Proliferation and differentiation potential of cryopreserved human skin-derived precursors

Objectives: Skin‐derived precursors are recognized to be a potentially autologous and accessible source of neural precursor cells for drug screening or cell‐based treatments, in many neurological disorders. Thus, it is necessary to investigate appropriate methods for cryopreservation of such human skin‐derived precursors (hSKPs). The aim of this study was to evaluate different cryopreservation techniques for retention of hSKPs to discover an optimized protocol. Materials and methods: We cryopreserved hSKPs treated with 0%, 10%, 20%, 30% and 40% foetal bovine serum (FBS) and three concentrations of dimethylsulphoxide (DMSO) 5%, 10% and 15%, with two different storage periods in liquid nitrogen (2 days: short‐term storage; and 2 months: long‐term storage). Then, we assessed survival and proliferation levels of the cells after freeze–thaw processes, by viability measurement and colony‐forming assay. For detecting hSKPs, we used immunocytochemistry and RT‐PCR assessments. Results: Our findings indicated that hSKPs cryopreserved in 5% DMSO without FBS, had better survival and proliferation potentials compared to other working formulations. With various concentrations of cryoprotectants over different time periods, hSKPs retained their differentiation potentiality and were able to differentiate into neurons (NFM and βΙΙΙ tubulin‐positive), glial cells (GFAP‐positive) and smooth muscle cells (SMA‐positive). Conclusions: Results revealed that in only 5% DMSO, hSKPs could be cryopreserved for long‐term storage with considerable survival and proliferation levels, without losing multipotency.     

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.     

Optimal conditions for heart cell cryopreservation for transplantation

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm³ for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.     

Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for cryopreservation of human erythrocytes

Red blood cells (RBCs) can be cryopreserved using glycerol as a cryoprotective agent, but one of the main disadvantages is the time-consuming deglycerolization step. Novel cryopreservation strategies for RBCs using nontoxic cryoprotective agents are urgently needed. The effect of DMPC, DOPC, and DPPC liposomes on survival of RBCs cryopreserved with trehalose and HES has been evaluated. DMPC caused hemolysis before freezing and affected RBC deformability parameters. DMPC treated RBCs displayed a strong increase in trehalose uptake compared to control cells, whereas DOPC treated liposomes only displayed a slight increase in trehalose uptake. High intracellular trehalose contents were observed after cryopreservation. The recovery of cells incubated with trehalose and liposomes, frozen in HES ranged between 92.6 and 97.4% immediately after freezing. Recovery values of RBCs frozen in HES, however, decreased to 66.5% after 96 h at 4°C compared to 77.5% for DOPC treated RBCs. The recovery of RBCs incubated and frozen in trehalose medium was 77.8%. After 96 hours post-thaw storage recovery of these cells was 81.6%. DOPC and DPPC treated RBCs displayed higher recovery rates (up to 89.7%) after cryopreservation in trehalose compared to control RBCs. Highest survival rates were obtained using a combination of trehalose and HES: 97.8% directly after thawing and 81.8% 96-h post-thaw. DOPC liposomes, trehalose and HES protect RBCs during cryopreservation in a synergistic manner. The advantage is that the protective compounds do not need to be removed before transfusion.     

玻璃化冷藏条件对连香树定芽细胞存活率及微结构的影响

选择6种保护剂、2种玻璃化方式和2种低温处理方式对连香树定芽的冷藏条件进行筛选,用TTC还原法测定冷藏定芽的细胞存活率,并用电子显微镜观察定芽细胞冷冻时的受损情况,以优选连香树定芽的冷藏技术条件。结果表明:(1)保护剂、玻璃化方式、冷藏温度均对莲香树定芽的细胞存活率具有显著影响,不同冷藏处理的定芽组织细胞内冰晶形状、数量不同,细胞破裂数也不同。(2)连香树定芽的最佳冷藏条件为:用含甘油35.0mL、乙烯乙二醇20.0mL、DMSO 15.0mL、甘氨酸1.0mg和3%蔗糖脱氨MS液体培养基组成的100mL保护剂于真空下浸泡30min,再经-20℃预冻处理6h后,于液氮中玻璃化和冷藏60d,其定芽细胞的存活率最高,相对存活率达95%。(3)细胞微结构观察显示,在该优选条件下明显抑制了连香树定芽细胞内冰晶体的形成,消除了冰晶对细胞的涨破和刺伤,其细胞结构完整无损伤。     

一种无血清冻存液在猪胎儿成纤维细胞上的应用研究

在猪胎儿成纤维细胞(porcine fetal fibroblasts, PFF)冻存过程中,血清品质常常制约着细胞的冻存效果。为了解决这个问题,本研究旨在开发一种无血清冻存液应用于猪胎儿成纤维细胞冻存。用3种不同冻存液冻存猪胎儿成纤维细胞,每种冻存10管。冻存30 d后复苏细胞,测定冻存细胞存活率,细胞增殖活力以及电转后细胞活性。结果显示:自制无血清细胞冻存液,冻存猪胎儿成纤维细胞后存活率达95.33%;细胞增殖活力以及电转后细胞活性均显著高于标准胎牛血清冻存液(p<0.05),与特级胎牛血清冻存液效果相当(p>0.05)。因此,自制冻存液冻存猪胎儿成纤维细胞效果稳定,能够替代含血清冻存液,有良好的推广应用前景。     

Secondary follicle growth and oocyte maturation by culture in alginate hydrogel following cryopreservation of the ovary or individual follicles

An option for fertility preservation for women facing a cancer diagnosis involves the cryopreservation of ovarian tissue for later re‐transplantation or in vitro culture, with in vitro culture preferred to avoid reintroduction of the cancer. Small, immature follicles survive the freeze‐thaw process, and can be matured through in follicle maturation (IFM) that involves an initial growth of the follicle and subsequent maturation of the oocyte. The ovarian tissue can be cryopreserved in two forms: (i) cortical strips consisting of follicles and surrounding stroma (Cryo‐Ov) or (ii) individually isolated follicles (Cryo‐In). The aim of this study was to assess the follicle growth and oocyte maturation for follicles that were cryopreserved either as strips or individually using a slow‐freezing cryopreservation method. The two follicle groups, together with non‐cryopreserved control follicles, were grown in an alginate‐based three‐dimensional culture system for 12 days. The overall survival, size increase and antrum formation rates were comparable among the three groups. At day 12 of culture, Androstenedione levels were decreased in the Cryo‐Ov group relative to the other two, and the ratio of progesterone to estradiol was increased in the two cryopreserved groups relative to the control. Both Gja1 (known as connexin 43) and Gja4 (known as connexin 37) mRNA expression were decreased at day 6 in the cryopreserved groups relative to controls, and by day 12, Gja1 was similar for all three groups. Moreover, Cryo‐In resulted in lower GVBD rate indicating some impaired oocyte development. Overall, the present study demonstrated that mouse preantral follicles, either within ovarian tissues or individually isolated, could be successfully cryopreserved by the slow‐freezing method, as evidenced by post‐thaw follicle development and steroidgenesis, oocyte maturation and molecular markers for oocyte and/or granulosa cells connection. Biotechnol. Bioeng. 2009;103: 378–386. © 2009 Wiley Periodicals, Inc.     

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of −150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.     

Cryopreservation of Specific Pathogen-Free (SPF) Pig Islet Cells: Effect of Culture Time before Cryopreservation and after Thawing

The aim of this study was to determine the optimal conditions (effect of culture time before and after cryopreservation) for cryopreservation of specific pathogen-free pig islet cells. METHODS: (1) Glucose-induced insulin secretion by fresh islet cells cultured for 10 days was compared to that by islet cells cryopreserved 7 days after isolation and cultured 3 days after thawing. (2) Islet cells were cryopreserved 1, 7, or 14 days after isolation and cultured 3, 7, 14, or 21 days after thawing. Islet cell number, insulin content, and insulin response under perifusion tests were investigated. RESULTS: (1) Insulin response by cryopreserved islet cells was identical to that by fresh islet cells (basal/stimulation index: 2. 13 +/- 0.19 vs 2.17 +/- 0.16, n = 4, NS), although the amount of secreted insulin was reduced by 40% (area under the curve: 2136 +/- 198 pM/10(4) cells/180 min vs 3564 +/- 636 pM/10(4) cells/180 min, P = 0.104). (2) Cell number 6 days after thawing was reduced by 54, 40, and 63% when cryopreservations were carried out at D1, D7, and D14. (3) Insulin content in cultured or cryopreserved islet cells increased between 7 and 14 days of culture. (4) Whatever the culture time before and after cryopreservation, insulin secretion in response to glucose was maintained. The insulin release was the highest for islet cells cryopreserved 14 days after isolation and cultured 14 days after thawing (stimulation index: 6.19 +/- 2.68). CONCLUSIONS: SPF pig islet cells remained functional after cryopreservation in polyethylene glycol and it may be important to culture islet cells over 14 days before and after cryopreservation.     

Proliferation, multipotency and neuronal differentiation of cryopreserved neural progenitor cells derived from the olfactory neuroepithelium of the adult rat

The use of olfactory neuroepithelium neural progenitor cells for transplantation has attracted attention in the treatment of many neurological disorders, which require efficient recovery methods and cryopreservation procedures. The purpose of this study was to evaluate different cryopreservation techniques for neural progenitor cells derived from the olfactory neuroepithelium (ONe NPCs) in adult rats. Initially, we compared the survival rates of cryopreserved ONe NPCs treated with six different cryoprotectants: dimethylsulfoxide (DMSO), ethylene glycol (EG) and glycerol, each with or without 10% FBS and with two different storage periods in liquid nitrogen (-196 degrees C), specifically 3 days short-term storage and 3 months long-term storage. We assessed the recovery efficiency of ONe NPCs after freezing and thawing by viability testing and colony-forming assay as well as immunocytochemistry under different conditions. No significant difference in the survival rate was observed among these different cryoprotectants. With these protocols, ONe NPCs retained their multipotency and differentiated into glial (GFAP-positive), neuronal (NeuN-positive) and oligodendroglia (Galc-positive) cells. Collectively, our results imply that, under optimal conditions, ONe NPCs might be cryopreserved for periods of >3 months without losing their proliferative and multipotency activities.     

Variation of the HES concentration for the cryopreservation of keratinocytes in suspensions and in monolayers

It has recently been shown that keratinocytes, both in suspension and in monolayers, can be successfully cryopreserved with hydroxyethyl starch (HES) (6, 9). HES is a nontoxic biodegradable macromolecule which is clinically approved as a plasma expander and which has already been used for the cryopreservation of red blood cells (10, 11). In this study we varied the HES concentration between 0 and 10 wt% in 2% steps for suspended cells and between 0, 4, 6, 8, and 10 wt% for monolayer cells in order to determine the effect on the survival rate and metabolic activity after cryopreservation. The experiments with the suspended cells were performed both with and without NCS. Cryopreserved keratinocytes can be transplanted onto patients for the treatment of deep dermal burns and leg ulcers. In this study, we achieved a survival rate of 80% for the suspended cells (10 wt% HES, 3 degrees C/min) and a survival rate of even 88% when the cells were cryopreserved as a monolayer using the same parameters. The addition of NCS did not improve the results for the suspended cells significantly.     

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 (3 weeks) and long-term (1 year) 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.     

Structure and functional alterations in lymphocytes induced by cryopreservation

Surface markers, Con A-induced capping, blastogenic transformation stimulated by PHA and allogeneic mononuclear cells, and natural killer activity of Ficoll — Hypaque-separated lymphocytes were studied before and after varying periods of cryopreservation. An increase was observed in the relative number of E rosetteforming cells and in the incorporation of [³H]thymidine into DNA, by the unstimulated cryopreserved cells after thawing. On the other hand, a substantial drop occurred in the Con A-induced capping and the natural killer activity of cryopreserved cells. The possible causes for the variation in the effects of cryopreservation on lymphocyte functions as reported by different investigators were discussed. It was concluded that until universally accepted, standardized procedures for the assessment of lymphocyte functions in vitro become available, each laboratory should establish the changes induced by cryopreservation in lymphocyte function with the methods employed locally to allow the observations made on cryopreserved lymphocytes to be meaningful.     

Cryopreservation of Human Oocytes and Ovarian Tissue

Oocyte cryopreservation has the potential to be an important adjunct to assisted reproductive technologies and bypasses some ethical, moral, and religious dilemmas posed by human embryo cryopreservation. The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Among the morphological factors, the maturity, quality, size of the oocyte, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. The main biophysical factor of cellular disruption during cryopreservation process in the intracellular ice formation that can be avoided by an adequate cell dehydration; thus reducing the intracellular water by increasing the dehydration process we can limit the damages of the cryopreservation procedure. The dehydration process can be affected by the presence and concentration of the cryoprotectants in the freezing solutions (equilibration and loading solutions), and by the freezing and thawing rate. Two additional properties of cryoprotectants help to protect cells during slow cooling, when the cells are very dehydrated and are surrounded by concentrated salts. The cryoprotectants appear to reduce damage caused by high levels of salt, a property known as salt buffering. Some events occurring to the oocyte during cryopreservation procedure has been found to be a premature exocitosis of cortical granules, leading to an intempestive zona hardening and consequently to a reduction of fertilization rate, and the cryoinjury to the zona pellucida leading to a polispermic fertilization. ICSI is an efficient method to by pass these two events and to achieve a satisfactory outcome in terms of normal fertilization of cryopreserved oocytes. The application of the ICSI to cryopreserved oocytes did not seem to increase the degeneration rate after insemination with respect to fresh oocytes. The increased oocyte survival rate and the use of ICSI have facilitated the recent increase in the number of pregnancies and live birth.     

Neuropeptides to replace serum in cryopreservation of mesenchymal stromal cells?

Background aimsThe therapeutic potential of human mesenchymal stromal cells (MSCs) has generated considerable interest in a wide variety of areas. MSC banking is feasible, but the optimal technique of cryopreservation remains to be determined.MethodsTo reduce dimethyl sulfoxide (DMSO) concentration in cryopreservation medium, DMSO was replaced with sucrose or trehalose. To increase cell survival and proliferation rates after thawing and to eliminate the need for fetal bovine serum (FBS), neuropeptides of the vasoactive intestinal peptide/glucose-dependent insulinotropic peptide/pituitary adenylate cyclase activating polypeptide family were added to the cryopreservation medium. Cell survival was analyzed by a trypan blue dye exclusion assay. Cell proliferation of cryopreserved MSCs was determined after 7 days of culture.ResultsNo significant differences in cell survival rates were detected between cryopreservation solutions with 5% and 10% DMSO, independently of the addition of trehalose or sucrose. Cell proliferation rates tended to be highest when MSCs were frozen in 5% DMSO + trehalose. FBS could be replaced by human albumin (HA) without loss in cell survival and proliferation potential. With FBS, the addition of neuropeptides could increase cell survival and proliferation rates. Without FBS or HA, cell survival and proliferation rates in the presence of neuropeptides were comparable to rates achieved with FBS or HA.ConclusionsClassic cryopreservation with 10% DMSO could be replaced by 5% DMSO + 30 mmol/L trehalose. FBS could be replaced by HA or neuropeptides without loss in cell survival and proliferation potential. The addition of neuropeptides in the cryopreservation medium containing FBS could increase the cell proliferation rate and consequently cellular output.     

Cryopreservation of periodontal ligament cells with magnetic field for tooth banking

The purpose of this study was to establish a long-term tooth cryopreservation method that can be used for tooth autotransplantation. Human periodontal ligament (PDL) cells were frozen in 10% dimethyl sulfoxide (Me₂SO) using a programmed freezer with a magnetic field. Cells were cryopreserved for 7 days at −150 °C. Immediately after thawing, the number of surviving cells was counted and the cells were cultured; cultured cells were examined after 48 h. Results indicated that a 0.01 mT of a magnetic field, a 15-min hold-time, and a plunging temperature of −30 °C led to the greatest survival rate of PDL cells. Based on these findings, whole teeth were cryopreserved under the same conditions for 1 year. The organ culture revealed that the PDL cells of cryopreserved tooth with a magnetic field could proliferate as much as a fresh tooth, although the cells did not appear in the cryopreserved tooth without a magnetic field. Histological examination and the transmission electron microscopic image of cryopreserved tooth with a magnetic field did not show any destruction of cryopreserved cells. In contrast, severe cell damage was seen in cells frozen without a magnetic field. These results indicated that a magnetic field programmed freezer is available for tooth cryopreservation.     

Effects of different cryoprotectants on the viability and biological characteristics of porcine preadipocyte

Effective techniques for the cryopreservation of porcine preadipocytes could increase the usefulness of these cells as a model in obesity studies. The objective of this study was to test the effects of the following cryoprotective agents (CPAs) on the cytotoxicity, post-thaw survival, proliferation and differentiation capacity of porcine preadipocytes: ethylene glycol (EG), dimethyl sulphoxide (Me2SO), polyvinylpyrrolidone (PVP), Me2SO+PVP, and no-CPA. In addition to the CPAs, the CPA medium contained 80% DMEM/F12 plus 10% FBS. Trypan blue exclusion tests showed that among the CPA treatments in this study, only EG was toxic to porcine preadipocytes. The highest survival rate (94.96%) and cell viability were obtained when preadipocytes were cryopreserved with 10% PVP. Morphologically, PVP cryopreserved preadipocytes resembled fibroblasts and most underwent attachment, proliferation, and growth arrest with subsequent accumulation of intracellular lipid droplets before becoming mature adipocytes. There were no significant differences in the GPDH activity between adipocytes in the PVP treatment and primary cells from days 3 to 10 of the culture. Analysis of RT-PCR confirmed that there was no significant difference of PPARgamma2 mRNA levels between the cells in the 10% PVP treatment and primary cells. In summary, porcine preadipocytes cryopreserved with DMEM/F12 medium containing 10% PVP and 10% FBS have high survival rate and proliferation potential. Furthermore, the cryopreserved cells synthesize a range of markers that are consistent with this cell type. We conclude that 10% PVP is a suitable CPA for porcine preadipocytes.     

Effects of sample collection and storage conditions on DNA damage in buccal cells from agricultural workers

Buccal cells are becoming a widely used tissue source for monitoring human exposure to occupational and environmental genotoxicants. A variety of methods exist for collecting buccal cells from the oral cavity, including rinsing with saline, mouthwash, or scraping the oral cavity. Buccal cells are also routinely cryopreserved with dimethyl sulfoxide (DMSO), then examined later for DNA damage by the comet assay. The effects of these different sampling procedures on the integrity of buccal cells for measuring DNA damage are unknown. This study examined the influence of the collection and cryopreservation of buccal cells on cell survival and DNA integrity. In individuals who rinsed with Hank's balanced salt solution (HBSS), the viability of leukocytes (90%) was significantly (p<0.01) greater than that of epithelial cells (12%). Similar survival rates were found for leukocytes (88%) and epithelial cells (10%) after rinsing with Listerine(?) mouthwash. However, the viability of leukocytes after cryopreservation varied significantly (p<0.01) with DMSO concentration. Cell survival was greatest at 5% DMSO. Cryopreservation also influenced the integrity of DNA in the comet assay. Although tail length and tail moment were comparable in fresh or cryopreserved samples, the average head intensity for cryopreserved samples was ～6 units lower (95% CI: 0.8-12 units lower) than for fresh samples (t(25)=-2.36, p=0.026). These studies suggest that the collection and storage of buccal samples are critical factors for the assessment of DNA damage. Moreover, leukocytes appear to be a more reliable source of human tissue for assessing DNA damage and possibly other biochemical changes.     

Characterization of rat and human Kupffer cells after cryopreservation

Kupffer cells (KC) are the resident macrophages of the liver and represent about 80% of the total fixed macrophage population. They are involved in disease states such as endotoxin shock, alcoholic liver diseases and other toxic-induced liver injury. They release physiologically active substances such as eicosanoids and inflammatory cytokines (IL-1, IL-6, TNFalpha), and produce free radical species. Thus, KC are attractive targets for anti-inflammatory therapies and potential candidates responsible for differences in inflammation in liver disease seen between different individuals. However, to perform parallel in vitro experiments with KC from different donors a suitable method for conservation of KC would be necessary. Therefore, the present study evaluated, whether rat and human KC can be frozen, stored and recovered without losing their functional integrity. Rat and human KC were isolated and either cultured under standard conditions (fresh KC) or cryopreserved in special freezing medium (cryopreserved KC). At least 24 h later, cryopreserved KC were thawed, brought into suspension and seeded in the same density as fresh cells for subsequent experiments. Viability of cultured KC was analyzed by trypan blue exclusion. LPS (or PBS as control) stimulation was performed at different time points and cytokine release was analyzed with IL-6 and TNFalpha ELISAs, respectively. Phagocytic capacity was investigated by using a specific phagocytosis assay and FACS analysis. The recovery rate after thawing was around 57% for rat and around 65% for human cryopreserved KC. The results indicate, that KC can successfully be cryopreserved with an adequate recovery rate of viable cells. The properties of fresh and frozen KC can also be compared after thawing. Freshly isolated and cryopreserved cultured KC showed near-normal morphology and did not differ in the cultivation profiles over a period of 72 h. One to three days after seeding, frozen rat or human KC also retained inducible functions such as the production of TNFalpha or IL-6 after LPS challenge. Finally, regardless if they were cryopreserved or not, no differences in the phagocytic activities of the cells were obtained. Taken together, it is concluded that cryopreservation of KC does not change the physiological characteristics of the cells in vitro. Therefore, the method used here for cryopreservation of especially human KC allows the accumulation of KC from several donors for parallel in vitro experiments.     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.