| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a Department of Obstetrics and Gynaecology, and
b Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore
Key Words. Closed straws • Cryovials • Human embryonic stem cells • Liquid nitrogen vapor Slow machine freezing • Undifferentiated • Vitrification
Correspondence: Ariff Bongso, D.Sc., Ph.D., Department of Obstetrics and Gynaecology, National University of Singapore, Kent Ridge, Singapore 119260. Telephone: 65-7724260; Fax: 65-7794753; e-mail: obgbongs{at}nus.edu.sg
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Several attempts at improving hESC culture conditions have been reported. These advances include the use of conditioned media together with MatrigelTM as an attachment substrate for hESC culture [1] and the derivation and propagation of hESC lines on human feeder layers [2–6]. These improvements are important steps forward in developing a CGMP-compliant protocol for the establishment of xeno-free clinically compliant hESC lines. The derivation of xeno-free CGMP-compliant hES lines also necessitates the development of a cryopreservation protocol that is effective and minimizes or restricts the possibility of cell line contamination in long-term liquid nitrogen (LN2) storage.
Numerous reports have been published describing the contamination of frozen blood and cells with adventitious agents, primarily viruses, in LN2 storage tanks [7]. The cross-contamination of frozen cell stock from one cell line with other cell types in LN2 storage tanks has also been well documented [8]. Several viruses, including the hepatitis B virus, have also been reported to survive well in LN2 [9, 10].
Two freezing protocols are currently used for hESCs. These include (a) the conventional slow stepwise programmed freezing method using cryovials (CVs) and storage in LN2 and (b) a snap-freezing vitrification method using an open pulled straw (OPS) and storage in LN2[11].
Although controlled rate freezing is popular for most somatic cell types, its use for hESCs has been shown to result in low thaw-survival rates and low plating efficiencies [11], presumably because of ice crystal formation during the cooling process that will disrupt cell-cell adhesion. Also, CVs are traditionally used in controlled-rate freezing methods, and LN2 seepage into such tightly sealed screw-cap CVs often occurs in long-term storage. Vitrification, on the other hand, which works on the principle of glass induction instead of ice crystal formation, is simple, quick, and inexpensive. It has also been proven to consistently yield higher plating efficiencies than controlled-rate freezing methods. However, the OPS vitrification method, although simple and efficient, is not an ideal protocol for cryopreserving CGMP-compliant hESC lines, because it is nonsterile [11]. This method involves direct contact of hESCs with LN2 via the open end of the pulled straw, thereby increasing the possibility of hESC contamination and infection. Sterile sources of LN2 are available, but maintaining aseptic conditions while working with LN2 is costly, cumbersome, and impractical.
Current cryopreservation methods also use fetal calf serum (FCS) containing xenoproteins in the formulation of freezing/vitrification and thawing/warming solutions. Therefore, the development of an effective, safe, and sterile cryopreservation protocol is a prerequisite for the long-term storage of CGMP- and GTCP-compliant xeno-free hESC lines in stem cell banks.
Hence, in this report, we sought to refine an established vitrification strategy for hESCs and compared its efficiency with the conventional controlled-rate freezing method. We document the successful vitrification of hESCs in sealed closed straws (CSs), their storage in the vapor phase of LN2 (VLN2), and the substitution of FCS with human serum albumin (HSA) as the major protein source in the cryoprotectant.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
.
|
Solutions for hESC Slow Freezing and Thawing andVitrification andWarming
Slow Freezing in CVs For slow freezing of hES colony fragments in CVs, conventional freezing medium comprising 90% FCS and 10% dimethylsulfoxide (DMSO; Hybrimax, sterile filtered, endo-toxin, and hybridoma tested, Sigma D2650) was used. Alternatively, commercially available cell culture freezing media (Gibco, catalog # 11101–011) was found to be a suitable substitute.
Vitrification in Straws The holding medium, ES-HEPES-HSA, consisted of 80% DMEM (vol/vol) (high-glucose DMEM, Invitrogen, catalog #11960–044) and 20% HSA (vol/vol) (HSA-solutionTM,Vitrolife, Goteberg, Sweden, #10064 containing purified HSA at 100 mg/ml), buffered to 20 mM HEPES (1 M HEPES solution, Invitrogen, catalog #15630–080). The holding medium with sucrose, ES-HEPES-HSA-sucrose, was comprised of 1 M sucrose solution in ES-HEPES-HSA medium (e.g., 3.42 g sucrose in 10 ml ES-HEPES-HSA). Vitrification solution 1 (VS1) was comprised of 80% ES-HEPES-HSA (vol/vol), 10% DMSO (vol/vol) (DMSO, hybrimax, sterile filtered, endotoxin and hybridoma tested, Sigma D2650), and 10% EG (vol/vol) (Ethylene Glycol, Sigma E9129). Vitrification solution 2 (VS2) was comprised of 30% ES-HEPES-HAS (vol/vol), 30% ES-HEPES-HSA-sucrose (vol/vol), 20% DMSO (vol/vol), and 20% EG (vol/vol). Warming solution 1 (WS1) was comprised of 80% ES-HEPES-HSA (vol/vol) and 20% ES-HEPES-HSA-sucrose (vol/vol). Warming solution 2 (WS2) was comprised of 90% ES-HEPES-HSA (vol/vol) and 10% ES-HEPES-HSA-sucrose (vol/vol).
All solutions were sterile filtered before use. HSA was substituted for FCS in experimental arms. FCS was retained as a protein source in controls. Sterile embryo straws, 250-mL volume (Pailette Cristal, 0459, #006433), were purchased from Cryo Bio System, Groupe I.M.V. Technologies, Paris. OPS straws were purchased from Demtek, Aarhus, Denmark. These commercial straws are sold sterile with one end sealed with a cotton plug.
Method for Slow Freezing and Thawing in CVs
Approximately 10 to 15 hES colony fragments were placed inside a 1-ml CV (Nunclon, Roskilde, Denmark) containing 500 µL of conventional freezing medium. The CV containing the hES colony fragments was then placed inside a programmable freezing machine (Planar, London, U.K.) equilibrated at a steady temperature of 4°C. The cooling cycle was set at a decrease of 1°C/min to –30°C. After a 5-minute holding period at –30°C, the CVs were plunged directly and stored in LN2. CVs were thawed rapidly by removing a CV from LN2 storage and plunging directly into a 37°C water bath.
Method for Vitrification and Thawing in OPSs
Protocols developed by Reubinoff et al. [11] for OPS vitrification and thawing were used.
Method for Vitrification and Warming in CSs
Vitrification Dissected colony fragments of hESCs were vitrified in clumps of approximately 300–400 cells as soon as possible to prevent the clumps from sticking together. After dissection, hES clumps were washed once in modified phosphate-buffered saline (PBS+) (Invitrogen) and then transferred into an organ culture dish containing holding medium to wash off PBS.
Vitrification was performed at room temperature in a four-well sterile tissue culture dish (Nunclon) containing holding medium and vitrification solutions (wells one through three), as shown in Figure 1. Five to eight colony fragments were first transferred to well one of the four-well dish containing holding medium using a sterile glass Pasteur pipette (colony clumps can be kept in this solution for at least 20–30 minutes). An embryo straw was then loaded through the open end with a column of VS2 (~20 mm) using the load ing/unloading syringe device (Fig. 2). A column of air (~5 mm) was then aspirated. The straw attached to the syringe device was then left on a pipette rest for later use. The hESC fragments were then transferred from well 1 to 2 (VS1; Fig. 1) and left for 1 minute. A 20-µl drop of VS2 solution was then aliquoted into the center of a 35-mm Falcon Petri dish. After the 1-minute exposure to VS1 in well two, the hES clumps were transferred quickly to VS2 in well three for 5 seconds (Fig. 1). Using a fresh glass pipette, the clumps were then immediately removed from well three with minimum VS2 and transferred to the fresh 20-µl drop of VS2 in the 35-mm Petri dish for immediate loading into the embryo straw (Fig. 2). This was followed by an air column (~5 mm) and then a column of WS1 (~20 mm; Fig. 2). A pair of cold forceps that had been chilled in ice was then used to hold the straw at the VS2-hESC column firmly while sealing both ends of the straw with a commercial plastic bag heat sealer. After the straw had been completely sealed, it was plunged and stored in either the LLN2 orVLN2.
|
|
Warming
Warming was performed in a four-well tissue culture dish (Nunclon) with thaw solutions and holding medium, as shown in Figure 3. Straws were removed from VLN2 or LLN2 storage and then plunged immediately into a beaker containing water at room temperature. After the columns in the straw had thawed (~5 seconds), the straw was removed from the beaker and swabbed with a 70% isopropanol sterile wipe. Sterile scissors were used to cut off the sealed ends of the straw, and the entire contents of the straw was expelled into WS1 in well one of another four-well dish (Fig. 3) for 1 minute at room temperature. The hESC fragments were then transferred into WS2 in well two (Fig. 3) for 5 minutes at room temperature. Finally, the hESCs were transferred into holding medium in well three (Fig. 3) for 5 minutes. The wash step was repeated in fresh holding medium in well four, and the hES colony fragments were transferred to a new feeder dish plated with mitomycin-C–treated D551/CCL 110 fetal skin fibroblasts.
|
Statistics
Chi-square analysis was used to determine if differences in the slow freezing and vitrification protocols were significant. The numbers of grade A and B colonies for all hESC lines in each of the seven experimental arms and control arm were pooled for the calculation of P values. The same was done for grade C and D colonies. Pooled grade A and B colonies of one experimental arm were tested against the pooled grade A and B colonies of the control using the chi-square statistic.
Characterization of Post-Thaw/Warmed hESC Colonies
SSEA-4, Tra-1-60, Tra-1-81, and Alkaline Phosphatase Cell-Surface Pluripotency Markers For immunofluorescence demonstration of stem cell surface markers Tra-1-60 and Tra-1-81, hES colonies were fixed in four-well slide flasks (Becton Dickinson) with 100% ethanol for 20 minutes. For SSEA-4 staining, hES colonies were fixed in 4% paraformaldehyde for 30 minutes. The sources of the monoclonal antibodies for the detection of the markers were as follows: SSEA-4 (MC-813-70), Development Studies Hybridoma Bank (Iowa City, IA); Tra-1-60 and Tra-1-81, gifts from Dr. Peter Andrews, University of Sheffield. Primary antibodies were diluted in PBS (Invitrogen) accordingly, and blocking was performed with 10% normal goat serum (Dako) for 20 minutes. Antibody localization was performed using rabbit anti-mouse immunoglobulin secondary antibody conjugated to flourescein isothiocyanate (Sigma).
Alkaline phosphatase activity was detected with the Vector Red Alkaline Phosphatase Substrate Kit I (Vector Labs, Inc., Burlingame, CA) and viewed with rhodamine excitation and emission filters.
hESCs were routinely tested for markers of pluripotency every 15 passages.
Teratoma Formation in Severely Combined Immunodeficient Mice Morphologically undifferentiated regions of postwarmed HES-2, HES-3, and HES-4 colonies grown on D551/CCL110 were mechanically dissected into clumps of approximately 300–400 cells each. Approximately 1 x 106 cells were injected with a sterile 27G needle into the thigh muscle of severely combined immunodeficient (SCID) mice. Two SCID mice were injected for teratoma formation for each hESC line. The mice were euthanized 8–12 weeks later, and tumors were dissected and fixed in 4% formaldehyde. Tumors were embedded in paraffin and examined histologically after hematoxylin and eosin staining.
Karyotyping hES colonies were incubated with 50 mg/ml colcemid solution (Invitrogen) for 2.5 hours at 37°C and in a 5% carbon dioxide in air atmosphere. Cells were trypsinized and washed with PBS (Invitrogen), and pellets were resuspended and incubated with 0.075 M KCl for 30 minutes at 37°C. After treatment with the hypotonic solution, cells were fixed with 3:1 methanol:glacial acetic acid three times and dropped onto precleaned chilled glass slides. Chromosome spreads were Giemsa banded and photographed. At least 20 metaphase spreads and five banded karyotypes were evaluated for chromosomal rearrangements by a qualified cytogeneticist.
Reverse Transcription Polymerase Chain Reaction Analysis Total RNA was extracted from hESCs with TRIzol reagent (Invitrogen) following the manufacturer’s protocol. First-strand synthesis was performed using the SuperScriptTM II first-strand synthesis system for reverse transcription polymerase chain reaction (RT-PCR) (Invitrogen). One microgram of first-strand reaction was used for each 50-ml PCR together with 50 pmol of forward and reverse primers. Initial denaturation was carried out at 94°C for 2 minutes and followed by 30 cycles of PCR (94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute) and a final extension cycle at 72°C for 5 minutes. One tenth of each PCR reaction was loaded on a 1.5% agarose gel and size fractionated. The following primers were used: ACTB: product 400 bp 5'-tggcaccacacctttctacaatgagc-3', 5'-gcacagcttctccttaatgt-cacgc-3; NANOG: product 493 bp 5'-ggcaaacaacccacttctgc-3', 5'-tgttccaggcctgattgttc-3'; OCT4: product 247 bp 5'-cgrgaagctggagaaggagaagctg-3', 5'-caagggccgcagcttaca-catgttc-3'; REX1: product 418 bp 5'-tctagtagtgctcacagtcc-3', 5'-tctttaggtattccaaggact-3'; SOX2: product 370 bp 5'-ccgcat-gtacaacatgatgg-3', 5'-cttcttcatgagcgtcttgg-3'
Real-Time RT-PCR Analysis
Quantitative real-time PCR was performed using TaqManTM probes from Applied BioSystems Assay (ABI) on DemandTM and Assay by DesignTM service. Total RNA was extracted from post-thaw hES colonies using TRIzol reagent and first-strand synthesis performed using the SuperScriptTM II first-strand synthesis system for RT-PCR. Markers of pluripotency assayed were OCT4, SOX2, and REX1, and early markers of differentiation assayed were AFP, ND1, and BMP4. Gene expression was normalized to 18S rRNA levels (ABI); equal amounts of input cDNA (25 ng) were used per reaction, and all reactions were performed in triplicate. Real-time PCR analysis was conducted using the ABI PRISM 7000 Sequence Detection System (ABI). These data are summarized in Figure 7 and Table 2. The 2–
Ct method was used to determine normalized target gene expression in post-thaw CS-HSA-VLN2 and OPS control hES colonies relative to fresh unfrozen hES colonies. Log transformation (base 10) was performed on 2–Ct values to facilitate graphical representation of relative gene expression data (Fig. 7). Mean CT values±standard error of the mean (SEM) values for target genes in fresh colonies, post-thaw CS-HSA-VLN2, and post-thaw OPS control hES colonies are summarized in Table 2.
|
|
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
.
Clearly, conventional slow freezing in CVs (CV-FCS-LLN2 and CV-FCS-VLN2) was the least effective method for hES cryopreservation and resulted in substantial post-thaw hESC differentiation and cell death (8.2±1.9% to 10.0±10.0% of grade A and B colonies) compared with the experimental and control vitrification arms (75.0±9.7% to 88.3±2.4%, p < .001; Table 1). All experimental vitrification protocols gave postwarm survival rates ranging from 75.0±9.7% to 88.3±2.4% (grade A and B colonies), which were as good as controls (79.9±5.0%, grades A and B; Table 1) and unfrozen stock hESC cultures [3].
All vitrified hES clumps that survived the thawing process using the CS approach attached to D551 feeders 24 hours after thaw with little fragmentation (Fig. 4). CS vitrification in LLN2 or VLN2 with HSA or FCS proved to be as effective as the OPS-FCS-LLN2 controls. Thaw-survival differentiation rates (grades A and B) were better when HSA was substituted for FCS in the holding media (80.1±7.7% to 88.3 ± 2.4% versus 75.0±9.7% to 81.7±2.1%). The CS-HSA- VLN2 ranked first (mean±SEM of 88.3±2.4% grade A and B colonies from three hESC lines; Table 1), suggesting that this method could replace the existing nonsterile OPS method. Postwarmed vitrified colonies for experimental and control arms had to be passaged a bit earlier at day 6 or 7 before differentiation, unlike unfrozen colonies (days 7 and 8).
|
), and strong alkaline phosphatase activity was also detected (Fig. 5). Teratomas comprising all three primordial germ layers were formed in SCID mice after intramuscular thigh injection of undifferentiated post-thawed hESC colony fragments, confirming pluripotency (Fig. 6). Post-thawed hESC colonies also displayed normal banded karyotypes (Fig. 6). Chromosomal breakages and other abnormalities were not detected at the first passage after warming as well as subsequent passages, and hESC colonies continued to express other hES-specific marker genes, such as POU5F1, SOX2, NANOG, and REX1 (Fig. 5).
|
|
). However, a slight decrease in OCT4, SOX2, and REX1 expression in post-thaw CS-HSA-VLN2 and OPS control colonies was evident when expression levels of these markers of pluripotency were compared with the expression levels of these genes in fresh, unfrozen hES colonies (Fig. 7). Post-thaw CS-HSA-VLN2 colonies also showed an increase in ND1 expression, indicating neural differentiation, whereas post-thaw OPS control colonies showed large increases in both AFP and ND1 (Fig. 7, Table 2). This suggested that colony differentiation was more extensive in post-thaw OPS control colonies than in post-thaw CS-HSA- VLN2 cultures. We could not harvest enough post-thaw colonies frozen by the CV-FCS-LLN2 and CV-FCS-VLN2 methods for real-time PCR analysis because of extensive cell death. Overall, quantitative real-time PCR results matched well with our morphological scoring scheme for colony differentiation. |
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The results of this study indicate that hESCs may be successfully cryopreserved using the CS vitrification approach. In vitrification, it is important to induce glass formation by bringing the cells into rapid contact with the LN2 once they are in the vitrification solutions. This is necessary because of the high concentration of cryoprotectants used, which can be toxic to the cells. Because heat-sealing of the straws delays this process, two steps were incorporated to reduce this toxicity in the CS method of this study. The column containing hESC fragments was held by a pair of prechilled (chilled in ice) broad forceps and sandwiched by VS2 and WS1 columns so as to protect the hESC fragments during heat sealing. Also, the prechilled forceps allow rapid permeation of the cold temperature through the wall of the straw.
Additionally, the WS1 column helps to flush out any hESC fragments remaining in the straw during the warming process. It must be noted that for this entire study, there was no loss of hESC fragments in the CS method after warming. Thus, for the CS method to work well, the correct brand of straw with the right wall thickness used, as well as the speed of sealing, may be important. The total time taken for transfer of the hESC fragments to VS2 and the sealing of the straws and the plunging into LN2 should not exceed 120 seconds. In this study, the average duration was 100 seconds. It is important that the straws be completely sealed to ensure sterility as well as prevent the danger of explosion due to the seepage of LN2.
The vitrification technique is quick and rapid, and unlike slow gradient freezing, it minimizes the exposure time of the cells to low temperatures outside the normal physiological temperature range. Vitrification also circumvents problems associated with ice formation, cell dehydration, and the control of freezing rates. Furthermore, vitrification totally eliminates the formation of ice crystals, unlike slow gradient freezing, where substantial ice nucleation and crystallization can occur outside the cells. Also, to minimize cryoprotectant toxicity, all procedures were carried out at room temperature.
The CS method is a safe and effective approach for hESC cryopreservation; the results are highly reproducible, as reflected by the consistency of the replicates, unlike some other vitrification protocols [15, 16]; and the method is cheap, not requiring expensive programmable freezing machines. CS-vitrified hESC colonies continued to express markers of pluripotency, formed teratomas in SCID mice, and maintained normal diploid karyotypes after warming and subsequent culture, confirming that they maintain their bonafide stem cell properties after vitrification warming. Post-thaw 6-day-old CS-vitrified hESCs did not show elevated AFP gene expression compared with unfrozen colonies, suggesting that CS vitrification and thawing procedure did not induce endodermal differentiation in hESCs. OCT4, SOX2, and REX1 expression levels in 6-day-old CS-vitrified hES colonies were similar to those of OPS controls but only slightly reduced compared with fresh unfrozen hES colonies. Zona-free hatched murine blastocysts have also been successfully vitrified in CSs sealed at both ends [17], and, more recently, rabbit zona-intact blastocysts were effectively vitrified in standard plastic straws sealed at both ends as a model for human blastocysts [18].
The CS vitrification method that we describe gives excellent recovery of hESC colonies after thawing with low differentiation rates, comparable with the results of Reubinoff et al. [11]. Vitrified-warmed colonies were observed to differentiate slightly earlier than unfrozen control colonies. These findings are consistent with those reported by Reubinoff et al. [11].
FCS is a major constituent of the cryoprotectants conventionally used in the OPS method [11] and other hESC-freezing protocols. It is also undesirable in a CGMP-compliant hESC culture protocol. This study showed that a human-based protein (HSA) can successfully be substituted for this mixture of xeno-proteins. The CS method in this study therefore describes a totally xeno-free approach for hESC cryopreservation.
The results also demonstrated that CS vitrification can be performed in either the LLN2 orVLN2. It has been well documented that LN2 not only serves as a refrigerant but can also act as a vehicle for the transmission of viruses, bacteria, fungi, and animal cells. Reports that infectious viruses were found in LN2 and thus should be treated as a biohazard [19] are indicative of the potential dangers of the liquid phase of LN2. As models for human and animal viral pathogens, three bovine viruses, bovine viral diarrhea virus (BVDV), bovine herpes virus (BHV), and bovine immunodeficiency virus (BIV), were used to study the potential for their transmission by experimentally contaminated LN2 to embryos frozen and stored in open freezing CVs [20]. Bovine embryos in a mixture of 20% ethylene glycol, 20% ME2SO, and 0.6% sucrose were vitrified in either unsealed standard 0.25-ml or modified pulled straws or in open plastic CVs and then plunged into the contaminated LLN2. Postwarmed testing of a pool of 83 batches of embryos showed that 13 of 61 (21.3%) exposed to BVDV and BHV tested positive for BVDV and BHV, whereas 22 batches exposed to BIV tested negative for BIV. All control embryos vitrified in sealed CVs and straws were free from viral contamination [20]. The submersion of OPS straws and screw-capped plastic CVs in the liquid phase allows for contact between contaminated LN2 and the sample. Condensation of the atmosphere within the tube creates a vacuum that can draw in the LN2, and any contaminants in the LN2 may thus contaminate the sample. OPS vitrification [11] involves direct contact of the hESC clumps with LN2, thus substantially elevating the risk of contamination with adventitious agents in LN2. However, CS vitrification in LN2 reduces the possibility of hESC contamination, because the straws are sealed at both ends, whereas vitrification of hESCs in the vapor phase above the levels of LN2 eliminates totally the possibility of the contamination of cells with adventitious agents and the cross-contamination with other cell types in the same storage vessel. This method also accommodates the temporary storage and shipping of hESCs in LN2 dry shippers. We have observed that the temperature of LN2 dry shippers is constantly below – 132°C, the theoretical glass transition temperature of water; cells stored below this temperature are estimated to survive for hundreds of years [7]. Therefore, the CS vitrification protocol with storage in LN2 vapor we describe is an improvement over previous hESCs vitrification protocols.
We note, however, that our method of passaging hESCs involved manual manipulation of individual colonies. A major drawback of this method is the difficulty in generating sufficient numbers of cells for clinical application. In addition, smaller volumes and fewer numbers of cells are cryo-preserved using the vitrification technique in straws. However, we feel that, in combination, these techniques will be particularly useful for the cryopreservation of critical very-early-passage hES stock and for hESC lines that are not amenable to bulk culture protocols.
The CS vitrification protocol described in this study for hESC cryopreservation may also work for future hES-directed tissues, such as clumps of islet-like cells, and as such will be very useful in complementing efforts in starting hESC banks and the creation of new xeno-free hESC lines for therapeutic application.
|
ACKNOWLEDGMENTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  X. Li, R. Krawetz, S. Liu, G. Meng, and D. E. Rancourt ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells Hum. Reprod., December 4, 2008; (2008) den404v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Martin-Ibanez, C. Unger, A. Stromberg, D. Baker, J.M. Canals, and O. Hovatta Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor Hum. Reprod., December 1, 2008; 23(12): 2744 - 2754. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Unger, H. Skottman, P. Blomberg, M. Sirac Dilber, and O. Hovatta Good manufacturing practice and clinical-grade human embryonic stem cell lines Hum. Mol. Genet., April 15, 2008; 17(R1): R48 - R53. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Li, C. Zhou, C. Liu, Q. Mai, and G. Zhuang Bulk vitrification of human embryonic stem cells Hum. Reprod., February 1, 2008; 23(2): 358 - 364. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Y. Ha, B. C. Jee, C. S. Suh, H. S. Kim, S. K. Oh, S. H. Kim, and S. Y. Moon Cryopreservation of human embryonic stem cells without the use of a programmable freezer Hum. Reprod., July 1, 2005; 20(7): 1779 - 1785. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
