glycol and/or postthaw culture medium significantly improved the postthaw viability of mouse embryonic stem cells. This effect, which was caused by a decrease in reactive oxygen species, was only induced by exposure of embryonic stem cells during cryopreservation. Adding a potent antioxidant, glutathione (GSH), to a cryoprotective solution consisting of dimethyl sulfoxide and ethylene glycol and/or postthaw culture medium significantly improved the postthaw viability of mouse embryonic stem cells. This effect, which was caused by a decrease in reactive oxygen species, was only induced by exposure of embryonic stem cells during cryopreservation. Stem cell banking is an important infrastructure for the clinical and industrial application of stem cell engineering, and various cryopreservation protocols for human (1Reubinoff B. Pera M. Vajta G. Trounson A. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method.Hum Reprod. 2001; 16: 2187-2194Crossref PubMed Scopus (280) Google Scholar, 2Ji L. de Pablo J.J. Palecek S.P. Cryopreservation of adherent human embryonic stem cells.Biotechnol Bioeng. 2004; 88: 299-312Crossref PubMed Scopus (122) Google Scholar, 3Richards M. Fong C. Tan S. Chan W. Bongso A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells.Stem Cells. 2004; 22: 779-789Crossref PubMed Scopus (120) Google Scholar, 4Zhou C.Q. Mai Q.Y. Li T. Zhuang G.L. Cryopreservation of human embryonic stem cells by vitrification.Chin Med J (Engl). 2004; 117: 1050-1055PubMed Google Scholar, 5Ha S. Jee B. Suh C. Kim H. Oh S. Kim S. et al.Cryopreservation of human embryonic stem cells without the use of a programmable freezer.Hum Reprod. 2005; 20: 1779-1785Crossref PubMed Scopus (65) Google Scholar, 6Suemori H. Yasuchika K. Hasegawa K. Fujioka T. Tsuneyoshi N. Nakatsuji N. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage.Biochem Biophys Res Commun. 2006; 345: 926-932Crossref PubMed Scopus (276) Google Scholar, 7Lee J, Lee J, Kim D, Yoon T, Chung H, Lee D. High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells. Fertil Steril. Published online Nov 18, 2008.Google Scholar, 8Li T. Zhou C. Liu C. Mai Q. Zhuang G. Bulk vitrification of human embryonic stem cells.Hum Reprod. 2008; 23: 358-364Crossref PubMed Scopus (44) Google Scholar, 9Li Y, Tan JC, Li LS. Comparison of three methods for cryopreservation of human embryonic stem cells. Fertil Steril. Published online December 22, 2008.Google Scholar, 10Li X. Meng G. Krawetz R. Liu S. Rancourt D.E. The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation.Stem Cells Dev. 2008; 17: 1079-1085Crossref PubMed Scopus (110) Google Scholar, 11Martin-Ibanez R. Unger C. Stromberg A.M. Baker D. Canals J.M. Hovatta O. Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor.Hum Reprod. 2008; 23: 2744-2754Crossref PubMed Scopus (111) Google Scholar, 12Li X.Y. Krawetz R. Liu S.Y. Meng G.L. Rancourt D.E. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells.Hum Reprod. 2009; 24: 580-589Crossref PubMed Scopus (139) Google Scholar, 13Martin-Ibanez R. Stromberg A.M. Hovatta O. Canals J.M. Cryopreservation of dissociated human embryonic stem cells in the presence of ROCK inhibitor.Curr Protoc Stem Cell Biol. 2009; (chapter 1:unit 1C.8)PubMed Google Scholar) and mouse (14Miszta-Lane H. Gill P. Mirbolooki M. Lakey J.R.T. Effect of slow freezing versus vitrification on the recovery of mouse embryonic stem cells.Cell Preserv Technol. 2007; 5: 16-24Crossref Scopus (5) Google Scholar, 15He X.M. Park E.Y.H. Fowler A. Yarmush M.L. Toner M. Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: a study using murine embryonic stem cells.Cryobiology. 2008; 56: 223-232Crossref PubMed Scopus (96) Google Scholar, 16Kashuba Benson C.M. Benson J.D. Critser J.K. An improved cryopreservation method for a mouse embryonic stem cell line.Cryobiology. 2008; 56: 120-130Crossref PubMed Scopus (38) Google Scholar, 17Kim G.A. Lee S.T. Lee E.J. Choi J.K. Lim J.M. Simplified slow freezing program established for effective banking of embryonic stem cells.Asian Aust J Anim Sci. 2009; 22: 343-349Google Scholar) embryonic stem cells (ESCs) have been developed. Recently, we suggested a simplified method for cryopreserving ESCs that does not employ a programmable freezer or a highly toxic cryoprotective solution (17Kim G.A. Lee S.T. Lee E.J. Choi J.K. Lim J.M. Simplified slow freezing program established for effective banking of embryonic stem cells.Asian Aust J Anim Sci. 2009; 22: 343-349Google Scholar). Optimizing this simplified protocol is necessary to further improve postthaw survival, which reaches a similar level to that of fresh ESCs. Optimizing the protocol may also minimize any unknown modification of stem cell activity that is important for self-renewal and differentiation. Long-term storage by cryopreservation induces mitochondria-specific damage (18Kim GA, Kim HY, Kim JW, Lee G, Lee E, Lim JM. Ultrastructural deformity of ovarian follicle induced by different cryopreservation protocols. Fertil Steril. Published online January 26, 2010.Google Scholar), which subsequently triggers the activation of apoptosis-related genes (19Parker G.C. Acsadi G. Brenner C.A. Mitochondria: determinants of stem cell fate?.Stem Cells Dev. 2009; 18: 803-806Crossref PubMed Scopus (62) Google Scholar) and the generation of reactive oxygen species (ROS) (20Bai J. Cederbaum A.I. Mitochondrial catalase and oxidative injury.Biol Signals Recept. 2001; 10: 189-199Crossref PubMed Scopus (107) Google Scholar, 21Du J. Daniels D.H. Asbury C. Venkataraman S. Liu J. Spitz D.R. et al.Mitochondrial production of reactive oxygen species mediate dicumarol-induced cytotoxicity in cancer cells.J Biol Chem. 2006; 281: 37416-37426Crossref PubMed Scopus (61) Google Scholar, 22Du C. Gao Z. Venkatesha V.A. Kalen A.L. Chaudhuri L. Spitz D.R. et al.Mitochondrial ROS and radiation induced transformation in mouse embryonic fibroblasts.Cancer Biol Ther. 2009; 8: 1962-1971Crossref PubMed Scopus (43) Google Scholar). In this study, we examined whether exposure of ESCs to an antioxidant during either cryopreservation or cryopreservation and postthaw culture could improve postthaw cell survival. All procedures for ESC manipulation followed the standard Seoul National University operation protocols. We added the potent antioxidant glutathione (GSH) as an antioxidant cryoprotectant. Dimethyl sulfoxide (DMSO) and ethylene glycol (EG) were also employed as cryoprotectants, and Dulbecco's minimal essential medium (DMEM) was used as the base medium for the cryoprotective solution (17Kim G.A. Lee S.T. Lee E.J. Choi J.K. Lim J.M. Simplified slow freezing program established for effective banking of embryonic stem cells.Asian Aust J Anim Sci. 2009; 22: 343-349Google Scholar) and postthaw ESC culture medium (23Cho M. Jang M. Lee E.J. Han J.Y. Lim J.M. An alternative method of deriving embryonic stem cell-like clones by aggregation of diploid cells with tetraploid embryos.Fertil Steril. 2006; 85: 1103-1110Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Postthaw cell viability and ROS production were monitored to evaluate cryopreservation efficiency. Two sets of experiments were conducted. First, ESCs were freeze-thawed and cultured at 37°C. The cryoprotective solution and ESC culture medium were concomitantly supplemented with or without 100 μM GSH, and cell viability was monitored at 0 and 72 hours after postthaw culture. Fresh ESCs were used as the control treatment. In the second set of experiments, ESCs were frozen in a GSH-free or GSH-containing cryoprotective solution, and GSH-free medium was used for postthaw culture. Postthaw cell viability and ROS generation were monitored at 72 hours after thawing. A generalized linear model (PROC-GLM) in the Statistical Analysis System software (SAS Institute, Cary, NC) was used, and P<.05 was considered statistically significant. In our study, E14 ESCs purchased from ATCC (Manassas, VA) were subcultured five times on mouse embryonic fibroblast (MEF) feeder layers in modified DMEM (GIBCO-Invitrogen, Carlsbad, CA), to which 15% (v/v) fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 1000 IU/mL of mouse leukemia inhibitory factor (LIF; Chemicon, Temecula, CA), 0.1 mM β-mercaptoethanol (GIBCO-Invitrogen), 1% (v/v) nonessential amino acids, 2 mM L-glutamine, and 1% (v/v) antibiotics solution were added. All medium substrates were purchased from Sigma-Aldrich Corp. (St. Louis, MO) unless stated otherwise. Medium osmolarity was adjusted within a range of 270–275 mOsm, and 5 × 103 ESCs were seeded in each well of multiwell plates containing MEFs. The medium for ESC culture was equilibrated at 37°C in 5% CO2 in air before use. After culturing, both ESCs and MEFs were treated with 0.25% (v/w) trypsin-EDTA (ethylene diaminetetra-acetic acid) solution for 5 minutes and were cryopreserved using different cryoprotective solutions. The DMSO (10%, v/v)–containing and EG (10%, v/v)–containing DMEM supplemented with 10% (v/v) FBS was used as a basic cryoprotective solution, to which 0 or 100 μM GSH was added. The ESCs exposed to 1 mL of cryoprotective solution were subsequently loaded into a 1.2-mL cryovial within 10 minutes after initial exposure, and the vials were placed at –80°C for 24 hours. The cryovials were then plunged directly into liquid nitrogen. For thawing, the cryovials were placed in a water bath at 37°C; immediately after thawing, the same volume (1 mL) of prewarmed medium was added to dilute the cryoprotectants. After the cell number was counted using a hemocytometer, ESC postthaw survival and proliferation were monitored with a colorimetric cell counting kit (CCK)-8 (Dojindo Molecular Technologies Inc., Gaithersburg, MD). Briefly, ESCs were separated from MEFs by discarding buoyant cells immediately after thawing, and the separated ESCs (3 × 103) were suspended in 0.2 mL of culture medium. After 4 hours of incubation in a well of a 96-well plate, the ESCs were treated with 10% (v/v) CCK-8 reagent for another 2 hours, and the color reaction was determined as absorbance at 490 nm using a microplate reader (Model-550; Bio-Rad Laboratories, Hercules, CA). To measure intracytoplasmic ROS production, ESCs harvested with trypsin/EDTA solution were incubated for 10 minutes with 2',7'-dichloro-dihydro-fluorescein diacetate (DCFHDA, 5 μg/mL) at 37°C (17Kim G.A. Lee S.T. Lee E.J. Choi J.K. Lim J.M. Simplified slow freezing program established for effective banking of embryonic stem cells.Asian Aust J Anim Sci. 2009; 22: 343-349Google Scholar). Treated cells (1 × 105) were placed in a 96-well plate, and the fluorescence intensity was measured at a 485-nm excitation wavelength and 530-nm emission wavelength using a multilabel microplate reader (Victor 3; Perkin Elmer, Waltham, MA). Adding GSH to both the cryoprotective solution and the postthaw culture medium significantly improved ESC viability; there was no significant difference in viability between fresh ESCs and the freeze-thawed ESCs at 0 hours (0.604 to 0.653; P>.1324) or 72 hours (0.626 to 0.671; P>.2998) after postthaw culture (Fig. 1A ). In contrast, ESCs that were freeze-thawed without GSH were less viable than fresh ESCs at both 0 hours (0.671 vs. 0.544; P<.0024) and 72 hours (0.742 vs. 0.590; P<.0138) (see Fig. 1B). As shown in Figure 1C, the beneficial effect of GSH on postthaw survival also occurred when ESCs were exposed to GSH only during cryopreservation and not during postthaw culture; no significant difference in viability was detected between fresh ESCs and the freeze-thawed ESCs exposed to GSH only during cryoprotection (0.665 vs. 0.612; P>.0841). However, there was a significant difference in viability between fresh ESCs and the freeze-thawed ESCs that were not exposed to GSH (0.665 vs. 0.535; P<.0002). The generation of ROS in GSH-treated (see Fig. 1D), freeze-thawed ESCs was statistically lower than that in freeze-thawed ESCs that were not exposed to GSH (2.249 vs. 3.101; P<.0001). Our data showing an increase in postthaw viability of ESCs contributes to optimizing this simplified method for ESC cryopreservation. 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Cryopreservation of adherent human embryonic stem cells.Biotechnol Bioeng. 2004; 88: 299-312Crossref PubMed Scopus (122) Google Scholar, 3Richards M. Fong C. Tan S. Chan W. Bongso A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells.Stem Cells. 2004; 22: 779-789Crossref PubMed Scopus (120) Google Scholar, 4Zhou C.Q. Mai Q.Y. Li T. Zhuang G.L. Cryopreservation of human embryonic stem cells by vitrification.Chin Med J (Engl). 2004; 117: 1050-1055PubMed Google Scholar, 5Ha S. Jee B. Suh C. Kim H. Oh S. Kim S. et al.Cryopreservation of human embryonic stem cells without the use of a programmable freezer.Hum Reprod. 2005; 20: 1779-1785Crossref PubMed Scopus (65) Google Scholar, 6Suemori H. Yasuchika K. Hasegawa K. Fujioka T. Tsuneyoshi N. Nakatsuji N. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage.Biochem Biophys Res Commun. 2006; 345: 926-932Crossref PubMed Scopus (276) Google Scholar, 7Lee J, Lee J, Kim D, Yoon T, Chung H, Lee D. High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells. Fertil Steril. Published online Nov 18, 2008.Google Scholar, 8Li T. Zhou C. Liu C. Mai Q. Zhuang G. Bulk vitrification of human embryonic stem cells.Hum Reprod. 2008; 23: 358-364Crossref PubMed Scopus (44) Google Scholar, 9Li Y, Tan JC, Li LS. Comparison of three methods for cryopreservation of human embryonic stem cells. Fertil Steril. Published online December 22, 2008.Google Scholar, 10Li X. Meng G. Krawetz R. Liu S. Rancourt D.E. The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation.Stem Cells Dev. 2008; 17: 1079-1085Crossref PubMed Scopus (110) Google Scholar, 11Martin-Ibanez R. Unger C. Stromberg A.M. Baker D. Canals J.M. Hovatta O. Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor.Hum Reprod. 2008; 23: 2744-2754Crossref PubMed Scopus (111) Google Scholar, 12Li X.Y. Krawetz R. Liu S.Y. Meng G.L. Rancourt D.E. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells.Hum Reprod. 2009; 24: 580-589Crossref PubMed Scopus (139) Google Scholar, 13Martin-Ibanez R. Stromberg A.M. Hovatta O. Canals J.M. Cryopreservation of dissociated human embryonic stem cells in the presence of ROCK inhibitor.Curr Protoc Stem Cell Biol. 2009; (chapter 1:unit 1C.8)PubMed Google Scholar). For a variety of cell lines, DMSO (1Reubinoff B. Pera M. Vajta G. Trounson A. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method.Hum Reprod. 2001; 16: 2187-2194Crossref PubMed Scopus (280) Google Scholar, 2Ji L. de Pablo J.J. Palecek S.P. Cryopreservation of adherent human embryonic stem cells.Biotechnol Bioeng. 2004; 88: 299-312Crossref PubMed Scopus (122) Google Scholar, 3Richards M. Fong C. Tan S. Chan W. Bongso A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells.Stem Cells. 2004; 22: 779-789Crossref PubMed Scopus (120) Google Scholar, 4Zhou C.Q. Mai Q.Y. Li T. Zhuang G.L. Cryopreservation of human embryonic stem cells by vitrification.Chin Med J (Engl). 2004; 117: 1050-1055PubMed Google Scholar, 5Ha S. Jee B. Suh C. Kim H. Oh S. Kim S. et al.Cryopreservation of human embryonic stem cells without the use of a programmable freezer.Hum Reprod. 2005; 20: 1779-1785Crossref PubMed Scopus (65) Google Scholar, 6Suemori H. Yasuchika K. Hasegawa K. Fujioka T. Tsuneyoshi N. Nakatsuji N. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage.Biochem Biophys Res Commun. 2006; 345: 926-932Crossref PubMed Scopus (276) Google Scholar, 7Lee J, Lee J, Kim D, Yoon T, Chung H, Lee D. High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells. Fertil Steril. Published online Nov 18, 2008.Google Scholar, 8Li T. Zhou C. Liu C. Mai Q. Zhuang G. Bulk vitrification of human embryonic stem cells.Hum Reprod. 2008; 23: 358-364Crossref PubMed Scopus (44) Google Scholar, 9Li Y, Tan JC, Li LS. Comparison of three methods for cryopreservation of human embryonic stem cells. Fertil Steril. Published online December 22, 2008.Google Scholar, 10Li X. Meng G. Krawetz R. Liu S. Rancourt D.E. The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation.Stem Cells Dev. 2008; 17: 1079-1085Crossref PubMed Scopus (110) Google Scholar, 11Martin-Ibanez R. Unger C. Stromberg A.M. Baker D. Canals J.M. Hovatta O. Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor.Hum Reprod. 2008; 23: 2744-2754Crossref PubMed Scopus (111) Google Scholar, 12Li X.Y. Krawetz R. Liu S.Y. Meng G.L. Rancourt D.E. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells.Hum Reprod. 2009; 24: 580-589Crossref PubMed Scopus (139) Google Scholar, 13Martin-Ibanez R. Stromberg A.M. Hovatta O. Canals J.M. Cryopreservation of dissociated human embryonic stem cells in the presence of ROCK inhibitor.Curr Protoc Stem Cell Biol. 2009; (chapter 1:unit 1C.8)PubMed Google Scholar, 14Miszta-Lane H. Gill P. Mirbolooki M. Lakey J.R.T. Effect of slow freezing versus vitrification on the recovery of mouse embryonic stem cells.Cell Preserv Technol. 2007; 5: 16-24Crossref Scopus (5) Google Scholar, 16Kashuba Benson C.M. Benson J.D. Critser J.K. An improved cryopreservation method for a mouse embryonic stem cell line.Cryobiology. 2008; 56: 120-130Crossref PubMed Scopus (38) Google Scholar) or a combination of DMSO and EG (5Ha S. Jee B. Suh C. Kim H. Oh S. Kim S. et al.Cryopreservation of human embryonic stem cells without the use of a programmable freezer.Hum Reprod. 2005; 20: 1779-1785Crossref PubMed Scopus (65) Google Scholar, 17Kim G.A. Lee S.T. Lee E.J. Choi J.K. Lim J.M. Simplified slow freezing program established for effective banking of embryonic stem cells.Asian Aust J Anim Sci. 2009; 22: 343-349Google Scholar) as cryoprotectants, cooling rates of 0.5° to 2.0°C (1Reubinoff B. Pera M. Vajta G. Trounson A. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method.Hum Reprod. 2001; 16: 2187-2194Crossref PubMed Scopus (280) Google Scholar, 2Ji L. de Pablo J.J. Palecek S.P. Cryopreservation of adherent human embryonic stem cells.Biotechnol Bioeng. 2004; 88: 299-312Crossref PubMed Scopus (122) Google Scholar, 3Richards M. Fong C. Tan S. Chan W. Bongso A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells.Stem Cells. 2004; 22: 779-789Crossref PubMed Scopus (120) Google Scholar, 4Zhou C.Q. Mai Q.Y. Li T. Zhuang G.L. Cryopreservation of human embryonic stem cells by vitrification.Chin Med J (Engl). 2004; 117: 1050-1055PubMed Google Scholar, 5Ha S. Jee B. Suh C. Kim H. Oh S. Kim S. et al.Cryopreservation of human embryonic stem cells without the use of a programmable freezer.Hum Reprod. 2005; 20: 1779-1785Crossref PubMed Scopus (65) Google Scholar, 6Suemori H. Yasuchika K. Hasegawa K. Fujioka T. Tsuneyoshi N. Nakatsuji N. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage.Biochem Biophys Res Commun. 2006; 345: 926-932Crossref PubMed Scopus (276) Google Scholar, 7Lee J, Lee J, Kim D, Yoon T, Chung H, Lee D. High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells. Fertil Steril. Published online Nov 18, 2008.Google Scholar, 8Li T. Zhou C. Liu C. Mai Q. Zhuang G. Bulk vitrification of human embryonic stem cells.Hum Reprod. 2008; 23: 358-364Crossref PubMed Scopus (44) Google Scholar, 9Li Y, Tan JC, Li LS. Comparison of three methods for cryopreservation of human embryonic stem cells. Fertil Steril. Published online December 22, 2008.Google Scholar, 10Li X. Meng G. Krawetz R. Liu S. Rancourt D.E. 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Fong C. Tan S. Chan W. Bongso A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells.Stem Cells. 2004; 22: 779-789Crossref PubMed Scopus (120) Google Scholar, 4Zhou C.Q. Mai Q.Y. Li T. Zhuang G.L. Cryopreservation of human embryonic stem cells by vitrification.Chin Med J (Engl). 2004; 117: 1050-1055PubMed Google Scholar, 5Ha S. Jee B. Suh C. Kim H. Oh S. Kim S. et al.Cryopreservation of human embryonic stem cells without the use of a programmable freezer.Hum Reprod. 2005; 20: 1779-1785Crossref PubMed Scopus (65) Google Scholar, 6Suemori H. Yasuchika K. Hasegawa K. Fujioka T. Tsuneyoshi N. Nakatsuji N. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage.Biochem Biophys Res Commun. 2006; 345: 926-932Crossref PubMed Scopus (276) Google Scholar, 7Lee J, Lee J, Kim D, Yoon T, Chung H, Lee D. High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells. Fertil Steril. Published online Nov 18, 2008.Google Scholar, 8Li T. Zhou C. Liu C. Mai Q. Zhuang G. Bulk vitrification of human embryonic stem cells.Hum Reprod. 2008; 23: 358-364Crossref PubMed Scopus (44) Google Scholar, 9Li Y, Tan JC, Li LS. Comparison of three methods for cryopreservation of human embryonic stem cells. Fertil Steril. Published online December 22, 2008.Google Scholar, 10Li X. Meng G. Krawetz R. Liu S. Rancourt D.E. The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation.Stem Cells Dev. 2008; 17: 1079-1085Crossref PubMed Scopus (110) Google Scholar, 11Martin-Ibanez R. Unger C. Stromberg A.M. Baker D. Canals J.M. Hovatta O. Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor.Hum Reprod. 2008; 23: 2744-2754Crossref PubMed Scopus (111) Google Scholar, 12Li X.Y. Krawetz R. Liu S.Y. Meng G.L. Rancourt D.E. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells.Hum Reprod. 2009; 24: 580-589Crossref PubMed Scopus (139) Google Scholar, 13Martin-Ibanez R. Stromberg A.M. Hovatta O. Canals J.M. Cryopreservation of dissociated human embryonic stem cells in the presence of ROCK inhibitor.Curr Protoc Stem Cell Biol. 2009; (chapter 1:unit 1C.8)PubMed Google Scholar, 14Miszta-Lane H. Gill P. Mirbolooki M. Lakey J.R.T. Effect of slow freezing versus vitrification on the recovery of mouse embryonic stem cells.Cell Preserv Technol. 2007; 5: 16-24Crossref Scopus (5) Google Scholar, 16Kashuba Benson C.M. Benson J.D. Critser J.K. An improved cryopreservation method for a mouse embryonic stem cell line.Cryobiology. 2008; 56: 120-130Crossref PubMed Scopus (38) Google Scholar, 17Kim G.A. Lee S.T. Lee E.J. Choi J.K. Lim J.M. Simplified slow freezing program established for effective banking of embryonic stem cells.Asian Aust J Anim Sci. 2009; 22: 343-349Google Scholar) have been widely used. However, conventional freeze-thawing methods are efficient for only some ESC lines (2Ji L. de Pablo J.J. Palecek S.P. Cryopreservation of adherent human embryonic stem cells.Biotechnol Bioeng. 2004; 88: 299-312Crossref PubMed Scopus (122) Google Scholar, 3Richards M. Fong C. Tan S. Chan W. Bongso A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells.Stem Cells. 2004; 22: 779-789Crossref PubMed Scopus (120) Google Scholar, 4Zhou C.Q. Mai Q.Y. Li T. Zhuang G.L. Cryopreservation of human embryonic stem cells by vitrification.Chin Med J (Engl). 2004; 117: 1050-1055PubMed Google Scholar, 5Ha S. Jee B. Suh C. Kim H. Oh S. Kim S. et al.Cryopreservation of human embryonic stem cells without the use of a programmable freezer.Hum Reprod. 2005; 20: 1779-1785Crossref PubMed Scopus (65) Google Scholar, 6Suemori H. Yasuchika K. Hasegawa K. Fujioka T. Tsuneyoshi N. Nakatsuji N. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage.Biochem Biophys Res Commun. 2006; 345: 926-932Crossref PubMed Scopus (276) Google Scholar, 7Lee J, Lee J, Kim D, Yoon T, Chung H, Lee D. High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells. Fertil Steril. Published online Nov 18, 2008.Google Scholar, 8Li T. Zhou C. Liu C. Mai Q. Zhuang G. Bulk vitrification of human embryonic stem cells.Hum Reprod. 2008; 23: 358-364Crossref PubMed Scopus (44) Google Scholar, 9Li Y, Tan JC, Li LS. Comparison of three methods for cryopreservation of human embryonic stem cells. Fertil Steril. Published online December 22, 2008.Google Scholar, 10Li X. Meng G. Krawetz R. Liu S. Rancourt D.E. The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation.Stem Cells Dev. 2008; 17: 1079-1085Crossref PubMed Scopus (110) Google Scholar, 11Martin-Ibanez R. Unger C. Stromberg A.M. Baker D. Canals J.M. Hovatta O. Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor.Hum Reprod. 2008; 23: 2744-2754Crossref PubMed Scopus (111) Google Scholar, 12Li X.Y. Krawetz R. Liu S.Y. Meng G.L. Rancourt D.E. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells.Hum Reprod. 2009; 24: 580-589Crossref PubMed Scopus (139) Google Scholar, 13Martin-Ibanez R. Stromberg A.M. Hovatta O. Canals J.M. Cryopreservation of dissociated human embryonic stem cells in the presence of ROCK inhibitor.Curr Protoc Stem Cell Biol. 2009; (chapter 1:unit 1C.8)PubMed Google Scholar, 14Miszta-Lane H. Gill P. Mirbolooki M. Lakey J.R.T. Effect of slow freezing versus vitrification on the recovery of mouse embryonic stem cells.Cell Preserv Technol. 2007; 5: 16-24Crossref Scopus (5) Google Scholar, 16Kashuba Benson C.M. Benson J.D. Critser J.K. An improved cryopreservation method for a mouse embryonic stem cell line.Cryobiology. 2008; 56: 120-130Crossref PubMed Scopus (38) Google Scholar, 17Kim G.A. Lee S.T. Lee E.J. Choi J.K. Lim J.M. Simplified slow freezing program established for effective banking of embryonic stem cells.Asian Aust J Anim Sci. 2009; 22: 343-349Google Scholar). In our previous study (17Kim G.A. Lee S.T. Lee E.J. Choi J.K. Lim J.M. Simplified slow freezing program established for effective banking of embryonic stem cells.Asian Aust J Anim Sci. 2009; 22: 343-349Google Scholar), the viability of mouse ESCs decreased from 90% to 60% within the first 3 days of postthaw culture, and osmotic shock (40Woods E. Liu J. Derrow C. Smith F. Williams D. Critser J. Osmometric and permeability characteristics of human placental/umbilical cord blood CD34 cells and their application to cryopreservation.J Hematother Stem Cell Res. 2000; 9: 161-173Crossref PubMed Scopus (56) Google Scholar, 41Liu J. Christian J.A. Critser J.K. 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