WO1999031977A1 - Procede de conservation d'oocytes - Google Patents

Procede de conservation d'oocytes Download PDF

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Publication number
WO1999031977A1
WO1999031977A1 PCT/US1998/027248 US9827248W WO9931977A1 WO 1999031977 A1 WO1999031977 A1 WO 1999031977A1 US 9827248 W US9827248 W US 9827248W WO 9931977 A1 WO9931977 A1 WO 9931977A1
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WIPO (PCT)
Prior art keywords
oocytes
temperature
medium
formation
ice
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PCT/US1998/027248
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English (en)
Inventor
John D. Biggers
Fouad S. Trad
Mehmet Toner
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President And Fellows Of Harvard College
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Publication date
Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Priority to AU20083/99A priority Critical patent/AU2008399A/en
Publication of WO1999031977A1 publication Critical patent/WO1999031977A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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% C0 2 in air.
  • SSS synthetic serum substitute
  • 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.
  • ICSI intracytoplasmic sperm injection
  • mice 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.
  • DPBS Dulbecco's phosphate buffered solution
  • PB I DPBS + glucose
  • DPBS + glucose DPBS + glucose
  • 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).
  • cryoprotectant solutions were used for the oocyte cryopreservation experiments.
  • PG, DMSO, and EG were supplemented with 20% SSS.
  • the freezing solutions were used without adding SSS.
  • 1.5M cryoprotectant solutions were used, except when a stepwise addition of 0.5M and 1.0M solutions of PG was needed.
  • 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.
  • 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.
  • 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).
  • Cooling rates of 0.2°C/minute .and 10°C/minute were used to investigate the role of dehydration on IIF.
  • 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.
  • 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.
  • IMV freezing basket
  • 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.
  • the freezing ramp was reactivated to reach - 4.5 °C. This temperature was held for 20 minutes.
  • 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.
  • 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).
  • the sigmoid curves shown in Figure 7 are estimates o ⁇ F 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):
  • cryoprotectants significantly depressed the temperatures of IIF in human and mouse oocytes.
  • the magnitude ofthe depression was noticeably different between the two species.
  • the addition of cryoprotectants depressed the T ⁇ by 19.6°C, 25.9°C and 25. 1 °C respectively.
  • 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.
  • FIGS. 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.
  • 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).
  • FIG. 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 i n 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.
  • mice 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.

Abstract

L'invention porte sur un procédé empêchant la formation de glace intracellulaire dans des cellules viables; sur un procédé améliorant le taux de survie des cellules après congélation et décongélation; sur une préparation de cellules viables immergées dans une solution comportant un cryoprotecteur. Ladite préparation est surgelée à une température inférieure à son point de congélation, puis la formation de glace extracellulaire est provoquée à une température supérieure à celle de formation de la glace intracellulaire. La préparation est finalement refroidie jusqu'à sa température de stockage.
PCT/US1998/027248 1997-12-22 1998-12-21 Procede de conservation d'oocytes WO1999031977A1 (fr)

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AU20083/99A AU2008399A (en) 1997-12-22 1998-12-21 Method for cryopreserving oocytes

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US6846297P 1997-12-22 1997-12-22
US60/068,462 1997-12-22

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003044440A2 (fr) * 2001-11-20 2003-05-30 Supachill Technologies Pty. Ltd. Solute preconditionne a utiliser dans des procedes cryogeniques
US6673607B2 (en) 2000-05-16 2004-01-06 Mehmet Toner Microinjection of cryoprotectants for preservation of cells
US7094601B2 (en) 2000-05-16 2006-08-22 The General Hospital Corporation Microinjection of cryoprotectants for preservation of cells
CN104745528A (zh) * 2015-03-13 2015-07-01 深圳普若赛斯生物科技有限公司 一种卵母细胞或胚胎冻存复苏的方法及其使用的复苏液

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0072225A2 (fr) * 1981-08-10 1983-02-16 Hoxan Corporation Procédé pour congeler des oeufs fécondés, des spermatozoides ou des produits similaires et appareil pour mettre en oeuvre ce procédé
WO1994019455A1 (fr) * 1993-02-23 1994-09-01 Genentech, Inc. Maturation in vitro d'oocytes en presence d'inhibine, d'activine ou de combinaisons d'inhibine/activine
WO1998010231A1 (fr) * 1996-09-06 1998-03-12 Interface Multigrad Technology Ltd. Procede et dispositifs de refroidissement et rechauffement d'echantillons biologiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0072225A2 (fr) * 1981-08-10 1983-02-16 Hoxan Corporation Procédé pour congeler des oeufs fécondés, des spermatozoides ou des produits similaires et appareil pour mettre en oeuvre ce procédé
WO1994019455A1 (fr) * 1993-02-23 1994-09-01 Genentech, Inc. Maturation in vitro d'oocytes en presence d'inhibine, d'activine ou de combinaisons d'inhibine/activine
WO1998010231A1 (fr) * 1996-09-06 1998-03-12 Interface Multigrad Technology Ltd. Procede et dispositifs de refroidissement et rechauffement d'echantillons biologiques

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6673607B2 (en) 2000-05-16 2004-01-06 Mehmet Toner Microinjection of cryoprotectants for preservation of cells
US7094601B2 (en) 2000-05-16 2006-08-22 The General Hospital Corporation Microinjection of cryoprotectants for preservation of cells
WO2003044440A2 (fr) * 2001-11-20 2003-05-30 Supachill Technologies Pty. Ltd. Solute preconditionne a utiliser dans des procedes cryogeniques
WO2003044440A3 (fr) * 2001-11-20 2004-02-26 Supachill Technologies Pty Ltd Solute preconditionne a utiliser dans des procedes cryogeniques
CN104745528A (zh) * 2015-03-13 2015-07-01 深圳普若赛斯生物科技有限公司 一种卵母细胞或胚胎冻存复苏的方法及其使用的复苏液

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