Post-thaw Culture Research Articles

An efficient, widely applicable cryopreservation of Lilium shoot tips by droplet vitrification

We report a straightforward and widely applicable cryopreservation method for Lilium shoot tips. This method uses adventitious shoots that were induced from leaf segments cultured for 4 weeks on a shoot regeneration medium containing 1 mg/l α-naphthaleneacetic acid and 0.5 mg/l thidiazuron. Shoot tips (1.5–2 mm in length) including 2–3 leaf primordia were precultured on Murashige and Skoog (MS; 1962) medium with 0.5 M sucrose for 1 day and then treated with a loading solution containing 0.4 M sucrose and 2 M glycerol for 20 min, followed by a Plant Vitrification Solution 2 (PVS2) treatment for 4 h at 0 °C. Dehydrated shoot tips were transferred onto 2.5 µl PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for shoot regrowth. Shoot regrowth levels ranged from 42.5 % for L. longiflorum × Oriental ‘Triumphator’ to 87.5 % for L. Oriental hybrid ‘Siberia’, with a mean shoot regrowth level of 67.1 % across the six diverse Lilium genotypes tested. Histological observations found that the survival patterns were similar in cryopreserved shoot tips of ‘Triumphator’ and ‘Siberia’. Assessments using inter-simple sequence repeat markers found no differences in regenerants recovered from the control stock cultures and from cryopreserved shoot tips in ‘Triumphator’ and ‘Siberia’. This Lilium droplet-vitrification cryopreservation method is efficient, simple and widely applicable for the long-term conservation of lily genetic resources.

Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants

A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me2SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me2SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me2SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3weeks) and long-term (1year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media.

Targeting Cryopreservation-Induced Cell Death: A Review

Despite marked developments in the field of cryopreservation of cells and tissues for research and therapeutic applications, post-thaw cell death remains a significant drawback faced by cryobiologists. Post cryopreservation apoptosis and necrosis are normally observed within 6 to 24 h after post-thaw culture. As a result, massive loss of cell viability and cellular function occur due to cryopreservation. However, in this new generation of cryopreservation science, scientists in this field are focusing on incorporation of apoptosis and necrosis inhibitors (zVAD-fmk, p38 MAPK inhibitor, ROCK inhibitor, etc.) to cryopreservation and post-thaw culture media. These inhibitors target and inhibit various proteins such as caspases, proteases, and kinases, involved in the cell death cascade, resulting in reduced intensity of apoptosis and necrosis in the cryopreserved cells and tissues, increased cell viability, and maintenance of cellular function; thus improved overall cryopreservation efficiency is achieved. The present article provides an overview of various cell death pathways, molecules mediating cryopreservation-induced apoptosis and the potential of certain molecules in targeting cryopreservation-induced delayed-onset cell death.

Increasing Follicular and Stromal Cell Proliferation in Cryopreserved Human Ovarian Tissue after Long-Term Precooling Prior to Freezing: In Vitro versus Chorioallantoic Membrane (CAM) Xenotransplantation

A positive effect on the future development of cells, which have been cooled to low suprazero temperatures and then thawed, has been observed before and is not new. The aim of this study was to test the effectiveness of postthawing culture of human ovarian tissue, which was either frozen just after operative removal or cooled after removal to 5°C for 24 h before cryopreservation. Ovarian fragments from six patients were divided into small pieces in the form of cortex with medulla and randomly divided into the following four groups: Group 1 consisted of pieces that just after removal had been cultured in vitro for 8 days in a big volume of medium with mechanical agitation; Group 2 included pieces cooled after operation to 5°C for 24 h and then cultured in vitro for 8 days; Group 3 was comprised of pieces frozen-thawed just after operation and then cultured for 5 days in the chorioallantoic membrane (CAM) culture system; and the pieces in Group 4 were cooled after operation to 5°C for 24 h, frozen-thawed, and then cultured in the CAM system for 5 days. The effectiveness of the tissue culture was evaluated by the development of follicles and by the intensiveness of proliferation in the tissue (by expression of cytokeratin and Ki-67). For Groups 1, 2, 3, and 4, the mean densities of follicles per 1 mm(3) was 12.9, 12.2, 12.4, and 16.1, respectively (p1-2>0.1; p3-4<0.05). For these groups, 87%, 95%, 71%, and 84% of the preantral follicles were morphologically normal (p1-2, 3-4<0.05). The immunohistochemical analysis showed increased proliferation after cooling of fresh and cryopreserved tissue. Long-term (24 h) cooling of ovarian tissue to 5°C before cryopreservation increases the viability of the cells in the tissue after thawing. Additionally, the efficacy of the CAM system for the culture of thawed human ovarian tissue was demonstrated.

Microbial screening of unrelated cord blood units in a Chinese cord blood bank

Given limited sample volume available for sterility testing, optimal testing methods and algorithms of cord blood (CB) have not been established according to standards. The volume of CB unit was reduced to 20 mL in a closed system. Hetastarch and cryopreservation solution was added to the processed CB unit in Class 100 environment. In the routine sterility testing, 20 mL of red blood cells (RBC) sample was cultured in aerobic and anaerobic culture bottles. In verification post-thaw culture of the final product, some of the discarded final product units were also sampled and cultured after thaw. All the culture bottles were incubated in the BacT/ALERT 3D system. In the routine sterility testing, 139 of 7032 CB units (1·98%) were contaminated with microorganism. In 84 of these 139 units (60·4%) only the anaerobic bottle was positive. Lactobacillus spp. were the most prevalent contaminant. Sixty-two discarded CB stem cell units were recultured after thaw. Of these, 10 of 48 units with a positive culturing of RBC sample were negative in the post-thaw reculture. One of 14 units with negative culturing of RBC sample was contaminated with Bifidobacterium breve in special verification test. Our study demonstrates the predominant organisms implicated in CB microbial contamination were part of the human intestinal and vaginal flora. The larger sample volume and anaerobic culture would significantly increase the detection rate of microbial contaminated CB. We also found that potential transfusion-transmitted bacterial infection risk still existed in final product although microbial screening was performed.

Use of embryoglue® as a post-thaw culture and transfer medium did not improve clinical outcome measures in FET cycles

EmbryoGlue® (VitroLife, Denver; CO) may act as a uterine-embryo receptor mediator to improve implantation; published trials demonstrate conflicting results when EmbryoGlue was used for the transfer medium, however efficacy may be realized in patients with multiple IVF failures. Null hypothesis: Clinical outcome metrics are not different using Embryo Glue or SOP culture media for post-thaw culture and embryo transfer in frozen embryo transfer cycles.

Human Wharton’s Jelly Stem Cell Conditioned Medium Enhances Freeze-Thaw Survival and Expansion of Cryopreserved CD34+ Cells

Hematopoietic stem cells (HSCs) from umbilical cord blood have been successfully used to treat blood disorders but one major hurdle is the relatively low cell dose available. Double cord blood unit transplantation results in elevated engraftment failure because one unit predominates over the other. Various approaches are thus being undertaken to expand HSCs ex vivo from single cord blood units. We report here a protocol involving slow freezing (-1 °C per minute to -120 °C) + freezing medium containing DMSO + FBS + 24 h-50% hWJSC-CM that enhances thaw-survival of CD34+ cells. Post-thawing, the fold, percentage and colony forming unit numbers of CD34+ cells were significantly increased (2.08 ± 0.3; 102 ± 1.17%; 1.07 ± 0.02 respectively) while the percentages of apoptotic, necrotic, dead and sub-G1 phase cells (91.06 ± 3.63%; 91.80 ± 5.01%; 95.6 ± 3.61 %; 86.1 ± 16.26% respectively) were significantly decreased compared to controls. Post-thaw culture in 24 h-50% hWJSC-CM+FBS for 72 h showed further significant increases in CD34+ cells (fold: 2.28 ± 0.17; percentage: 153.3 ± 21.99%, CFU: 1.6 ± 0.19) and significant decreases in apoptotic, necrotic, dead and sub-G1 cells (49.2 ± 3.59%; 62.0 ± 4.30%; 56.6 ± 5.06%; 28.6 ± 5.74 % respectively) compared to controls. We hypothesize that these improvements are probably related to the high levels of cytokines, cell adhesion molecules and growth factors in hWJSC-CM that help to preserve cell membrane integrity during freezing and stimulate mitosis post-thaw. A 24 h-50% hJWSC-CM may be a useful supplement for freezing CD34+ cells in cord blood banks.

Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification

This study was carried out to compare the post-thaw cryosurvival rate and the level of apoptosis in vitro produced zona-free cloned buffalo blastocysts subjected to slow freezing or vitrification in open-pulled straws (OPS). Zona-free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re-expansion rate following post-thaw culture for 22-24h. The post-thaw re-expansion rate was significantly (p<0.05) higher following vitrification in OPS (71.2±2.3%) compared with that after slow freezing (41.6±4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen-thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non-cryopreserved cloned embryos (422.6±67.8) and those cryopreserved by slow freezing (376.4±29.3) or vitrification in OPS (422.8±36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8±2.0) or OPS vitrification (13.3±1.8), was significantly (p<0.05) higher than that for the control non-cryopreserved cloned embryos (3.4±0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona-free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.

Shoot tip cryopreservation by vitrification in Kaempferia galanga L. An endangered, overexploited medicinal plant in Tropical Asia

Shoot tips from the established in vitro shoot cultures of Kaempferia galanga L., an endangered medicinal plant were successfully cryopreserved using the vitrification procedure. Effect of concentration of sucrose for preculture, vitrification treatment with PVS2(Plant vitrification solution 2) and recovery medium on cryopreservation of shoot tip meristems has been analyzed. Overnight preculturing of the dissected shoot tips in MS medium containing 0.4 M sucrose, osmoprotection with loading solution for 20 minutes, dehydration with PVS2 for 20 minutes at 0 oC were found to be optimum among the various treatments tested. The cryoprotected shoot tips upon rapid freezing in LN (liquid nitrogen) followed by rapid thawing produced 50-60% survival and 30-40% regeneration rates. Incorporation of GA3 (giberellic acid) in the post-thaw culture medium was essential to induce shoot emergence in the initial phase. Subculturing of the recovered shoot tips in MS medium supplemented with 1.0 mgl -1 BA (6-benzyl adenine) and 0.5 mgl -1 NAA (α-naphthaleneacetic acid) resulted plantlet production. The regenerated shoots grew to mature plants were free of any morphogenetic variation upon field transfer.

Frozen-thawed embryo transfer is associated with a significantly reduced incidence of ectopic pregnancy

To compare the incidence of ectopic pregnancy (EP) after fresh ET and thawed ET. Retrospective cohort study. Private fertility center. This retrospective study included 2,150 blastocyst transfers, including all 1,460 fresh autologous blastocyst transfers and all 690 transfers of autologous blastocysts derived from post-thaw extended culture of thawed bipronuclear oocytes in the 8-year study period 2004-2011. None. Visualized EP and treated persistent pregnancy of unknown location. The rate of visualized EP was 1.5% in pregnancies in fresh autologous cycles, which was significantly more than the rate of 0 with autologous post-thaw extended culture. The rates of treated persistent pregnancy of unknown location were 2.5% and 0.3% in these two groups, respectively, a difference that was also statistically significant (relative risk 7.3, 95% confidence interval 1.7-31.0). Relative to fresh transfer, thawed ET was associated with significantly reduced incidence of EP. These findings are consistent with ovarian stimulation increasing the risk of EP.

Influence of post-thaw culture on the developmental potential of human frozen embryos

PurposeApart from freezing/thawing related cryodamage, several additional factors have been identified as major players in the reduction of success rates after frozen embryo transfers. The post-thaw culture is particularly relevant as it may amplify environmental influences over a stressed embryo. In the present study the influence of the post-thaw culture duration on the implantation and developmental potential of cleavage stage embryos was evaluated.MethodsIn this retrospective evaluation, that spanned an 8-year period, 631 frozen-thawed embryos were allocated to one of two study groups, depending on their post-thaw culture period: 1) the long (18–24 h), or 2) the short (2–5 h) culture group. Groups were compared regarding implantation rate and live birth rate per embryo transferred. This comparison was corrected for the most common confounding factors such as maternal age at oocyte pick-up, number of transferred embryos, developmental day at freezing, blastomere survival after thawing, catheter used for transfer and year of procedure.ResultsImplantation and live birth rate per embryo transferred were inversely related to the duration of the post-thaw culture, as diminishing this period significantly increased both rates. Moreover, no advantage could be found for a long post-thaw culture period, even for embryos with observed mitotic activity.ConclusionThis retrospective analysis indicates that a short post-thaw culture period is associated with higher implantation and live birth rates per embryo. This study supports selection of frozen-thawed embryos strictly based on blastomere cryosurvival and raises the hypothesis that environmental factors may have an important role on embryo implantation and developmental potential during post-thaw culture.

Blastocyst transfer in frozen-thawed cycles.

ObjectiveIt is well known that fresh blastocyst transfer results in better pregnancy outcomes with a smaller number of transferred embryos compared with cleavage stage embryo transfer. However, in terms of frozen-thawed blastocyst transfer, only a few studies are available. We aimed to evaluate clinical outcomes of frozen-thawed embryo transfer (FET) with blastocysts.MethodsRetrospective analysis of FET cycles with blastocysts (B-FET) between Jan 2007 and June 2009 was performed. Age-matched FET cycles with cleavage stage embryos (C-FET) during the same period were collected as controls. A total of 58 B-FET cycles were compared with 172 C-FET cycles and also compared with those of post-thaw extended culture blastocysts from frozen pronuclear stage embryos (22 cycles).ResultsThere was no difference in the patient characteristics of each group. The embryos' survival rates after thawing were comparable (>90%) and there was no difference in the implantation rate or clinical and ongoing pregnancy rate among the three groups.ConclusionIn FET, blastocyst transfers may not present better pregnancy outcomes than cleavage stage embryo transfers. A further large-scale prospective study is needed.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Post-thaw culture in presence of insulin-like growth factor I improves the quality of cattle cryopreserved embryos

The goal of this study was to examine the effect of insulin-like growth factor I (IGF-I; added during post-thaw culture (48 h)) on the preimplantation viability and quality of cryopreserved bovine in vivo recovered embryos. The morula stage embryos, non-surgically recovered from superovulated dairy cows of Czech Fleckvieh cattle breed, had previously been cryopreserved by a slow freezing technique and stored in liquid nitrogen since 1989-1990. Following thawing, the embryos were cultured for 48 h either alone (no IGF-I) or in the presence of IGF-I (10 or 100 ng/ml); non-cultured embryos served as a control. Thereafter, the embryos were analyzed for cleavage to the blastocyst stage, apoptosis (TUNEL), embryo cell number and quality of actin cytoskeleton. Following post-thaw culture 41% of embryos developed to advanced blastocysts. IGF-I increased this per cent and, at a higher dose, essentially reduced the per cent of degenerated embryos. In cultured embryos, IGF-I at both doses elevated the cell number compared with non-cultured embryos. However, in comparison with embryos cultured without IGF-I, only the higher IGF-I dose resulted in elevating the embryo cell number. The TUNEL index was significantly lowered by IGF-I treatment. Thawed embryos were mostly of the grade III actin type and fewer (12%) had grade II actin, whilst no grade I actin embryos were noted. The addition of IGF-I resulted in the appearance of grade I actin embryos (8.33 and 6.9% for 10 and 100 ng/ml, respectively). These observations indicate that the addition of IGF-I during post-thaw culture can improve the quality of bovine cryopreserved embryos.

In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

The development of efficient and robust methods for the cryopreservation of human embryonic stem cells (hESCs) is important for the production of master and working cell banks for future clinical applications. Such methods must meet requirements of good manufacturing practice (GMP) and maintain genetic stability of the cell line. We investigated the culture of four Shef hESC lines in gas permeable 'culture cassettes' which met GMP compliance. hESCs adhered rapidly to the membrane and colonies displayed good proliferation and expansion. After 5-7 days of culture, hESCs were cryopreserved in situ using 10% dimethyl sulphoxide in foetal calf serum at approximately 1 degrees C/min. This method was compared with a control of standard flask culture and cryopreservation in vials. Post-thaw cassette culture displayed relative proliferation ratios (fold increase above flask/cryovial culture) of 114 (Shef 4), 8.2 (Shef 5), 195 (shef 6) and 17.5 (Shef 7). The proportion of cells expressing pluripotency markers after cryopreservation was consistently greater in cassette culture than for the control with the markers SSEA3 and SSEA4 exhibiting a significant increase (P> or =0.05). The efficiency of cell line culture in cassette was associated with the overall passage number of the cell line. The procedure enables cryopreservation of relatively large quantities of hESCs in situ, whilst returning high yields of viable, undifferentiated stem cells, thereby increasing capacity to scale up with greater efficacy.

Improved viability of freeze-thawed embryonic stem cells after exposure to glutathione

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.

An efficient and easy-to-use cryopreservation protocol for human ES and iPS cells

Here we describe a simple and efficient human embryonic stem (ES) and induced pluripotent stem (iPS) cells cryopreservation protocol. This protocol involves the use of Rho-associated kinase (ROCK) inhibitor, Y-27632, for the feeder-free dissociated cells. The addition of ROCK inhibitor to both pre- and post-thaw culture media enhanced the cloning efficiency. The presence of Y-27632 in Matrigel further increased the cloning efficiency. As compared with other available protocols for human ES and iPS cells cryopreservation, our protocol differs in the technical simplicity, high cloning efficiency and post-thawing passaging. We believe that this protocol could be a generally applicable and robust platform for feeder-free cryopreservation and the expansion of present and future applications of human ES and iPS cells. The treatment with ROCK inhibitor, cell harvesting and the freezing-thawing process usually takes about 2 h excluding overnight incubation at -80 degrees C.

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved ( P < 0.05) by the addition of 10 μM Y-27632 (94.9 ± 2.4%, mean ± SEM) compared to a control (78.0 ± 6.0%). Conversely, after 48 h of culture, there were no significant differences in hatching rate (62.8 ± 11.1 vs. 59.6 ± 9.4%) and mean total cell number (135.2 ± 13.1 vs. 146.7 ± 13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7 ± 3.8 vs. 54.7 ± 8.9%, P < 0.05), with no difference in mean total cell number of blastocysts (230.0 ± 23.0 vs. 191.2 ± 22.2, P = 0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5 ± 2.1, 31.4 ± 2.3, 36.2 ± 3.2, and 28.6 ± 6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.

In vitroanalysis of cryopreserved alginate-poly-l-lysine-alginate-microencapsulated human hepatocytes

The availability of well-characterized human hepatocytes that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after microencapsulated cryopreservation and investigated whether these cryopreserved microencapsulated hepatocytes can be used for clinical applications. Adult hepatocytes of 18 separate donors were isolated with a two-step extracorporeal collagenase perfusion technique. After pre-incubation at 4 degrees C for 12-24 h in HepatoZYME-SFM, hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules. The microencapsulated hepatocytes were transferred to a complete medium containing 10% dimethyl sulphoxide. They were immediately placed into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the post-thawing culture period, albumin secretion, urea synthesis, cell cycle, mRNA and protein levels, as well as the morphology and pathology structure of pre-incubation before microencapsulated cryopreservation (PMC) groups were analysed. Compared with the immediate cryopreservation (IC) groups, we found significant improvement in the mRNA and protein levels in the attached cells, and higher secretion of albumin and urea levels after thawing. In the attached cultured human cryopreserved/thawed hepatocytes from the PMC group, albumin production was not significantly different from those of the direct culture groups on days 2, 3 and 4. The preserved morphology in the PMC group compared with the IC group was obvious. The results of the present study suggested recovery of the functional and morphological integrity of human hepatocytes after pre-incubation at 4 degrees C for 12-24 h before microencapsulated cryopreservation. These studies offer the possibility for clinical applications in pharmacotoxicology, bioartificial liver and cell therapy in humans.

Enhancement of cell recovery for dissociated human embryonic stem cells after cryopreservation

Due to widespread applications of human embryonic stem (hES) cells, it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation, and further investigated the role of the combination of Rho-associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow-freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture, we found out that hES cell recovery was significantly enhanced by around 30 % (P < 0.05) by the new freezing solution. Moreover, at the first day of post-thaw culture, the presence of 10 μM ROCK inhibitor (Y-27632) and 1 μM pifithrin-μ together further significantly improved cell recovery by around 20% (P < 0.05) either for feeder-dependent or feeder-independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore, this protocol is a scalable cryopreservation method for handling large quantities of hES cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010