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US20080286863A1 - Method and solutions for cryopreserving oocytes, especially fresh human oocytes - Google Patents

Method and solutions for cryopreserving oocytes, especially fresh human oocytes Download PDF

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US20080286863A1
US20080286863A1 US12/146,826 US14682608A US2008286863A1 US 20080286863 A1 US20080286863 A1 US 20080286863A1 US 14682608 A US14682608 A US 14682608A US 2008286863 A1 US2008286863 A1 US 2008286863A1
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Prior art keywords
oocytes
solution
solutions
proh
cryopreserving
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Raffaella Fabbri
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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
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
    • 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
    • A01N1/10—Preservation of living parts

Definitions

  • This invention relates to an improved method for cryopreserving oocytes, especially fresh human oocytes.
  • This invention also relates to solutions particularly suitable for cryopreservation of oocytes, especially fresh human oocytes.
  • cryopreservation of unfertilised human ooccytes is a technique which offers several advantages, especially whenever oocytes are to be preserved of patients who are at risk of ovarian hyperstimulation and can not transfer embryos during the in vitro fertilization treatment.
  • cryopreservation of oocytes, especially of fresh human oocytes has run into greater technical difficulties than preservation of male gametes or embryos because of oocytes cytological peculiarity.
  • oocytes survival rate in cryopreservation depends, as well as on oocytes size, also on cryoprotectant used (composition, concentration and exposure time) and on freezing/thawing rate.
  • oocytes size is a very important parameter affecting the survival rate because the large quantity of water in ooplasm causes intracellular ice formation during the freezing process: intracellular ice is one of the main responsible factors for intracellular structure damages.
  • Oocytes cryopreservation protocols usually include the following steps:
  • a permeating cryoprotectant e.g. 1,2-propanediol (PROH)
  • PROH 1,2-propanediol
  • oocytes subsequently exposing for a time of 2-5 min. the oocytes to a so-called loading solution including a mixture of a permeating cryoprotectant and a non permeating cryoprotectant (e.g. sucrose) to increase oocytes dehydration;
  • a so-called loading solution including a mixture of a permeating cryoprotectant and a non permeating cryoprotectant (e.g. sucrose) to increase oocytes dehydration;
  • cryopreserving oocytes provide for using loading/thawing solutions including sucrose at a concentration of 0.1M or 0.2M.
  • cryopreserving oocytes In a method for cryopreserving oocytes, according to the present invention, it is provided for using loading/thawing solutions including sucrose at a concentration of 0.3M or greater.
  • sucrose at a concentration of 0.3M or greater outside the cell membrane highly increases cell dehydration/rehydration with an improvement of cryopreserved oocytes survival rate.
  • the present method provides for exposing oocytes to a loading solution including at least 0.3M sucrose for a time of 15 min before starting the freezing process.
  • PBS solution Dulbecco's Phosphate Buffered Saline Solution w/o sodium bicarbonate
  • composition of the above solutions is as follows:
  • solutions are used in a slow freezing program.
  • the solutions are prepared, mixed, filtered and conserved at +4° C. It is better to maintain the solutions at room temperature for 15 min before using.
  • a Petri dish is prepared with 2.1 ml of PBS solution+0.9 ml SSS (Synthetic Serum Substitute). In this solution all the oocytes are washed from their culture medium before transferring in the equilibration solution. Then a suitable dish provided with a plurality of wells is prepared: some of the wells contain 0.350 ml of equilibration solution (1.5M PROH)+0.150 ml of SSS (Synthetic Serum Substitute). In this solution 1 or 2 oocytes/well are transferred and maintained for 10 min before transferring in loading solution.
  • SSS Synthetic Serum Substitute
  • the others wells contain 0.350 ml of loading solution (1.5M PROH+0.3M sucrose)+0.150 ml of SSS (Synthetic Serum Substitute).
  • the oocytes (taken from the equilibration solution) are transferred in the loading solution and rapidly loaded in plastic straws. Afterwards the straws are placed in a programmable freezer, e.g. a Kryo 10 series III (Planer Kryo 10/1.7 GB).
  • the oocytes before starting the freezing program the oocytes are exposed to the loading solution (including sucrose at a concentration of at least 0.3M) for 13-15 min and preferably for about 15 min.
  • the loading solution including sucrose at a concentration of at least 0.3M
  • a slow freezing method is applied to the oocytes as follows.
  • the initial chamber temperature is 20° C.
  • the temperature is slowly (2° C./min) reduced to ⁇ 7° C. at which temperature ice nucleation is manually induced.
  • the straws are cooled slowly (0.3° C./min) to ⁇ 30° C. and then rapidly (50° C./min) to ⁇ 150° C.
  • the straws are transferred into liquid nitrogen tanks and stored until thawing.
  • sucrose solution composed of:
  • solutions are utilized in a rapid thawing program.
  • the solutions are prepared, mixed, filtered and conserved at +4° C. It is better to maintain the solutions at room temperature for 15 min before using.
  • the straws are initially air-warned for 30 sec and then immersed in a 30° C. water bath for 40 sec until all traces of ice have disappeared
  • the first well contains 0.350 ml of solution A (1.0M PROH solution+0.3M sucrose)+0.150 ml of solution E SSS (Synthetic Serum Substitute). The content of the straw is expelled in this solution and the oocytes are equilibrated for 5 min at room temperature.
  • solution A 1.0M PROH solution+0.3M sucrose
  • E SSS Synthetic Serum Substitute
  • the second well contains 0.350 ml of solution B (0.5M PROH solution+0.3M sucrose)+0.150 ml SSS (Synthetic Serum Substitute).
  • solution B 0.5M PROH solution+0.3M sucrose
  • SSS Synthetic Serum Substitute
  • the third well contains 0.350 ml of solution C (0.3M sucrose solution)+0.150 ml of SSS solution (Synthetic Serum Substitute).
  • the oocytes, taken from the second well are transferred to this solution and maintained for 10 min at room temperature.
  • the fourth well contains 0.350 ml of solution D (PBS solution)+0.150 ml of SSS solution (Synthetic Serum Substitute).
  • the oocytes takes from the third well, are transferred to this solution and maintained for 10 min at room temperature and for additional 10 min at 37° C.
  • oocytes can be transferred to a conventional culture medium before insemination.

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  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

In a method for cryopreserving fresh human oocytes, freezing and thawing solutions are used, which solutions include 1,2 propanediol (PROH) and sucrose at a concentration of at least 0.3M. The oocytes are exposed for 13-15 minutes to a solution including 1.5M PROH and 0.3M sucrose before starting the freezing process.

Description

  • This application is a continuation of co-pending application ser. no. 11/323,250; filed Dec. 30, 2005, which is a divisional of application ser. no. 10/251,624; filed Sep. 20, 2002, now U.S. Pat. No. 7,011,937, issued Mar. 14, 2006, which is a continuation of International Application No. PCT/IT01/00139; filed Mar. 20, 2001.
    • FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT Not Applicable BACKGROUND OF THE INVENTION
  • 1. Technical Field
  • This invention relates to an improved method for cryopreserving oocytes, especially fresh human oocytes. This invention also relates to solutions particularly suitable for cryopreservation of oocytes, especially fresh human oocytes.
  • 2. Background Art
  • It is well known that cryopreservation of unfertilised human ooccytes is a technique which offers several advantages, especially whenever oocytes are to be preserved of patients who are at risk of ovarian hyperstimulation and can not transfer embryos during the in vitro fertilization treatment. However, cryopreservation of oocytes, especially of fresh human oocytes, has run into greater technical difficulties than preservation of male gametes or embryos because of oocytes cytological peculiarity.
  • The low number of births from human cryopreserved oocytes, reported in literature, shows the technical difficulties of cryopreserving oocytes. Up to now researches carried out on oocytes cryopreservation provide contrasting results regarding the more suitable and less damaging methods for maintaining cellular integrity and for getting a higher rate of viable oocytes.
  • As yet, a definitive protocol for cryopreserving human oocytes has not been established and the number of oocytes utilised up to now is still too low to determine a definitive methodology to be applied.
  • Human oocytes survival rate in cryopreservation depends, as well as on oocytes size, also on cryoprotectant used (composition, concentration and exposure time) and on freezing/thawing rate.
  • In the cryopreservation process, oocytes size is a very important parameter affecting the survival rate because the large quantity of water in ooplasm causes intracellular ice formation during the freezing process: intracellular ice is one of the main responsible factors for intracellular structure damages.
  • Oocytes cryopreservation protocols usually include the following steps:
  • a) initially exposing the oocytes to a solution including a permeating cryoprotectant (e.g. 1,2-propanediol (PROH)), which aim is to reduce to a minimum intracellular structure damages caused by water crystallization;
  • b) subsequently exposing for a time of 2-5 min. the oocytes to a so-called loading solution including a mixture of a permeating cryoprotectant and a non permeating cryoprotectant (e.g. sucrose) to increase oocytes dehydration;
  • c) slowly cooling to −150° C.;
  • d) storing in liquid nitrogen (−196° C.);
  • e) thawing;
  • f) diluting and removing the cryoprotectants by exposure to so-called thawing solutions and returning to the physiological environment for further manipulations.
  • Cryoprotectants benefit are related to
  • I) their concentration,
  • II) exposure times;
  • III) the temperature at which they are added to oocytes.
  • Known methods for cryopreserving oocytes provide for using loading/thawing solutions including sucrose at a concentration of 0.1M or 0.2M.
    • DISCLOSURE OF INVENTION
  • In a method for cryopreserving oocytes, according to the present invention, it is provided for using loading/thawing solutions including sucrose at a concentration of 0.3M or greater.
  • In fact, it has been surprisingly noticed that the presence of sucrose at a concentration of 0.3M or greater outside the cell membrane highly increases cell dehydration/rehydration with an improvement of cryopreserved oocytes survival rate.
  • Furthermore, in a particular embodiment of the invention, the present method provides for exposing oocytes to a loading solution including at least 0.3M sucrose for a time of 15 min before starting the freezing process.
  • In this latter case, a morphological analysis, effected by an inverted microscope, has shown an oocyte cytoplasmatic volume reduction of about 30% with further dehydration; this also reduces the possibility of intracellular ice formation.
    • BEST MODE FOR CARRYING OUT THE INVENTION
  • The essential phases of a process, according to the present invention, for cryopreserving fresh human oocytes are described hereinafter.
  • The following solutions are used in the freezing process:
  • a PBS solution (Dulbecco's Phosphate Buffered Saline Solution w/o sodium bicarbonate);
  • an equilibration solution (1.5M PROH);
  • a loading solution (1.5M PROH+0.3M sucrose);
  • a SSS solution (Synthetic Serum Substitute)
  • The composition of the above solutions is as follows:
  • PBS solution
  • 8 ml PBS
  • Equilibration solution (1.5M PROH)
  • 6.79 ml PBS
  • 1.21 ml PROH (1,2-propanediol)
  • Loading solution (1.5M PROH+0.3M sucrose)
  • 6.79 ml PBS
  • 1.21 ml PROH
  • 1.128 gr sucrose
  • These solutions are used in a slow freezing program. The solutions are prepared, mixed, filtered and conserved at +4° C. It is better to maintain the solutions at room temperature for 15 min before using.
  • When the above described solutions are ready, a Petri dish is prepared with 2.1 ml of PBS solution+0.9 ml SSS (Synthetic Serum Substitute). In this solution all the oocytes are washed from their culture medium before transferring in the equilibration solution. Then a suitable dish provided with a plurality of wells is prepared: some of the wells contain 0.350 ml of equilibration solution (1.5M PROH)+0.150 ml of SSS (Synthetic Serum Substitute). In this solution 1 or 2 oocytes/well are transferred and maintained for 10 min before transferring in loading solution.
  • The others wells contain 0.350 ml of loading solution (1.5M PROH+0.3M sucrose)+0.150 ml of SSS (Synthetic Serum Substitute). The oocytes (taken from the equilibration solution) are transferred in the loading solution and rapidly loaded in plastic straws. Afterwards the straws are placed in a programmable freezer, e.g. a Kryo 10 series III (Planer Kryo 10/1.7 GB).
  • According to a particularly advantageous embodiment of the present method, before starting the freezing program the oocytes are exposed to the loading solution (including sucrose at a concentration of at least 0.3M) for 13-15 min and preferably for about 15 min.
  • Then a slow freezing method is applied to the oocytes as follows. The initial chamber temperature is 20° C. Then the temperature is slowly (2° C./min) reduced to −7° C. at which temperature ice nucleation is manually induced. After a hold time of 10 min at −7° C., the straws are cooled slowly (0.3° C./min) to −30° C. and then rapidly (50° C./min) to −150° C. After 10-12 min of stabilisation temperature, the straws are transferred into liquid nitrogen tanks and stored until thawing.
  • The following solutions are used in the thawing process:
  • a mother solution 1.0M PROH composed of
  • 14.35 ml PBS
  • 1.65 ml PROH
  • and utilized to prepare
  • A) 1.0M PROH solution+0.3M sucrose, composed of
  • 8 ml of mother solution (1.0M PROH)
  • 1.128 gr sucrose
  • B) 0.5M PROH solution+0.3M sucrose, composed of
  • 4 ml of mother solution (1.0M PROH)
  • 4 ml PBS
  • 1.128 gr sucrose.
  • The following solutions are also used:
  • C) 0.3M sucrose solution, composed of:
  • 8 ml PBS
  • 1.128 gr sucrose
  • D) PBS solution, composed of:
  • 8 ml PBS
  • E) SSS solution (Synthetic Serum Substitute)
  • These solutions are utilized in a rapid thawing program. The solutions are prepared, mixed, filtered and conserved at +4° C. It is better to maintain the solutions at room temperature for 15 min before using.
  • To thaw, the straws are initially air-warned for 30 sec and then immersed in a 30° C. water bath for 40 sec until all traces of ice have disappeared
  • Then, for each straw, a four wells dish is prepared for removing cryoprotectant by stepwise dilution of PROH in the thawing solutions.
  • More particularly, the first well contains 0.350 ml of solution A (1.0M PROH solution+0.3M sucrose)+0.150 ml of solution E SSS (Synthetic Serum Substitute). The content of the straw is expelled in this solution and the oocytes are equilibrated for 5 min at room temperature.
  • The second well contains 0.350 ml of solution B (0.5M PROH solution+0.3M sucrose)+0.150 ml SSS (Synthetic Serum Substitute). The oocytes, taken from the first well, are transferred to this solution and maintained for additional 5 min at room temperature.
  • The third well contains 0.350 ml of solution C (0.3M sucrose solution)+0.150 ml of SSS solution (Synthetic Serum Substitute). The oocytes, taken from the second well are transferred to this solution and maintained for 10 min at room temperature.
  • The fourth well contains 0.350 ml of solution D (PBS solution)+0.150 ml of SSS solution (Synthetic Serum Substitute). The oocytes, takes from the third well, are transferred to this solution and maintained for 10 min at room temperature and for additional 10 min at 37° C.
  • Finally the oocytes can be transferred to a conventional culture medium before insemination.
  • Experimental data have shown a very high survival rate (82%) of fresh human oocytes cryopreserved with loading/thawing solutions including sucrose at a concentration of 0.3M. A higher survival rate is also observed when the oocytes are exposed to the loading solution for 13-15 min instead of 2-5 min.

Claims (10)

1. Use of a solution for cryopreserving human oocytes, characterized in that said solution comprises a permeating cryoprotectant and sucrose at a concentration of at least 0.3M.
2. Use of a solution according to claim 1, further characterized in that the permeating cryoprotectant comprises 1,2-propanediol (PROH) at a concentration of 1.5M, and wherein the solution is useable as a loading solution in cryopreserving processes of oocytes.
3. Use of a solution according to claim 1, further characterized in that the permeating cryoprotectant comprises 1,2-propanediol (PROH) at a concentration of 1.0M, and wherein the solution is useable as a thawing solution in cryopreserving processes of oocytes.
4. Use of a solution according to claim 1, further characterized in that the permeating cryoprotectant comprises 1,2-propanediol (PROH) at a concentration of 0.5M, and wherein the solution is useable as a thawing solution in cryopreserving processes of oocytes.
5. Use of a solution according to claim 1, further characterized in that the solution is used as a thawing solution in cryopreserving processes for oocytes.
6. Use of a solution according to claim 1, wherein the oocytes are fresh human oocytes.
7. Use of a solution according to claim 2, wherein the oocytes are fresh human oocytes.
8. Use of a solution according to claim 3, wherein the oocytes are fresh human oocytes.
9. Use of a solution according to claim 4, wherein the oocytes are fresh human oocytes.
10. Use of a solution according to claim 5, wherein the oocytes are fresh human oocytes.
US12/146,826 2000-03-20 2008-06-26 Method and solutions for cryopreserving oocytes, especially fresh human oocytes Abandoned US20080286863A1 (en)

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US12/146,826 US20080286863A1 (en) 2000-03-20 2008-06-26 Method and solutions for cryopreserving oocytes, especially fresh human oocytes

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ITIT-PI2000A000020 2000-03-20
IT2000PI000020A IT1317301B1 (en) 2000-03-20 2000-03-20 SACCHAROSE CONCENTRATIONS AND DURATION OF THE EXPOSURE OF THE Oocyte LOADING SOLUTIONS IN THE CRYOPRESERVATION PROCEDURE OF
PCT/IT2001/000139 WO2001070020A2 (en) 2000-03-20 2001-03-20 Method and solutions for cryopreservingoocytes
US10/251,624 US7011937B2 (en) 2000-03-20 2002-09-20 Method and solutions for cryopreserving oocytes, especially fresh human oocytes
US11/323,250 US20060105317A1 (en) 2000-03-20 2005-12-30 Method and solutions for cryopreserving oocytes, especially fresh human oocytes
US12/146,826 US20080286863A1 (en) 2000-03-20 2008-06-26 Method and solutions for cryopreserving oocytes, especially fresh human oocytes

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US11/323,250 Abandoned US20060105317A1 (en) 2000-03-20 2005-12-30 Method and solutions for cryopreserving oocytes, especially fresh human oocytes
US12/146,826 Abandoned US20080286863A1 (en) 2000-03-20 2008-06-26 Method and solutions for cryopreserving oocytes, especially fresh human oocytes

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US11/323,250 Abandoned US20060105317A1 (en) 2000-03-20 2005-12-30 Method and solutions for cryopreserving oocytes, especially fresh human oocytes

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US (3) US7011937B2 (en)
EP (1) EP1267613B1 (en)
AT (1) ATE329486T1 (en)
AU (1) AU2001246812A1 (en)
DE (1) DE60120658T2 (en)
IT (1) IT1317301B1 (en)
WO (1) WO2001070020A2 (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060003309A1 (en) * 2004-07-02 2006-01-05 Akin James W Method of frozen donor egg banking
US20080113432A1 (en) * 2006-11-15 2008-05-15 Mariposa Biotechnology, Inc. Methods and compositions for reanimating cryopreserved oocytes
US20080113431A1 (en) 2006-11-15 2008-05-15 Mariposa Biotechnology, Inc. Methods and compositions for cryopreserving oocytes
JP7672127B2 (en) * 2018-03-06 2025-05-07 ゼノジェンファーマ株式会社 Cell cryopreservation solution and its use
CN114521550A (en) * 2021-03-23 2022-05-24 上海慧存医疗科技有限公司 Biological preservation solution and application thereof in oocyte and ovary tissue preservation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5985538A (en) * 1997-08-01 1999-11-16 Saint Barnabas Medical Center Cryopreservation and cell culture medium comprising less than 50 mM sodium ions and greater than 100 mM choline salt

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5985538A (en) * 1997-08-01 1999-11-16 Saint Barnabas Medical Center Cryopreservation and cell culture medium comprising less than 50 mM sodium ions and greater than 100 mM choline salt

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US7011937B2 (en) 2006-03-14
ITPI20000020A1 (en) 2001-09-20
EP1267613A2 (en) 2003-01-02
DE60120658D1 (en) 2006-07-27
US20030077567A1 (en) 2003-04-24
WO2001070020A3 (en) 2002-03-21
WO2001070020A2 (en) 2001-09-27
ATE329486T1 (en) 2006-07-15
EP1267613B1 (en) 2006-06-14
US20060105317A1 (en) 2006-05-18
DE60120658T2 (en) 2007-05-31
AU2001246812A1 (en) 2001-10-03
IT1317301B1 (en) 2003-06-16
ITPI20000020A0 (en) 2000-03-20

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