Cryopreservation of Rainbow Trout (Oncorhynchus mykiss) Blastomeres: Influence of Embryo Stage on Postthaw Survival Rate

The cryopreservation of isolated fish blastomeres is likely to provide a valid alternative to embryo cryopreservation, the results of which are still discouraging. A repeatable technique for the cryopreservation of rainbow trout blastomeres has been established and the effect of embryonic developmental stage on freezing tolerance evaluated. Embryos at Ballard 6A, 6B, and 6C stages were dechorionated and left to dissociate in a Ca2+- and Mg2+-free medium. Cryoprotection was provided by step-wise addition of 1.4 M 1,2-propanediol. Cells were loaded into the middle of 250-μl straws and slowly frozen to -80 degreesC before being plunged into LN2. A low thawing rate was adopted, followed by step-wise removal of the cryoprotectant. Morphological evaluation was by microscopy and video recording. Metabolic activity and survival rate were determined by FDA and PI staining, by recovery of the ability to reassociate after 24 h culture in Leibovitz L15 + 2% Ultroser, and by measuring DNA synthesis in 6B cells by the method of BrdU incorporation. Survival rates were 53 +/- 9.3, 88 +/- 1.7, and 95 +/- 0.5% for stage 6A, 6B, and 6C cells, respectively. While 6A cells reassociated into clumps of cells, 6B and 6C cells formed holoblastic morulas in 24 h; proliferation of 6B cells was comparable to fresh control cells. The relationship between freezing tolerance and the physiological events occurring during early embryonic development is discussed in light of these results and conclusions are drawn that envisage the transfer of frozen-thawed blastomeres into recipient embryos. Copyright 1998 Academic Press.     

Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by improper cryopreservation

Ty1 is a retrotransposon of the yeast Saccharomyces cerevisiae whose transposition at new locations in the host genome is activated by stress conditions, such as exposure to UV light, X-rays, nitrogen starvation. In this communication, we supply evidence that cooling for 2 h at +4 °C followed by freezing for 1 h at −10 °C and 16 h at −20 °C also increased Ty1 transposition. The mobility of Ty1 was induced by cooling at slow rates (3 °C/min) and the accumulation of trehalose inside cells or the cooling at high rates (100 °C/min) inhibited significantly the induction of the transposition. The freeze-induced Ty1 transposition did not occur in mitochondrial mutants (rho⁻) and in cells with disrupted SCO1 gene (Δsco1 cells) evidencing that the Ty1 transposition induced by cooling depends on the mitochondrial oxidative phosphorylation. We also found that the freeze induced Ty1 transposition is associated with increased synthesis and accumulation of superoxide anions (O⁻₂) into the cells. Accumulation of O⁻₂ and activation of Ty1 transposition were not observed after cooling of cells with compromised mitochondrial functions (rho⁻, Δsco1), or in cells pretreated with O⁻₂ scavengers. It is concluded that (i) elevated levels of reactive oxygen species (ROS) have a key role in activation the transposition of Ty1 retrotransposon in yeast cells undergoing freezing and (ii) given the deleterious effect of increased ROS levels on cells, special precautions should be taken to avoid ROS production and accumulation during cryopreservation procedures.     

State and Phase Transition Behaviors ofQuercus rubraSeed Axes and Cotyledonary Tissues: Relevance to the Desiccation Sensitivity and Cryopreservation of Recalcitrant Seeds

Freezing and melting transitions of cellular water in embryonic axes and cotyledonary tissues of recalcitrantQuercus rubra(red oak) seeds were compared under slow and rapid cooling conditions. The relevance of desiccation sensitivity (critical water content) and state/phase transition behaviors to cryopreservation was examined. Under a slow to intermediate cooling condition (≤10°C min⁻¹), unfrozen water content in the tissues decreased to less than the critical water content, resulting in a dehydration damage. Under a rapid cooling condition (>100°C min⁻¹) using liquid nitrogen (LN₂), freeze-induced dehydration damage could be avoided if the initial water content was >0.50 g g⁻¹dry wt. However, at water content >0.50 g g⁻¹dry wt, the vitrified cellular matrix was highly unstable upon warming at 10°C min⁻¹. These results offered a theoretical explanation on the difficulty for successful cryopreservation of recalcitrant red oak embryonic axes. A complete state/phase transition diagram for red oak axes was constructed, and a vitrification-based cryopreservation protocol that employed predehydration and rapid cooling was examined. State/phase transition behaviors of cellular water are important parameters for cryopreservation; however, vitrification alone was not sufficient for seed tissues to survive the cryopreservation condition.     

Natural cryoprotectants combinations of l-proline and trehalose for red blood cells cryopreservation

Cryopreservation of red blood cells (RBCs) holds great potential benefits for supplying transfusion timely in emergencies. Currently, glycerol is the main cryoprotectant permitted in clinical therapy for RBCs cryopreservation, but its broad application is limited by the toxicity and complex deglycerolization process. Successful cryopreservation of RBCs using more effective materials should be studied to reduce freezing damage, increase biocompatibility, and save processing time. Herein, a simple protocol using natural cryoprotectants combinations of l-proline and trehalose attains a low degree of hemolysis (11.2 ± 2.73%) after thawing compared to glycerol. Furthermore, the morphology of RBCs and the activities of Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase maintain well. Further mechanism study shows that l-proline plays an important role in decreasing the freezing points and inhibiting the growth of ice crystal by permeating into cells during the freezing process. While trehalose works as an inhibitor of ice growth in the freezing process and ice recrystallization in the thawing process. This simple l-proline & trehalose combinations protocol is a promising method to replace current time-consuming and labor-intensive cryopreservation methods of RBCs.     

Tolerance of human embryonic stem cell derived islet progenitor cells to vitrification-relevant solutions

We have previously shown that human embryonic stem cell derived islet progenitors (hESC-IPs), encapsulated inside an immunoprotective device, mature in vivo and ameliorate diabetes in mice. The ability to cryopreserve hESC-IPs preloaded in these devices would enhance consistency and portability, but traditional ‘slow freezing’ methods did not work well for cells encapsulated in the device. Vitrification is an attractive alternative cryopreservation approach. To assess the tolerance of hESC-IPs to vitrification relevant conditions, we here are reporting cell survival following excursions in tonicity, exposure to fifteen 40% v/v combinations of 4 cryoprotectants, and varied methods for addition and elution. We find that 78% survival is achieved using a protocol in which cells are abruptly (in one step) exposed to a solution containing 10% v/v each dimethyl sulfoxide, propylene glycol, ethylene glycol, and glycerol on ice, and eluted step-wise with DPBS + 0.5 M sucrose at 37 °C. Importantly, the hESC-IPs also maintain expression of the critical islet progenitor markers PDX-1, NKX6.1, NGN3 and NEURO-D1. Thus, hESC-IPs exhibit robust tolerance to exposure to vitrification solutions in relevant conditions.     

Purification and Properties of Cold-active Metalloprotease from Curtobacterium luteum and Effect of Culture Conditions on Production

Curtobacterium luteum, a gram-positive psychrotrophic bacterium, secreting an extracellular protease was isolated from the soil of Gangotri glacier, Western Himalaya. The maximum enzyme production was achieved when isolate was grown in a pH-neutral medium containing skim milk at 15oC over 120 hour. The metal ions such as Zn2+ and Cr2+ enhanced enzyme production. The specific activity of purified enzyme was 8090 u/mg after 34.1 fold purification. The 115 kD enzyme was a metalloprotease (activity inhibited by EDTA and EGTA) and showed maximum activity at 20oC and pH 7. The enzyme was active over a broad pH range and retained 84% of its original activity between pH 6–8. There was no loss in enzyme activity when exposed for 3 hours at 4oC-20oC. However, lost 65% of activity at 30oC, and was almost inactivated at 50oC, but was resistant to repeated freezing and thawing. The enzyme activity was stimulated by manganese ions; however, it was inactivated by copper ions.     

Cryoprotective effects of antifreeze proteins delivered into zebrafish embryos

Fish embryo cryopreservation, which is useful in aquaculture or biodiversity conservation, is still far from being achieved. Structural barriers reduce the entrance of cryoprotectants into embryo compartments. Previous studies demonstrated a better ability for freezing in Arctic species which naturally express antifreeze proteins (AFPs). In this study, AFPs were delivered in early zebrafish embryos by incubation in media containing protein. Their cryoprotective effects were then analyzed. Chilling sensitivity was evaluated at 4 °C and −10 °C. Survival rates significantly increased in embryos incorporating AFPI and kept at −10 °C. To analyze their effects on cryopreservation, 5-somite embryos were vitrified. Incorporation of AFPI reduced the percentage of embryos that collapsed at thawing (14.2% of AFPI-treated embryos and 48.9% of controls). Cellular damage caused by vitrification was assessed after thawing by cell dissociation and further analysis of cell survival in culture (SYBR-14/IP labeling). The percentage of viable cells at thawing ranged from 25 to 50%, considered incompatible with embryo development. Cells recovered from frozen-control embryos did not survive in culture. However, the incorporation of AFPs allowed survival similar to that of cells recovered from non-frozen embryos. Blastomere cryopreservation trials incorporating AFPI in the extender also demonstrated a significant increase in viability after freezing. Our findings demonstrated that delivery of AFPs into zebrafish embryos by incubation in media containing protein at early stages is a simple and harmless method that increases cryoprotection of the cellular compartment. This beneficial effect is also noticed in blastomeres, encouraging their use in further protocols for embryo cryopreservation.     

Cryopreservation of isolated fish blastomeres: effects of cell stage, cryoprotectant concentration, and cooling rate on postthawing survival

The toxicity of the cryoprotectant dimethyl sulfoxide (Me(2)SO) to isolated blastomeres was examined in three fish species representative of distinct environments: marine (whiting, Sillago japonica); estuarine (pejerrey, Odontesthes bonariensis); and freshwater (medaka, Oryzias latipes). The effects of embryonic stage, Me(2)SO concentration, and cooling rate on the cryopreservation of blastomeres were also studied. Whiting sheds small planktonic eggs whereas the other two species shed large demersal eggs. Isolated blastomeres from the three species tolerated Me(2)SO concentrations up to 9% relatively well for over 5 h but lost viability rapidly at 18%. Cells from later embryonic stages (512 or 1024 cells) were more tolerant of Me(2)SO than those from earlier stages (128 or 256 cells). The three factors examined, alone or in combination, had a significant effect on the survival of blastomeres after freezing and thawing, but the extent of the effect and the optimum conditions varied with the species. In general, the highest rates of successful cryopreservation were observed with older rather than younger blastomeres, slower rather than faster cooling, and with 9-18% rather than 0% Me(2)SO. Survival rates for blastomeres cryopreserved under the most effective combination of the three factors examined for each species were 19.9 +/- 10.1% for whiting, 34.1 +/- 8.5% for medaka, and 67.4 +/- 12.8% for pejerrey. Copyright 1999 Academic Press.     

Factors affecting survival of deep frozen bovine embryos in vitro: The effect of freezing container and method of removing cryoprotectant

The effect of freezing container and method of glycerol removal on in vitro survival of frozen-thawed Day 7 bovine embryos was investigated. Two hundred and fifteen embryos were frozen in ampules or straws, in either vertical or horizontal position and at a cooling rate of 0.3 degrees C/minute from -7 degrees C to -35 degrees C, before being plunged into liquid nitrogen. Samples were thawed in a water bath at +35 degrees C and glycerol was removed by either step-wise dilution (increments 0.25 M) or by exposure to 1.0 M sucrose for 10 minutes. A total of 197 embryos was recovered post-thaw (91%) with an overall survival after 1, 3, 6 and 24 hours in culture of 87, 81, 71, and 23%, respectively. Embryonic quality and percent survival, as assessed morphologically, did not change significantly between 1 and 3 hours but decreased significantly between 6 and 24 hours in culture (p < 0.05). Survival at 24 hours was significantly higher after removal of the cryoprotectant with sucrose when compared to the step-wise glycerol dilution (p < 0.05). Overall, embryonic survival in straws equaled that in ampules; freezing orientation of straws did not affect results. Further, glycerol removal with sucrose tended to yield survival superior to that provided by a step-wise dilution technique.     

Effect of egg yolk, cryoprotectant, and various sugars on semen cryopreservation in endangered Cuvier's gazelle (Gazella cuvieri)

Cryopreservation of spermatozoa from endangered species is a valuable tool for genetic management. Previous studies showed the feasibility of cryopreservation of spermatozoa from various endangered gazelles but have also revealed difficulties with available protocols for semen freezing in Cuvier's gazelle (Gazella cuvieri). Experiments were carried out to investigate the effect of (a) 5% or 20% egg yolk or 4% or 6% glycerol, and (b) addition of sugars (glucose, fructose, lactose and raffinose) on cryopreservation using a Tes-Tris-based diluent (TEST). A diluent containing 13.5% raffinose, 5% or 20% egg yolk, and 6% glycerol (REYG) was also evaluated. Semen was obtained by electroejaculation from 22 G. cuvieri males. Diluted samples were loaded into 0.25 ml straws, cooled to 5 °C over 1.5 h (−0.16 °C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapours for 10 min and plunged into liquid nitrogen. Subsamples were assessed for motility and acrosome integrity upon collection, after refrigeration–equilibration, after freezing and thawing, and 2 h after thawing. Use of TEST with 20% egg yolk or with 4% glycerol led to worse motility preservation, whereas TEST with 5% egg yolk and 6% glycerol led to better results. Addition of fructose, lactose or raffinose to TEST resulted in similar or worse preservation of motility than inclusion of glucose. On the other hand, use of a raffinose-based medium with 20% egg yolk and 6% glycerol (REYG) afforded better preservation of motility than use of TEST. With REYG, 20% egg yolk was better than 5% egg yolk for motility preservation. Differences were noted between males in their responses to cryopreservation when using different egg yolk or glycerol concentrations. Moreover, spermatozoa from most males exhibited better cryopreservation with REYG although some were better cryopreserved in TEST. The raffinose-based diluent thus represents an improvement over previous results but more work is needed to better characterize cryopreservation conditions for future routine banking of Cuvier's gazelle spermatozoa.     

Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling

This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to −196 °C using controlled slow cooling. Stage III oocytes (>0.5 mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2 M DMSO (both prepared in Hank’s medium) for 30 min at 22 °C before being loaded into 0.5 ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10 min, and thawed by immersing straws into a 27 °C water bath for 10 s. Thawed oocytes were washed twice in Hank’s medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 °C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4 MβNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5 min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2 M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to −196 °C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2 M DMSO and 2 M methanol. Trypan blue staining showed that 63.0 ± 11.3% and 72.7 ± 5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30 min at 22 °C, respectively, whilst 14.9 ± 2.6% and 1.4 ± 0.8% stayed intact after freezing in DMSO and methanol to −196 °C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk mobilisation.     

Freezing damage of bovine erythrocytes: Simulation using glycerol concentration changes at subzero temperatures

When a cell is frozen and thawed, it is exposed to (i) lowered temperature, (ii) increased solute concentration during freezing, and (iii) decreased solute concentration during thawing. Without actually freezing the cells, an attempt has been made to simulate physical-chemical changes to which bovine erythrocytes are exposed when frozen and thawed in glycerol solutions. Experimentally, the study consisted of suspending erythrocytes in 1, 2, or 3 glycerol at 20 °C for various times and then exposing them to each of several dilution sequences. The dilution sequences were: (i) transfer from the initial glycerol concentration at 20 °C into the same concentration at −5 °C, (ii) transfer into an increased glycerol concentration at 20 °C, (iii) transfer into an increased followed by a decreased glycerol concentration at 20 °C, (iv) transfer into an increased glycerol concentration at −5 °C, and (v) transfer into an increased followed by a decreased glycerol concentration at −5 °C. This last sequence is analogous to the exposure that cells undergo at subzero temperatures to increased solute concentration during freezing and decreased solute concentration during thawing. This dilution sequence yielded a survival pattern very similar to that obtained when bovine erythrocytes are frozen and thawed, and thus does appear to mimic freezing damage. It is concluded that a major factor in freezing damage is the extent to which a cell must shrink or swell to achieve osmotic equilibrium at subzero temperatures in partially frozen or thawed solutions.     

Determination of surface immunoglobulin density on frozen-thawed mononuclear cells analyzed by the fluorescence-activated cell sorter

Recent data have demonstrated that differences in sIg density on B lymphocytes distinguish functionally distinct subpopulations of these cells. Other reports suggest that cyropreservation may change the frequency of sIg-bearing lymphocytes. To determine if cryopreservation alters either the frequency of sIg cells or the distribution of sIg density, PBM from normals and patients with CLL and LCL were analyzed using the FACS. Aliquots of Ficoll-Hypaque-separated PBM were controlled-rate frozen (1 °C/min) in 7.5% Me₂SO in RPMI 1640 and thawed in a 37 °C water bath on the same day. Fresh and frozen-thawed PBM aliquots were labeled with fluorescein conjugates of F(ab′) fragments of affinity chromatography-purified anti-Fab or class-specific anti-μ, anti-δ, anti-γ, or anti-α. Histograms of relative cell fluorescence, reflecting sIg density, were prepared for each aliquot with the FACS. The frequency of sIg-bearing PBM labeled with each reagent was not significantly altered by freezing. Likewise, FACS profiles demonstrated that the distribution of sIg density on normal and CLL PBM was unchanged after freezing. However, the fluorescence peak produced by frozen-thawed unlabeled cells was occasionally slightly broader than that of fresh cells, suggesting increased autofluorescence induced by freezing. These data indicate that frozen cell preparations may be utilized for the study of B-lymphocyte subsets as determined by sIg density.     

Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use

Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120 h after tooth extraction, and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC, ending with development of procedures for banking. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me₂SO at a concentration between 1 and 1.5 M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen, at least up to 2 × 10⁶ cells/mL. It was further established that DPSC can be stored at −85 °C or −196 °C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues, with digestion and culture performed post-thaw. A recovery of cells from >85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free, defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions.     

Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures

Brassica napus suspension-cultured cells can be hardened to a lethal temperature for 50% of the sample of −20°C in eight days at room temperature with abscisic acid. During the induction of freezing tolerance, changes were observed in the electrophoretic pattern of [³⁵S]methionine labeled polypeptides. In hardening cells, a 20 kilodalton polypeptide was induced on day 2 and its level increased during hardening. The induction of freezing tolerance with nonmaximal hardening regimens also resulted in increases in the 20 kilodalton polypeptide. The 20 kilodalton polypeptide was associated with a membrane fraction enriched in endoplasmic reticulum and was resolved as a single spot by two-dimensional electrophoresis. In vitro translation of mRNA indicate alteration of gene expression during abscisic acid induction of freezing tolerance. The new mRNA encodes a 20 kilodalton polypeptide associated with increased freezing tolerance induced by either abscisic acid or high sucrose. A 20 kilodalton polypeptide was also translated by mRNA isolated from cold-hardened B. napus plants.     

Extracellular calcium-inhibited alpha-amylase of Bacillus coagulans B 49

Extracellular α-amylase (EC 3.2.1.1) from Bacillus coagulans B 49 was purified to homogeneity by ion-exchange chromatography and gel filtration. The optimum pH and temperature for dextrinizing activity were 6–7 and 70°C and for saccharolytic activity were 7 and 60°C, respectively. Calcium inhibited α-amylase activity even at low concentrations (10 m ), and most of its activity could be restored by dialysis against EDTA. Other cations such as Mg²⁺, Fe²⁺, and Hg²⁺ also inhibited amylase activity, while Mn²⁺ exhibited a slight stimulatory effect. The activity of the enzyme was not affected by ethylenediaminetetraacetic acid (EDTA).     

Cryopreservation of human bone marrow cells by a modification of the two-step cooling technique

The first attempt to freeze human bone marrow cells with a two-step cooling method is reported. A simple and reliable way of obtaining stable first-step subzero freezing baths is described. One-milliliter samples each containing 20 × 10⁶ bone marrow cells and 10% Me₂SO were frozen in polypropylene cryotubes. Using these experimental conditions, the optimal freezing temperature was found to be in the range of −36 to 37.5 °C for BM progenitor cell (GM-CFC, CFU_E, and BFU_E) survival. Such temperatures were easily obtained in stable sludges of anisole or K₂CO₃ eutectic solution in water. The optimal holding time was 20 min before plunging tubes into liquid nitrogen. Similar or improved progenitor cell recoveries were observed compared with the conventional cooling technique. Adaptation of this two-step technique for the freezing of large volumes of BM cells for autografting is under investigation.     

Effect of cryopreservation on mitochondrial DNA of zebrafish (Danio rerio) blastomere cells

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programmes for endangered species. However, despite the growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells very often remains poor. Many studies have been devoted to the mechanisms of cryodamage. It is known that cryopreservation causes extensive damage to membranes; reduce the metabolic activity of cells; and disturbs the mitochondrial bioenergetical processes of cells. But few investigations on the genetic stability of cells during cryopreservation have been performed, and the role of any genetic impact cryopreservation needs to be determined. Some indirect data in the literature suggests that progress in this field might come from investigating freezing damage to mitochondrial DNA (mtDNA), nuclear DNA and other genome-related structures. In this study, zebrafish (Danio rerio) blastomeres were treated in three different ways: control suspension of blastomere cells in phosphate buffered saline; equilibration of blastomeres with 2M dimethyl sulfoxide (Me2SO) for 1h at room temperature and cryopreservation using Me2SO as a cryoprotectant. Mitochondrial DNA was analysed in fresh cells and after the different treatments. Two different loci of mtDNA were amplified with the help of PCR and sequenced. The sequences were analysed and nuclear base substitutions were counted for both control and treated samples. The results showed that cryopreservation significantly increased the frequency of mutations (0.78+/-0.27% in comparison to 0.16+/-0.25% of control), whilst 2M Me2SO treatment did not bring a significant increase in frequency of mutations (0.24+/-0.28%). The distributions of the mutation locations were analysed. More investigations are needed to determine whether optimisation of cryopreservation protocol is possible to reduce these adverse effects; whether such mutations interfere with overall function of the cells; whether similar changes also occur in the nuclear DNA and whether such mutations happen in other species. Meanwhile, it is important to be cautious in making judgements of the effect of cryopreservation technique in assisted reproduction. This is the first report on the effect of cryopreservation on mtDNA.     

Cryopreservation of human adipose tissues

Scientific studies on cryopreservation of adipose tissues have seldom been performed. The purpose of our present study is conducted both in vitro and in vivo to develop a novel cryopreservation method that can be used successfully for long-term preservation of human adipose tissues for possible future clinical application. In this study, samples of adipose aspirates were obtained from 36 adult white female patients after liposuction and collected from the middle layer after centrifugation. In the in vitro study, suitable cryoprotectant agents (CPAs) and their concentrations and possible combinations were selected from our preliminary experiment. A combination of dimethyl sulfoxide (Me₂SO) and trehalose as CPA with the optimal concentration (0.5 M Me₂SO and 0.2 M trehalose) was chosen and then used throughout the study. In addition, maximal recovery of adipose tissues was achieved after cryopreservation using slow cooling without seeding (1–2 °C/min to −30 °C, followed by plunging to −196 °C for storage) and fast warming (in 40 °C water bath, averaging 35 °C/min). Fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were evaluated by integrated adipocyte counts and histology. In the in vivo study, fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were injected into a nude mouse. The retained adipose aspirates (fat grafts) were harvested in each animal at 4 months and their weight, volume, and histology was assessed. In the in vitro study, significantly higher integrated viable adipocyte count (2.06 ± 0.54 × 10⁶ mL⁻¹ vs. 1.07 ± 0.41 × 10⁶ mL⁻¹, p < 0.0011) of adipose aspirates was found in Group 3 compared with Group 2. Group 3 had only a marginally lower integrated viable adipocyte count compared with Group 1 (2.06 ± 0.54 ×10⁶ mL⁻¹ vs. 2.57 ± 0.56 × 10⁶ mL⁻¹, p = 0.083). Histologically, more tissue shrinkage was evident in Group 2 compared with Group 3. In the in vivo study, various degrees of absorption of injected fat grafts were seen in all 3 groups. However, Group 3 had significantly more retained weight and volume of the injected fat grafts than Group 2 (both p < 0.0001) but had significantly less retained weight and volume than Group 3 (weight, p = 0.009178; volume, p = 0.007836). Histologically, a large amount of tissue fibrosis was seen in Group 2, and reasonably well maintained fatty tissue with only a small amount of tissue fibrosis was seen in Group 3. The results from the present in vitro and in vivo studies, for the first time, demonstrate that our preferred cryopreservation method, the combination of 0.5M Me₂SO and 0.2 M trehalose as CPA in addition to the controlled slow cooling and fast rewarming protocol, appears to provide the maximum recovered results in cryopreservation of human adipose tissues and may become a real option after further refinements for cryopreservation of human adipose aspirates in a clinical setting.     

The embryonic development of the triclad <Emphasis Type="Italic">Schmidtea polychroa</Emphasis>

Triclad flatworms are well studied for their regenerative properties, yet little is known about their embryonic development. We here describe the embryonic development of the triclad Schmidtea polychroa, using histological and immunocytochemical analysis of whole-mount preparations and sections. During early cleavage (stage 1), yolk cells fuse and enclose the zygote into a syncytium. The zygote divides into blastomeres that dissociate and migrate into the syncytium. During stage 2, a subset of blastomeres differentiate into a transient embryonic epidermis that surrounds the yolk syncytium, and an embryonic pharynx. Other blastomeres divide as a scattered population of cells in the syncytium. During stage 3, the embryonic pharynx imbibes external yolk cells and a gastric cavity is formed in the center of the syncytium. The syncytial yolk and the blastomeres contained within it are compressed into a thin peripheral rind. From a location close to the embryonic pharynx, which defines the posterior pole, bilaterally symmetric ventral nerve cord pioneers extend forward. Stage 4 is characterized by massive proliferation of embryonic cells. Large yolk-filled cells lining the syncytium form the gastrodermis. During stage 5 the external syncytial yolk mantle is resorbed and the embryonic cells contained within differentiate into an irregular scaffold of muscle and nerve cells. Epidermal cells differentiate and replace the transient embryonic epidermis. Through stages 6–8, the embryo adopts its worm-like shape, and loosely scattered populations of differentiating cells consolidate into structurally defined organs. Our analysis reveals a picture of S. polychroa embryogenesis that resembles the morphogenetic events underlying regeneration.Edited by D. Tautz