1 Department of Obstetrics and Gynecology, College of Medicine, 2 Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, 110744 and 3 Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, 463707, Korea
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam, Kyunggi-do, 463707, Korea. Email: suhcs{at}snu.ac.kr
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Abstract |
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Key words: cryopreservation/dimethylsulphoxide/ethylene glycol/human embryonic stem cells
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Introduction |
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Recently, human ES cells were derived from human blastocysts (Thomson et al., 1998; Reubinoff et al., 2000
), and were found to be pluripotent and to have the ability to self-renew. These human ES cells are expected to have far-reaching applications in the areas of regenerative medicine, pharmacology, and basic scientific research (Reubinoff et al., 2001
). Human ES cells offer tremendous potential for clinical applications as an unlimited source of cells for transplantation and tissue regeneration. In addition, they offer the possibilities of studying basic developmental science and cell signalling.
In order to use human ES cells in research, satisfactory cryopreservation technologies are crucial. Moreover, efficient cryopreservation is essential for the establishment of a human ES cell bank (Gearhart, 1998). Several researchers have suggested that the characteristics of human ES cells may change during long-term culture (Andrews, 2004
; Buzzard et al., 2004
). Therefore, the maintenance of early passage human ES cells is important, and it may be possible to preserve stocks of early passage cells by effective freezing and thawing.
Two methods are used to cryopreserve ES cell lines: slow freezingrapid thawing (Whittingham et al., 1972; Trounson and Mohr, 1983
; Kaufman et al., 1995
) and vitrification (Rall and Fahy, 1985
; Karlsson, 2002
). The slow freezingrapid thawing method has been commonly used to cryopreserve mouse ES cells (Robertson, 1987
). Slow freezing protocols are straightforward and can be easily applied to the cryopreservation of ES cells or embryos. In addition, a large cell volume could be frozen in one vial. Moreover, when large volumes of cells are required for applications such as drug screening or clinical applications, slow freezing may be beneficial. In addition, although these two methods are effective for murine ES cells (Kaufman et al., 1995
; Reubinoff et al., 2001
), it has been observed that the survivals of human ES cells subjected to slow freezingrapid thawing are poor (Reubinoff et al., 2001
; Kim et al., 2004
). Sometimes, the conventional slow freezing method requires a programmable embryo freezing module designed to freeze mammalian embryos at a controlled rate of 0.40.6 °C/min (Shaw et al., 1995
). However, programmable freezers are relatively expensive, and not always available.
Vitrification is a simple cryopreservation method and has been widely used to cryopreserve mammalian embryos. Recently, some groups have reported on the cryopreservation efficiencies of human ES cells that have been exposed to modified vitrification procedures (Reubinoff et al., 2001; Kim et al., 2004
; Richards et al., 2004
; Zhou et al., 2004
). However, vitrification is not an easy option because the steps required are more complicated than those of the slow freezing method. In addition, only small cell volume can be cryopreserved using this technique, and therefore it is unsuitable for clinical applications. Moreover, Reubinoff et al. (2001)
reported that vitrified human ES cells showed considerable cell death and spontaneous differentiation after thawing or plating.
Thus in the present study, we undertook to develop a simple and convenient mass cryopreservation method using a freezing container instead of an expensive programmable freezer. Although combination of dimethylsulphoxide (DMSO) and ethylene glycol (EG) was shown to be efficient in vitrification of human ES cells (Reubinoff et al., 2001) we examined the survival efficiency of human ES cells using various combinations of cryoprotectants, namely DMSO, EG and glycerol. During this work we tried to reduce the serum content based on the belief that it acts as a differentiation stimulator of human ES cells. The developed freezing method can cryopreserve large numbers of human ES cells using simple steps.
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Materials and methods |
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Human ES cell culture
The human ES cell line, SNUhES-3, was used to evaluate the efficiency of the mass cryopreservation of human ES cells. Human ES cells were cultured on an STO feeder layer in gelatin-coated tissue culture dishes. The culture medium consisted of DMEM and F-12 (1:1) supplemented with 20% Knock-out serum replacement (SR), 0.1 mmol/l -mercaptoethanol, 1% non-essential amino acids, 50 IU/ml penicillin and 50 µg/ml streptomycin (all from Gibco, USA) and 0.4 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen, USA). The colonies were dissociated into several clumps by mechanical slicing using a glass pipette and subcultured on fresh STO feeder layers every 7 days.
Cryopreservation of human ES cells
Human ES cell colonies were dissected into several small clumps with a glass pipette 5 days after replating. After removing the culture medium by brief centrifugation, 100 clumps of human ES cells were transferred into a 2 ml cryo-vial (Sarstedt, Germany) containing 1 ml of pre-cooled (4 °C) freezing medium. The cryo-vials were then immediately placed into a freezing container (Mr. Frosty; Nalgene, USA), and cooled to 70 °C within a deep freezer. Although the temperature within the deep freezer dropped at a rate of 1 °C/min, the real cooling rate of the freezing container was 0.5 °C/min, as indicated in the manufacturer's manual. After keeping overnight, the cryo-vials were plunged into and stored in liquid nitrogen. One week later, the cryo-vials were rapidly thawed in a 37 °C water bath. The freezing medium was gradually diluted with 4 ml of ES culture medium, and then the human ES cell clumps were washed by gentle pipetting. After removing the suspension by brief centrifugation, ES culture medium was added to the clumps, which were then plated onto a fresh STO feeder layer. The growth medium was renewed on the 3rd day after thawing and then daily.
Several freezing media were prepared to compare survival rates. Initially, three cryoprotectants were compared; 5 and 10% dimethylsulphoxide (DMSO; Sigma, USA), 5% and 10% ethylene glycol (EG; Sigma, USA), or 5% glycerol (Sigma, USA). The mix was made up to 100% with FBS (vol/vol). In this experiment, 5% DMSO showed the highest survival rate in the presence of 95% FBS (Table I, Experiment I). Subsequently, the effects of different FBS concentrations were tested, i.e. 95, 50 or 5%; the mix was made up to 100% with DMEM/F-12, the basic human ES cell growth medium. After determining the optimal FBS content, we prepared freezing medium mixtures by adding either EG or glycerol to 5% DMSO + optimal FBS.
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Characterization of cryopreserved human ES cells
Immunocytochemistry
Three passages after thawing, surface markers for human ES cells were defined. Colonies that had originated from cryopreservedthawed human ES cells were fixed in 4-well culture dishes containing 4% paraformaldehyde for 30 min at room temperature. The primary antibodies used for immunocytochemistry were stage-specific embryonic antigens (SSEA) and undifferentiated ES cell surface markers, i.e. SSEA-1, SSEA-3 and SSEA-4, and tumour rejection antigens (TRA)-1-60 and TRA-1-81 (all from Chemicon, USA). Antibody localization was detected by using Vectastain ABC reagent and a DAB kit (Vector Laboratories, Inc., USA).
Alkaline phosphatase (AP) activity was demonstrated at the same passages as above. Cryopreservedthawed human ES cells were fixed in a 4-well culture dish using citrateacetate formaldehyde fixative and incubated with FRV-alkaline, sodium nitrite and naphtol AS-BI alkaline solution mixture for 15 min at room temperature (Sigma, USA).
RT-PCR of Oct-4 and Nanog
Total RNA was extracted using a QIAGEN RNeasy kit (QIAGEN, USA). Standard reverse transcription reactions were performed with 300 ng of total RNA with random hexamers using SuperScriptTM First-Strand Synthesis System (Invitrogen, USA). The PCR was performed using a rtaq PCR master mix (TaKaRa, Japan). Primer sequences are shown in Table II. After incubation for 5 min and initial denaturation for 30 s at 94 °C, 30 amplification cycles were performed (annealing for 30 s at 61 °C (Oct-4) or 56 °C (Nanog), extension for 30 s at 72 °C), and this was followed by a final extension for 7 min at 72 °C. Successful PCR was confirmed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.
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The differentiation potentials of cryopreserved human ES cells were confirmed by RTPCR. Total RNA extraction, cDNA systhesis and PCR were carried out using the conditions previously described for the RTPCR of Oct-4 and Nanog. To detect the differentiation potential of EB to the three germ layers, -fetoprotein (AFP), Brachyury and PAX-6 primer were used as representative markers of the three embryonic germ layers, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard (Table II).
Statistical analysis
The MannWhitney U-test in SPSS for Windows (version 10.0) was used to compare means, and the 2-test was used to compare proportions. P<0.05 (two-tailed) was considered statistically significant.
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Results |
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Impact on survival rate of adding EG or glycerol to 5% DMSO+50% FBS
EG has been successfully used as a cryoprotectant for the vitrification of mammalian embryos or ES cells. However, the majority of previous studies have used only EG for vitrification. Thus, we examined the uses of EG or glycerol for the slow freezing of human ES cells. At 10 days after thawing, the mean survival rate was significantly higher for 5% DMSO+50% FBS+10% EG compared to other combinations (Table I, Experiment III). The rate was also significantly higher than that in 5% DMSO+50% FBS. Moreover, the survival rates obtained for 5% DMSO+50% FBS+10% EG were rather consistent at: 31, 25, 31, 26, 34 and 34%. In contrast, no colonies were observed after adding 20% EG or 520% glycerol to 5% DMSO+50% FBS.
Characterization of cryopreservedthawed human ES cells
In vitro colony morphology of human ES cells
Three days after thawing, human ES cells started to form small colonies, and at 10 days after thawing, the sizes of the colonies were similar to day 7 human ES cell colonies that had not been frozen (Figure 1).
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Discussion |
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We compared the survival rates of the cells treated with DMSO, EG and glycerol containing basic cryoprotectants. In our experiment, DMSO showed the highest survival rate, which is consistent with other reports concerning the superiority of DMSO (Chen et al., 2001; Chi et al., 2002
). Furthermore, 5% DMSO showed a better survival rate compared with 10% DMSO, which has been commonly used in slow freezing protocols of human ES cells.
Serum is a complex mixture and probably contains various compounds that are beneficial and detrimental to human ES cells. Moreover, different serum batches vary widely in their ability to support the vigorous undifferentiated proliferation of human ES cells. Thus, replacing serum with defined components should reduce the experimental variability associated with serum batch variations (Amit et al., 2000). To maintain the undifferentiated state of human ES cells, most have used SR instead of serum (Chiu and Rao, 2003
). In our experiment, we reduced the content of FBS from 95% to 50% or 5%. In this trial, 95% FBS showed the highest survival rate. However, no significant difference was observed between the survival rates of 95% FBS- and 50% FBS-treated cells. Serum is essentially needed for the cryopreservation of human ES cells, and freezing ES cells without FBS is associated with a poor survival rate. According to our results, the optimal content of FBS may be
50%, and here we considered 50% FBS as sufficient for human ES cell cryopreservation.
Ethylene glycol, a rapid permeable cryoprotectant, appears to have a low toxic effect on mice and human embryos (Ali and Shelton, 1993; Chi et al., 2002
). It is also used in human ES cell vitrification solutions combined with DMSO (Reubinoff et al., 2001
). In our experiment, an improved survival rate was obtained only after adding 10% EG to freezing medium containing 5% DMSO+50% FBS. Mean survival rate increased 3-fold versus 5% DMSO+50% FBS and this was consistently achieved. This finding indicates that cryopreservation efficiency was both remarkably increased and stabilized by adding 10% EG. Since equimolar combinations of DMSO and EG have been commonly used in vitrification of human ES cells, we also tested the efficiency of 5% DMSO combined with 5% EG. However, an improvement was not observed in survival rate. Furthermore, no colonies were observed after adding 520% glycerol to 5% DMSO.
We concluded that 5% DMSO with 10% EG is a more effective cryoprotectant for slow freezing of human ES cells even at 50% FBS levels. It is not certain why 5% DMSO with 10% EG is superior to 5% DMSO alone or in other combinations. It is likely that EG may work efficiently with a lower level of DMSO and FBS in slow freezing methods using a freezing container.
A recent report indicates that loading human ES cells with the disaccharide trehalose prior to cryopreserving in a DMSO-containing cryoprotectant solution improves cell viability, although the cells adherent to, or embedded in, a Matrigel matrix are cultured and cryopreserved (Ji et al., 2004). Trehalose is a high mol. wt sugar and one of the non-penetrating cryoprotectants. The use of trehalose could reduce the toxic effect of DMSO by dehydrating cells. It will be further investigated whether the treatment of trehalose could be useful in the slow freezing method of human ES cells.
In summary, we applied slow freezing to cryopreserve human ES cells using a mixture of cryoprotectants without using a programmable freezer. Our results indicate that the addition of 10% EG to 5% DMSO+50% FBS produced satisfactory survival rates using our developed technique for human ES cells. Our results also show that the mass cryopreservation of human ES cells may be possible without the cells contacting liquid nitrogen directly. The key properties of human ES cells, namely, proliferative ability and pluripotency, were maintained after cryopreservation using the developed method. Since this method is similar to those used for other mammalian cells, it can be easily performed in many laboratory settings. The method could be used to store stocks of early ES cell passages, and transfer cells to other laboratories, and thus we hope that it contributes to the widespread use of human ES cell lines.
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Acknowledgements |
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This study was also supported by the research fund of the College of Medicine, Seoul National University (Grant No. 8002002116).
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References |
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Submitted on January 15, 2005; resubmitted on February 11, 2005; accepted on February 18, 2005.
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