Hum. Reprod. Advance Access originally published online on December 14, 2007
Human Reproduction 2008 23(2):358-364; doi:10.1093/humrep/dem386
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Bulk vitrification of human embryonic stem cells
Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, People's Republic of China
1 Correspondence address. Tel: +86-20-87755766-8362; Fax: +86-20-87755766-8365; E-mail: zhoucanquan{at}gmail.com
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Abstract |
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BACKGROUND: The traditional vitrification method cannot keep up with the increased culture and propagation efficiency required to cryopreserve large quantities of vigorously proliferating human embryonic stem (HES) cells. In this study, we describe a newly invented vitrification carrier for cryopreserving large amount of HES cells and evaluate whether this bulk vitrification (BV) method is as effective as the popular open-pulled straw (OPS) vitrification method.
METHODS: HES cell clumps were harvested after passage and transferred to a cell strainer; only those clumps with a diameter more than 70 µm were included in the study and randomly selected to be cryopreserved by the BV method, OPS vitrification or slow freezing method. HES cell survival, growth and pluripotency were analyzed after thawing.
RESULTS: Bulk vitrification method with cell strainer could cryopreserve 136 ± 23.4 cell clumps at one time (round), which was 30 times as high as those for OPS method (4 ± 1.5). After thawing, bulk-vitrified HES cells exhibited high survival rate up to 94.3%, comparable with the OPS method. All surviving cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of post-thawed, bulk-vitrified HES cell clumps, confirming pluripotency.
CONCLUSIONS: This new BV method could cryopreserve a large quantity of HES cell clumps at one time, which not only would satisfy routine cryopreservation of HES cell during daily culture process but also guarantee researchers have large quantity of efficiently cryopreserved HES cells ready for a scheduled study at any time.
Key words: cryopreservation/human embryonic stem cells/vitrification/cell strainer
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementReferences |
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Given the unlimited self-renewal potential of human embryonic stem (HES) cells, combined with the recently improved culture system (culture medium improvement, propagation method modification) (Inzunza et al., 2005
; Kim et al., 2005; Ding et al., 2006; Lu et al., 2006; Rajala et al., 2007), they can be split 1:3
1:6 every 4–7 days and thus produce a large amount of HES cells from only two to three continued passages. If not directly put into a study, these precious cells would be carefully cryopreserved for later use whenever possible. Moreover, these cryopreserved cells could be transferred between research centers, promoting scientific collaboration and facilitating widespread use of the cells for research and clinical application (Gearhart, 1998; Pera and Trounson, 2004).
As they are highly sensitive to cryo-injury, the HES cell cryopreservation method has been extensively studied at recent years (Reubinoff et al., 2001; Richards et al., 2004; Ha et al., 2005; Zhou et al., 2004; Heng et al., 2006; Yang et al., 2006). Among the methods, open-pulled straw (OPS) vitrification method has been shown to be highly efficient and reliable since 2001, compared with conventional slow freezing and rapid thaw protocol with cryovials (CV) (Reubinoff et al., 2001). It has proved to be valuable for the cryopreservation of the very low number of HES cell clones at earlier stages of HES cell research, when most of the cell clones were propagated by mechanical cutting, and fetal bovine serum (FBS) included in the culture media often induced unexpected differentiation during the culture and passage process, leaving a limited number of cell clones available for harvesting and cryopreservation. However, as they were originally designed for the cryopreservation of a very limited number of oocytes and embryos (Vajta et al., 1998; Lane et al., 1999), the mini-straw and other vitrification carriers, such as cryoleaf, cryoloop, etc., were too small to hold more than 10 cell clumps (Reubinoff et al., 2001; Liebermann and Tucker, 2002; Mavrides and Morroll, 2002). Furthermore, it would take extra time to transfer cell clumps by Pasteur pipettes to different freezing/thawing media if more than five of them were to be cryopreserved at one time, which would cause extra damage to the cryopreserved cells. Thus at the present time, the traditionally used vitrification method is no longer suitable for the daily cryopresevation of HES cells. It could not keep up with the increased culture and propagation efficiency required to meet the increasing needs of cryopreserved HES cells for further study. This meant that the relatively inefficient slow freezing with CV was the only practical choice for routinely cultured HES cell clones.
In this study, a cell strainer was used to cryopreserve HES cells by the vitrification method. Since it is made of nylon mesh and cup-shaped, the cell strainer can hold not only small numbers but also a large quantity of HES cell clumps, and cools down very quickly when put into liquid nitrogen. This method combines the large holding volume of the CV with the high efficiency of the vitrification method. Herein we show that this method satisfactorily harvests and efficiently cryopreserves vigorously proliferating HES cells during routine culture and propagation processes and ensures sufficient HES cells available for study at any time.
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Materials and Methods |
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HES cell culture
The HES cell line used in this study was
-thalassemia carrier embryonic stem cell (-ES-C), newly established in our research centre, and derived from an -thalassemia embryo diagnosed by preimplantation genetic diagnosis (Jiao et al., 2003; Li et al., 2005; Deng et al., 2006). Culture medium consisted of 80% knockout Dulbecco's modified Eagle's medium (KO-DMEM) (no pyruvate, Gibco/BRL, Invitrogen, Carlsbad, CA, USA) supplemented with 20% knockout serum replacement (KSR, Gibco/BRL), 1 mmol/l β-mercaptoethanol (Sigma, USA), 1% non-essential amino acid stock (Gibco/BRL) and 4 ng/ml human recombinant basic fibroblast growth factor (bFGF). -ES-C colonies were grown on mitomycin-C-inactivated mouse embryo fibroblasts (MEFs) and were propagated every 4–5 days by a 1:6 split after collagenase IV treatment (Li et al., 2005).
Cryopreservation of HES cells
HES cell colonies were passaged as mentioned above, and cell clumps were collected for cryopreservation. Approximately 250–300 -ES-C cell clumps per 35 mm dish (Falcon 3001, Becton Dickinson, Lincoln, Park, NJ, USA) were harvested and randomly selected batches were cryopreserved using our bulk vitrification (BV) method, OPS vitrification or slow freezing.
Bulk vitrification protocol
Three vitrification solutions (VSs) were used: basic medium (BM), vitrification solution 1 (VS1) and vitrification solution 2 (VS2). The BM was a medium routinely used to culture HES cells. VS1 was BM supplemented with 10% dimethylsulfoxide (DMSO, Sigma D2650), 10% ethylene glycol. And VS2 was a BM supplemented with 20% DMSO, 20% ethylene glycol (EG, Sigma E9129) and 0.5 mol/l sucrose. The thawing solutions were stepwise solutions consisting of BM supplemented with 0.2 mol/l sucrose (TS1), 0.1 mol/l sucrose (TS2) and finally no sucrose. All freezing and thawing procedures were performed on a heating stage at 37°C.
Before freezing, vitrification medium was added to dishes (cat. no. 3001; 3 ml of BM, VS1 or VS2 per dish; one medium per dish) and incubated at 37°C, 5% CO2. Cell clumps were harvested as described above for routine passage and transferred as a suspension of clumps in BM to our cell strainer. Small clumps (diameter <70 µm) were washed through our cell strainer (70 µm nylon, 352350, BD FalconTM) and therefore excluded from cryopreservation. The cell strainer was first transferred to VS1 medium with a tweezer and incubated for 1 min and then to VS2 for 25 s and finally to liquid nitrogen directly (Fig. 1). After 2 days in liquid nitrogen, the cell strainer was retrieved. At 10 s after removal from liquid nitrogen, the cell strainer was submerged in TS1 for 1 min, then TS2 for 5 min and finally BM twice for 5 min each before being plated onto a fresh feeder layer.
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Slow freezing protocol and OPS method
All slow freezing procedures were performed at room temperature. Two kinds of media were used: holding medium (i.e. 50% KO-DMEM plus 50% KSR) and freezing medium (i.e. 20% DMSO and 80% KSR).
Cell clumps were harvested after routine passage and washed through a cell strainer. Only those clumps with a diameter more than 70 µm were frozen. With the slow conventional method, HES cell clumps were first suspended in holding medium for 5 min and then mixed with the same volume of pre-cooled (4°C) freezing medium. Cell clumps were transferred to a 2 ml CV (Nalge Nunc, Naperville, IL, USA). The vials were slowly cooled (1°C/min) in a freezing container (Nalgene; Nalge Nunc) to –80°C and then plunged into and stored in liquid nitrogen. Two days later, the vials were rapidly thawed in a water bath at 37°C. The freezing medium was gradually diluted with HES cell culture medium. Thawed HES cell clumps were then transferred to a 15 ml tube and centrifuged at 20g/min for 5 min. The supernatant was discarded. The pellet was suspended gently in HES culture medium and immediately plated onto a fresh feeder layer.
The OPS vitrification method were carried out according to that of Reubinoff et al. (2001).
Assessment of HES cell survival, growth and differentiation
Cryopreserved HES cells in three groups were thawed 2 days after freezing. All of the thawed cell clumps were plated onto fresh feeder layers regardless of the clump size. After 48 h of culture, those cells/cell clumps still floating and not attached to the feeder were regarded as dead cells and discarded. To evaluate the influence of different cryopreservation protocols on the survival of HES cells, the plating efficiency or survival rate (the number of attached clumps/the number of thawed clumps) was calculated for each method.
Post-thaw colonies were observed and scored for differentiation by visual inspection. The growth characteristics of HES cell colonies in terms of shape, thickness, fragility and extent of differentiation were carefully recorded at low and high magnifications. The growth and differentiation status of each colony in the three groups were evaluated on the seventh day after plating. Undifferentiated HES cells have a high ratio of nucleus to cytoplasm and prominent nucleoli, and they form flat colonies of individual distinct cells. Differentiated HES cells have a large volume of cytoplasm and a relatively small nucleus. According to the levels of differentiation and viability, colonies were regarded as grade A if they remained >80% undifferentiated, grade B if 50–80% undifferentiated, grade C if <50% undifferentiated and grade D if either dead or lysed with no growth. Only grades A and B colonies were suitable and used for additional serial passaging.
Identifying the pluripotence of vitrified HES cells
At passage 6 after BV, ES cell surface markers including SSEA-1 (MC-480), SSEA-3 (MC-631), SSEA-4 (MC-813-70) and TRA-1-80, TRA-1-60 (related reagents from Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA) were determined by immunohistochemical tests. Reverse transcription (RT) was carried out to investigate the presence of undifferentiation-specific transcription factors of HES cell, such as OCT-4, Rex-1 and Nanog, along with markers of different germ lineages expressed in spontaneously differentiated HES cells (embryo bodies). Imprinted genes were also checked to investigate the effects of vitrification on vitrified HES cells. Polymerase chain reaction (PCR) was carried out using the primers as described in Table I.
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To test the capacity for differentiation in vivo, bulk-vitrified
-ES-C cells at passage 9 on MEFs were injected subcutaneously into three mice with severe combined immunodeficiency disease (SCID). Two teratomas were isolated 12 weeks after injection and subjected to histological analysis.
Statistical analysis
Student's t-test and 
2 analysis were carried out using the SPSS 10.0 statistical package. The data are presented as mean ± standard error. A P-value of <0.05 was considered statistically significant.
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Results |
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In all, 1546 HES cell clumps with diameter more than 70 µm were harvested from HES cell colonies cultured in six dishes (35 mm) and cryopreserved for 2 days. In the BV group, the cryo-container/cell strainer was loaded with 136 ± 23.4 cell clumps each (Table II), which was 30 times higher than that loaded in a pulled straw in the OPS group (4 ± 1.5). In other words, a large number of HES cell clumps (250–300) from a 35 mm dish could be loaded in only two cell strainers and cryopreserved with two rounds of freezing protocol, taking no more than 5 min.
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Furthermore, cell clumps could be observed directly under the dissecting microscope once they were taken out of liquid nitrogen and no transfer procedure with Pasteur pipette was needed during the thawing process. So the recovery rate was high in the BV group no matter how many cell clumps were frozen. In the present study, the recovery rate was not significantly different between three groups. However, when more HES cell colonies were to be frozen, the real advantage of high recovery rate with cell strainer was shown.
As expected, conventional slow freezing with CV was the least effective method for HES cell cryopreservation, although large quantity of HES cell colonies could also be frozen by this method (Table II). Only 38.6% of cell clumps survived after thawing on Day 2, which was significantly lower than that of BV and OPS group (P < 0.0001). Of the cells, 28.3% of the cells showed high level of differentiation (grade C), significantly higher than the BV group (P < 0.001), but not significantly different from the OPS group (P = 0.141). Furthermore, only 7.6% was undifferentiated (grades A and B), much lower than that of both BV and CV group (P < 0.0001) (Tables II and III). Therefore, cryopreserving a small number of HES cell colonies with this method is risky because it is highly possible that no HES colony will develop after plating.
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In contrast, a dramatically improved outcome was observed when our BV method was used. Survival/plating efficiency up to 94.3% was achieved and this was not different from that of OPS group (P = 0.922) (Table II), which means that most of bulk-vitrified HES cell clumps attached to MEFs 48 h after thawing with little fragmentation. All attached cell clumps generated HES cell colonies (the majority of them were undifferentiated grades A and B colonies). Only grades A and B colonies could be passaged further, and the percentage of them could indicate efficiency of different freezing method. In the study, the percentage of grade A, grade B and grade A plus grade B between the BV and OPS groups was not significantly different, P = 0.613, P = 0.485, P = 0.114, respectively. Even the differentiation rate (grade C, grade C+D) was a little lower using the strainer method compared with the OPS method (not significant, P = 0.217, P = 0.123, respectively, Table III). Bulk vitrification using the cell strainer proved to be as effective as OPS vitrification and at the same time allowed the large holding volume of CV as used in slow freezing.
Bulk and OPS vitrification was also associated with some cell death, which was evident on Day 2 by the presence of dark cells scattered throughout attached colonies and the significantly reduced mean area of the colonies at Days 4 and 7 after plating, compared with unfrozen control colonies. However, an additional day in culture was sufficient to overcome the vitrification-induced cell deficit as reported by Reubinoff et al. (2001). After the first passage, there was no significant difference in cell morphology, growth rate and differentiation status between bulk-vitrified-thawed HES cells and unfrozen control cells.
Post-thawed HES cell colonies maintained expression of undifferentiated cell markers, such as OCT-4, Rex-1 and Nanog, and embryonic bodies (EBs) derived from frozen-thawed -ES-C cells expressed markers of their characteristic three germ layers indicating their differentiation capacity, as determined by TaqMan RT–PCR analysis (Fig. 2A), Furthermore, they also correctly expressed paternally derived imprinted genes (IGF-2), and maternally derived imprinted genes (UBE3A and H19) (Fig. 2B) Post-thawed HES cell colonies expressed surface markers for undifferentiated HES cells, such as SSEA-4 (Fig. 3), TRA-1-60 and TRA-1-81 (Fig. 3), predominantly in the colonies but not in the differentiated stroma-like cells. Teratomas comprising all three primordial germ layers were formed in SCID mice after subcutaneous injection of undifferentiated post-thawed -ES-C colony fragments, confirming pluripotency (Fig. 4).
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Discussion |
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Stem cell research has become an area of constant progress and improvement in recent years (Chin et al., 2007
; Skottman et al., 2007; Watanabe et al., 2007). The characteristics of HES cells have become valuable not only for research in human development, gene control, pharmacological testing and other basic research approaches, but also for future therapeutic indications. However, all these potential scientific benefits would not have been possible without a highly efficient cryopreservation method available in the routine culture process.
As they are highly sensitive to cryo-injury, a vitrification method is a better choice for HES cell cryopreservation than conventional slow freezing and rapid thawing (Reubinoff et al., 2001; Kuleshova and Lopata, 2002; Richards et al., 2004). With the vitrification approach, a glass-like solidification of the freezing solution is achieved by using a high concentration of cryopretectant and rapid cooling. Although this approach can eliminate cell injury due to ice crystal formation, the high concentration of cryopretectant may induce significant toxic and osmotic damage. Therefore, to minimize the cryopretectant damage, maximizing the cooling rate was prerequisite for successful vitrification. A maximized cooling rate has currently been achieved by plunging small-volume samples held on tiny carries, such as thin-walled OPS (Reubinoff et al., 2001), electron microscopy grids, and small nylon loops (Mavrides and Morroll, 2002), directly into liquid nitrogen.
Although these tiny carriers have given promising results for oocyte and embryo cryopreservation, and are quite efficient for very small number of HES clones (Reubinoff et al., 2001; Richards et al., 2004), they are not suitable for the daily cryopreservation of HES cells at the present time. They cannot even hold surplus HES cell colonies during routine passage, let alone those large amounts of HES cells propagated and cryopreserved for a scheduled study. Moreover, transferring cell clumps from one dish to another dish using Pasteur pipettes is time-consuming, and would cause toxic and osmotic damage to cells.
To overcome these limitations, we used a cell strainer (70 µm nylon, 352350, BD Falcon TM) to freeze HES cells. This cell strainer was cup-shaped and made of nylon mesh. After being submerged in liquid nitrogen, the cells cool down very quickly, just as in vitrification with much larger electron microscopy grids. In this study, each cell strainer was loaded with cell colonies harvested from half of a 35 mm dish (136 ± 23.4 cell clumps), which was 30 times that loaded in a pulled straw in OPS group (4 ± 1.5). Considering it might decrease the cooling rate, we have not tried to freeze more cell clumps in a cell strainer.
As it is cup-shaped, it is much easy to hold many cell clumps without the use of Pasteur pipettes when transferring cells from one freezing/thawing medium to another. Additionally, this little 'cup' has a small handle that can easily be held by tweezers. The step-by-step transfer of the cell strainer loaded with HES cells between freezing/thawing media and put into liquid nitrogen or retrieved from liquid nitrogen (in the case of thawing) can be carried out with tweezers. The need for cell clumps transfer by Pasteur pipette was eliminated and transfer time and cell exposure to highly toxic cryoprotectants was minimized. It was a much easier, quicker and simpler way to freeze cells. Furthermore, this little handle could be dyed different colors to indicate particular cell lines and could assist the search for HES cells stored in the liquid nitrogen freezer. More importantly, the size of the cell strainer permits both large and small amounts of HES cells to be frozen at one time. In this study, 8–15 HES cell clumps were cryopreserved in a strainer. Cell clumps can be easily found and picked from the strainers, for transfer to dishes, under a dissecting microscope. Finally, the method is inexpensive (i.e. does not require expensive programmable freezing machines).
With this BV method, recovery of HES cell colonies after thawing was excellent with a high survival rate and low differentiation rate, comparable with those of the OPS method. Bulk-vitrified HES cell colonies continued to express markers of pluripotency and formed teratomas in SCID mice after warming and subsequent culture, confirming that they maintain their bona fide stem cell properties after vitrification and warming. The results are highly reproducible, as reflected by the precision of the replicates.
Unlike the OPS, the cell strainer retained only those cell clumps larger than 70 µm during the freezing and thawing process. However, collagenase IV treatment disaggregated HES cell colonies into different sized cell clumps. Although small cell clumps (<70 µm) could pass through the cell strainer, the number of these was minimized by gentle pipetting after collagenase IV treatment. In this study, only cell clumps with diameter >70 µm were used to compare the efficiency of different cryopreservation methods. The small cell clumps (diameter <70 µm) were plated onto fresh feeder layers for use in later BVs.
Although this BV method is easy and efficient to perform, it has limitations. Cells had to be in direct contact with liquid nitrogen, increasing the possibility of contamination and cell infection (Tedder et al., 1995; Hawkins et al., 1996). At present, although sterile sources of liquid nitrogen are available, maintaining aseptic conditions while working with liquid nitrogen is costly and cumbersome. Thus, modification of this protocol is needed to solve this problem.
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Funding |
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Guangdong Natural Science Foundation (No. 06300778); National Natural Science Foundation of China (No. 30571956).
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Acknowledgement |
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We would like to thank Dr Renli Zhang for his assistance in the preparation of the figures.
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References |
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Submitted on June 15, 2007; resubmitted on September 17, 2007; accepted on September 20, 2007.
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