Physical problems with the vitrification of large biological systems

Vitrification is an attractive potential pathway to the successful cryopreservation of mature mammalian organs, but modern cryobiological research on vitrification to date has been devoted mostly to experiments with solutions and with biological systems ranging in diameter from about 6 through about 100 microns. The present paper focuses on concerns which are particularly relevant to large biological systems, i.e., those systems ranging in size from approximately 10 ml to approximately 1.5 liters. New qualitative data are provided on the effect of sample size on the probability of nucleation and the ultimate size of the resulting ice crystals as well as on the probability of fracture at or below Tg. Nucleation, crystal growth, and fracture depend on cooling velocity and the magnitude of thermal gradients in the sample, which in turn depend on sample size, geometry, and cooling technique (environmental thermal history and thermal uniformity). Quantitative data on thermal gradients, cooling rates, and fracture temperatures are provided as a function of sample size. The main conclusions are as follows. First, cooling rate (from about 0.2 to about 2.5 degrees C/min) has a profound influence on the temperature-dependent processes of nucleation and crystal growth in 47-50% (w/w) solutions of propylene glycol. Second, fracturing depends strongly on cooling rate and thermal uniformity and can be postponed to about 25 degrees C below Tg for a 482-ml sample if cooling is slow and uniform. Third, the presence of a carrier solution reduces the concentration of cryoprotectant needed for vitrification (CV). However, the CV of samples larger than about 10 ml is significantly higher than the CV of smaller samples whether a carrier solution is present or not.     

Localization of Drosophila retinal degeneration B, a membrane- associated phosphatidylinositol transfer protein

The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160- kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium.     

Conventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rabbit embryos

Three different methods of cryopreservation viz., conventional slow freezing, vitrification and open pulled straw vitrification were compared for their ability to support post thaw in vitro and in vivo development of rabbit embryos. Morula stage rabbit embryos were collected from super-ovulated donor does. They were randomly allocated to different freezing methods and stored up to 3 months in liquid nitrogen. After thawing and removal of cryoprotectants, embryos exhibiting intact zona pellucida and uniform blastomeres were considered suitable for in vitro culture and/or transfer. Three to five cryopreserved embryos placed in approximately 1 ml of culture medium (TCM 199 supplemented with foetal calf serum and antibiotics) were incubated for up to 72 h under humidified atmosphere of 5% CO2 in air at 39 degrees C. Development to hatched blastocyst stage was considered the initial indicator of success of cryopreservation of embryos. Of the embryos cryopreserved by programmed freezing, open pulled straw vitrification, vitrification-55 h pc and vitrification-72 h pc 55, 71, 17 and 48%, respectively, developed into hatched blastocysts. Similarly 19, 29, and 4% of embryos cryopreserved by programmed freezing, open pulled straw vitrification and vitrification -72 h pc developed into live offspring on transfer to recipient does. This is the first report on open pulled straw vitrification of rabbit embryos. Present results, suggest that (a) open pulled straw vitrification supports better in vitro survival of frozen thawed rabbit morulae; (b) both programmed freezing and OPS are similar but superior to vitirification in supporting in vivo survival of frozen thawed rabbit embryos.     

Development of membrane- and calcium-gradients during pollen germination of Lilium longiflorum

Chlorotetracyclin (10^-4M) has been used to observe the distribution of membrane-associated calcium during pollen germination of Lilium longiflorum. For comparison, the general membrane distribution has been determined with 4·10^-5 M fluorescamine. The pollen grains show a calcium gradient with either weak or strong chlorotetracycline-fluorescence intensity, but always increasing toward the germination colpus. This gradient intensifies during germination, reaching a maximum before the pollen tube emerges. The typical tip-to-base calcium gradient of the tube does not change during growth. Independent of the developmental stage, the pollen grains show a flat fluorescamine-fluorescence gradient with the highest intensity in one half of the grain. Pollen tubes reveal a tip-to-base membrane gradient, independent of their length. As an additional marker for membrane distribution, the distribution of phosphorus, measured by proton-induced X-ray emission in chemically fixed tubes, has been used. A tip-to-base phosphorus gradient, distinct from the calcium gradient measured with the same method, was detected.Abbreviation CTC chlorotetracycline     

Freezing Injury in Onion Bulb Cells: II. Post-thawing Injury or Recovery

Onion (Allium cepa L.) bulbs were subjected for 12 days to either a moderate freeze (−4 C) or a severe freeze (−11 C). They were then thawed slowly over ice. During 7 to 12 days following the thaw, the injury progressed with time in the severely frozen bulbs, but appeared completely repaired in the moderately frozen bulbs. This was shown by the following post-thawing changes.     

Physiological Response of Neurospora Conidia to Freezing in the Dehydrated, Hydrated, or Germinated State

This study concerned the response to freezing of Neurospora crassa conidia in four different states: air-dry, hydrated in water, hydrated in Vogel medium lacking only sucrose, or hydrated in complete Vogel medium. All hydrated conidia were incubated in one of the above media for various times before freezing and were then washed and frozen in distilled water. Viability was estimated by three techniques, and the agreement among them was good. Hydration of air-dry conidia was found to be very rapid and, once hydrated, the conidia were much more sensitive to rapid freezing than they were before hydration. Rapidly cooled conidia survived freezing to a much higher extent when the warming rate was rapid than when it was slow; slowly cooled conidia showed little or no dependence on the warming rate. This sensitivity to rapid cooling and slow warming was attributed to the effects of intracellular ice. The sensitivity to freezing could be reversed by dehydrating the conidia in vacuo before freezing; thus, it was concluded that the presence or absence of water is the determining factor in the initial sensitivity due to freezing. In water, the sensitivity remained constant from 2 min to 15 days after hydration. Although conidia hydrated in growth medium lacking sucrose remained metabolically inactive, their sensitivity to rapid freezing decreased as a function of time in the medium before freezing. The reason for this decreased sensitivity is not understood. Conidia hydrated in complete growth medium (i.e., containing sucrose) became metabolically active and, after the initial sensitivity associated with hydration, became increasingly more sensitive to freezing as a function of their time in the medium. Drying itself was deleterious to metabolically active conidia, and those that survived dehydration did not exhibit a large absolute increase in resistance to subsequent freezing. The increase in sensitivity to freezing and to drying seems associated with the presence of metabolic activity; however, the precise cause of the sensitization remains obscure.     

Numerical simulation of cooling rates in vitrification systems used for oocyte cryopreservation

Oocyte cryopreservation is of key importance in the preservation and propagation of germplasm. Interest in oocyte cryopreservation has increased in recent years due to the application of assisted reproductive technologies in farm animals such as in vitro fertilization, nuclear transfer and the need for the establishment of ova/gene banks worldwide. However, the cryopreservation of the female gamete has been met with limited success mainly due to its small surface-area:volume ratio.In the past decade, several vitrification devices such as open pulled straws (OPS), fine and ultra fine pipette tips, nylon loops and polyethylene films have been introduced in order to manipulate minimal volumes and achieve high cooling rates. However, experimental comparison of cooling rates presents difficulties mainly because of the reduced size of these systems. To circumvent this limitation, a numerical simulation of cooling rates of various vitrification systems immersed in liquid nitrogen was conducted solving the non-stationary heat transfer partial differential equation using finite element method.Results indicate the nylon loop (Cryoloop®) is the most efficient heat transfer system analyzed, with a predicted cooling rate of 180,000 °C/min for an external heat transfer coefficient h = 1000 W/m² K when cooling from 20 to −130 °C; in contrast, the open pulled straw method (OPS) showed the lowest performance with a cooling rate of 5521 °C/min considering the same value of external heat transfer coefficient. Predicted cooling rates of Miniflex® and Cryotop® (polyethylene film system) were 6164 and 37,500 °C/min, respectively, for the same heat transfer coefficient.     

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 degrees C/min to -5 degrees C, seeded and further cooled at 0.3 degrees C/min to -15, -20, -25, -30 and -35 degrees 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 degrees 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((R)) straw treatments, cooled to -6 degrees C at 1 degrees C/min, seeded, cooled at 0.5 degrees C/min to -35 degrees 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.     

Cryopreservation of immature Parkia speciosa Hassk. zygotic embryonic axes following desiccation or exposure to vitrification solution

This work reports on the cryopreservation of immature zygotic embryonic axes (EA) of petai (Parkia speciosa Hassk.) for the first time. Two cryopreservation protocols, namely desiccation and vitrification method were tested individually using excised EA. Desiccation of EA to lower moisture content (MC) reduced the survival percentage but a drastic decline in survival percentage (~20 %) was recorded at 16 % MC prior to exposure to LN, rendering the EA to be sensitive to desiccation. Cryopreservation of EA after desiccation, irrespective of the MC, did not result in any survival. On the other hand, post-cryopreservation survival was obtained when the EA were exposed to plant vitrification solution-2 (PVS2) for 75–105 min. The best results were obtained when the EA were exposed to PVS2 for 90 min with an average recovery of 55.5 %. EA recovery into whole plantlets was obtained when the EA were cultured on MS medium supplemented with 2 gl^?1 activated charcoal and 0.1 mgl^?1 of the plant growth regulators α-naphthalene acetic acid, 6-benzylaminopurine and gibberellin A₃, each. EA, exposed for less than 75 min and more than 105 min to PVS2, did not show any survival after cryopreservation. The optimization of exposure time is necessary to increase survival. This study has shown that the employment of suitable method is important for conservation using cryopreservation.     

Acrosomal integrity, viability, and DNA damage of sperm from dasyurid marsupials after freezing or freeze drying

Dasyurids are a diverse group of Australian native carnivores and insectivores that contains several threatened species. Despite successful cryopreservation of sperm from several marsupials, only 3% postthaw motility is reported for dasyurid marsupials. This study examined sperm preservation in the fat-tailed dunnart (Sminthopsis crassicaudata), an experimental model, with supplementary observations on the eastern quoll (Dasyurus viverrinus) and northern quoll (Dasyurus hallucatus). In S. crassicaudata, a toxicity trial demonstrated that incubation with up to 40% glycerol did not reduce sperm viability, suggesting that glycerol is not toxic to dasyurids. On the basis of this finding, S. crassicaudata, D. viverrinus, and D. hallucatus sperm were extended to a final concentration of 20% or 40% glycerol in Tris-citrate fructose and frozen in liquid nitrogen vapor. Postthaw sperm from all three species were nonmotile, and vital staining (SYBR14 and propidium iodide) indicated that sperm were nonviable. However, there was no evidence suggesting disruption of normal gross morphology or loss of acrosomal integrity when assessed by Bryan's staining. After freeze drying, Bryan's staining indicated that approximately 80% of S. crassicaudata sperm had normal acrosomes and no head loss. Despite being nonviable, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling showed that S. crassicaudata sperm frozen in 40% glycerol or freeze-dried had no DNA damage compared with fresh controls. This study has described a method for preservation of the dasyurid sperm nuclei, but continued studies are required to achieve viable motile sperm and establish tools for the long-term storage of dasyurid sperm.     

Cryopreservation of mouse half-morulae and chimeric embryos by vitrification.

Mouse half-morulae were cryopreserved less than or equal to 1, 3, 6, and 12 hr after bisection by the vitrification method using 25% glycerol and 25% 1,2-propanediol as cryoprotectant. The developmental rates of the frozen-thawed half-embryos to blastocysts in vitro were 77.8% (63/81), 82.0% (41/50), 92.1% (117/127), and 0% (0/37), respectively. Sixty-one of the half-embryos that had been vitrified 6 hr after the bisection followed by transfer to five recipients resulted in a total of ten (16.4%) normal fetuses. Chimeric mouse embryos constructed by two half-morulae were also vitrified 6 and 16 hr after aggregation. Survivors were obtained from the former case: 40 (80.0%) of 50 frozen-thawed embryos developed in vitro to blastocysts, and, after transfer, six chimeric offspring were obtained from the 34 vitrified chimeric embryos. These results showed that mouse half-morulae and chimeric embryos could be cryopreserved by the vitrification method. It seems possible to manufacture a chimeric mouse embryo of defined genotypic composition that can be analyzed during its frozen state using the identical half-embryos of the components.     

Cryopreservation of Hyoscyamus niger adventitious roots by vitrification

An auxin-independent adventitious root culture of Hyoscyamus niger was established, and the roots were successfully cryopreserved with a high regeneration rate of 93.3 percnt; by vitrification method. The root tips were cultured for 12 to 14 days in phytohormone-free Murashige and Skoog (MS) liquid medium, and were excised and precultured on Woody Plant (WP) solid medium supplemented with 0.3 mol/L sucrose at 25 °C in the dark. After 1 day, they were treated with MS-based loading solution for 10 min, followed by soaking in MS-based PVS2 for 10 min at 0 °C. The treated root tips were immersed in liquid nitrogen (-196 °C). For recovery, the root tips were thawed rapidly at 40 °C and washed with MS medium containing 1 mol/L sucrose prior to plating onto WP solid medium. The regenerated roots were evaluated by their growth and tropane alkaloid production. The growth and alkaloid content of regenerated roots analyzed using HPLC were found to be almost the same as those of non-treated roots.     

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.