Cryoprotectant Treatment Research Articles

Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation

Shoot-tips from in vitro cultured Hypericum perforatum L. genotypes were subjected to assessments of developmental competence, genetic stability, and biosynthetic ability to identify critical points during cryopreservation. Survival rate, chromosome number stability, alteration in VNTR sequences and hypericin content were evaluated, in plants after pre-culture, and two subsequent cryogenic steps (cryoprotection and cooling) and those recovered from cryopreserved meristems. Pre-culture and cryoprotection treatments, did not reveal any significant differences, in these studied characteristics. Genetic stability was assessed by chromosome counts and analysis of variability in the VNTR sequences. No changes in chromosome number were detected in comparison with the untreated control but minor alterations were revealed in non-coding sequences. The content of hypericin after the recovery of cryopreserved meristems remained comparable with the unfrozen control. The controlled rate freezing technique used for cryopreservation was relevant for restoration of genetic and biochemical stability in Hypericum perforatum L. shoot-tips.

CRYOPRESERVATION OF BLACK-LIP PEARL OYSTER (PINCTADA MARGARITIFERA, L.) SPERMATOZOA: EFFECTS OF CRYOPROTECTANTS ON SPERMATOZOA MOTILITY

Cryopreservation of sperm is seen as an important step in developing effective hatchery culture techniques for the black-lip pearl oyster, Pinctada margaritifera. As a preliminary investigation into cryopreservation of the gametes of this species we tested 5 cryoprotectant agent combinations for their ability to retain sperm motility: (1) 1 M trehalose and 5, 10 and 15% dimethyl sulphoxide (DMSO); (2) Hanks calcium-free balanced salt solution (C-F HBSS) and 5%, 10% and 15% DMSO; (3) C-F HBSS and 5, 10 and 15% propylene glycol (PG); (4) 1 M trehalose and an equal combination of DMSO and PG making up 5, 10, 15% total volume; and (5) C-F HBSS and an equal combination of DMSO and PG making up 5%, 10% and 15% total volume. Total, rapid and progressive sperm motilities were estimated through computer assisted sperm analysis (CASA). Sperm cryopreserved in 1 M trehalose and 5% DMSO retained the highest total (86.0 ± 1.2% SE), progressive (46.0 ± 1.2% SE) and rapid (25.1 ± 0.6% SE) motilities of all cryoprotectant solutions, whereas those cryopreserved with PG generally retained poor motility. Although the 1 M trehalose and 5% DMSO treatment compared favorably with that of fresh sperm for total motility (P < 0.01), all cryoprotectant treatments were poor at retaining the proportion of original rapid and progressively moving sperm. This study highlights the potential for cryopreservation of gametes from P. margaritifera, which will benefit selective breeding and conservation programs with this important commercial species.

Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection

Plant shoot tips do not survive exposure to liquid nitrogen temperatures without cryoprotective treatments. Some cryoprotectant solutions, such as plant vitrification solution 2 (PVS2), dehydrate cells and decrease lethal ice formation, but the extent of dehydration and the effect on water freezing properties are not known. We examined the effect of a PVS2 cryoprotection protocol on the water content and phase behavior of mint and garlic shoot tips using differential scanning calorimetry. The temperature and enthalpy of water melting transitions in unprotected and recovering shoot tips were comparable to dilute aqueous solutions. Exposure to PVS2 changed the behavior of water in shoot tips: enthalpy of melting transitions decreased to about 40 J g H 2O −1 (compared to 333 J g H 2O −1 for pure H 2O), amount of unfrozen water increased to ∼ 0.7 g H 2O g dry mass −1 (compared to ∼0.4 g H 2O g dry mass −1 for unprotected shoot tips), and a glass transition ( T g) at −115 °C was apparent. Evaporative drying at room temperature was slower in PVS2-treated shoot tips compared to shoot tips receiving no cryoprotection treatments. We quantified the extent that ethylene glycol and dimethyl sulfoxide components permeate into shoot tips and replace some of the water. Since T g in PVS2-treated shoot tips occurs at −115 °C, mechanisms other than glass formation prevent freezing at temperatures between 0 and −115 °C. Protection is likely a result of controlled dehydration or altered thermal properties of intracellular water. A comparison of thermodynamic measurements for cryoprotection solutions in diverse plant systems will identify efficacy among cryopreservation protocols.

Effects of Various Extenders and Permeating Cryoprotectants on Cryopreservation of Cynomolgus Monkey (Macaca fascicularis) Spermatozoa

The cryoprotective effects of 11 different extenders, TTE, DM, mDM, LG-DM, G-DM, TCG, TEST, TSM, Test-M, Test-H, and LM, on sperm cryopreservation of cynomolgus monkey (Macaca fascicularis) have been compared with glycerol as cryoprotectant. Sperm motility, plasma membrane, and acrosomal integrity were examined to evaluate frozen-thawed sperm function. The results showed that TTE, DM, mDM, LG-DM, G-DM, and TCG exhibited the best and similar protective efficiencies for cynomolgus monkey sperm cryopreservation in terms of sperm motility and plasma membrane integrity (P > .05). The acrosomal integrity for spermatozoa cryopreserved in TCG was statistically lower than that of TTE, DM, mDM, LG-DM, and G-DM (P < .05) but was significantly higher than that of TEST, TSM, Test-M, Test-H, and LM (P < .05). The postthaw sperm motility for 5 other extenders (TEST, TSM, Test-M, Test-H, and LM) did not exceed 30%, and the 3 sperm parameters evaluated for them were significantly lower than that of TTE, DM, mDM, LG-DM, G-DM, and TCG (P < .05). On the basis of these findings, 5 commonly used permeating cryoprotectants, glycerol, ethylene glycol, dimethyl sulfoxide, acetamide and propylene glycol have further been tested for their effectiveness on sperm cryopreservation in extenders of TTE, DM, mDM, LG-DM, G-DM, and TCG. The results showed that the sperm cryoprotective efficiencies of glycerol and ethylene glycol were similar and best among 5 permeating cryoprotectant treatments (P > .05). Dimethyl sulfoxide or acetamide resulted in average cryoprotection for cynomolgus monkey spermatozoa: poorer than glycerol or ethylene glycol but better than that of propylene glycol (P < .05). In addition, the action of permeating cryoprotectant appeared to be independent of extenders. The results in the present study demonstrate that 1) TTE, DM, mDM, LG-DM, G-DM, and TCG are excellent extenders and suitable for cynomolgus monkey sperm cryopreservation; 2) the mechanism of action of permeating cryoprotectants are not affected by extender composition; 3) ethylene glycol has a similar cryoprotective efficacy to glycerol that makes it a successful cryoprotectant for sperm cryopreservation in cynomolgus monkeys.

CRYOPRESERVATION OF SPERM OF RED ABALONE (HALIOTIS RUFESCENS)

Abalone culture, a developing industry in Baja California, Mexico, would benefit from genetic improvement and controlled breeding. The use of cryopreserved sperm would allow germplasm availability, and this study was designed to develop sperm cryopreservation protocols for red abalone Haliotis rufescens. The acute toxic effects of the cryoprotectants dimethyl sulfoxide (DMSO), propylene glycol (PG), and glycerol (GLY) were assessed after suspending sperm in different concentrations, whereby cryoprotectant treatments of 10% DMSO and 10% GLY equilibrated for 10 min yielded the highest range of motile sperm in preliminary freezing trials and were used for cryopreservation studies. To determine effective cooling rates, three freezing chambers were tested. Replicate samples of sperm from 4 males were placed in 0.5-mL French straws and frozen using a commercial freezing chamber (CFC) used for bull sperm, a programmable rate chamber (PRC), and a manually controlled styrofoam chamber (MCC). For the CFC, the cooling rate was 16°C/min, from 4°C to −140°C. For the PRC and MCC, it was 1°C/min, from −20°C to −30°C. The samples were held at −30°C for 5 min before being plunged into liquid nitrogen (−196°C) for storage, and each sample was thawed in a water bath at 45°C for 8 s. The quality of thawed sperm was determined by estimating percent motility, evaluating membrane integrity using a dual-staining technique and flow cytometry, and estimating fertilization rate. Statistical analyses were performed using 2-way ANOVA where chamber and treatment were the independent variables. Sperm quality parameters were independent. For motilities, a significant interaction was noted between the cryoprotective treatment and the chamber type, whereby motilities for DMSO and GLY were higher (P = 0.0055) using MCC. Membrane integrities were significantly lower after using the PRC than the CFC or the MCC (P = 0.0167). The highest post-thaw motility (48 ± 7%) was found using sperm suspended in 10% glycerol and frozen in the MCC. The highest percent of intact membranes (56 ± 11%) was for sperm suspended in 10% glycerol and frozen in the CFC. The highest fertilization rate (29 ± 10%) was with samples frozen with 10% glycerol in the CFC. The use of cryopreserved sperm from red abalone provides an alternative breeding option for culture and the protocols delineated are the first developed for this species.

91IMPACT OF PROPYLENE GLYCOL V. ETHYLENE GLYCOL ON OOCYTE MATURATION, SPINDLE INTEGRITY, FERTILIZATION AND EMBRYO DEVELOPMENT IN VITRO IN THE DOMESTIC CAT

A thorough characterization of cryoprotectant (CPA) sensitivity is required to formulate a successful cryopreservation protocol for any biomaterial. The aim of this study was to characterize the toxic impact of various CPA types, concentrations, and exposure temperatures on the immature domestic cat oocyte. In Experiment 1, grade I immature oocytes (n=561) were exposed (30min; 25°C or 0°C) to 0M, 0.75M, 1.5M, or 3M of propylene glycol (PrOH) or ethylene glycol (EG) in PBS+20% fetal calf serum (v/v). After exposure, CPA was removed step-wise by subjecting oocytes to decreased CPA concentrations. Oocytes were cultured (30h; 38.5°C, 5% CO2) in IVM medium as reported previously (Wolfe and Wildt 1996 J. Reprod. Fertil. 106, 135–141). Oocytes were then fixed and stained to examine nuclear status (Hoechst 33342) and spindle integrity (FITC-labeled anti-α-tubulin antibodies; Sigma Chemical Co., St. Louis, MO). Experiment 2 was designed on the basis of Experiment 1 results to assess the impact of the spindle abnormalities on subsequent embryo development. Oocytes (n=776) were exposed to CPA conditions yielding optimal nuclear maturation with either high (0.75M or 3.0M PrOH or 1.5M EG at 25°C) or low (1.5M PrOH at 25°C) proportions of abnormal spindle. After IVM, oocytes were inseminated with thawed semen (5×105 motile sperm mL−1 ) in Ham’s F-10 (Irvine Scientific, St-Anna, CA). At 16h post-insemination, oocytes were fixed and stained (Hoechst 33342) to assess IVF success (pronuclear formation) or cultured in vitro for 7 days to assess embryo development. Data were analyzed by ANOVA and Tukey’s multiple comparison test. In Experiment 1, CPA treatment had no effect (NS) on meiotic progression to metaphase I. However, percentage of oocytes reaching metaphase II (MII) was reduced (P&amp;lt;0.05) in 3.0M PrOH at 0°C (29.3±8.3%; mean±SD), 3.0M EG at 25°C (33.7±8.9%), and 0°C (29.4±11.0%) compared to all other conditions examined (range, 52.0% to 62.0%). All CPA treatments also increased (P&amp;lt;0.05) spindle abnormalities at MII (range, 40.3% to 75.9%) compared to control (13.8±8.6%), except 1.5M PrOH at 25°C (20.7±10.1%). None of the CPA treatments in Experiment 2 influenced IVF success (range, 55% to 63%; NS). However, percentage of cleaved embryos was reduced (P&amp;lt;0.05) in 0.75M PrOH (32.1±4.1%), 1.5M EG (33.4±4.0%), and 3.0M PrOH (29.3±3.8%) compared to control (50.1±4.0%) or 1.5M PrOH (50.6±4.9%). Developmental competence (number of blastocysts relative to number of cleaved embryos) also was impaired (P&amp;lt;0.05) in 1.5M EG (16.5±7.4%) and 3.0M PrOH (14.9±7.8%) compared to the other conditions (range, 32.5% to 38.5%), including 1.5 PrOH at 25°C (32.5±7.8%). In conclusion, exposure of immature oocytes to 1.5M PrOH at 25°C does not adversely impact oocyte maturation, MII spindle, fertilization, or embryo development in vitro in the domestic cat.

Cryopreservation for the elimination of cucumber mosaic and banana streak viruses from banana (Musa spp.)

The utilisation of cryopreservation for the eradication of cucumber mosaic virus (CMV) or banana streak virus (BSV) from Musa spp. was investigated. Banana plants, cv. Williams (AAA, Cavendish subgroup), were mechanically infected with CMV or naturally infected with BSV, and proliferating meristems were produced from the infected plants. Excised meristematic clumps were cryopreserved through vitrification using PVS-2 solution. The health status of regenerated in vitro plants was first checked by means of ELISA. The putative virus-free material was subsequently tested a second time following greenhouse acclimatisation. The frequency of virus eradication for CMV and BSV was 30% and 90%, respectively, following cryopreservation. In comparison, the frequency of virus-free plants regenerated directly from highly proliferating meristems, corresponding to a spontaneous eradication rate, reached 0% and 52% for CMV and BSV, respectively. The conventional meristem culture resulted in 0% CMV-free plants and 76% BSV-free plants, while the cryoprotective treatment resulted in 2% CMV-free plants and 87% BSV-free plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the structure of the meristem tips by light microscopy. The cryopreservation method used only allowed survival of small areas of cells located in the meristematic dome and at the base of the primordia.

Differential scanning calorimetry studies of intraembryonic freezing and cryoprotectant penetration in zebrafish (Danio rerio) embryos.

Nucleation temperatures of intraembryonic water and cryoprotectant penetration in zebrafish embryos were studied using differential scanning calorimetry. The effects of embryo developmental stage, dechorionation, partial removal of yolk, cooling rate, and cryoprotectant treatment on the temperatures of intraembryonic freezing were investigated. Embryo stages were found to have a significant effect on the nucleation temperatures of intact embryos. Freeze onset temperatures of -11.9 +/- 1.5, -15.6 +/- 0.3, and -20.5 +/- 0.1 degrees C were obtained for intact embryos at 6-somite, prim-6, and high-pec stages, respectively. After dechorionation, the freeze onset temperatures of intraembryonic water shifted to significantly lower temperatures, being -23.5 +/- 0.8, -18.7 +/- 0.7, -24.9 +/- 0.8 degrees C for 6-somite, prim-6, and high-pec stages, respectively. Yolk-reduced high-pec stage embryos showed significantly lower nucleation temperatures with an average onset at -27.9 +/- 0.4 degrees C. The effect of cryoprotectant treatment on the nucleation temperatures of intraembryonic water varies among different embryo stages and different cryoprotectants. Thirty-minute treatment with 2 M methanol significantly decreased the nucleation temperatures of dechorionated 6-somite embryos whilst no temperature decrease was observed for prim-6 or yolk-reduced high-pec embryos. Thirty-minute exposure to 1 M propylene glycol did not significantly affect the nucleation temperatures of dechorionated 6-somite, prim-6, or yolk-reduced high-pec embryos. In order to increase the permeability of embryos to cryoprotectants, the yolk sacs of dechorionated embryos at 6-somite or prim-6 embryos were punctured with a sharp micro-needle before exposure to cryoprotectants. The punctured prim-6 embryos showed significantly lower temperatures of intraembryonic freezing after 30 min of exposure to 2 M methanol following the multi-punctures. The nucleation temperatures of punctured 6-somite or prim-6 embryos were also decreased significantly after exposure to 1 M propylene glycol for 30 min. These results suggested that in intact embryos, intraembryonic freezing appeared to be seeded by the external ice in the perivitelline fluid and that in dechorionated embryos (in the absence of external water) intraembryonic freezing was more likely a consequence of heterogeneous nucleation. Methanol was demonstrated to show a limited degree of penetration into prim-6 stage embryos, but it did not penetrate later-stage embryos such as prim-6 and yolk-reduced high-pec. No propylene glycol permeation was observed for embryos at all stages. However, multi-punctures of yolk resulted in the permeation of both cryoprotectants into prim-6 embryos and propylene glycol permeation into 6-somite embryos. These findings may have important implications in overcoming the problem associated with the low membrane permeability of zebrafish embryos to cryoprotectants.

Genetic fidelity of cryopreserved embryogenic cultures of open-pollinated Abies cephalonica

The aim of the present study was to apply cryopreservation for the embryogenic cultures of open-pollinated Abies cephalonica, as well as to test the genetic fidelity of the cryopreserved cell lines using random amplified polymorphic DNAs, i.e. RAPD markers. Dimethylsulfoxide (DMSO) and two mixtures of polyethylene glycol, glucose and DMSO (PGD I and PGD II), were used as cryoprotectants and the cryoprotectant treatments were performed with and without storage in liquid nitrogen. All the tested cryopreservation methods, i.e. DMSO, PGD I or PDG II used as cryoprotectant combined with slow cooling and fast thawing, proved to be applicable for A. cephalonica SE cultures. The best results, measured as survival percentage and mean growth ratio, were achieved using the PGD I treatment. The RAPD assays performed revealed considerable genetic variation in the DMSO treated but non-frozen samples, i.e. 16.8% of the produced RAPD profiles showed intraclonal variation while background variation was seen in 1.7% of the control amplifications. The present results show that cryoprotectants may cause a risk for genetic fidelity of plant material and thus, monitoring it is important. This is particularly true in the breeding of the long-living conifers, since the effects of occasional mutations or genetic rearrangements may not be readily observed in young plants but expressed substantially later on in mature trees.

Cryopreservation of embryogenic cultures of Scots pine

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures.

Cryopreservation of horse-chestnut (Aesculus hippocastanum L. ) somatic embryos using three different freezing methods

Cryopreservation of somatic embryos of Aesculus hippocastanum L. cultured on nutritive media containing abscisic acid (ABA) at concentrations of 0.75 μM, 7.5 μM and 75.0 μM was evaluated for three cooling methods: (i) slow freezing with cryoprotectants, (ii) fast freezing with cryoprotectants, and (iii) fast freezing with desiccation techniques. The ‘cryoprotectant’ freezing techniques included the embryo pretreatment on ABA containing medium for 4 days, followed by cryoprotective treatment in liquid medium containing 0.5 M dimethylsulfoxide, 0.5 M glycerol, 1.0 M sucrose, and cooled at slow, and rapid rates. Embryos pretreated on a medium containing 0.75 μM ABA, and cooled to −35 °C at 1°C /min, held for 30 min at this transfer temperature and then immersed in liquid nitrogen (LN) had the best embryo recovery (43%). The ‘desiccation’ method involved an air drying step of similar ABA-pretreated, non-cryoprotected embryos followed by rapid cooling. Embryos precultured on 0.75 μM ABA, then subjected to a 4 h period of air desiccation (water content reduction to 13%) showed about the same level of survival (46%) as found with the ‘cryoprotectant’ slow freezing technique. The air-dry ‘desiccation’ method is easier to apply than the more complicated ‘cryoprotectant’ method.

Cryopreservation of spermatozoa of the edible mud crabScylla serrata (Forskal)

Biotechnique for programmed freezing of the spermatozoa of the edible mud crab Scylla serrata and the procedures for assessing viability of the frozen-thawed spermatozoa are described. Free spermatozoa recovered from spermathecae and those released from the spermatophores were subjected to cryoprotectant treatment at different physiological temperatures (30°C, 23°C, 15°C), frozen to subzero temperatures in a rate-controlled programmable freezer Kryo 10/1.7 and subsequently plunged into liquid nitrogen. Post-thaw viability, after 8 hr of storage at −196°C, was assessed by artificial induction of acrosome reaction using divalent cation ionophore A23187. Hypo/hyperosmotic sensitivity tests were used to assess the membrane integrity of the spermatozoa before and after cryopreservation experiments. Viability tests using dye exclusion methods were discontinued owing to variations in incorporation of different stains into the spermatozoa. The limitations of the dye exclusion method as an indicator of sperm viability are discussed. Dimethyl sulfoxide, ethylene glycol and glycerol offered cryoprotection at concentrations ranging from 5% to 15% v/v. Post-thaw survival was highest (52%) with 12.5% glycerol at a cooling rate of −5°C/min. J. Exp. Zool. 277:326–336, 1997. © 1997 Wiley-Liss, Inc.

Serial Thick, Frozen, Gallocyanin Stained Sections of Human Centrl Nervous System

AbstractA rapid method for macroscopic and microscopic investigation of human CNS is proposed. After formalin fixation, gelatin or agarose embedding, and cryoprotective treatment, frozen human spinal cords, brainstems, or hemispheres can be serially cut into 0.7 mm thick slices. Stained with gallocyanin- chromalum, these slices facilitate cytoarchitectonic, neuropathologic, and quantitative examination. Regions of interest from parallel formalin-stored unstained slices can be embedded into paraffin and stained by any immunocytologic and histologic stain compatible with formalin fixation and paraffin embedding. (The J Histotechnol 14:167, 1991)

Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos

Two studies were conducted to evaluate the influence of cryoprotectant, cooling rate, container and cryopreservation procedure on the post-thaw viability of sheep embryos. In Study 1, late morula- to blastocyst-stage embryos were exposed to 1 of 10 cryoprotectant (1.5 M, glycerol vs propylene glycol)-plunge temperature treatments. Embryos were placed in glass ampules and cooled at 1°C/min to −5°C, seeded and further cooled at 0.3°C/min to −15, −20, −25, −30 and −35°C before rapid cooling by direct placement in liquid nitrogen (LN 2). Post-thaw embryo viability was improved (P<0.01) when embryos were cooled to at least −30°C before LN 2 plunging. Although there were no overt differences in embryo viability between cryoprotectant treatments (each resulted in live offspring after embryo transfer), there was a lower (P<0.01) incidence of zona pellucida damage using propylene glycol (4%) compared to glycerol (40%). In Study 2, embryos were equilibrated in 1.5 M propylene glycol or glycerol or a vitrification solution (VS3a). Embryos treated in propylene glycol or glycerol were divided into ampule or one-step ® straw treatments, cooled to −6°C at 1°C/min, seeded, cooled at 0.5°C/min to −35°C, held for 15 minutes and then transferred to LN 2. Embryos vitrified in the highly concentrated VS3a (6.5 M glycerol + 6% bovine serum albumin) were transferred from room air to LN 2 vapor, and then stored in LN 2. Propylene glycol- and glycerol-treated embryos in straws experienced lower (P<0.05) degeneration rates (27%) and yielded more (P<0.05) hatched blastocysts (73 and 60%, respectively) at 48 hours of culture and more (P<0.05) trophoblastic outgrowths (67 and 53%, respectively) after 1 week than vitrified embryos (47, 40 and 20%, respectively). In vitro development rate for VS3a-treated embryos was similar (P>0.10) to that of ampule controls, which had fewer (P<0.05) expanded blastocysts compared to similar straw treatments. Live offspring were produced from embryos cryopreserved by each straw treatment (propylene glycol, 3 of 7; glycerol, 1 of 7; VS3a, 2 of 7). In summary, freeze-preservation of sheep embryos was more effective in one-step straws than glass ampules and propylene glycol tended to be the optimum cryoprotectant. Furthermore, these findings demonstrate, for the first time, the biological competence of sheep embryos cryopreserved using the simple and rapid procedure of vitrification.

Effect of cooling rate, cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with −30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at − 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and − 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.

Cryopreservation of Coleus blumei Suspension and Callus Cultures

Callus and suspension cultures of Coleus blumei Benth. were cryopreserved and stored for various periods of time in liquid nitrogen. Sorbitol was the only additive used during preculture and during cryoprotection treatments. The best results were obtained using cells from the early growth period. The duration of storage was 3, 6, 12 and 15 months, respectively. Cell viability could be improved considerably either by thawing the samples rapidly in a 60 °C water bath (instead of 45 °C) or by thawing them in a microwave oven. After a short period of recovery growth on semi-solid agar medium, the cells were cultivated under standard conditions. The growth characteristics and the accumulation of rosmarinic acid, a compound of pharmaceutical value, were found to be in the range of untreated controls.

EFFECTS OF CRYOPROTECTANTS IN MINIMIZING PHYSICOCHEMICAL CHANGES OF BOVINE NATURAL ACTOMYOSIN DURING FROZEN STORAGE

Physicochemical changes in bovine natural actomyosin extracted from prerigor semimembranosus muscle were investigated during frozen storage at −28°C as affected by the addition of cryoprotectants (5.6% Polydextrose® or 5.6% mixture (1:1) of sucrose and sorbitol). Proteins were destabilized during freezing and frozen storage as reflected by decreases in protein solubility, the visual appearance of aggregates in protein sols, decrease in intensity of flow birefringence, intrinsic viscosity and ATPase activity, and changes in size, shape, or charge of the protein (especially myosin) as evidenced by nondenaturing electrophoresis. These effects were reduced to some extent by the two cryoprotectant treatments.

Fine structure of intramembranous particle aggregates in ADH-treated frog urinary bladder and skin: influence of glutaraldehyde and N-ethyl maleimide.

The fine structure of ADH-induced intramembrane particle aggregates has been studied in different tissues and under different experimental conditions. Particle aggregates similar to those previously observed in the amphibian urinary bladder and in the mammalian collecting duct were also found in the frog skin, another ADH target tissue. In the frog urinary bladder, typical aggregates were observed in the absence of glutaraldehyde fixation. Two experimental approaches were used 1) the absence of both fixative and cryoprotectant treatments and b) the absence of only glutaraldehyde treatment. In the latter case the reversal of hydrosmotic action was prevented by exposing the preparations to N-ethyl maleimide. In specimens of frog urinary bladder conventionally fixed with glutaraldehyde, two fracture levels could be observed in the aggregates, suggesting that the aggregated particles span an appreciable part of the membrane thickness.

Gap junction dynamics: reversible effects of hydrogen ions.

Reversible crystallization of intramembrane particle packings is induced in gap junctions isolated from calf lens fibers by exposure to 3 x 10(-7) M or higher [H+] (pH 6.5 or lower). The changes from disordered to crystalline particle packings induced by low pH are similar to those produced in junctions of intact cells by uncoupling treatments, indicating that H+, like divalent cations, could be an uncoupling agent. The freeze-fracture appearance of both control and low pH-treated gap junctions is not altered by glutaraldehyde fixation and cryoprotective treatment, as suggested by experiments in which gap junctions of both intact cells and isolated fractions are freeze-fractured after rapid freezing to liquid N2 temperature according to Heuser et al. (13). In junctions exposed to low pH, the particles most often form orthogonal and rhombic arrays, frequently fused with each other. A number of structural characteristics of these arrays suggest that the particles of lens fiber gap junctions may be shaped as tetrameres.

Gefrier-Ultramikrotomie: Entwicklungsstand und Anwendungsmöglichkeiten (mit Beispielen aus der Hefecytologie)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.