WO2010141317A1 - Populations d'ovocytes sensiblement spheriques a volume reduit - Google Patents

Populations d'ovocytes sensiblement spheriques a volume reduit Download PDF

Info

Publication number
WO2010141317A1
WO2010141317A1 PCT/US2010/036390 US2010036390W WO2010141317A1 WO 2010141317 A1 WO2010141317 A1 WO 2010141317A1 US 2010036390 W US2010036390 W US 2010036390W WO 2010141317 A1 WO2010141317 A1 WO 2010141317A1
Authority
WO
WIPO (PCT)
Prior art keywords
embryos
oocytes
blastocysts
population
partially
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2010/036390
Other languages
English (en)
Inventor
Fred Burbank
Michael Jones
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mariposa Biotechnology Inc
Original Assignee
Mariposa Biotechnology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mariposa Biotechnology Inc filed Critical Mariposa Biotechnology Inc
Publication of WO2010141317A1 publication Critical patent/WO2010141317A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients

Definitions

  • the present invention relates generally to the field of cryopreservation.
  • the present invention relates to methods and compositions for the cryopreservation and/or reanimation of substantially spherical oocytes, embryos or blastocysts.
  • conventional manual movement of oocytes among cryopreservation solution baths can impart osmotic and thermal shock.
  • formation of ice crystals within the oocyte can cause intracellular damage in the oocyte.
  • Oocytes undergoing conventional cryopreservation techniques can also experience a loss of sphericity and undesirable changes in volume. Such effects may result in structural damage in addition to toxicity, thereby significantly diminishing the viability of the oocyte and ultimately reducing the probability of successful fertilization.
  • Human involvement and conventional preservation techniques greatly contribute to the lack of consistency in cryopreservation of oocytes, embryos or blastocysts and results in an undesirably low fertilization success rate.
  • This invention provides a method for producing a substantially spherical, partially cryopreserved oocyte, embryo or blastocyst.
  • the substantially spherical shape of the partially cryopreserved oocyte, embryo or blastocyst is provided by contacting an oocyte, embryo or blastocyst with a cryoprotecting solution having an initial osmolarity under continuous process conditions.
  • the continuous process conditions comprise gradually increasing the osmolarity of the cryoprotecting solution over a predetermined period of time such that a portion of cytoplasmic water of the oocyte, embryo or blastocyst is replaced with cryoprotectant from the solution, thereby producing a substantially spherical, partially cryopreserved oocyte, embryo or blastocyst.
  • the substantially spherical shape of the oocyte, embryo or blastocyst reduces or alternatively substantially eliminates the stress on the cell walls and the interior proteins that are key to maintaining viability of the oocyte, embryo or blastocyst after reanimation.
  • This invention also provides a method for producing a partially reanimated oocyte, embryo or blastocyst comprising contacting a cryopreserved oocyte, embryo or blastocyst with a reanimating solution having an initial osmolarity under continuous process conditions.
  • the continuous process conditions comprise gradually decreasing the osmolarity of the reanimating solution over a predetermined period of time such that a portion of cytoplasmic cryoprotectant of the oocyte, embryo or blastocyst is replaced with water from the solution, thereby producing a substantially spherical partially reanimated oocyte, embryo or blastocyst.
  • a population of substantially spherical, partially cryopreserved oocytes, embryos or blastocysts characterized in that a portion of their cytoplasmic water has been replaced with a cryoprotectant while maintaining their substantially spherical shape.
  • the population provides for a viable pregnancy at a ratio of less than about 10: 1, or alternatively less than 8:1 or alternatively less than about 5:1. The ratio is a number of cryopreserved and reanimated oocytes, embryos or blastocysts used to produce a single viable pregnancy.
  • a population of partially reanimated oocytes, embryos or blastocysts characterized in that a portion of their cytoplasmic cryoprotectant has been replaced with water.
  • the population provides for a viable pregnancy at a ratio of less than about 10: 1, or alternatively less than 8:1 or alternatively less than about 5:1. The ratio is a number of cryopreserved and reanimated oocytes, embryos or blastocysts used to produce a single viable pregnancy.
  • Figure 1 shows a graphic representation of the gradual increase in osmolality of the cryoprotecting solution during the cryopreservation of mouse embryos using the methods of Example 1.
  • the X-axis represents time (min), whereas the Y-axis represents the osmolality (mM) of the cryoprotecting solution.
  • Figure 2 shows a graphic representation of the gradual decrease in osmolality of the reanimating solution during the reanimation of mouse embryos using the methods of Example 2.
  • the X-axis represents time (min), whereas the Y-axis represents the osmolarity (mM) of the reanimating solution.
  • Figure 3 shows a photograph of mouse embryos following cryopreservation and reanimation of mouse embryos using the methods of Example 1.
  • Figure 4 shows a photograph of the mouse embryos of Figure 3 following 1 day of growth.
  • Figure 5 shows a photograph of the mouse embryos of Figure 3 following 2 days of growth.
  • a oocyte includes a plurality of oocytes, including populations thereof.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the composition or method.
  • Consisting of shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated methods steps or compositions (consisting of).
  • a "population” refers to a composition of at least two individual oocytes, embryos, blastocysts or equivalents thereof.
  • a “population” refers to at least three, or alternatively at least four, or alternatively at least five, or alternatively at least six, or alternatively at least seven, or alternatively at least eight, or alternatively, at least nine, or alternatively at least ten individual oocytes, embryos, blastocysts.
  • substantially spherical refers to an oocyte, embryo or blastocyst which has no more than ⁇ 40% change in its surface area as compared to the oocyte prior to introduction of a cryoprotectant during freezing or cryopreserving or prior to introduction of water during thawing or reanimating.
  • the change in the surface area of the oocyte, embryo or blastocyst can be a decrease in surface area due to shrinkage or an increase in surface area due to the introduction of undulations or other surface deformities which arise during shrinkage.
  • the oocyte, embryo or blastocyst has no more than about ⁇ 30% change, no more than about ⁇ 20% change, no more than about ⁇ 15% change, no more than about ⁇ 10% change, or no more than about ⁇ 5% change in its surface area during shrinkage.
  • substantially non- spherical refers to an oocyte, embryo or blastocyst which has more than ⁇ 40% change in its surface area as compared to the oocyte prior to introduction of a cryoprotectant during freezing or cryopreserving or prior to introduction of water during thawing or reanimating.
  • the change in the surface area of the oocyte, embryo or blastocyst can be a decrease in surface area due to shrinkage or an increase in surface area due to the introduction of undulations or other surface deformities which arise during shrinkage.
  • the term "partially cryopreserved" refers to an oocyte, embryo or blastocyst having a portion of its cytoplasmic water replaced with cryoprotectant prior to freezing.
  • the portion of cytoplasmic water that has been replaced with water is more than about 1 v/v%, or alternatively more than about 10 v/v%, or alternatively more than about 50 v/v%, or alternatively more than about 60 v/v%, or alternatively more than about 70 v/v%, or alternatively more than about 80 v/v%, or alternatively more than about 90 v/v%.
  • substantially all of the cytoplasmic water of the oocyte, embryo or blastocyst (90-100 v/v%) is replaced with the cryoprotectant.
  • the portion of cytoplasmic water of the oocyte, embryo or blastocyst that has been replaced with cryoprotectant is sufficient to protect the oocyte, embryo or blastocyst. It is understood that one of skill in the art will be able to readily ascertain the amount of cryoprotectant necessary to protect the oocyte, embryo or blastocyst.
  • cryopreserved oocytes, embryos or blastocysts refers to frozen oocytes, embryos or blastocysts, comprising a cryoprotectant, which are preserved by cooling to low sub-zero temperatures, such as, but not limited to, 77 K or -196 0 C (the boiling point of liquid nitrogen).
  • the term "partially reanimated" refers to an oocyte, embryo or blastocyst having at least a portion of the cytoplasmic cryoprotectant that is replaced with water after being thawed.
  • the portion of the cytoplasmic cryoprotectant that is replaced with water is more than about 1 v/v%, or alternatively more than about 10 v/v%, or alternatively more than about 50 v/v%, or alternatively more than about 60 v/v%, or alternatively more than about 70 v/v%, or alternatively more than about 80 v/v%, or alternatively more than about 90 v/v%.
  • all of the cytoplasmic cryoprotect of the oocyte, embryo or blastocyst (100 v/v%) is replaced with the water.
  • the cytoplasmic cryoprotectant of the "partially reanimated" oocyte, embryo or blastocyst has been replaced with water that is sufficient to reanimate the oocyte, embryo or blastocyst. It is understood that one of skill in the art will be able to readily ascertain the amount of water necessary to reanimate the oocyte, embryo or blastocyst.
  • the term "reanimated oocytes, embryos or blastocysts” refers to thawed oocytes, embryos or blastocysts which are capable of fertilization and/or embryo development.
  • oocyte refers to an unfertilized freshly harvested or mature oocyte.
  • freshly harvested oocyte means that the oocyte was harvested from the animal donor within 8 hours of initiation of the stabilization/cryopreservation process, or alternatively within about 4 hours of initiation of the stabilization/cryopreservation process, or alternatively within about 1 hour of initiation of the stabilization/cryopreservation process, or alternatively within about 0.1 hour of initiation of the stabilization/cryopreservation process.
  • the mature oocytes mean harvested oocytes which are graded on a maturation scale as "mature stage -MIL" This scale further identifies harvested oocytes as “intermediate stage - (MI)” or “immature stage - (GV)".
  • MI immediate stage -
  • GV millimmature stage -
  • stabilized oocytes refer to mature oocytes still retaining the cumulus mass (granulosis cells) which permit maturation of the oocyte by nutrient intake through gap junctions in the cumulas mass.
  • the mature oocyte is characterized by formation of the meiotic spindle in conjunction with extrusion of the first polar body while maintaining the integrity/activity of the intracellular proteins.
  • blastocyst refers to a fertilized egg freshly harvested from about 5 days after fertilization up to implantation in the uterus.
  • freshly harvested blastocyst means that the blastocysts were harvested from the animal donor within about 8 hours of initiation of the stabilization/cryopreservation process, or alternatively within about 4 hours of initiation of the stabilization/cryopreservation process, or alternatively within about 1 hour of initiation of the stabilization/cryopreservation process, and alternatively within about 0.1 hour of initiation of the stabilization/cryopreservation process.
  • embryo refers to a fertilized egg freshly harvested from between the time of the first division to two cells to about 5 days after fertilization.
  • freshly harvested embryo means that the embryos were harvested from the animal donor within about 8 hours of initiation of the stabilization/cryopreservation process, preferably within about 4 hours of initiation of the stabilization/cryopreservation process, more preferably within about 1 hour of initiation of the stabilization/cryopreservation process, and even more preferably within about 0.1 hour of initiation of the stabilization/cryopreservation process.
  • stabilization process refers to the incubation of the oocytes, embryos or blastocyst in a stabilization solution, which provides the oocytes, embryos or blastocyst an opportunity to stabilize in a solution of low to intermediate osmolality prior to incubation in a cryoprotecting solution having gradually increasing osmolality.
  • cryoprotecting solution refers to a solution having at least one cryoprotectant.
  • a "cryoprotectant” permeates across the cell wall of the oocyte, embryo or blastocyst typically by osmotic methods and promotes survival and retention of viability of the oocyte, embryo or blastocyst during the process of cry opre serving as well as in the cryopreserved state and/or during reanimation.
  • cryoprotectants are well known in the art and include, by way of example only, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol (1,2-propanediol), glycerol, as well as mixtures of 2 or more of such cryoprotectants, and the like.
  • DMSO dimethyl sulfoxide
  • ethylene glycol propylene glycol (1,2-propanediol)
  • glycerol glycerol
  • the cryoprotecting solution has an initial osmolarity.
  • the "cryoprotecting solution” comprises a dehydrating agent.
  • the "cryoprotecting solution” has a cryoprotectant concentration, osmolarity and/or a dehydrating agent concentration as described in Tables 1 or 2.
  • Osmolarity refers to the amount of solute (dissolved chemical) per liter of total solution and is typically measured in milliosmoles per liter (mOsmol/L).
  • cryoprotecting solutions useful in methods of the invention further comprise at least one or more components such as, but not limited to, sterile water, HEPES, sodium bicarbonate, sodium hydroxide, sodium chloride, potassium chloride, calcium chloride, potassium phosphate, magnesium sulfate, dextrose, sodium lactate solution, sodium pyruvate, gentamicin sulfate, and human serum albumin.
  • the cryoprotecting solution of the invention does not comprise alpha globulin or beta globulin.
  • the cryoprotecting solution of the invention comprises component concentrations as described in Tables 1 or 2.
  • dehydrating agent refers to an agent that facilitates dehydration of the intra-cytoplasmic water in the oocyte, embryo or blastocyst during cryopreservation. In one aspect, such agents do not osmotically traverse the cellular wall of the oocyte.
  • Dehydrating agents include, sucrose, dextrose, trehalose, lactose, raffinose, and the like.
  • reanimating solution refers to a solution having at least one cryoprotectant and water.
  • a “reanimating solution” allows water to permeate across the cell wall of the oocyte, embryo or blastocyst, typically by osmotic methods and promotes survival and retention of viability of the oocyte, embryo or blasocycst during the process of reanimating.
  • the "reanimating solution” has an initial osmolarity.
  • the "reanimating solution” comprises a dehydrating agent.
  • the "reanimating solution” has a cryoprotectant concentration, osmolarity and/or dehydrating agent concentration as described in Tables 1 or 2.
  • reanimating solutions of the invention further comprise at least one or more components such as, but not limited to, sterile water, HEPES, sodium bicarbonate, sodium hydroxide, sodium chloride, potassium chloride, calcium chloride, potassium phosphate, magnesium sulfate, dextrose, sodium lactate solution, sodium pyruvate, gentamicin sulfate and human serum albumin.
  • the reanimating solution of the invention does not comprise alpha globulin or beta globulin.
  • the reanimating solution of the invention comprises component concentrations as described in Tables 1 or 2.
  • the term “gradually” refers to proceeding by fine or incremental steps or degrees.
  • the phrase “gradually increasing” refers to increasing the amount of a component in a solution by no more than about 0.001%, or alternatively no more than about 0.01%, or alternatively no more than about 0.1%, or alternatively no more than about 1%, or alternatively no more than about 5%, or alternatively no more than about 10%.
  • the osmolarity of a solution is "gradually increased” at a given rate, for example, from about 90 mOsmol/L per 1 minute to about 110 mOsmol/L per 1 minute.
  • the phrase "gradually decreasing” refers to decreasing the amount of a component in a solution by no more than about 0.001%, or alternatively no more than about 0.01%, or alternatively no more than about 0.1%, or alternatively no more than about 1%, or alternatively no more than about 5%, or alternatively no more than about 10%.
  • the osmolarity of a solution is "gradually decreased” at a given rate, for example, from about 30 mOsmol/L per 1 minute to about 50 mOsmol/L per 1 minute.
  • the temperature of a solution is "gradually" increased or decreased from one temperature to another temperature over a predetermined period of time. In one aspect, the above gradual changes occurs under continuous process conditions.
  • continuous process conditions refers to the uninterrupted conditions used in a process of making a product.
  • the “continuous process conditions” comprise contacting of an oocyte, embryo or blastocyst or a population thereof with one or more solutions described herein.
  • the “continuous process conditions” comprise gradually increasing the osmolarity of a solution.
  • the “continuous process conditions” comprise gradually decreasing the osmolarity of the solution.
  • the “continuous process conditions” comprise controlling one or more characteristic of the solution, such as temperature, concentration of a component, pH level, pressure or flow rate of the solution contacting the oocyte, embryo or blastocyst.
  • viable pregnancy refers to the implantation of an embryo into the uterine wall of a subject.
  • Methods for identifying a viable pregnancy include, but are not limited to, detection of fetal cardiac activity and/or fetal development using ultrasound or an equivalent thereof or detection of ⁇ -human chorionic gonadotropin ( ⁇ -hCG) in the subject's blood and/or urine.
  • ⁇ -hCG ⁇ -human chorionic gonadotropin
  • the viability of a pregnancy is determined by serial quantitative blood tests done about 2 to 3 days apart, wherein if the blood serum ⁇ -hCG level of a subject is below 1,200 mlU/ml and increases by about 50% to about double every 48 to 72 hours, or alternatively, if the blood serum ⁇ -hCG levels of a subject is between 1,200 and 6,000 mlU/ml and doubles about every 72-96 hours, or alternatively if the blood serum ⁇ -hCG level of a subject is above 6,000 mlU/ml and the ⁇ -hCG takes more than about four days to double, all of the above are an indication of a viable pregnancy.
  • a "successive viable pregnancy” is a viable pregnancy in a subject which is subsequent to a preceding viable pregnancy in the same subject following termination of the preceding pregnancy. Termination of a pregnancy includes, but is not limited to, successful delivery of a neonate, miscarriage of a fetus, or any other action with removes a developing neonate or fetus from the subject.
  • predetermined period of time refers to the amount of time in which the oocyctes, embryos or blastocysts are contacted with the solutions described herein to obtain the desired portion of a cryoprotectant or water in the oocyctes, embryos or blastocysts thereby producing a population of substantially spherical, partially cryopreserved or partially reanimated oocyctes, embryos or blastocysts, respectively.
  • the term "progressive rate” refers to advancing a measured quantity with respect to another measured quantity.
  • the osmolality of a solution is increased at a "progressive rate” such as, but not limited to, mOsmol/L per minute, or alternatively from an initial osmolarity of a solution to a final osmolarity of a solution over a predetermined amount of time.
  • the osmolarity of solution is decreased at a "progressive rate" such as, but not limited to, mOsmol/L per minute, or alternatively from an initial osmolarity of a solution to a final osmolarity of a solution over a predetermined amount of time.
  • a progressive rate such as, but not limited to, mOsmol/L per minute, or alternatively from an initial osmolarity of a solution to a final osmolarity of a solution over a predetermined amount of time.
  • the "progressive rate" is substantially linear, wherein the advancing measured quantity is equal throughout the other measured quantity.
  • the "substantially linear progressive rate” will vary by no more than about ⁇ 1%, or alternatively no more than about ⁇ 5%, or alternatively no more than about ⁇ 10%, or alternatively no more than about ⁇ 15%, or alternatively no more than about ⁇ 20%, or alternatively no more than about ⁇ 25%, or alternatively no more than about ⁇ 30%, or alternatively no more than about ⁇ 40% from a desired rate.
  • the "progressive rate” is substantially non-linear, wherein the advancing measured quantity is not equal throughout the other measured quantity.
  • a non-limiting example of this substantially non-linear progressive rate is an initial increase in osmolarity of a solution that is lower than the later rate of increasing osmolarity or vise- versa, all within a predetermined period of time.
  • This invention provides a method for producing a substantially spherical, partially cryopreserved oocyte, embryo or blastocyst comprising contacting an oocyte, embryo or blastocyst with a cryoprotecting solution having an initial osmolarity under continuous process conditions, wherein the conditions comprise gradually increasing the osmolarity over a predetermined period of time, such that a portion of cytoplasmic water of the oocyte, embryo or blastocyst is replaced with cryoprotectant from said solution, thereby producing a substantially spherical, partially cryopreserved oocyte, embryo or blastocyst.
  • the portion of cytoplasmic water replaced with cryoprotect from the solution is sufficient to protect the oocyte, embryo or blastocyst. It is understood that one of skill in the art will be able to readily ascertain the amount of cryoprotectant necessary to protect the oocyte, embryo or blastocyst. In a further aspect, more than about 10% of the cytoplasmic water is replaced with cryoprotectant.
  • the oocyte, embryo or blastocyst is freshly harvested and stabilized to in vitro conditions prior to the initiation of cryopreservation.
  • the oocyte, embryo or blastocyst is incubated or stored in one or more stabilization solutions for at least about 1 hour, or alternatively at least 2 hours, or alternatively at least 3 hours after harvesting.
  • the stabilization solutions are intended to permit further maturation of the oocytes in vitro.
  • the stabilization solutions are designed to mimic the environment within the follicle from which the eggs were removed.
  • the follicle supports ongoing processes of the oocyte maturation in vivo which in one aspect of the invention are duplicated when the oocyte is removed from the follicle and placed into the stabilization solution.
  • the stabilization solution also provides nutrients for further meiotic development and supports metabolic processes continuing within the oocyte, embryo or blastocyst as well as supports the cell membrane.
  • a further function of the stabilization solution is to remove biological wastes occurring from metabolism within the oocyte, embryo or blastocyst and to stabilize the intra-oocyte or intra-cellular pH.
  • the stabilization solution maintains the integrity and activity of these proteins/polypeptides so that after cry opre serving and subsequent reanimating, these proteins/polypeptides can be employed within the oocyte, embryo or blasocycst for their intended use.
  • the stabilization solution does not include any cryoprotectant, as described herein.
  • Stabilization solutions useful in this invention include, by way of the example, Global ® media (available from Life Global, IVF Online), Global ® media supplemented with SSS (available from Irvine Scientific, Santa Ana, California, USA), human tubal fluid (HTF - available from Irvine Scientific, Santa Ana, California, USA) optionally supplemented with SSS and/or an antibiotic (e.g., gentamicin) and modified HTF (HTF with HEPES (mHTF) - available from Life Global, IVF Online) optionally supplemented with SSS and/or an antibiotic (e.g., gentamicin), phosphate buffered saline (PBS), sodium depleted PBS (e.g., sodium hydrogen phosphate (H 2 NaPO 4 )) or equivalents thereof.
  • GSS available from Irvine Scientific, Santa Ana, California, USA
  • HTF - human tubal fluid
  • an antibiotic e.g., gentamicin
  • mHTF modified HTF
  • SSS phosphate buffere
  • One such stabilization method includes incubation or storage of the oocyte, embryo or blastocyst in Global media supplemented with about 5% to about 20%
  • Synthetic Serum Substitute SSS
  • Global media supplemented with about 10% human serum albumin (HSA), or alternatively human tubal fluid (HTF), or alternatively modified human tubal fluid (mHTF), which includes HTF and HEPES buffer.
  • HSA human serum albumin
  • HEF human tubal fluid
  • mHTF alternatively modified human tubal fluid
  • the stabilization of the oocyte, embryo or blastocyst will include the incubation or storage of the oocyte, embryo or blastocyst in two or more stabilization solutions.
  • the stabilization solutions are maintained at a temperature of from about 33 0 C to about 38 0 C, or alternatively from about 35 0 C to about 38 0 C, or alternatively from about 36.5 0 C to 37.5 0 C for a sufficient period to permit the oocyte, embryo or blastocyst to be stabilized to its in vitro conditions.
  • the osmolarity of the cryoprotecting solution is increased at a progressive rate throughout the predetermined period of time.
  • the progressive rate is substantially linear or substantially non-linear.
  • the substantially linear progressive rate is from about 50 mOsmol/L per 1 minute to about 200 mOsmol/L per 1 minute, or alternatively from about 75 mOsmol/L per 1 minute to about 150 mOsmol/L per 1 minute, or alternatively from about 90 mOsmol/L per 1 minute to about 110 mOsmol/L per 1 minute, or alternatively about 100 mOsmol/L per 1 minute.
  • the osmolarity of the cryoprotecting solution is increased from the initial osmolarity of about 300 mOsmol/L to a final osmolarity of at least about 1300 mOsmol/L within the predetermined period of time.
  • the cryoprotectant concentration of the cryoprotecting solution is gradually increased from substantially no cryoprotectant to at least 1.5 M cryoprotectant within the predetermined period of time.
  • the predetermined period of time is from about 5 minutes to about 20 minutes, or alternatively from about 8 minutes to about 18 minutes, or alternatively from about 12 minutes to about 16 minutes, or alternatively about 13 minutes.
  • the increase in the cryoprotectant concentration and/or the osmolality of the cryoprotecting solution comprises mixing of at least a first and a second solution, wherein the first solution comprises substantially no cryoprotectant and an osmolality lower than the second solution and the second solution comprises more cryoprotectant than the first solution and has an osmolality higher than the first solution.
  • the cryoprotecting solution does not contain alpha globulin or beta globulin.
  • the cryoprotecting solution further comprises a dehydrating agent.
  • the concentration of the dehydrating agent is gradually increased from substantially no dehydrating agent to at least 0.1 M dehydrating agent within the predetermined period of time.
  • the continuous process conditions comprise maintaining the cryoprotecting solution at a temperature of about 22 0 C to about 26 0 C, or alternatively the cryoprotecting solution is gradually decreasing the temperature of the cryoprotecting solution from about 38 0 C to about 22 0 C within the predetermined period of time.
  • the oocytes, embryos or blastocysts are transferred into a sealed container and cryopreserved. Cryopreservation of the prepared oocyte proceeds via conventional techniques including slow freezing of the oocytes in a manner similar to that described by Fabbri, et al., U.S. Patent No. 7,011,937, which patent is incorporated herein by reference in its entirety.
  • the oocytes, embryos or blastocysts are loaded into a suitable container such as a 0.25 mL plastic straw. The straws are sealed at both ends and placed in an automated biological vertical freezer such as the Kryo 10 Series III (Planer 10/1.7 GB).
  • the straws are chilled at a controlled cooling rate to about -7 0 C whereupon "seeding" is performed to induce the water molecules to undergo crystallization.
  • the straws are cooled at a rate of from about -0.5 0 C per minute to about -4 0 C; or alternatively the straws are cooled at a rate of from about -I 0 C per minute to about -3 0 C; or alternatively the straws are cooled at a rate of about -2 0 C per minute.
  • cooling is resumed at a controlled rate until the straw reaches about -33 0 C which allows diffusion of the cryoprotectant into the oocytes, embryos or blastocysts.
  • the straws are cooled at a rate of from about -0.1 0 C per minute to about -I 0 C; or alternatively the straws are cooled at a rate of from about -0.2 0 C per minute to about -0.5 0 C; or alternatively the straws are cooled at a rate of about -0.3 0 C per minute.
  • the straws are then plunged into liquid nitrogen for storage.
  • this invention provides a method for producing a partially reanimated oocyte, embryo or blastocyst comprising contacting a cryopreserved oocyte, embryo or blastocyst with a reanimating solution having an initial osmolality under continuous process conditions, wherein said conditions comprise gradually decreasing the osmolality over a predetermined period of time, thereby producing a partially reanimated oocyte, embryo or blastocyst.
  • the portion of the cytoplasmic cryoprotect replaced with water is sufficient to reanimate the oocyte, embryo or blastocyst.
  • the osmolarity of the reanimating solution is increased at a progressive rate throughout the predetermined period of time.
  • the progressive rate is substantially linear or substantially non-linear.
  • the substantially linear progressive rate of the osmolarity of the reanimating solution is from about 10 mOsmol/L per 1 minute to about 70 mOsmol/L per 1 minute, or alternatively from about 20 mOsmol/L per 1 minute to about 60 mOsmol/L per 1 minute, or alternatively from about 30 mOsmol/L per 1 minute to about 50 mOsmol/L per 1 minute, or alternatively about 40 mOsmol/L per 1 minute.
  • the osmolarity of the cryoprotecting solution is gradually decreased from an initial osmolarity of at least about 1100 mOsmol/L to a final osmolarity of about 300 mOsmol/L within the predetermined period of time.
  • the concentration of cryoprotectant in the reanimating solution is gradually decreased from at least 1.0 M cryoprotectant to substantially no cryoprotectant within the predetermined period of time.
  • the predetermined period of time is from about 15 minutes to about 60 minutes, or alternatively from about 30 minutes to about 50 minutes, or alternatively from about 40 minutes to about 45 minutes, or alternatively about 42 minutes.
  • the decrease in the cryoprotectant concentration and/or the osmolality of the reanimating solution comprises mixing of at least two solutions, wherein the first solution comprises substantially no cryoprotectant and an osmolarity lower than the second solution and the second solution comprises more cryoprotectant than the first solution and has an osmolarity higher than the first solution.
  • the reanimating solution does not contain alpha globulin or beta globulin.
  • the reanimating solution further comprises a dehydrating agent.
  • the dehydrating agent concentration of the reanimating solution is gradually decreased from at least 0.1 M dehydrating agent to substantially no dehydrating agent within the predetermined period of time.
  • the continuous process conditions comprise gradually increasing the temperature of the reanimating solution from about 3O 0 C to about 38 0 C within the predetermined period of time.
  • the oocyte, embryo or blastocyst is placed into a stabilization solution prior to fertilization and/or implantation.
  • the stabilization solutions useful in this invention include, by way of the example, Global ® media (available from Life Global, IVF Online), Global ® media supplemented with SSS (available from Irvine Scientific, Santa Ana, California, USA), human tubal fluid (HTF - available from Irvine Scientific, Inc., Santa Ana, California) optionally supplemented with SSS and/or an antibiotic (e.g., gentamicin) and modified HTF (HTF with HEPES (mHTF) - available from Irvine Scientific, Inc., Santa Ana, California) optionally supplemented with SSS and/or an antibiotic (e.g., gentamicin), phosphate buffered saline (PBS), sodium depleted PBS (e.g., sodium hydrogen phosphate (H 2 NaPO 4 )) and the like.
  • GBS phosphate buffered saline
  • PBS sodium deplete
  • This solution stabilizes the oocyte, embryo or blastocyst relative to physiological temperature while providing a nutrient rich environment.
  • a reanimated oocyte is immersed in a reanimation stabilization solution to reestablish the processes relating to formation of meiotic spindle required for fertilization as well as enhancing mitochondria energy production and reestablishing the mRNA processes and protein activity.
  • the oocyte, embryo or blastocys is maintained in this solution until stabilized.
  • stabilization occurs within about 2 hours. At this point the oocytes are ready for fertilization and/or implantation.
  • a cross-sectional area of the oocyte, embryo or blastocyst is measured during the process of cryopreservation and/or reanimation.
  • the measuring method is, by way of example only, birefringence imaging and NADH measurement.
  • the oocyte, embryo or blastocyst is observed under a microscope to determine a substantially spherical shape of the oocyte, embryo or blastocyst.
  • a sample of an oocyte, embryo or blastocyst or a population of oocytes, embryos or blastocysts is imaged under magnification to investigate structural elements within the oocyte, embryo or blastocyst. Images obtained under magnification can be recorded while the sample is mounted in a microscope. A variety of different imaging techniques can be used to obtain the images. For example, broad- or narrow- bandwidth light sources can be used to illuminate the sample, and light transmitted through the sample can be viewed by an operator through a microscope eyepiece and/or can be directed to a detector such as a CCD camera.
  • the oocytes can be graded as immature, mature, and post mature before in vitro fertilization. Embryos and blastocysts are typically graded by symmetry of the cells and sphericity. Damaged embryos or blastocysts will typically be aspherical or in the case of embryos, composed of a odd number of cells. Grading of the oocytes can be performed at the time of oocyte pickup under a stereomicro scope fitted with a warm stage and at, for example, xl5 and x 40 magnification.
  • the oocytes can be graded based on the status of a first polar body, size of the perivitelline space, and/or presence of cytoplasmic inclusions.
  • the evaluation of the morphology of the oocyte includes, but not limited to, variations such as shape of the oocyte, color, granularity, and homogeneity of the cytoplasm, size of perivitelline space, debris in the perivitelline space, vacuolization, inclusions and abnormalities of the first polar body or zona pellucida.
  • the grading of the oocytes as described in P. Xia, Human Reproduction, 1997, 12(8), 1750-1755 is incorporated herein by reference in its entirety.
  • birefringence imaging techniques can be used to measure the substantially spherical shape of the oocyte, embryo or blastocyst.
  • the birefringence imaging techniques can provide contrast between structural elements of interest and the rest of the sample of an oocyte, embryo or blastocyst or a population of oocytes, embryos or blastocysts.
  • birefringence imaging techniques will provide contrast even when the elements appear approximately transparent in images recorded using other techniques, e.g., broadband illumination.
  • input light is incident on the sample with a selected polarization, which is then altered by the presence of birefringence in the sample.
  • Output polarization analyzers can then be used to create contrast (e.g., by polarization-dependent attenuation) in an image of the sample. For example, different regions of the sample through which corresponding portions of the input light pass exhibit different amounts of retardation, and thereby changes to the state of polarization of the different input light portions being imaged.
  • Birefringence imaging systems are disclosed, for example, in U.S. Pat. Publ. No. 2007-0231784, entitled “Quantitation of Oocytes and Biological Samples Using Birefringent Imaging," by Clifford C. Hoyt and Cathy Boutin, filed on April 4, 2006; U.S. Pat. No. 5,521,705 entitled “Polarizied Light Microscopy” by Rudolf Oldenbourg and Guang Mei, filed on May 12, 1994; and U.S. Pat. No. 6,924,893 entitled "Enhancing Polarized Light
  • the substantially spherical shape of the oocyte, embryo or blastocyst is measured using a method of NADH measurement.
  • the method involves reducing the endogenous NADH concentration of the oocyte, embryo or blastocyst by placing the oocyte, embryo or blastocyst in a control medium and obtaining at least one control NADH fluorescence measurement for the nutrient-deprived oocyte. Thereafter, the oocyte is contacted with a nutrient for a period of time sufficient for the oocyte to acquire (i.e., take up and metabolically process) the nutrient. At least one post-nutrient NADH fluorescence measurement is obtained.
  • the quality of the oocyte, embryo or blastocyst is assessed by comparing the control NADH fluorescence measurement(s) to the post-nutrient NADH fluorescence measurement(s). Higher quality oocytes, embryos or blastocysts are those for which the post-nutrient NADH fluorescence measurement is significantly (i.e., at least 2%) greater than the control NADH fluorescence measurement.
  • the NADH measurement is disclosed, for example, in U.S. Pat. No. 5,541,081, entitled, "Process for Assessing Oocyte and Embryo Quality", by R. Ian Hardy, David E. Golan, and John D. Biggers, filed March 22, 1994, the entire content of which is incorporated herein by reference.
  • the NADH measurement method can be used for evaluating membrane integrity and function (and obtaining metabolite transfer rates). The method involves measuring the time of onset of NADH fluorescence increase following nutrient perfusion.
  • oocyte, embryo or blastocyst include, by way of example only, a development rating system (Cummins, J. et al, (1986) /. IVF-ET 3(5), 284), fluorescein diacetate fluorescence measurement (Mohr, L. et al, (1980) /. Reprod. Fertil. 58, 189), determination of immunosuppressive activity in pre- implantation culture media (Clark, D. et al, (1989) /. IVF-ET 6(1), 51-58), and measurement of immunoactive factors in culture media including progesterone (Hardy, R. et al, (1993) Soc. Gyn. Invest.
  • compositions [0065]
  • a population of substantially spherical, partially cryopreserved oocytes, embryos or blastocysts characterized in that a portion of their cytoplasmic water has been replaced with a cryoprotectant while maintaining their substantially spherical shape.
  • This population provides for a viable pregnancy at a ratio of less than about 10: 1, or alternatively less than 8:1, or alternatively less than 5:1, wherein said ratio is a number of cryopreserved and reanimated oocytes, embryos or blastocysts used to produce a single viable pregnancy.
  • the viable pregnancy is at least a second, or alternatively at least a third, or alternatively at least a fourth successive viable pregnancy resulting from the same population of cryopreserved oocytes, embryos or blastocysts.
  • the oocytes, embryos or blastocysts are animal, including a human, or non-human animal including, for example, a rodent, such as a rat and mouse, a canine, such as a dog, a leporid, such as a rabbit, livestock, sport animal or pet.
  • a rodent such as a rat and mouse
  • a canine such as a dog
  • a leporid such as a rabbit, livestock, sport animal or pet.
  • the portion of cytoplasmic water replaced with cryoprotectant is sufficient to protect the oocytes, embryos or blastocycts. In a further aspect, more than about 10 % of the cytoplasmic water is replaced with cryoprotectant.
  • a population of partially reanimated oocytes, embryos or blastocysts characterized in that a portion of their cytoplasmic cryoprotectant has been replaced with water.
  • This population provides for a viable pregnancy at a ratio of less than about 10:1, or alternatively less than 8:1, or alternatively less than 5:1, wherein said ratio is a number of cryopreserved and reanimated oocytes, embryos or blastocysts used to produce a single viable pregnancy.
  • the viable pregnancy is at least a second, or alternatively at least a third, or alternatively at least a fourth successive viable pregnancy resulting from the same population of cryopreserved oocytes, embryos or blastocysts.
  • the oocytes, embryos or blastocysts are animal, including a human, or non-human animal including, for example, a rodent, such as a rat and mouse, a canine, such as a dog, a leporid, such as a rabbit, livestock, sport animal or pet.
  • a rodent such as a rat and mouse
  • a canine such as a dog
  • a leporid such as a rabbit, livestock, sport animal or pet.
  • the portion of their cytoplasmic cryoprotectant that has been replaced water is sufficient to reanimate the oocytes, embryos or blastocycts. In a further aspect, more than about 10% of the cytoplasmic cryoprotectant has been replaced with water.
  • the methods and solutions disclosed herein produce a substantially spherical oocyte or embryo.
  • the benefit of this spherical entity is to minimize stress on the cell walls and the interior proteins that are key to further development of the oocyte or embryo.
  • Table 1 lists a theoretical osmolality and a measured osmolality as measured with a Wescor, model 5500, vapor pressure osmometer.
  • FIG. 3 The images in Figure 3, Figure 4 and Figure 5 show the results obtained from a continuously changing osmolality solution in the cryopreservation and subsequent return to normal osmolality of a batch of mouse embryos. These embryos were processed on an automated instrument and held within an egg/embryo holder.
  • HSA human serum albumin
  • the cryopreservation process was initiated on an automated system and proceeded from an initial osmolality of approximately 300 mOsmol/L in the Base Solution (Table 1) to a final osmolality of about 1325 mOsmol/L (measured) in the E- F2 solution (Table 1).
  • An example of the osmolality curve for this cryopreservation program is shown in Figure 1. As the E-F2 solution was increased in its volumetric flow rate the Base solution was decreased. This program used a total volumetric flow rate of about 70 microliters per minute and proceeded at a temperature of 23.5 0 C.
  • the embryo holders were removed and the ends were sealed.
  • the sealed embryo holders were placed into an automated freezer and cooled to -7 0 C where upon "seeding" was performed to induce crystallization. After holding at - 7 0 C for approximately 15 minutes, the cooling resumed to -33 0 C at -0.3°C/minute. Once the -33 0 C was achieved the holders were plunged into liquid nitrogen for storage.
  • the reanimation process was initiated by removing the frozen embryo holder from liquid nitrogen and warming in room temperature air for 30 seconds followed by 37 0 C water for 30 seconds. The ends were cut and then the embryo holder was installed on the automated instrument.
  • the solutions used in this process were the Base solution and the E-Tl solution of Table 1.
  • the initial fluid introduced was the E-Tl solution at an approximate osmolality of 1169 mOsmol/L (measured) and then proceeded by mixing the two solutions to a final osmolality of 300 mOsmol/L for the Base solution.
  • An example of the osmolality curve for this reanimation program is shown in Figure 2. This program used a total volumetric flow rate of about 70 microliters per minute.
  • the temperature of the solutions was initially 23.5 0 C and proceeded for 31.5 minutes at this temperature and then rised to 37.O 0 C in about 1 minute for the remainder of the time.
  • Table 2 lists a theoretical osmolality and a measured osmolality as measured with a Wescor, model 5500, vapor pressure osmometer.
  • the oocytes were harvested and stabilized to in vitro conditions by storage in a stabilization solution for at least about 1 hour after harvesting from the ovaries.
  • a stabilization solution for at least about 1 hour after harvesting from the ovaries.
  • One such method is Global media supplemented with 5 to 20% Synthetic Serum Substitute (SSS). This is so common that one might consider it industry practice.
  • SSS Synthetic Serum Substitute
  • the cryopreservation process for oocytes was initiated on an automated system and proceeded from an initial osmolality of approximately 300 mOsmol/L in the Base Solution (Table 2) to a final osmolality of about 1638 mOsmol/L (measured) in the O-F2 solution (Table 2).
  • the osmolality curve for this cryopreservation program was similar to Figure 1, except for the final osmolality exceeding 1600 mOsmol/L and the time interval being about 20 minutes.
  • This program used volumetric flow rate of about 70 microliters per minute.
  • the temperature was controlled during this process and ranged from 37 0 C at the start of the process and decreased to 25 0 C over the course of the fluid exchange time period. The combination of temperature and osmolality were used to control the fluid exchange from the solution into the oocyte and vice versa.
  • the oocyte holders were removed and the ends were sealed.
  • the sealed oocyte holders were placed into an automated freezer and cooled to -7 0 C where upon "seeding" was performed to induce crystallization. After holding at - 7 0 C for approximately 15 minutes, the cooling resumed to -33 0 C at -0.3°C/minute. Once the -33 0 C was achieved the holders were plunged into liquid nitrogen for storage.
  • the reanimation process was initiated by removing the frozen oocyte holder from liquid nitrogen and warming in room temperature air for 30 seconds followed by 37 0 C water for 30 seconds. The ends were cut and then the oocyte holder was installed on the automated instrument.
  • the solutions used in this process were the Base solution and the O-Tl solution of Table 2.
  • the initial fluid introduced was the O-Tl solution at an approximate osmolality of 1362 mOsmol/L and then proceeded by mixing the two solutions to a final osmolality of 300 mOsmol/L for the Base solution.
  • the temperature during this thawing and reanimation process started around 3O 0 C and raised slowly to 37 0 C over the course of approximately 15 minutes.
  • This program used volumetric flow rate of about 70 microliters per minute. This flow rate can range from 35 to 150 microliters per minute, but is preferred to be in the 50 to 100 micoliters per minute.
  • the automated instrument used 10 ml syringes and a syringe pump to drive the individual solutions into a manifold where they were mixed and temperature controlled.
  • the embryo or oocyte holder of either Example 1 or 2 were constructed from a Surlyn® extrusion which is drawn to narrow it in an area and had a polycarbonate membrane filter which had approximately 10 micron pores to allow fluid through, but retain the embryos or oocytes.
  • the solutions include removing all or substantially all of the sodium ions from the solutions by substituting choline chloride for sodium chloride as there are examples of sodium toxicity during the cryopreservation process as the wave front of solidification proceeds through the fluid during the "seeding" process.
  • This sodium toxicity is thought to cause changes in the membrane due to "sodium pump” mechanism in the cell wall.
  • Other chemicals could be exchanged such as potassium hydroxide for sodium hydroxide and also potassium bicarbonate for sodium bicarbonate. This would essentially remove substantially all sodium ions from the solution.
  • Barritt et al. used fetal heartbeats as a measure of pregnancy.
  • the other literature citations used ⁇ -hCG levels.
  • cryopreservation methods of this invention and/or the reanimation methods of this invention, provide for a high success rate for producing a viable pregnancy.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

La présente invention concerne des procédés et des compositions utiles pour le maintien d'ovocytes, d'embryons ou de blastocystes sensiblement sphériques pendant la cryoconservation et la réanimation.
PCT/US2010/036390 2009-06-01 2010-05-27 Populations d'ovocytes sensiblement spheriques a volume reduit Ceased WO2010141317A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US18297609P 2009-06-01 2009-06-01
US61/182,976 2009-06-01

Publications (1)

Publication Number Publication Date
WO2010141317A1 true WO2010141317A1 (fr) 2010-12-09

Family

ID=43298051

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/036390 Ceased WO2010141317A1 (fr) 2009-06-01 2010-05-27 Populations d'ovocytes sensiblement spheriques a volume reduit

Country Status (1)

Country Link
WO (1) WO2010141317A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9642352B2 (en) 2008-07-23 2017-05-09 Mariposa Biotechnology, Inc. Method of preparing embryos or blastocysts for cryopreservation
US12426594B2 (en) 2020-09-24 2025-09-30 Everest Medical Innovation GmbH Cryoprotective compositions and methods for protection of a surgical site during cryosurgery
US12453805B2 (en) 2020-09-24 2025-10-28 Everest Medical Innovation GmbH Cryoprotective compositions, surgical kits, and methods for protection of a surgical site during cryosurgery

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080145930A1 (en) * 2006-11-15 2008-06-19 Mariposa Biotechnology, Inc. Methods and compositions for reanimating cryopreserved oocytes
US20080213887A1 (en) * 2006-11-15 2008-09-04 Mariposa Biotechnology, Inc. Methods and compositions for cryopreserving oocytes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080145930A1 (en) * 2006-11-15 2008-06-19 Mariposa Biotechnology, Inc. Methods and compositions for reanimating cryopreserved oocytes
US20080213887A1 (en) * 2006-11-15 2008-09-04 Mariposa Biotechnology, Inc. Methods and compositions for cryopreserving oocytes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MAGLI ET AL.: "Cryopreservation of biopsied embryos at the blastocyst stage.", HUM. REPROD., vol. 21, no. 10, 2006, pages 2656 - 2660 *
SILLS ET AL.: "Ovarian hyperstimulation syndrome and prophylactic human embryo cryopreservation: analysis of reproductive outcome following thawed embryo transfer.", J. OVARIAN RES., vol. 1, no. 1, 2008, pages 1 - 6, XP021046396, DOI: doi:10.1186/1757-2215-1-7 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9642352B2 (en) 2008-07-23 2017-05-09 Mariposa Biotechnology, Inc. Method of preparing embryos or blastocysts for cryopreservation
US12426594B2 (en) 2020-09-24 2025-09-30 Everest Medical Innovation GmbH Cryoprotective compositions and methods for protection of a surgical site during cryosurgery
US12453805B2 (en) 2020-09-24 2025-10-28 Everest Medical Innovation GmbH Cryoprotective compositions, surgical kits, and methods for protection of a surgical site during cryosurgery

Similar Documents

Publication Publication Date Title
Ruffing et al. Osmometric behavior, hydraulic conductivity, and incidence of intracellular ice formation in bovine oocytes at different developmental stages
US6673607B2 (en) Microinjection of cryoprotectants for preservation of cells
Pedro et al. Permeability of mouse oocytes and embryos at various developmental stages to five cryoprotectants
Hafez Preservation and cryopreservation of gametes and embryos
Suzuki et al. Fertilization and Development of Frozen–Thawed Germinal Vesicle Bovine Oocytes by a One-Step Dilution Methodin Vitro
US20160102286A1 (en) Methods For The Cryopreservation Of Mammalian Cells
Lim et al. The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization
AU2001261644A1 (en) Microinjection of cryoprotectants for preservation of cells
JP2002543041A (ja) 生体試料のガラス化方法
US8633023B2 (en) Method of liquid nitrogen surface vitrification
Isachenko et al. Modified vitrification and cooling of human pronuclear oocytes: efficacy and effect on ultrastructure
JP2000197481A (ja) 細胞の凍結保存方法
WO2010141317A1 (fr) Populations d'ovocytes sensiblement spheriques a volume reduit
EP1131998A1 (fr) Procédé de cryoconservation de cellules
JP2011036196A (ja) 生物材料用ガラス化液、ガラス化キット、及びその利用
US20080113432A1 (en) Methods and compositions for reanimating cryopreserved oocytes
US20080213887A1 (en) Methods and compositions for cryopreserving oocytes
Yang et al. Optimization of ethylene glycol concentrations, freezing rates and holding times in liquid nitrogen vapor for cryopreservation of rhesus macaque (Macaca mulatta) sperm
WO2008061148A9 (fr) Procédés et compositions de cryopréservation d'ovocytes
Rall Cryobiology of gametes and embryos from nonhuman primates
EP1516923A2 (fr) Procédé de mise en culture de cellules
Gosden Techniques for slow cryopreservation of embryos
OKADA et al. Viability of cryopreserved and vitrified embryos and fertility after direct transfer in ewes
RUFFING et al. Osmometric Behavior, Hydraulic Conductivity, and Incidence of Intracellular lce Formation in Bovine Oocytes at Different
Porcu Slow freezing of human oocytes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10783839

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10783839

Country of ref document: EP

Kind code of ref document: A1