Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification

The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.Abbreviations DMSO dimethyl sulfoxide - PVS2 vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - BA 6-benzylaminopurine - MT Murashige-Tucker basal medium - INAA naphthaleneacetic acid     

Cryopreservation of small leaf squares-bearing adventitious buds of <Emphasis Type="Italic">Lilium</Emphasis> Oriental hybrid ‘Siberia’ by vitrification

We report a new cryopreservation method for Lilium Oriental hybrid ‘Siberia’. Adventitious buds were induced from leaf segments cultured for 12 days on adventitious bud induction medium composed of half-strength Murashige and Skoog medium (MS) supplemented with 1 mg L^?1 α-naphthalene acetic acid and 0.5 mg L^?1 thidiazuron. Small leaf squares (SLSs, 3?×?4 mm), each bearing at least one adventitious bud, were cut from leaf segments, precultured on medium with 0.5 M sucrose for 1 day, and then treated for 20 min with a loading solution containing 0.4 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 for 7 h at 0 °C. Dehydrated SLSs were directly immersed in liquid nitrogen for 1 h. Cryopreserved SLSs were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, 85% survival and 72% shoot regrowth were achieved following cryopreservation. The use of SLSs bearing adventitious buds for cryopreservation reported in the present study eliminates the time-consuming and labour-intensive step of shoot tip excision, and has great potential to facilitate cryopreservation in other plant species.     

Cryopreservation of wheat suspension culture and regenerable callus

Wheat (Triticum aestivum L. cv. Norstar) suspension cultures and regenerable calli initiated from immature embryos can be cryopreserved in liquid nitrogen temperature (–196°C) by slow freezing (0.5°C/min) in the presence of a mixture of DMSO and sucrose or sorbitol. Cold hardening or ABA treatment before cryopreservation increased the freezing resistance and improved the survival of wheat suspension culture in liquid nitrogen. Callus culture, established from immature embryos, prefrozen in 5% DMSO and 0.5M sorbitol survived liquid nitrogen storage and resumed plant regeneration after thawing. The results confirm the feasibility of long term preservation of wheat embryo callus by cryopreservation and retention of plant regeneration ability.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - LN Liquid nitrogen - TTC 2,3,5-triphenyltetrazolium chloride NRCC No. 23850.     

Systematic factor optimization for sperm cryopreservation of tetraploid Pacific oysters, Crassostrea gigas

The availability of tetraploid Pacific oysters provides a unique opportunity for comparative studies of sperm cryopreservation between diploids and tetraploids. In parallel to studies with sperm from diploid oysters, this study reports systematic factor optimization for sperm cryopreservation of tetraploid oysters. Specifically, this study evaluated the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), and straw size. Similar to sperm from diploids, the optimal cooling rate was 5 degrees C/min to -30 degrees C, followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations showed that a combination of the cryoprotectants 6% polyethylene glycol/4% propylene glycol and 6% polyethylene glycol/4% dimethyl sulfoxide yielded consistently high post-thaw motility. A long equilibration (60 min) yielded higher percent fertilization, and confirmed that extended equilibration could be beneficial when low concentrations of cryoprotectant are used. There was no significant difference in post-thaw motility between straw sizes of 0.25 and 0.5 mL. Despite low post-thaw fertilization (<10%) in general for sperm from tetraploids, optimized protocols in the present study effectively retained post-thaw motility for sperm from tetraploid oysters. This study confirmed that sperm from tetraploid Pacific oysters were more negatively affected by cryopreservation than were those of diploids. One possible explanation is that sperm from these two ploidies are different in their plasma membrane properties (e.g., structure, permeability, and elasticity), and the plasma membrane of sperm from tetraploids is more sensitive to cryopreservation effects. The fact that combinations of non-permeating and permeating cryoprotectants improved post-thaw motility in sperm from tetraploids provided presumptive evidence for this interpretation.     

Impact of freezing/thawing procedures on the post-thaw viability of cryopreserved human saphenous vein conduits

BACKGROUND: Cryopreserved human blood vessels are important tools in reconstructive surgery. However, patency of frozen/thawed conduits depends largely on the freezing/thawing procedures employed. METHODS: Changes in tone were recorded on rings from human saphenous vein (SV) and used to quantify the degree of cryoinjury after different periods of exposure at room temperature to the cryomedium (Krebs-Henseleit solution containing 1.8M dimethyl sulfoxide and 0.1M sucrose) and after different cooling speeds and thawing rates following storage at -196 degrees C. RESULTS: Without freezing, exposure of SV to the cryomedium for up to 240 min did not modify contractile responses to noradrenaline (NA). Pre-freezing exposure to the cryomedium for 10-120 min attenuated significantly post-thaw maximal contractile responses to NA, endothelin-1 (ET-1) and potassium chloride (KCl) by 30-44%. Exposure for 240 min attenuated post-thaw contractile responses to all tested agents markedly by 62-67%. Optimal post-thaw contractile activity was obtained with SV frozen at about -1.2 degrees C/min and thawed slowly at about 15 degrees C/min. In these SV maximal contractile responses to NA, ET-1 and KCl amounted to 66%, 70% and 60% of that produced by unfrozen controls. Following cryostorage of veins for up to 10 years the responsiveness of vascular smooth muscle to NA was well maintained. CONCLUSION: Cryopreservation allows long-term banking of viable human SV with only minor loss in contractility.     

Ultrastructural and cytochemical characterization of follicular cell types in bovine (Bos taurus) cumulus-oocyte complexes aspirated from small and medium antral follicles during the estrus cycle

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.     

Strategies for the cryopreservation of microencapsulated cells

The major challenge in developing cryopreservation protocols for microencapsulated cells is that the relatively large size (300-400 microm) and the fragile semipermeable membrane of microcapsules makes them particularly prone to cryodamage. Rapid-cooling cryopreservation protocols with high DMSO concentrations (3.5M, 25% v/v) resulted in low post-thaw cell viability (<10%), which did not improve with higher concentrations (4.5M, 32% v/v) and longer exposure to DMSO, even though the majority of microcapsules (60-80%) remained intact. Subsequent investigations of slow cooling with a range of DMSO and EG concentrations resulted in a much higher post-thaw cell viability (80-85%), with the majority of the microcapsules remaining intact ( approximately 60%) when DMSO was used at a concentration of 2.8M (20% v/v) and EG at a concentration of 2.7M (15% v/v). The presence of 0.25M sucrose significantly improved post-thaw cell viability upon slow cooling with 2.8M (20% v/v) DMSO, although it had no effect on microcapsule integrity. Multistep exposure and removal of sucrose did not significantly improve either post-thaw cell viability or microcapsule integrity, compared to a single-step protocol. Ficoll 20% (w/v) also did not significantly improve post-thaw cell viability and microcapsule integrity. Hence, the optimal condition for microcapsule cryopreservation developed in this study is slow cooling with 2.8M (20% v/v) DMSO and 0.25M sucrose.     

Cryopreservation of human umbilical vein and porcine corneal endothelial cell monolayers

Cryopreservation of endothelium is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (Me₂SO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of endothelial monolayers. The addition of 2% chondroitin sulfate to 5% Me₂SO and 6% HES and cooling at 0.2 or 1 °C/min led to high membrane integrity (97.3 ± 3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9 ± 3.7% membrane integrity) with metabolic activity 12 h post-thaw comparable to unfrozen control.     

Survival of mouse embryos after being frozen in glycerol-sucrose mixture

Random bred female albino mice (6-8 weeks old) were used as a source of embryos. 8- to 16 cell embryos were dehydrated in glycerol-sucrose mixture in 0.25 ml straws at room temperature. Straws were cooled at the rate of 5 degrees C/min to -7 degrees C. Seeding was induced by touching the out side of the straw at -7 degrees C. Straws were further cooled at 0.5 degree C/min down to -35 degrees C and then plunged into liquid N2. Thawing of straws was done by direct transfer into water at 35 degrees C. Frozen-thawed embryos were cultured in a CO2 incubator maintained at 39 degrees C. Out 190 embryos (8-16 cell) initially frozen, 169 (88.94%) were recovered on thawing. 158 (93.5%) out of 169 were apparently normal and used for culture. 75 (47.46%) developed to morulae/early blastocysts and 72 (45.56%) to expanded blastocysts on 24 and 48 hr culture respectively. In conclusion, the incorporation of sucrose in the freezing medium at a concentration of 0.25 M has led us to propose a freezing, thawing and transfer method without dilution of glycerol. The technique being quite simple is worth trying in farm animals where importance of this technique in non-surgical transfer of frozen-thawed embryos will be a boon.     

A comparative study of mouse embryo freeze-preservation including the examination of a thermoelectric freezing device

In Study 1 over 2000 4- to 8-cell mouse embryos were randomly pooled and assigned to 1 of 12 treatment groups. A 2 X 2 X 3 factorial design was used to analyze two types of cryoprotectant/post-thaw (PT) dilutions (dimethyl sulfoxide [Me2SO]/stepwise dilution versus glycerol/sucrose dilution), two storage containers (glass ampoules versus plastic straws), and three cooling treatments. Two commercial, controlled-rate freezing machines were examined, employing either nitrogen gas (Planer) or thermoelectric (Glacier) cooling. Embryos were cooled slowly (0.5 degrees C/min) to -35 or -80 degrees C and then cooled rapidly by transfer into liquid nitrogen (LN2). Thawed embryos were cultured for 24 hr after which developmental stage, post-thaw survival (PTS), embryo degeneration rate (EDR), quality grade (QG), and fluorescein diacetate viability grade (VG) were assessed. Overall, PTS and EDR were similar (P greater than 0.05) among the three freezing unit/plunge temperature treatments. Cumulative results of container and cryoprotectant/PT dilution treatments consistently demonstrated greater PTS, QG, and VG ratings and lower EDR values when embryos were frozen in ampoules using glycerol/sucrose dilution. Embryos treated with Me2SO/stepwise dilution were particularly sensitive to freezing damage when stored in plastic straws and plunged into LN2 at -35 degrees C. Study 2 was directed at determining whether Study 1 methods for diluting Me2SO-protected embryos markedly affected PTS rates. Post-thaw culture percentages were no different (P greater than 0.05) for four- to eight-cell Me2SO-treated embryos frozen in ampoules (using the forced-LN2 device), thawed, and diluted either conventionally in reduced concentrations of Me2SO or in the sucrose treatment normally accorded glycerolated embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of in vitro matured,in vitro fertilized domestic cat embryos following cryopreservation,culture and transfer

The ability of embryos to successfully survive cryopreservation is dependent on both morphological and developmental characteristics. Domestic cat oocytes matured in vitro exhibit alterations in nuclear and cytoplasmic maturation that may affect developmental competence, particularly after cryopreservation. In Experiment 1, we evaluated the developmental competence of in vitro produced (IVM/IVF) cat embryos after cryopreservation on Days 2, 4 or 5 of IVC. In Experiment 2, in vivo viability was examined by transfer of cryopreserved embryos into recipient queens. Oocytes recovered from minced ovaries were cultured in TCM-199 with hCG/eCG and EGF at 38 degrees C in 5% O(2), 5% CO(2), 90% N(2) for 24h. In Experiment 1, after IVM/IVF, on Day 2 (n=56), Day 4 (n=48) and Day 5 (n=42) of IVC, embryos were equilibrated for 10 min at 22 degrees C in HEPES (15m M) Tyrode's (HeTy) with 1.4M propylene glycol (PG), 0.125 M sucrose (S), 10% dextran and 10% FBS, loaded into 0.25 ml straws, cooled at 2.0 degrees C/min to -6.0 degrees C and held for 10 min. After seeding, cooling resumed at 0.3 degrees C/min to -30 degrees C and after a 10 min hold, straws were plunged into liquid nitrogen (LN(2)). Straws were thawed in air for 2 min and cryoprotectant was removed by a five-step rinse consisting of 3 min each in HeTY with 0.95 M PG/0.25 M S; 0.95 M PG/0.125 M S; 0.45 M PG/0.125 M S; 0 PG/0.125 M S; 0 PG/0.0625 M S. Contemporary IVM/IVF embryos were used as nonfrozen controls (Day 2, n=14; Day 4, n=26; Day 5, n=35). After 8 days of IVC, the number of embryos developing to blastocysts was recorded and blastocyst cell numbers were counted after staining with Hoechst 33342. In Experiment 1, developmental stage did not affect the survival rate after thawing (Day 2=79%, Day 4=90%, Day 5=98%) and was not different from that of controls (Day 2=89%, Day 4=88%, Day 5=96%). Blastocyst development was similar among days both after cryopreservation (Day 2=59%, Day 4=54%, Day 5=63%) and in controls (Day 2=55%, Day 4=54%, Day 5=58%). Mean (+/-S.D.) cell number of blastocysts was slightly lower (NS) in cryopreserved embryos (Day 2=152+/-19, Day 4=124+/-20, Day 5=121+/-24) than in controls (Day 2=141+/-25, Day 4=169+/-21, Day 5=172+/-19). In Experiment 2, embryos frozen on Day 2 (n=68), Day 4 (n=49) or Day 5 (n=73) were thawed and cultured for 3, 1, or 0 days before transfer by laparotomy to 5 (mean=12.6+/-2.4), 4 (mean=12.2+/-3.7) and 6 (mean=12.0+/-1.6) recipients, respectively. Four recipients were pregnant on Day 21; two from embryos frozen on Day 4 and two from Day 5. Two live kittens were born at 66 days, a third kitten died during parturition at 64 days and a fourth pregnancy aborted by Day 45. In summary, we have shown that a controlled rate cryopreservation technique can be successfully applied to cat embryos produced by IVM/IVF. In vitro development to the blastocyst stage was not affected by the age of embryos at cryopreservation. The births of live kittens after ET of cryopreserved embryos is additional validation of progress toward applying assisted reproductive technology to preservation of endangered felids.     

Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.     

Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas

Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lacking, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 degrees C/min to -30 degrees C followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.     

Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii

In order to develop cryopreservation techniques for Japanese pearl oyster spermatozoa, the effects of various cryopreservation conditions on post-thaw motility were examined. Spermatozoa cryopreserved with 10% methanol (MET), dimethylformamide or dimethylacetamide plus 90% diluent comprising 80% seawater and 20% fetal bovine serum (FBS) showed higher percentages of post-thaw motility than those cryopreserved with 10% dimethylsulfoxide or glycerol. When spermatozoa were cryopreserved with various concentrations (0-20%) of MET and 100-80% diluent, 10% MET showed the highest percentages of post-thaw motility. When spermatozoa were cryopreserved with 10% MET and 90% diluent comprising various concentrations (0-100%) of FBS or Ringer solution mixed with seawater, the percentages of post-thaw motility peaked at 20% FBS or Ringer solution, and were significantly higher for 20% FBS than for 20% Ringer solution. The percentages of post-thaw motility increased with increasing dilution ratios from 2.5- to 50-fold. Spermatozoa cooled to -50 degrees C and then immersed in liquid nitrogen (LN) showed higher post-thaw motility than those cooled to -30 degrees C or -40 degrees C. When spermatozoa were cryopreserved to -50 degrees C at various cooling rates by changing the sample height above the LN surface, the post-thaw motilities of spermatozoa cooled at 10 cm (cooling rate: -21.3 degrees C/min) and 12.5 cm (-15.6 degrees C/min) from the LN surface were higher than those at 5, 7.5 or 15 cm. These results indicate that 10% MET plus 90% diluent comprising 80% seawater and 20% FBS is a suitable extender for cryopreservation of Japanese pearl oyster spermatozoa and that samples should be cooled to -50 degrees C at a cooling rate between -15 and -20 degrees C/min for efficient storage.     

Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour

The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20 degrees C, exposure to less than or equal to 1.0 M-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36 degrees C, however, greater than or equal to 1.0 M-sucrose significantly reduced the developmental potential (P less than 0.001). In the freezing experiments the embryos were placed in 0.5 ml straws containing 40 microliters freezing medium separated by an air bubble from 440 microliters sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20 degrees C than at 36 degrees C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P less than 0.001). The best survival was obtained when the freezing medium contained 3.0 M-glycerol + 0.25 M-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20 degrees C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0.5 M, 69%; 1.0 M, 73%; 1.5 M, 64%). The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.     

Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel.

During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.     

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

Functional assessment of cryopreserved pig aortas for pharmaceutical research

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached.After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.     

Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).