Effects Of Different Cryoprotectants Research Articles

Role of Cryoprotectants on the Viability and Functional Properties of Probiotic Lactic Acid Bacteria during Freeze Drying

Effect of different cryoprotectants on acid/bile tolerance, β-galactosidase activity, and cholesterol assimilation properties of freeze dried Pediococcus acidilactici, Lactobacillus plantarum, and Lactobacillus salivarius were evaluated. The retention of probiotic properties and viability of the above three cultures freeze dried with lactose, skimmed milk, and maltodextrin were found to be significant (P ≤ 0.05). In another approach, lactic acid bacteria (LAB) were subjected to cold shock first and then freeze dried. Effect of cold shock treatment on retention of probiotic properties of freeze dried LAB cultures was also investigated. The cold shock treatment resulted in an increase of 6–8%, both in viability and probiotic properties, compared with LAB freeze dried without cold shock treatment. The survival of freeze dried L. plantarum, L. salivarius, and P. acidilactici was investigated over a period of 60 d storage at 4°C. The above three cultures freeze dried with maltodextrin exhibited 83%, 68%, and 60% retention in survival percentage, respectively, in comparison with initial values.

Cryopreservation of swine ovarian tissue: Effect of different cryoprotectants on the structural preservation of preantral follicle oocytes

The present study aimed to test different cryoprotectants on cryopreservation of pig ovarian tissue. Pig ovaries ( n = 3) were collected at a local slaughterhouse. From each ovary, ten cortex samples were taken. One was immediately fixed (control) and another placed in short-term tissue incubation (STTI control). The other 8 samples were cryopreserved, in pairs, using 4 different cryoprotectants: dimethyl sulphoxide (Me2SO – 1.5 M), ethylene glycol (EG – 1.5 M), propanediol (PROH – 1.5 M) and glycerol (GLY – 10%), all with 0.4% sucrose. Samples were slow cooled and stored in liquid nitrogen for 7 days. After thawing and cryoprotectant removal, one sample from each treatment was immediately fixed and the other was placed in short-term tissue incubation (STTI) for 2 h and then fixed. Samples were processed for histology and transmission electron microscopy. The percentages of morphologically normal follicles (MNF) in cryopreserved tissue using Me2SO (67.0 ± 4.9), EG (81.8 ± 1.4) and PROH (55.9 ± 9.9) were significantly lower ( P < 0.05) than observed in fresh control tissue (97.7 ± 1.2). When ovarian tissue was cryopreserved with GLY, no morphologically normal follicles could be found (0%). After STTI, PROH showed a significantly lower percentage of MNF when compared with all other treatments and the control. After ultrastructural analysis, follicles cryopreserved with Me2SO and EG showed some small alterations, but no signs of advanced degeneration. Overall, these were similar to follicles from the control group. In conclusion, it is possible to cryopreserve preantral follicles from pig ovarian tissue using Me2SO or EG.

Comparative Study between Slow Freezing and Vitrification of Mouse Embryos Using Different Cryoprotectants

The objective of this study was to evaluate the effects of different cryoprotectants and different cryopreservation protocols on the development of mouse eight-cell embryos. Mouse eight-cell embryos were cryopreserved by using propylene glycerol (PROH), ethylene glycerol (EG), dimethyl sulfoxide (DMSO) or glycerol (G) as cryoprotectant with slow-freezing or Vit-Master vitrification protocol. After thawing, the survival rate, blastocyst formation rate and blastocyst hatching rate of the embryos were compared. When the mouse eight-cell embryos were cryopreserved by the slow-freezing, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with PROH were significantly higher than those of DMSO and G (p < 0.05, respectively), but not significantly different among those of DMSO, G and EG (p > 0.05, respectively), and not significantly different between those of PROH and EG (p > 0.05, respectively). When the mouse eight-cell embryos were cryopreserved by Vit-Master vitrification, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with EG were significantly higher than those of PROH, DMSO and G (p < 0.05, respectively). Yet, there were no significant differences among those of PROH, DMSO and G (p > 0.05, respectively). In conclusion, PROH was the optimal cryoprotectant for the cryopreservation of mouse eight-cell embryos by slow-freezing protocol. EG was the optimal cryoprotectant for the cryopresevation of mouse eight-cell embryos by Vit-Master vitrification protocol, which may be commonly used in clinical and laboratory practice.

Effect of type of cryoprotectant on morphology and developmental competence of in vitro-matured buffalo (Bubalus bubalis) oocytes subjected to slow freezing or vitrification

The present study examined the effects of different cryoprotectants on morphology and developmental competence of in vitro-matured buffalo oocytes after slow freezing or vitrification. After slow freezing in dimethyl sulfoxide (DMSO), ethylene glycol (EG) or 1,2-propanediol (PROH), at 1.0 or 1.5 m each, the proportion of morphologically normal oocytes recovered was significantly higher (P < 0.05) with 1.5 than 1.0 m for all cryoprotectants and was highest (P < 0.05) for 1.5 m DMSO. Following vitrification, the percentage of morphologically normal oocytes recovered was lower (P < 0.01) for 40% EG than for 40% DMSO, 20% EG + 20% DMSO or 20% EG + 20% PROH. The most common damage, irrespective of the cryopreservation method, was loss of cumulus mass. The cleavage rate and the proportion of vitrified-warmed oocytes that developed to morulae/blastocysts were significantly higher (P < 0.01) for 20% EG + 20% DMSO than for the other groups. A higher proportion of oocytes developed to morulae (11.5% v. 4.3%) or blastocysts (5.4% v. 0.6%) after vitrification in 20% EG + 20% DMSO than after slow freezing in 1.5 m DMSO. In conclusion, vitrification was more effective than slow freezing for the cryopreservation of in vitro-matured buffalo oocytes.

Effects of pH, Sodium Bicarbonate, Cryoprotectants and Foetal Bovine Serum on the Cryopreservation of European Eel Sperm

The main objective of the present study was to evaluate the influence of pH and bicarbonate concentration in the activation or inhibition of European eel (Anguilla anguilla) spermatozoa and to evaluate the effect of different cryoprotectants: dimethyl sulphoxide (DMSO), acetamide, ethylene glycol, propanol, glycerol and methanol (MeOH). The effect of these factors was evaluated comparing the percentage of motile cells, the percentage of alive cells (by Hoechst staining) and the spermatozoa morphometry pre- and post-cryopreservation (by computer-assisted morphology analysis). Based on the above findings, three cryoprotectants (DMSO, MeOH and glycerol) were chosen and evaluated in two media (P1 and P1 modified) with different concentrations of NaHCO(3) and in the presence or absence of foetal bovine serum (FBS). The effect of these factors was evaluated comparing the percentage of alive and motile cells post-cryopreservation. DMSO was the cryoprotectant showing better results in relation to the percentage of spermatic alive cells post-freezing and caused a smaller modification of the head spermatozoa morphology. The combination of P1-modified medium with DMSO and containing FBS increased slightly but significantly the percentage of motile spermatozoa post-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 (Me 2SO), polyvinylpyrrolidone (PVP), Me 2SO+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 PPARγ2 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.

Cryopreservation of cultured periosteum: Effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me 2SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at −196 °C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 °C for 30 min with Me 2SO, under agitation, showing 63.3 ± 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me 2SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me 2SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.

Effect of different cryoprotectants on the structural preservation of follicles in frozen zebu bovine ( Bos indicus) ovarian tissue

Cryopreservation of ovarian tissue is a new and promising technique for germ-line storage. The objective of this study was to evaluate the effect of four cryoprotectants (at two concentrations each) on the preservation of zebu bovine preantral follicles after ovarian cryostorage. Strips of ovarian cortex were cryopreserved using glycerol (GLY; 10 or 20%), ethylene glycol (EG), propanediol (PROH) or dimethylsulphoxide (DMSO; 1.5 or 3 M). In addition, a toxicity test was performed for each cryoprotectant by exposing the ovarian tissue to them without freezing. Tissues were analyzed by histology and transmission electron microscopy. Ovarian tissue frozen in either concentration of DMSO or PROH or in 10% GLY retained a higher percentage of morphologically normal follicles (73–88%) than tissue frozen in 20% GLY or in either concentration of EG (16–52%). In the toxicity test, exposure of tissues to DMSO, PROH or GLY resulted in higher percentages of normal follicles (80–97%) than exposure to EG (49%). Electron microscopy revealed damage to the ultrastructure of follicles frozen in 10% GLY, while follicles cryopreserved in DMSO and PROH at either concentration exhibited normal ultrastructure. In conclusion, DMSO and PROH were the most effective cryoprotectants for zebu ovarian tissue, preserving the structural integrity of somatic and reproductive cells within the ovary.

Effect of Different Cryoprotectants on the Survivability and the Development of Bovine Oocytes Matured In vitro

Tsuzuki. Y., Kusao. T., Ashizawa, K. and Fujihara, N. 2000. Effect of different cryoprotectants on the survivability and the development of bovine oocytes matured in vitro. J. Appl. Anim. Res., 18: 15–24. To determine the optimum combination of cryoprotectant and freezing method, for improving the developmental, competence of in vitro matured bovine oocytes, three cryoprotectants (1.5 M dimethyl-sulfoxide + 0.25 M sucrose: DMSO; 0.9 M ethylene glycol + 0.8 M propylene glycol + 0.1 M sucrose: EG + PG; 0.8 M PG + 0.8 M DMSO: PG + DMSO) and two freezing methods [the oocytes were cooled from 0C to −6.5C at 1 C/min, seeded and cooled to −30C at 0.3C/min (method A); the oocytes were cooled at −8C directly, seeded and cooled to −35C at 1 C/min (method B)] were used. The survivability and developmental competence of the oocytes thawed from the liquid nitrogen were determined by the dye extrusion assay with eosin and embryonic development after in vitro fertilization, respectively. Without freezing, the survivability of the oocytes equilibrated with and removed from each cryoprotectant were not decreased compared with that in the control group. However, the per cent of the oocyte developed to the blastocyst stage in all of the cryoprotectant treated groups were significantly lower (P<0.05) than that of the control group. DMSO group frozen with method A and EG + PG group frozen with method B maintained their survivability compared to the each control group. However, cleavage rates in all groups were significantly lower (P<0.05)than those of the control groups. No blastocyst was observed in all frozen groups. These results suggest that the combination of DMSO with freezing method A and EG + PG with freezing method B seemed to be more suitable compared with the other combinations.

Surface changes associated with ram sperm cryopreservation revealed by counter-current distribution in an aqueous two-phase system effect of different cryoprotectants

Ram sperm was frozen in the presence of the most commonly used cryoprotectants. After thawing, the overall cell surface changes provoked by freezing were assessed by centrifugal counter-current distribution (CCCD). In addition, cell membrane integrity (viability) of all the treated sperm was estimated by fluorescent staining. Fresh and refrigerated sperm were used as controls. Our results show no improvement of the cooling-induced cell surface damage by freezing in the presence of bovine seminal plasma, proline, glycine-betaine and phosphatidylcholine. Better results were obtained with vitamin E and cholesterol. However, the best protective effects were found by employing seroalbumin and lactalbumin. Furthermore, freezing in the presence of bovine lactalbumin resulted in a good maintenance of the cellular viability and of the CCCD heterogeneity in respect to fresh cells.

Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes

Cumulus cell-enclosed bovine oocytes in germinal vesicle (GV) and in metaphase II (MII) stages were cryopreserved. Different concentrations (1 M; 1.5 M) of various cryoprotectants (glycerol, PROH, DMSO) were tested. After thawing, the oocytes were exposed to various carbohydrates (sucrose, lactose, trehalose) at a concentration of 0.1 M and 0.25 M for cryoprotectant removal. Developmental capacity of the frozen-thawed oocytes was studied by in vitro maturation, fertilization and culture. We found no difference in subsequent development using glycerol or PROH for GV and MII oocytes. The DMSO treatment led to significantly better cleavage and development up to 4-cell stage in MII oocytes. Development beyond the 8-cell stage was obtained only when unmatured oocytes were frozen. No difference in the efficiency of the 3 cryoprotectants was detected in MII oocytes. However, in GV oocytes, glycerol and PROH yielded significantly better cleavage and 4-cell rate compared to DMSO (P<0.001). Influence of the concentration of a cryoprotectant on development was not observed in GV or MII oocytes. Among the 3 cryoprotectants, DMSO was less suitable, at both concentrations, than PROH and glycerol for the development of 6- to 8-cell stage embryos in the GV group. In the MII group, 1.5 M DMSO was as efficient as PROH and as glycerol at a 1.5-M concentration, and it was more efficient than 1 M glycerol. The use of carbohydrates during rehydration did not render a beneficial effect at either of the 2 concentrations, and when no carbohydrates were used in the MII group the oocytes cleaved better than GV oocytes.