WO1999031977A1

WO1999031977A1 - Method for cryopreserving oocytes

Info

Publication number: WO1999031977A1
Application number: PCT/US1998/027248
Authority: WO
Inventors: John D. Biggers; Fouad S. Trad; Mehmet Toner
Original assignee: President And Fellows Of Harvard College
Priority date: 1997-12-22
Filing date: 1998-12-21
Publication date: 1999-07-01
Also published as: AU2008399A

Abstract

A method for inhibiting the formation of intracellular ice formation in viable cells is disclosed. A method of increasing survival rates of the cells after freezing and thawing is also disclosed. A preparation of viable cells immersed in a solution having a cryoprotectant is prepared. The preparation is then supercooled to a temperature below its freezing point. Then the formation of extracellular ice is promoted at a temperature above that at which intracellular ice formation occurs in the cells. The preparation is then cooled to a final storage temperature.

Description

METHOD FORCRYOPRESERVING OOCYTES

This application claims the benefit of U.S. Provisional Application Serial No. 60/068,462, filed December 22, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention Embodiments of the present invention relate in general to the freezing of cells for later storage at prolonged periods of time. Embodiments of the present invention also relate to methods of preparing cells for freezing so as to increase cell viability upon thaw. Embodiments of the present invention further relate to methods of inhibiting intracellular ice formation within cells at temperatures at which intracellular ice formation normally occurs.

2. Description of Related Art

Routine human oocyte cryopreservation remains an elusive objective. The need for a method of cryopreserving oocytes has become more pressing with the increase of donor oocyte programs and the potential risk of disease transmission. Women who are at risk of losing their ovarian function to surgery, cancer treatment or premature menopause will also benefit from oocyte banking. Cryopreservation of oocytes should also circumvent many ethical and legal objections to human embryo cryopreservation. Good fertilization and cleavage rates (Chen, 1988; Kazem, 1995; Gook et al, 1994, 1995; Toth et al, 1994) as well as a few pregnancies (Van Uem et al, 1987; Chen, 1988; Tucker et al, 1996) have been reported using human oocytes that survived cryopreservation. Although post thaw survival rates ranging from 20 to 80% have been achieved, these freezing protocols have not been robust enough to translate into a reproducible, clinically useful technique. Pre-freezing events, such as oocyte exposure to cryoprotectant solutions and simple cooling have been shown to be relatively innocuous, and do not inhibit fertilization and development (Hunter et al, 1991 ; Bernard et al, 1992 and Gook et al, 1995). Thus, the step that limits the efficiency of this multistep process seems to be confined to the freeze and thaw procedure. SUMMARY OF THE INVENTION

Embodiments of the present invention are directed to inhibiting the formation of intracellular ice formation in viable cells and to methods of increasing survival rates of the cells after freezing and thawing. Certain embodiments of the present inventions are directed to methods of preserving viable cells which includes the steps of providing a preparation of viable cells immersed in a solution having a cryoprotectant. The preparation is then supercooled to a temperature below its freezing point. Then the formation of extracellular ice is promoted at a temperature above that at which intracellular ice formation occurs in the cells. The preparation is then cooled to a final storage temperature. Embodiments of the present invention are further directed to methods of preserving viable oocytes by altering the oocytes in a manner to reduce a temperature at which intracellular ice formation occurs. The oocytes are then included in a liquid medium which is then supercooled to a first temperature above the temperature at which intracellular ice formation occurs. The formation of extracellular ice within the medium is then promoted, and the medium is then cooled to a final storage temperature. Embodiments of the present inventions are also directed to methods of dehydrating a viable cell for later freezing by altering the cell in a manner to reduce a temperature at which intracellular ice formation occurs. The cell is then included in a liquid medium which is then supercooled to a first temperature above the temperature at which intracellular ice formation occurs. The medium is maintained at the first temperature for a time sufficient to allow the cell to dehydrate in a manner to inhibit intracellular ice formation. Certain embodiments of the present invention are directed to methods of distributing oocytes throughout an ice crystal matrix for later freezing in which the oocytes are altered in a manner to reduce a temperature at which intracellular ice formation occurs. The oocytes are then included in a liquid medium which is then supercooled to a first temperature above the temperature at which intracellular ice formation occurs. The medium is then maintained at the first temperature for a time sufficient to allow the cell to dehydrate in a manner to inhibit intracellular ice formation. The oocytes are located in a discreet region of the medium. Extracellular ice crystals are then seeded at a location away from the discreet region such that formation of the extracellular ice crystals occurs in a direction toward the oocytes. The oocytes, in turn, are migrated in a direction toward the extracellular ice crystal formation and are trapped in hyperosmolar channels located between the growing ice crystals. Certain embodiments of the present invention are also directed to methods of optimizing cryoprotection of oocytes by providing a plurality of preparations of oocytes, wherein the oocytes of each preparation are immersed in a liquid medium that includes a cryprotectant. The plurality of samples are then supercooled to a first temperature that is at or below the freezing point of the liquid medium and above the temperature at which intracellular ice formation occurs. The oocytes are located in a discreet and corresponding region of each member of the plurality of preparations. The preparations are maintained at the first temperature for a time sufficient to allow the cell to dehydrate in a manner to inhibit the formation of intracellular ice. The preparations are then gradually cooled below the first temperature and then extracellular ice crystals are seeded in the plurality of preparations at a corresponding plurality of temperatures, where the seeding occurs at a location remote from the discreet region. The preparations are then cooled to a final storage temperature. The oocytes are then examined in order to detect levels of formation of intracellular ice as an indication of optimized cryoprotection.

BRIEF DESCRIPTIONS OF THE DRAWINGS

In the course of the detailed description of certain preferred embodiments to follow, reference will be made to the attached drawings, in which, Fig. 1 is a proportional, notched box plot of the IIF temperatures of human oocytes frozen in the presence of DPBS and of PB1 supplemented with the cryoprotectants PG, DMSO and EG at a rate of 120°C/minute.

Fig.2 is a proportional, notched box plot of the IIF temperatures of mouse oocytes frozen in the presence of DPBS and of PB1 supplemented with the cryoprotectants PG, DMSO and EG at a rate of 120°C/minute.

Fig. 3 are cryostage pictures combining 8 large human oocytes (right) and 15 smaller mouse oocytes (left) with temperatures measured at the tip of the thermocouple.

The oocytes were frozen in PG at a rate of 120°C/minute. (A) Temp. -6.5 °C: seeding is occurring with the ice front rapidly moving across the stage. (B) Temp. - 14.4°C: IIF (sudden darkening of the cytoplasm) occurred in 7/8 human oocytes and none of the mouse oocytes. (C) Temp. -21.1 °C: IIF occurred in all human oocytes and in 2/15 mouse oocytes. (D) Temp. -37J°C: IIF occurred in 14/15 of the mouse oocytes.

Fig. 4 is a proportional, notched box plot of the IIF temperatures of mouse and human oocytes frozen simultaneously in 1.5M PG at a rate of 120°C/minute. Fig. 5 is a micrograph showing the response of a human oocyte to 1.5M PG. (A)

0 seconds, (B) 30 seconds: maximal shrinkage due to rapid diffusion of water extracellularly, (C) 2 minutes: partial re-expansion due to diffusion of PG into the oocyte .and reduction of the osmotic gradient, (D) 6 minutes: resumption of original size due to equalization of the concentration of PG inside and outside the cell .and elimination of the osmotic gradient.

Fig. 6 is a graph of frequency distributions of the incidence of human oocyte IIF and irreversible damage at different seeding temperatures.

Fig. 7 is a graph of logistic regression curves of the 24 hours post thaw survival rate and the incidence of IIF on the seeding temperature in human oocytes. The bar represents the "safe window" for seeding temperatures in human oocytes.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The principles of the present invention may be applied with particular advantage to produce novel methods for inhibiting the formation of intracellular ice in viable cells, particularly oocytes. According to the present invention, biophysical characteristics of oocytes under sub-zero conditions such as water transport and intracellular ice formation, are determined by using a cryomicroscope. The parameters for water transport are known for bovine (Myers, 1987) .and mouse oocytes at supra-zero (Leibo, 1980) and sub-zero temperatures (Toner et al, 1990a). Ice nucleation parameters are known for both mouse oocyte (Toner et al, 1990b; Karlsson et al, 1996) and macaque (Younis et al, 1996) oocyte. Some water transport parameters have been determined in human oocytes (Hunter et al, 1992), but not those concerned with ice nucleation parameters.

The biophysical characteristics are then used in a theoretical model to determine optimum freezing conditions for inhibiting intracellular ice formation and to increase viability of frozen oocytes. According to one embodiment of the present invention, the oocytes are allowed to dehydrate either before, during or after extracellular ice formation, and before being subjected to temperatures at which ice nucleation occurs. The dehydration occurs during a programmed temperature drop, with the diffusion of intracellular water to the hyperosmotic extracellular media generated by the incorporation of pure water in the growing ice crystals. The rate of temperature drop is fast enough to minimize the long exposure of oocytes to deleterious freezing conditions such as high electrolyte concentrations, excessive cell dehydration, mechanical effects of external ice, etc., but slow enough to avoid the damaging effect of intracellular ice formation within the oocytes.

The following examples are set forth as representative of the present invention. These examples are not to be construed as limiting the scope of the invention as these and other equivalent embodiments will be apparent in view of the present disclosure, figures, tables and accompanying claims.

EXAMPLE I Ovarian Hyperstimulation. Oocyte Retrieval and Culture Protocol Human oocytes were obtained from patients undergoing in vitro fertilization

(IVF) at the Center for Reproductive Medicine, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital. Only those oocytes not useful clinically were used for this study. Ovarian stimulation was achieved using various combinations of gonadotropin and gonadotropin releasing hormone agonists depending on each patient. Follicular growth was monitored by ultrasound and estradiol- 17β measurements. Human chorionic gonadotropin (hCG) was administered after three follicles reached a diameter of 18 to 20mm. Oocyte retrieval was performed transvaginally 36 hours later. Oocyte-cumulus complexes were individually cultured in Ham's F-10 without hypoxanthine (Sigma Chemical Company, St Louis, MO, USA, N3389) supplemented with 10%> synthetic serum substitute (SSS) (Irvine Scientific, Irvine, CA, USA, 99193) at 37.2°C in an atmosphere of 5.5% C0₂ in air.

Human oocytes included the following categories: (1) Fresh oocytes with a germinal vesicle. These were retrieved from patients scheduled for intracytoplasmic sperm injection (ICSI) who had their oocytes evaluated for maturity prior to injection. The cumulus mass was completely removed 4 hours after the retrieval with a small bore glass pipette following a 30 seconds exposure to 80IU/ml hyaluronidase (Sigma, H3506) in Hepes-buffered HTF (Irvine Scientific, 9963). The immature eggs with a germinal vesicle were selected for the study.

(2) Oocytes after exposure to spermatozoa. Oocyte-cumulus complexes were inseminated 4 to 6 hours after retrieval using 150,000 spermatozoa/ml. The sperm was processed with a Percoll column (Punjabi et al, 1990). Fertilization was assessed at 14 to 19 hours post insemination. Two categories of these oocytes were selected for study. (i) Failed-to-fertilize. Unfertilized oocytes were evaluated for maturity and classified as metaphase I (MI) and metaphase II (Mil) oocytes, based on the presence or absence of a polar body. Eggs were used within 24 to 30 hours of the retrieval. Because of their scarce number, unfertilized oocytes with a germinal vesicle were not used for this study.

(ii) Polyspermic embryo. Fertilized oocytes with .an abnormal number of pronuclei were used while still in the uncleaved stage. Eggs were used 24 to 30 hours after retrieval.

Mouse Oocytes

The procedure for the collection of mouse oocytes has been described previously (Jackson et al., 1989). Four to six week old BDF mice were obtained from the Jackson Laboratories (Bar Harbor, ME, USA). Female mice were induced to superovulate by intraperitoneal (i.p.) injection of 5IU of pregnant mare serum gonadotropin (Sigma, G-4877), followed 48 hours later by an i.p. injection of 5IU of hCG (Sigma, CG-5). Twelve to fifteen hours later, metaphase 11 oocytes were collected and used for experiments.

Cryoprotectant Solutions The following saline solutions were used in this work: Dulbecco's phosphate buffered solution (DPBS) (Gibco, 450-1300EC) and PB I (DPBS + glucose) (Gibco 4501300eb). Both solutions were supplemented with penicillin G (0.095gm/liter) and streptomycin (0.05gm/liter). All freezing solutions were prepared using PB 1. The following cryoprotectant solutions were prepared: (1) 1.5M propylene glycol (Sigma, P1009) in PB I (PG), (2) 1.5M dimethyl sulfoxide (Sigma, 5879) in PB I (DMSO), and (3) 1.5M ethylene glycol (Sigma, E-9129) in PB I (EG). For the oocyte cryopreservation experiments, PG, DMSO, and EG were supplemented with 20% SSS. For the cryomicroscopy experiments, the freezing solutions were used without adding SSS. Only 1.5M cryoprotectant solutions were used, except when a stepwise addition of 0.5M and 1.0M solutions of PG was needed. For the removal of cryoprotectant solutions from oocytes, 1.08M sucrose (Sigma, S0389) in PB I supplemented with 20% SSS was used for both conventional and modified cryopreservation protocols. It is important to understand that the scope of the invention is not limited to the particular cryoprotectants mentioned above, but that other useful cryoprotectants can be readily identified by those skilled in the art based on this disclosure.

Oocyte Cryomicroscopy

The occurrence of IIF in oocytes, in the presence and absence of a cryoprotectant, was observed using the cryomicroscopy system described by Cosman et al, (1989). The equipment used included a programmable thermal microscope stage (Thermascope, Interface Technique, Cambridge, MA, USA) connected to a video microscopy system. Depending on availability, 2 to 15 human oocytes were equilibrated in the cryoprotectant solutions for 20 minutes and then placed in a 50 microliter drop on the cryomicroscope stage under a coverslip sealed with silicone grease. The initiation of the sample freezing was achieved by slightly supercooling the solution to -6.5 °C and manually triggering seeding extracellular ice by contacting the edge of the sample with a chilled forceps. Controlled cooling at a rate of 120°C/minute to -60 °C was initiated as soon as the growing ice front had engulfed all oocytes in the field of view. A rapid cooling rate of 120°C/minute was chosen to minimize water efflux, thus reducing the effect of water transport on ice nucleation. Under bright field illumination, IIF was manifested by a sudden darkening of the cytoplasm believed to be caused by the light scattering due to microscopic ice crystals and/or bubbles in the oocyte. The temperature at which IIF occurred in each oocyte was determined by reviewing the video recordings of each freezing experiment. The observed IIF temperature for each individual oocyte was corrected for the thermal gradient across the stage by subtracting the corresponding temperature of the ice front during the thaw and adding the melting point of the solution (Toner et al, 1990b). In subsequent experiments, the effect of varying the cooling rates and extracellular ice seeding temperatures on IIF was also determined. Cooling rates of 0.2°C/minute .and 10°C/minute were used to investigate the role of dehydration on IIF. Also, at a cooling rate of 0.2°C/minute, the extracellular ice seeding temperature was modified by supercooling the solution to -4.5, -5.5, -6.0, -6.5 .and -8°C before contacting the edge of the sample with a chilled forceps. The incidence of IIF for each experimental setting was recorded.

Oocyte Cryopreservation Viability of frozen-thawed oocytes was determined as follows. Control experiments were conducted using the one step method for human embryo cryopreservation derived from a bovine embryo freezing protocol designed by Leibo (1984) and modified by Nowrouzi et al. (1991) for human use.

Cryopreservation Protocol A Biocoll freezer (FTS System, Stone Bridge, NY, USA) was used, with a reagent grade alcohol at 95% (Thomas Scientific, 4916-E52) and a TPC-44.E programmable controller set for the following protocol: starting temperatures -6°C and -8°C, ramp rate -0.4° C/minute, holding temperature -40°C, holding time 15-30 minutes. In preparation for the freezing protocol, the oocytes were transferred to a PB1 solution for 10 minutes followed by PG equilibrated at room temperature for a maximum of 20 minutes. The 0.25ml straws (IMV, France, A101) were loaded as follows: 10mm of PG, 10mm of air, 10mm of PG containing the oocytes, 10mm of air, 65mm of sucrose solution, 10mm of air. The air was drawn up until the first PG column moistened the PVA powder between the two plugs and no more air could be drawn in. Both ends of the straw were sealed by heat and the plugged end was fitted with a 0.5ml straw (IMV, A102) to be used as a handle. The straws were maintained in a horizontal position until they were briefly plunged into the alcohol bath at -6°C and -8°C. The top of the sucrose column and the bottom of the PG column not containing the oocytes were then seeded using a chilled forceps. All the straws were placed perpendicularly in a freezing basket (IMV, M001) suspended in the 95% alcohol bath and held for 15 minutes at the extracellular ice seeding temperature. After confirming that the straws were frozen, the ramp was activated as described above. At the end ofthe 15 minutes holding time at -40°C, the straws were quickly transferred to a liquid nitrogen canister, .and then moved to a storage tank for 1 to 7 days. For thawing, the straw was removed from the liquid nitrogen tank and laid at room temperature for 2 minutes with both ends supported by a slant rack. If, after wiping with a Kim wipe, condensation formed, 30 more seconds were allowed. After taking the handle off, and holding the unplugged end, the straw was shaken a few times like a thermometer until there was only one air space at this end. The straw was then placed plugged end down in 15ml centrifuge tube containing MiUi-Q water in a 37 °C water bath for 3 minutes. The straw was then turned over, plugged end up, and placed in a 15ml centrifuge tube containing Milli-Q water at room temperature for 1 minute. The straw was emptied on a dry dish under direct vision using a dissecting microscope. The oocytes were washed twice in PB1 at room temperature and allowed to equilibrate for 10 minutes before transfer to a culture dish in the conditions described above.

To induce seeding of extracellular ice at -4.5 °C, the following modifications were introduced to the protocol described above. The Biocool unit was programmed as follows: segment 1 : start temperature -3.5°C, ramp 0.2°C/minute, holding temperature -15 °C, holding time 0 minute; segment 2: ramp 1 °C/minute, holding temperature -40°C, holding time 15-3 0 minutes. The IMV straws were loaded as follows: 10mm of PG, 10mm of air, 20mm of PG containing the oocytes, 10mm of air, 55mm of sucrose and 10mm of air. The unplugged end was fitted with the 0.5ml IMV straw. The straws were held perpendicularly, plugged end up, until all the oocytes migrated to the lower meniscus of the 20mm PG column. Their exact position was checked under a dissecting microscope. Meanwhile, the freezing program was activated from the starting temperature of -3.5°C, then put on hold to maintain the alcohol bath temperature at 4°C. The straw was inverted, and briefly dipped in the alcohol bath for 10 to 15 seconds at a 45 °C angle. All the temperatures were measured at the tip of a temperature probe located at the level ofthe 20mm PG column. The lower meniscus ofthe 20mm PG column was seeded in a location opposite to the oocytes, and replaced promptly in the alcohol bath in the slanted position. Simultaneously, the freezing ramp was reactivated to reach - 4.5 °C. This temperature was held for 20 minutes. After checking for seeding of the sucrose column without removing the straw from the alcohol bath, we proceeded with the protocol as above. Attention to the following details was important to insure adequate extracellular ice seeding. Because seeding of extracellular ice in an adjacent column at higher temperatures did not insure proper ice formation in the cryoprotectant column containing the oocytes, the latter was seeded directly. Consequently, and to prevent accidental exposure of the eggs to ice seeding, the size of the cryoprotectant column containing the oocytes was doubled to 20mm. The oocytes were also moved as far as possible from the seeding area by placing the straw perpendicularly, and their location was verified at the opposite meniscus prior to seeding of extracellular ice. The straw was placed in a 45 ° slanted position in the alcohol bath to allow the oocytes to move slowly toward the frozen area. In a perpendicular position, the oocytes reached the bottom of the PG column in 2 to 5 minutes, as opposed to 10 to 15 minutes in the slanted position. Using the slanted position allowed new ice to grow upward from the frozen region and trap the oocytes migrating downward in hyperosmolar channels located between the growing ice crystals.

Evaluation of Oocyte Survival

Oocyte survival was assessed at 2 and 24 hours post-thaw. Signs of oocyte damage included zonal fracture, discolored ooplasm, pyknosis and cytoplasmic disruption. A very good correlation was previously reported between delayed (24 hours) morphological assessment, and vital staining when demonstrating viability of frozen- thawed human oocytes (Pensis et al, 1989).

Biometrical Methods The distributions ofthe temperature of IIF under various experimental conditions were plotted as proportional notched box plots to show the 10^th, 25^th, 50^th (median), 75^th, 90^th, percentiles (Kafadar, 1985). Observations that fall outside the 10 a d 90 percentile were considered outliers. The width ofthe box is proportional to the number of observations. The notches were constructed using the formula 1.57 x (75^th percentile -25^th percentile)/n. If the notches do not overlap, the medians are considered significantly different at a significance level of p = 0.05. The box plots were computed using the Number Cruncher Statistical System (J.L. Hintze, Kaysville, UT, USA). Two sets of data were examined by a logistical regression analysis (Hosmer and Lemechow, 1989). A logistic linear equation ofthe form: y=b₀+b,T (1) was fitted to each set, where y = logit, p = In (p / ( 1-p)), p is the proportion responding, T is the extracellular ice seeding temperature, b₀ is the intercept, b, , is the slope of the linear regression. The goodness of fit was tested by the Hosmer-Lemechow statistic. All computations were done using the LogXact for Windows computer package (Cytel Software Corporation, Cambridge, MA, USA).

In a population of n oocytes, hypothetically each oocyte will undergo IIF as a result of extracellular ice seeding at some threshold temperature (T_π). Likewise the oocyte will suffer irreversible damage and die as the result of seeding at some other threshold temperature (T_jD). These two temperatures are not necessarily the same, although they may be highly correlated. In both cases frequency distributions [f(T_ID), f(Tn)] of threshold values can be used to characterize the population of oocytes. As a model, it was assumed that these two distributions can be represented by the logistic distribution which is very close to the normal distribution and has properties that make it relatively simple to use (Malik, 1985). Finney (1947) has described a comparable use of the logistic distribution, including the computations involved, in a discussion of tolerance distributions which arise in the field of biological assay. The logistic distributions can be estimated by substituting equation (1) in formula (49) given by Finney: /(T)=b, / (π(l + (b₀ + b,T)²)) (2)

Thus the equations fitted to the data in Table I and II have been substituted in (2) to estimate the two frequency distributions ofthe threshold temperatures that cause IIF and irreversible damage plotted in Figure 6. Two related cumulative distributions related to (2) are of interest and are plotted in Figure 7. These are the cumulative distributions of survivors (F(T_ID)) and IIF (F(T_n), given by: F(T_ID) = I f(T_ID) dTπ,, and F (T ) = J f (T„ ) dT„, respectively. (3)

The sigmoid curves shown in Figure 7 are estimates oϊF_ID(T) and F_n(T) computed from the data in Tables 1 and 2. They can be computed by transforming the fitted linear equations from the logit response scale to the proportion response scale using the inverse of equation (1):

p=exp(b₀+ b, T) / (1 - exp (b₀ +b, T)) (4)

EXAMPLE II Effect of Cryoprotectant on Intracellular Ice Formation

Preliminary experiments with human oocytes showed marginally significant differences in the median temperature of IIF (T^_D) in fresh oocytes with a germinal vesicle, unfertilized MI or Mil oocytes and polyspermic oocytes (data not shown). However, these differences were sufficiently small to justify pooling the data from all types of human oocytes when analyzing all subsequent experiments. The effect of PG, DMSO, and EG on the distribution of the temperatures of IIF in human and mouse oocytes are shown in Figs. 1 and 2, respectively. The distributions of IIF vary considerably in location and scatter. The

in human and mouse oocytes frozen without cryoprotectant are -8.5 °C and -12.5 °C, respectively. The addition of the cryoprotectants (PG, DMSO and EG) significantly depressed the temperatures of IIF in human and mouse oocytes. However, the magnitude ofthe depression was noticeably different between the two species. In mouse, the addition of cryoprotectants (PG, DMSO and EG) depressed the T^ by 19.6°C, 25.9°C and 25. 1 °C respectively. In human, the effect of PG, DMSO .and EG are much less, depressing the T_MEQ by only 5.0°C, 8.2°C and 6.5 °C, respectively. This interspecies difference in ice nucleation temperature is further emphasized by the absence of overlap in the range of temperatures (-8°C to -20 °C) at which 75%) of IIF occurred in human oocytes frozen in the presence of PG, DMSO or EG, compared to the corresponding range in mouse oocytes (-29 °C to -4 1 °C) (Figures 1 and 2). EXAMPLE III Simultaneous Freezing of Human and Mouse Oocytes

The large difference between human and mouse oocytes in the depression of T^u produced by the addition of cryoprotectant has been confirmed by freezing the oocytes of the two species together. Figures 3 A, B, C and D show a representative set of 8 human and 15 mouse oocytes during seeding at -6.5 °C, and subsequent cooling at 120° C/minute in PG. The micrograph depicts the behavior of oocytes at -14.4°C, -21.1 °C and -37J°C, respectively. When the temperature reached -14.4°C, IIF had been observed in 7/8 human oocytes and in 0/15 mouse oocytes. Mouse oocytes only began to undergo IIF when the temperature reached -21.1 °C (2/15). When the temperature reached -37J°C, IIF was observed in 14/15 mouse oocytes. Figure 4 shows the distributions of the temperatures of IIF in all the human and mouse oocytes frozen together in PG. The location ofthe two distributions is highly significantly different with T_MED iⁿ mouse of -3 2J°C and in human of - 14.4°C. In an attempt to further probe the difference in IIF temperatures of mouse and human oocytes, the following series of experiments were done.

Uptake of cryoprotectant (PG). Given the larger size of human oocytes, the equilibration of PG with human oocytes was determined from their volumetric behaviour in PG. Specifically, five human oocytes were placed in 1.5M PG. They shrank rapidly within 30 seconds and then, relatively slowly, returned to their original volume (6 minutes). The response of one of these oocytes is shown in Figure 5 which is the classical physiological response of a cell exposed to a permeable solute, and clearly demonstrates that human oocytes were fully equilibrated with 1.5 M PG during cryomicroscopy experiments. Cytoplasmic damage due to osmotic shock. To rule out the possibility that osmotic damage to human oocytes during one-step addition of 1.5 M PG was causing elevated IIF temperatures, thirty-three human oocytes were first exposed sequentially to 0.5M PG, 1.0M PG and 1.5M PG for 5 minutes each and then frozen at a rate of 120° C/minute in 1.5M PG. The T_UED was - 12J°C, very similar to previous estimates of - 13.5°C. Furthermore, gross membrane damage after prolonged exposure (90 minutes) of five human oocytes to PG could not be demonstrated in the form of superficial blebs.

Rate of freezing. Forty-seven human oocytes in PG were frozen at a rate of 10°C/minute. Although the T,^ decreased to -17J°C, potentially accounting for minimal water diffusion, it remained well above nucleation temperatures of mouse oocytes (below -30°C).

Oocyte culture. Since hum.an oocytes were cultured for approximately 24 hours prior to the cryomicroscopy experiments, the effect of culture on mouse oocyte IIF was also investigated. Twelve mouse oocytes cultured for 24 hours were frozen in PG at 120°C/minute. The observed T^_D of -28.8°C was similar to the 1^ determined in fresh mouse oocytes of -32.1 °C.

EXAMPLE IV Effect of seeding temperature on IIF

Groups of human oocytes were frozen on the cryomicroscope at a rate of 0.2°C/minute, until the temperature was -40°C. The groups were seeded at -8.0, -6.5, -6.0, -5.5 and -4.5°C in 1.5M propylene glycol. The numbers of oocytes undergoing IIF were determined at the end of each run. The pooled results are shown in Table 1 below with a fitted logistic regression y =-8.95 - 1.36t and a goodness of fit (Hosmer-Lemshow test) of p= 0.285.

TABLE I

A logistic regression analysis showed that the incidence of IIF was very significantly dependent on the seeding temperature (b, = -1.36, sem = 0.25, p < 10^"5). Intracellular ice formed in almost all oocytes seeded at -8.0 °C, while IIF did not occur when the seeding temperature was -4°C.

EXAMPLE V Effect of Freezing Protocol on Survival After Thawing

The importance of increasing the extracellular ice seeding temperature was verified by comparing frozen-thawed oocyte survival rates after 2 and 24 hours in a freezing protocol with seeding temperatures of -6JC and -8JC, and also in a freezing protocol with ice seeding temperature of -4.5 °C. The pooled results are shown in Table II below with a fitted logistic regression of y = 6.02 + 0.88t and a goodness of fit (Hosmer-Lemeshow test) of p = 0.055.

TABLE II

A logistic regression analysis showed that the post-thaw survival rate of human oocytes was very significantly dependent on the seeding temperature (b, = 0.88, sem = 0. 17, p < 10^"5). Thus, reasonably high survival rates in human oocytes were obtained using the highest seeding temperature for our instrument.

When mouse oocytes were frozen at 120° C/minute to minimize the influence of water diffusion across the cell membrane, the T_MEΌ was found to be -12.8 °C when cryoprotectant was absent, and -38.2 °C when DMSO was added (Figure 2). These results are very close to other published values (Toner et al, 1991; Karlsson et al, 1996). When human oocytes were frozen with no cryoprotectant, the T^,-, was found to be -8.5 °C (Figure 1). This result is similar to the T^,-, served when macaque oocytes were frozen without cryoprotectant (Younis et al, 1996). The closeness ofthe T^u in mouse and macaque to the T^_D observed in the examples, provides indirect evidence that the failed-fertilized and polyspermic oocytes used in the examples have intact cell membranes that regulate transport of water in and out of the oocyte, an important function in the freezing process. The results also provide another example of the usefulness of these types of human oocytes in research (Winston, 1993). When human oocytes were frozen at 120° C/minute in the presence of cryoprotectant, the

slightly depressed (~ 6.5 °C) (Figure 1). The fall in the IIF temperature was far less than that which occurs in mouse oocytes (~23'C) (Figure 2; Karisson et al, 1996). The reason for these differences may be related to the three-fold difference in volume between the small mouse oocyte and the large human oocyte. Consistent with this possibility, a comparable relatively small depression of IIF (~ 4C) produced by glycerol was observed in the large cow oocyte (Myers et al, 1987).

In contrast, the published literature on the nucleation temperature of ice in water droplets of diameters comparable to those of mouse and human oocytes (80 to 129 m) does not support this explanation, because of a very weak measured dependence of nucleation on droplet volume. However, it is import.ant to note that these water droplet studies .are performed under either homogeneous or relatively ineffective heterogeneous nucleation conditions at temperatures below -30 °C [review: Hobbs (1974)]. On the other hand, human oocytes underwent IIF at much higher temperatures, indicating a very effective heterogeneous catalysis of IIF. In fact, a model based on the modified classical nucleation theory predicts that the likelihood of IIF in mouse oocytes is a strong function of the surface area of the cell (thus, cell volume) under consideration (Toner, 1993). Other cell-specific factors may also be involved since the depression ofthe temperature for IIF produced by DMSO in several cell types from different species was not correlated with volume (Hubel et al, 1991).

In a typical freezing protocol with a freezing rate less than 2° C/minute, most mouse oocytes will loose 90% of their water before reaching the critical ice nucleation temperature zone between -30°C and -40°C, and will thus avoid lethal IIF (Mazur, 1977). In the human oocyte, it has been shown that lowering the rate of freezing to 0.2° C/minute is not successful in preventing IIF, presumably because the cell has not dehydrated sufficiently when it enters the critical IIF temperature zone. The insufficient dehydration is probably related to the remarkably higher ice nucleation temperatures observed in the human oocytes. More specifically, extracellular ice seeding at -6°C and -8°C, customary in most freezing protocols, triggers IIF in a significant number of human oocytes during the holding period (Table 1). According to the present invention, undesirable IIF can be prevented by increasing the temperature of extracellular ice seeding as close as possible to the melting point ofthe solution, i.e. -4.5 °C when using propylene glycol. Presumably the higher extracellular ice seeding temperature allows the human oocyte to dehydrate extensively prior to reaching the critical temperature zone of IIF, thereby preventing the occurrence of IIF. According to the present invention, a freezing rate of 0.2° C/minute down to minus 15°C was empirically adopted to guarantee near equilibrium conditions between cytoplasmic and extracellular water as the oocyte approaches the IIF temperature range so as to insure maximal dehydration. Thereafter, the freezing rate was increased to 1 ° C/minute to reduce the exposure time to the potentially damaging extracellular electrolyte and solute concentrations which develop during freezing (Karlsson et al., 1996). Since these settings were arbitrarily determined, this protocol could be further optimized by using the theoretical model described elsewhere (Toner et al, 1991; Karlsson et al, 1996). The effects of extracellular ice seeding temperature and cooling rate on IIF was theoretically investigated with mouse oocytes (Toner et al., 1993). This study showed a very strong dependence of IIF behavior of mouse oocytes on the extracellular ice seeding temperatures. For a cooling rate of 1 ° C/minute, the cumulative frequency of IIF increased from zero to one over extracellular seeding temperatures of -2.5 °C to -7°C, respectively. For slightly higher cooling rates (1.5 to 3 °C/minute), the cumulative frequency of IIF increased drastically for a given extracellular ice seeding temperature because less time is available for the small mouse oocyte to dehydrate at faster cooling rates. The theoretical predictions by Toner et al. (1993) for the effect of extracellular ice seeding temperature on mouse oocytes are in qualitative agreement with experimental studies of red blood cells (Diller, 1975), granulocytes (Schwartz and Diller, 1984), and hepatocytes (Toner et al., 1992). All these studies clearly indicate the important role ofthe extracellular ice seeding temperature on the fate of cells with respect to undergoing IIF.

In the case of human oocytes, minimal depression of IIF in the presence of cryoprotectants requires using both a very slow cooling rate and a high extracellular ice seeding temperature to afford enough cellular dehydration to minimize (or prevent) IIF. The logistical distributions ofthe threshold temperatures of IIF and irreversible damage, calculated from the data in Tables I and II, respectively, using equations 1 and 4, are shown in Figure 6. A prominent feature of both distributions is the variability of the two parameters over a wide range of extracellular ice seeding temperatures. A few oocytes are very sensitive to the extracellular ice seeding temperatures and undergo IIF. As a result, these sensitive oocytes suffer irreversible damage at relatively high extracellular ice seeding temperatures (-4.5 °C). On the other hand, a few other oocytes are very insensitive to the extracellular ice seeding temperature and do not undergo IIF. These oocytes suffer irreversible damage at only relatively low extracellular ice seeding temperatures (< -8°C). Yet IIF and oocyte death occurs more frequently at very similar temperatures of -6.6°C and -6.8°C, respectively. The relation is similar to the high correlation observed between the incidence of IIF and cell survival on cooling rates (Toner, 1993). The cause of the high variability associated with the effects of extracellular ice seeding temperatures on the incidence of IIF and irreversible cell damage is unknown. Hunter et al. (1992), in making measurements of the membrane water permeability and inactive volume of human oocytes, also encountered very high variability relative to that seen in the mouse. They also were unable to identify the cause ofthe variability. Finally, these distribution curves .are obtained from a mixed population of human oocytes. The proportions of oocytes undergoing IIF and survivors at different extracellular ice seeding temperatures, defined by equations (3), have been estimated from the data of Tables I and II using equation (4). There is an almost symmetrical inverse relationship between the cumulative proportion of oocyte survivors and the cumulative proportion of oocytes undergoing IIF over an extracellular ice seeding temperature range of -8°C and -4.5 °C. From a practical point of view, these results show that the initiation of the freezing protocol by seeding the extracellular ice at -8°C results in the loss of many human oocytes. On the other hand, seeding extracellular ice at -6°C will result in a significantly higher proportion of oocytes surviving. Nevertheless, particularly sensitive oocytes will still be lost. The extracellular ice seeding temperature, therefore, should be raised to a window between -4°C and -4.5 °C to minimize the occurrence of IIF and save the more sensitive oocytes (Figure 7). In contrast, the cumulative incidence of survival of mouse embryos decreases steeply only after the seeding temperature is lowered beyond -7°C (Wittingham, 1977) or -8°C (Miyamoto, 1981). Thus, the acceptable window for extracellular ice seeding in this species is somewhat wider (3 to 4°C), allowing much more flexibility in the choice of seeding temperatures. It is to be understood that the embodiments ofthe present invention which have been described are merely illustrative of some ofthe applications ofthe principles ofthe invention. Numerous modifications may be made by those skilled in the art based upon the teachings presented herein without departing from the true spirit and scope of the invention.

Claims

What is claimed is: L A method of preserving viable oocytes comprising the steps of a) providing a preparation of oocytes immersed in a solution having a cryoprotectant, the preparation having a freezing point, b) supercooling the preparation to a temperature below the freezing point, c) promoting extracellular ice formation at a temperature above that at which intracellular ice formation occurs in the oocytes, and d) cooling the preparation to a final storage temperature.

2. The method of claim 1 wherein the oocytes are human oocytes.

3. The method of claim 1 wherein the cryoprotectant is selected from the group consisting of propylene glycol, dimethyl sulfoxide and ethylene glycol.

4. The method according to claim 1 wherein the promoting of extracellular ice formation in step c) is performed at a temperature between about -4.0 ┬░C to about -5.0┬░C.

5. A method of preserving viable oocytes comprising the steps of a) altering oocytes in a manner to reduce a temperature at which intracellular ice formation occurs; b) including the oocytes in a liquid medium having a freezing point, c) supercooling the medium to a first temperature above the temperature at which intracellular ice formation occurs; d) promoting the formation of extracellular ice within the medium, and e) cooling the medium to a final storage temperature.

6. The method of claim 5 in which the step of altering the oocytes includes raising the ionic strength, osmolarity, or effective concentration of non-aqueous components in oocyte cytoplasm.

7. The method of claim 5 in which the liquid medium includes a cryoprotectant.

8. The method of claim 5 in which the step of altering the oocytes comprises introducing a cryoprotectant into the oocyte.

9. The method of claim 5 in which the step of altering the oocytes comprises immersing the oocytes into a liquid cryoprotectant.

10. The method of claim 5 wherein the step of altering the oocytes comprises reducing the amount of water within the oocytes.

11. The method of claim 5 wherein the step of supercooling the medium to a first temperature includes supercooling the medium to a temperature at or slightly below the freezing temperature ofthe medium.

12. The method of claim 1 wherein the step of promoting the formation of extracellular ice within the medium includes seeding extracellular ice crystals at a location within the medium such that formation ofthe extracellular ice crystals occurs in a direction toward the oocytes.

13. The method of claim 12 wherein the step of promoting the formation of extracellular ice within the medium further includes migrating the oocytes in a direction toward extracellular ice crystal formation.

14. The method of claim 1 further comprising the step of maintaining the medium at the first temperature for a time sufficient to allow the oocytes to dehydrate in a manner to inhibit intracellular ice formation.

15. The method of claim 14 wherein the oocytes dehydrate concurrently with extracellular ice crystal formation.

16. The method of claim 5 wherein the step of cooling to a final storage temperature includes cooling the medium below the first temperature to a second temperature at a rate sufficient to allow dehydration ofthe oocytes in a manner to inhibit intracellular ice formation.

17. The method of claim 5 wherein the medium is rapidly cooled from the second temperature to the final storage temperature.

18. A method of dehydrating a viable cell for later freezing comprising a) altering a cell in a manner to reduce a temperature at which intracellular ice formation occurs, b) including the cell in a liquid medium having a freezing point, c) supercooling the medium to a first temperature above the temperature at which intracellular ice formation occurs; d) maintaining the medium at the first temperature for a time sufficient to allow the cell to dehydrate in a manner to inhibit intracellular ice formation.

19. A method of distributing oocytes throughout an ice crystal matrix for later freezing comprising a) altering a cell in a manner to reduce a temperature at which intracellular ice formation occurs, b) including the cell in a liquid medium having a freezing point, c) supercooling the medium to a first temperature above the temperature at which intracellular ice formation occurs, d) maintaining the medium at the first temperature for a time sufficient to allow the cell to dehydrate in a manner to inhibit intracellular ice formation, e) locating the oocytes in a discreet region ofthe medium, f) seeding extracellular ice crystals at a location away from the discreet region such that formation ofthe extracellular ice crystals occurs in a direction toward the oocytes, and g) migrating the oocytes in a direction toward extracellular ice crystal formation.

20. A method of optimizing cryoprotection of oocytes, comprising a) providing a plurality of preparations of oocytes, wherein the oocytes of each preparation are immersed in a solution that comprises a cryprotectant and has a freezing point, b) supercooling the plurality of samples to a first temperature that is at or below the freezing point of the liquid medium and above the temperature at which intracellular ice formation occurs, c) locating the oocytes in a discreet and corresponding region of each member of the plurality, d) maintaining the preparations of oocytes at the first temperature for a time sufficient to allow the cell to dehydrate in a manner to inhibit the formation of intracellular ice, e) gradually cooling the plurality of samples below the first temperature, f) seeding extracellular ice crystals in the plurality of preparations at a corresponding plurality of temperatures, where the seeding occurs at a location remote from the discreet region, . g) cooling the preparation to a final storage temperature, and h) examining the oocytes in order to detect the formation of intracellular ice, wherein its reduction is indicative of optimized cryoprotection.

21. The method of claim 20, wherein the cooling of step e) is performed at a rate of 0.2 to 2 ┬░C per second.

22. The method of claim 20, wherein the plurality of temperatures of step h) are spaced at intervals of 1 to 5 ┬░C.