WO2018122011A1 - Methode de vitrification en une etape - Google Patents
Methode de vitrification en une etape Download PDFInfo
- Publication number
- WO2018122011A1 WO2018122011A1 PCT/EP2017/083123 EP2017083123W WO2018122011A1 WO 2018122011 A1 WO2018122011 A1 WO 2018122011A1 EP 2017083123 W EP2017083123 W EP 2017083123W WO 2018122011 A1 WO2018122011 A1 WO 2018122011A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- vitrification
- biological material
- cells
- solution
- embryos
- Prior art date
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
- A01N1/02—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
-
- 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
-
- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F25—REFRIGERATION OR COOLING; COMBINED HEATING AND REFRIGERATION SYSTEMS; HEAT PUMP SYSTEMS; MANUFACTURE OR STORAGE OF ICE; LIQUEFACTION SOLIDIFICATION OF GASES
- F25B—REFRIGERATION MACHINES, PLANTS OR SYSTEMS; COMBINED HEATING AND REFRIGERATION SYSTEMS; HEAT PUMP SYSTEMS
- F25B19/00—Machines, plants or systems, using evaporation of a refrigerant but without recovery of the vapour
- F25B19/005—Machines, plants or systems, using evaporation of a refrigerant but without recovery of the vapour the refrigerant being a liquefied gas
Definitions
- the invention relates to a method for cryopreservation of biological material, more particularly a method for vitrification of biological material in one step.
- a cryopreservation method is a method of conserving biological material at a very low temperature, typically 77K or -196 ° C, the boiling temperature of liquid nitrogen.
- the vitrification method is a transformation method without crystallization, from a liquid to an amorphous solid. It allows the biological material and their medium to cool down to a temperature of -196 ° C without the appearance of intra- and extracellular ice crystals.
- the vitrification method involves a sudden dive into the liquid nitrogen of the previously conditioned biological material, which, depending on the thermal inertia related to the material itself and its container, leads to cooling rates ranging from 900 to 20,000 ° C per minute
- any method of cryopreservation involving slow cooling rates and of the order of 0.5 to 4 ° C per minute is a slow freezing technology that involves the crystallization of extracellular water.
- the cryopreservation method in particular the vitrification method, generally applies to biological material of the human, animal or plant cell type, and more particularly to high-value individual cells such as embryonic cells, germ cells, cells strains, induced pluripotent cells, genetically modified cells, tool cells used for applications such as screening, diagnostics, toxicological studies, therapeutics such as vaccines or similar applications, but also to tissues, organs, embryos, gametes and their precursors or any other type of biological material.
- the use of high-value cells is booming in the areas of regenerative therapy, gene therapy, medically assisted reproduction, diagnostics, pharmaceutical research and vaccine production.
- the cryopreservation of these cells is essential for their storage, transport, screening and expansion both in the fields of research and biobanks, and for industrial players or users in the therapeutic or reproductive fields. medically assisted.
- the cryopreservation method must allow to be effective, a high recovery rate, a stability of biological characteristics regardless of the storage time in the cooling medium such as liquid nitrogen (LN2), be chemically and (micro-) biologically safe, easy to implement, automatable and guaranteeing optimal health safety. Indeed, the efforts of storage, transport, screening and expansion prior to their use depend on it.
- the quality and safety constraints applicable to the cryopreservation of therapeutic cells are also set out in European Directives (2004/03 / EC, 2006/17 / EC, 2006/86 / EC).
- the purpose of all methods of cryopreservation and thus methods of vitrification of biological material is to obtain and maintain intracellular conditions compatible with obtaining a vitreous amorphous state during the cooling and heating stages.
- a vitrification solution consists of different types of solutes. It can comprise one or more different cryoprotectants, for example propylene glycol, ethylene glycol, Ficoll, dimethyl sulfoxide (DMSO), glycerol, monosaccharides (sucrose or diose, trehalose, glucose, fructose, sucrose, mannose, sucrose, ... or their derivatives) or their mixture.
- cryoprotectants for example propylene glycol, ethylene glycol, Ficoll, dimethyl sulfoxide (DMSO), glycerol, monosaccharides (sucrose or diose, trehalose, glucose, fructose, sucrose, mannose, sucrose, ... or their derivatives) or their mixture.
- a vitrification solution may also comprise solutes intended to maintain the integrity of the biological material, for example phosphate buffers such as KH 2 PO 4 or K 2 HPO 4 in the presence of KCl, NaCl or other salts, but also the above mentioned sugars such as glucose. , sucrose, dextrose, trehalose or their derivatives.
- solutes intended to maintain the integrity of the biological material, for example phosphate buffers such as KH 2 PO 4 or K 2 HPO 4 in the presence of KCl, NaCl or other salts, but also the above mentioned sugars such as glucose. , sucrose, dextrose, trehalose or their derivatives.
- a vitrification solution may also comprise solutes such as animal or human serum, such as Bovine Fetal Serum (FBS), fetal calf serum (FCS), bovine serum albumin (BSA) used for their protein intake and their cryoprotective effect.
- solutes such as animal or human serum, such as Bovine Fetal Serum (FBS), fetal calf serum (FCS), bovine serum albumin (BSA) used for their protein intake and their cryoprotective effect.
- FBS Bovine Fetal Serum
- FCS fetal calf serum
- BSA bovine serum albumin
- a hypertonic or hyperosmotic vitrification solution is referred to as another solution of the hypotonic or hypoosmotic intracellular medium separated from the vitrifying solution by a biological or semi - permeable membrane; if the solute concentration of the hypertonic vitrifying solution is such that it exerts a pressure lower than that exerted by the solution of the intracellular (hypotonic) medium on this membrane. This results in a middle water call
- An isotonic solution can be described as normotonic to intracellular media under physiological conditions.
- a two-step vitrification process is described in accordance with the usual practice according to the state of the art.
- the biological material namely oocyte or embryo, is first subjected to a non-vitrifying solution containing penetrating cryoprotectants, and then is exposed to a 2nd vitrifying solution comprising high concentrations of penetrating and non-penetrating cryoprotectants.
- the oocytes are immersed for example in a 10% (v / v) ethylene glycol solution and 10% DMSO (v / v) in a phosphate buffer containing 18% Fetal Bovine Serum (FBS), then in a second vitrifying solution comprising 20% (v / v) ethylene glycol, 20% (v / v) DMSO and 0.3M trehalose in a phosphate buffer containing 18% fetal bovine serum (FBS).
- FBS Fetal Bovine Serum
- Cooling must be a hypertonic vitrifying solution (VS), ie a solution containing a mixture of penetrating and / or non-penetrating CPs.
- VS hypertonic vitrifying solution
- the role of SV is to coat the biological material such as cell, embryo or other in a vitrifying sheath inhibiting the appearance of extracellular ice crystals.
- most vitrification methods include multistage exposure of biological material such as cells or embryos, consisting of solutions CPs penetrating increasingly concentrated before final exhibition in the VS solution Thus Vanderzwalmen et al.
- nVSi non-vitrifying solutions
- penetrating CPs of the order of 3 to 4 M
- VS vitrifying solution
- CPs cryoprotective agents
- the mixture of penetrating and non-penetrating CPs in VS is responsible for the final cell dehydration that concentrates the intracellular components including salts, proteins, organelles, polysaccharides and possibly CPs that have already entered the cell during the previous steps.
- cryoprotective agent (CPs) non-penetrating include e.g. Ficoll ®, sucrose, trehalose, lactose, mannitol, maltose, mannose and any molecule belonging to the family of di- and trioses or polysaccharides or polyalcohols or other derived molecules or the like.
- cryoprotective agents there will be for example dimethylsulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), polyethylene glycol, triethylene glycol, glycerol and other derivative molecules or the like.
- DMSO dimethylsulfoxide
- EG ethylene glycol
- PG propylene glycol
- polyethylene glycol triethylene glycol
- glycerol triethylene glycol
- other derivative molecules or the like.
- cryoprotectants CPs
- CPs cryoprotectants
- Their toxicity depends on the concentration used, the temperature as the duration of exposure, the tonicity of the medium, the mode of contact of the cells with the products and finally the type of cell.
- DMSO dimethylsulfoxide
- P OH propylene glycol
- DMSO have a toxic effect by destabilizing membrane proteins and moving bound water associated therewith. If everyone agrees that all CPs are toxic and that it would be beneficial to reduce their intracellular concentration, there is no real consensus about how to cryopreserve cells or embryos without use them.
- the present invention indeed relates to a method of cryopreservation, more particularly to a method of vitrification in a single step with exposure of the biological material for a time limited in time and before the
- a single vitrifying solution comprising penetrating and non-penetrating CPs agents.
- the duration of exposure is preferably less than 90 sec, and preferably between 30 sec and 90 sec.
- the cooling is carried out at a cryopreservation temperature of the biological material.
- the cryopreservation temperature may for example be the temperature of the liquid nitrogen.
- Non-penetrating cryoprotectants (CPs) are present in
- concentration ranging from 10% (v / v) to 60% (v / v), preferably 60% in the vitrifying solution (VS).
- the CPS penetrating cryoprotective agents are present in concentrations ranging from 5 to 50% (v / v) in the vitrification solution (VS), preferably 20% (v / v).
- Animal or human serum such as albumin is also used as a cryoprotective agent in low concentrations ranging from 0.1 to 1% (v / v), preferably 0.6% in the vitrifying solution (VS).
- this method of vitrification in one step provides results equivalent or superior to those obtained with vitrification methods according to the state of the art combining successive exposures to nVSi and VS.
- This method also has the advantage of eliminating the toxic effects associated with prolonged exposure to CPs.
- the biological material according to the invention may be, for example, any type of cells or tissues or organs or a single or multicellular organism.
- the biological material is an embryo, cells
- the preferred biological material is the embryo or normal mammalian cell (e.g. a mesenchymal stem cell) or genetically modified cell (e.g., induced pluripotent cell).
- the biological material according to the invention also comprises, at the level of the embryos, their zygotes, morulas or blastocysts; at the level of cells derived from embryos, embryonic stem cells, trophoblastic cells; at the level of adult stem cells or differentiated cells of different origins, umbilical cord blood cells, cells from different tissues such as blood - Peripheral Blood Monocytes -, muscle - myocytes or myoblasts, satellite cells -, ligaments and tendons -tenocytes, mesenchymal stem cells-, bones - osteoblasts, osteocytes, osteoclasts -, cartilage -chondroblasts and chondrocytes-, heart -cardiomyocytes, cardiomyoblasts-, lungs and airways - pneumocytes, hair cells-, liver - hepatocytes-, pancreas- alpha and beta cells, exocrine pancreatic-cells, spleen -splenocytes, den
- the method according to the invention makes it possible, before cooling for cryopreservation, to expose biological material such as embryos or cells to a single vitrification solution (VS) for a short period, preferably less than 90 seconds and preferably between 30 sec and 90 sec to induce optimal dehydration without passing through intermediate solutions (nVSi) usually used in conventional vitrification techniques.
- VS vitrification solution
- nVSi intermediate solutions
- the solution hypertonic also called hyper osm otic responsible for this rapid dehydration allows the disappearance of intracellular free water and the survival of biological material following vitrification induced by cooling.
- the method according to the invention it is no longer necessary to use cryoprotective agents (CPs) which enter the intracellular space, which has the advantage of eliminating toxic effects, including genotoxic, known or unknown, in the short, medium or long term, related to prolonged intracellular exposure to CPs.
- CPs cryoprotective agents
- the cooling rate appears much less critical than in the conventional method according to the state of the art.
- the method according to the invention further comprises a step of vitrification of the biological material on support by immersion in a cooling medium.
- the cooling medium may for example be liquid nitrogen.
- the cryopreservation method according to the invention preferably comprises the following steps: a) bringing the biological material into contact with the hypertonic or hyper osmotic vitrifying solution (VS) for a limited period of time, preferably less than 90 sec; b) depositing on the support biological material from step a); c) vitrification of the biological material on support from step b) in the cooling medium which is preferably liquid nitrogen.
- VS hypertonic or hyper osmotic vitrifying solution
- the biological material can thus be preserved for example in liquid nitrogen as a cooling medium in aseptic or non-aseptic conditions for an unlimited period.
- a non-aseptic vitrification the biological material is deposited on a support, preferably in the form of a gutter and then immersed directly in the cooling medium, which is preferably liquid nitrogen, after a short-term exposure to the breast. of the vitrifying solution (VS).
- the biological material is deposited on a support after exposure to the vitrifying solution (VS) and is introduced into a container or protective straw sealed at one end.
- the protective straw is sealed at its other end when it is immersed in the cooling medium, which is preferably liquid nitrogen.
- Protective straw must be sterile and resistant to low temperature storage.
- the volume of the straw can vary between 250 ⁇ and 500 ⁇ . It is preferably 250 ⁇ .
- the vitrifying solution (VS) is previously cooled to a temperature between 5 and 1 ° C, preferably 4 ° C before being brought into contact with the biological material.
- the biological material After staying in the cooling medium such as liquid nitrogen, the biological material is then recovered by warming to room temperature.
- the method according to the invention comprises only a single step of brutal heating of the biological material having undergone the treatment. vitrification stage. This abrupt heating at the start of the cooling temperature, such as that of liquid nitrogen, at ambient temperature of the order of 18 to 25 ° C. is carried out at a speed ranging from 10,000 to 30,000 degrees per minute and preferably 20,000 degrees by minute.
- Warming is performed by immersing the biological material in a normotonic solution such as an M2 embryo rinse solution (washing medium according to Quinn, J. eprod.Fert 1982, Sep: 66 (1): 161-8 ). Brief description of the figure
- Figure 1 Is a photo of a litter of 9 chimeric mice derived from the injection of one-step vitreous mRNAs RI according to the invention into C57BL / 6 blastocysts.
- mice Female inbred mice, 5 weeks old, were superovulated by the injection of 5 international units (ui) of gonadotropin present in pregnant mare serum (Pregnant Mare Serum Gonadotrophin). , PMSG) intraperitoneally (ip) followed 46 h later by an ip injection of 5 ui human chorionic gonadotropin (human Chorionic Gonadotrophin, hCG). Injection of hCG was followed immediately by mating treated females with males of identical strain. The day after mating, mice with a vaginal plug were euthanized by cervical dislocation and their zygotes collected.
- ui human chorionic gonadotropin
- each manipulation included a non-cryopreserved control group and a vitrified group according to the so-called “classical” protocol, also called the protocol according to the state of the art described by Vanderzwalmen et al in Human Reproduction (vol 28, 2101-2110, p 1-10 , 2013), in addition to the test groups.
- vitrification according to the conventional protocol consists in exposing the embryos for 2 times 3 minutes and at room temperature to the non-vitrifying solutions 1 and 2 (nVS1 and nVS2), before washing them in vitrification solution (VS). - Cooled to 4 ° C and put on their support. This last step does not exceed 1 minute.
- the test groups were directly exposed to pre-cooled VS before being vitrified and / or reheated according to different protocols as described below (in Examples 1-4). The evaluation of the survival of embryos is performed one hour after warming, while the growth rate is measured at day 5 of culture in vitro counts obtained blastocysts.
- mESCs Murine embryonic stem cell culture
- Murine embryonic stem cells (mESCs) of the RI line were cultured in gelatinized culture dishes without cell feeders, in medium and in conditions that preserve their pluripotency and their potential for multiplication.
- the medium used is as described in Table 1 below.
- Table 1 Composition of culture medium of mESCs.
- the medium is changed daily.
- development requires it (70% confluence) the cells are distributed at a density consistent with the needs of the experiment in new boxes of crops.
- the cultures are rinsed with PBS without Calcium or Magnesium, and then harvested with trypsin-EDTA to the incubator for 3 to 5 minutes to obtain a homogeneous cell suspension.
- the activity of trypsin is stopped using 6-8 volumes of a wash solution as described in Table 2 below.
- Table 2 composition of the mESCs washing medium
- the washed cell suspension is centrifuged and the pellet is resuspended in culture medium.
- the cells are distributed in new culture dishes at a density according to the needs of the experiment. 4. Cryoprotective solutions used in the examples according to the invention
- nVSi cryoprotectant solutions used in the examples were prepared from a D-PBS buffer solution (Sigma D-4031) supplemented with 10% fetal calf serum (FCS) for cell culture (from commercial sources (by Bovine Fetal Serum Gibco ® Serum)
- FCS fetal calf serum
- FCS fetal calf serum
- the nVS1 and nVS2 solutions used for the control group are conventionally prepared according to the state of the art and described by Vanderzwalmen et al in Human Reproduction (Vol 28, 2101-2110, p. 1-10, 2013)
- the nVS1 solution contains 5% (v / v) dimethylsulfoxide (DMSO) and 5% (v / v) ethylene glycol (EG), while the nVS2 solution contains 10% (v / v). v) DMSO and 10% (v / v) EG.
- the hyper osmotic solution VS used after the nVSi solutions according to the conventional method of the state of the art, or alone in the method according to the invention is consisting of 20% (v / v) DMSO, 20% (v / v) EG, 0.5M sucrose (Sigma S-1888) and 25 ⁇ Ficoll (Sigma F-8636).
- the sucrose solutions (S-1888) used are prepared from D-PBS buffer (Sigma D-4031) supplemented with 10% fetal calf serum. 5 ° Cell counts after warming.
- the mESCs are counted using a Neubauer cell immediately, 24 and 48 hours after their warming. Mortality was estimated using a trypan blue exclusion test.
- the embryos in groups of 4 to 6 are taken from their culture medium and are moved in a drop of 0.5 ml VS previously cooled to 4 ° C. After exposures of varying durations in VS (30; 90; 120; 150 and 180 seconds for Experiment 1; 30 versus 150 seconds for Experiments 2; 3 and 4b; 50 seconds for Experiments 3 and 4b); embryos are placed on the gutter of the vitrification support: VitriPlug (Vitrimed, Austria) in the case of non-aseptic vitrifications (examples 1, 2, 3, 4a and control groups), and VitriSafe (Vitrimed, Austria) for aseptic vitrifications ( examples 3 and 4b).
- VitriPlug Vitrimed, Austria
- VitriSafe Vitrimed, Austria
- aseptic vitrification vitrification without direct contact of the medium and embryos with liquid nitrogen.
- this VitriSafe support is introduced into a 0.3 ml protection straw (CryoBioSystem) previously identified, weighted and sealed at one end (there are 2 compartments in 0.3 ml straws). separated by a cotton piston
- the large compartment of 0.3 ml is intended to receive the VitriSafe ® while the other is intended to receive a ballast (stainless steel rod coated with a plastic film of color) surrounded by a label
- Vitrification is performed aseptically for vitrification according to the state of the art and for vitrification in one step according to the invention.
- Cells in culture are harvested as described above, a cell suspension aliquot is then used for cell counting, and optionally divided into fractions according to their count.
- the cell suspension is then centrifuged, and the pellet is resuspended in 250 ⁇ l of VS solution previously cooled to 4 ° C. for 50 seconds. During this incubation, the cell suspension is aspirated into a 250 ⁇ straw which is sealed at both ends before being immersed in liquid nitrogen in the same manner as explained above for embryos.
- Example 1 One-step vitrification according to the invention of stage I, II and morulas embryos: effect of the duration of exposure to the VS
- Embryos are harvested at the stage of a cell (zygote, stage 1), according to the method of production described in "Manipulating the mouse embryo, a laboratory manual” 4th Edition, 2013 Behringer et al, CSH Press. Part of these embryos will be assigned to the non-cryopreserved control group and serve as a reference for the experiment. All the experiments for which this reference group gave us a percentage of blastocysts less than 90% were invalidated.
- one group is treated according to the conventional method of the state of the art described in Vanderzwalmen et al (Human Reproduction, 2013) and five other groups of embryos are exposed directly to the vitrification solution ( VS) described in point 3 for 30 sec, 90 sec, 120 sec, 150 sec and 180 sec respectively before cooling.
- the embryos are placed in groups of five (+ or - 1) on a non-aseptic support (from Vitrimed ® ) and immersed directly in liquid nitrogen to be stored there for a prolonged period, usually from one day to one day. week.
- the experimental survival rate is calculated after one hour of culture. On the other hand, the rate of development is evaluated on day 5 of the
- Table 1 presents the observed survival rate after one hour of warming and blastocyst percentages observed in 5 th day of development after vitrification step zygote, stages II and morulae.
- Example 1 a total of 681 zygotes were harvested from 46 superovulated females.
- Table 1 Survival rate 1 hour after warming (T0) and percentages of blastocysts observed after vitrification in one step depending on the stage of
- One-step short and brutal dehydration according to the invention induces intracellular vitrification and is no more deleterious than stepped dehydration which is each time followed by an entry of CPs followed by water.
- survival of mouse embryos is possible after exposures to SV as short as 30 seconds. It also means that embryos survive vitrification despite a
- Example 2 One-step vitrification of stages I, II and morulas: effects of one-step dilution of VS in medium without sucrose (medium M2 alone) during heating, inducing instantaneous cytoplasmic rehydration
- CPs enter the cell during the various exposures to nVSi solutions that precede exposure to VS and cooling.
- nVSi solutions that precede exposure to VS and cooling.
- sucrose an osmotically active agent used to counteract an abrupt and excessive entry of water into the dehydrated cytoplasm containing the penetrating CPs entered into the cell during the nVSi exposure steps.
- sucrose an osmotically active agent used to counteract an abrupt and excessive entry of water into the dehydrated cytoplasm containing the penetrating CPs entered into the cell during the nVSi exposure steps.
- mice embryos are vitrified in the zygote, 2-cell and morula stages.
- a non-cryopreserved control group serves as a reference for the experiment and control groups are vitrified and heated by the prior art method described in Vanderzwalmen et al (Vol 28, 2101-2110, p 1-10, 2013).
- Two other groups of embryos are vitrified according to the invention by direct exposure to the vitrification solution (VS) for 30 sec or 150 sec.
- the embryos are placed in groups of five (+ or -1) on a non-aseptic support (VitriPlug, from Vitrimed ® ) and immersed directly in liquid nitrogen to be stored there for an extended period of time, usually one day. to a week.
- a non-aseptic support VitriPlug, from Vitrimed ®
- vitrified control group according to the state of the art
- a total of 526 zygotes were harvested from 25 mice and used in Example 2.
- Table 2 Survival rate after 1 hour (T0) and blastocysts at D5 after vitrification in one step and immediate rewarming / rehydration in medium M2 without sucrose (warming in one step).
- this experiment shows that the vitrification process in one step followed by the one-step heating according to the invention is at least as effective as vitrification according to the state of the art with regard to concerns the viability and development of cells, while ensuring a reduction or even a suppression of their content in potentially deleterious cryoprotectants (CPs).
- CPs cryoprotectants
- Example 3 Vitrification and warming in one step: effect of the nature of the support: aseptic or non-aseptic.
- the non-aseptic carrier used heretofore in Examples 1 and 2 allows direct contact of the biological sample with the liquid nitrogen. This results in cooling rates of the order of +/- 20,000 ° C per minute.
- the aseptic support does not allow direct contact of the biological sample with the liquid nitrogen since the support bearing the embryos is placed in a protective straw. It results from the presence of this straw that the cooling rate is slowed down and is only +/- 1000 ° C per minute.
- mice zygotes are harvested and directly vitrified at this stage.
- a non-cryopreserved control group serves as reference and control groups are vitrified by the conventional method of the state of the art described by Vanderzwalmen et al. (Human Reproduction, vol 28, 2101-2110, p 1-10, 2013). Other groups of embryos are exposed directly to VS for 50 sec or 150 sec.
- the embryos are placed in groups of five (+ or - 1) on either a non-aseptic support (VitriPlug ® , from Vitrimed ® ) or on an aseptic support (Vitrisafe ® , from Vitrimed ® ) and immersed directly in liquid nitrogen for a period of time usually ranging from one day to one week.
- a non-aseptic support VitriPlug ® , from Vitrimed ®
- an aseptic support Vitrimed ®
- the vitrified control group according to the conventional method according to the state of the art (Vanderzwalmen et al, Human Reproduction, vol 28, 2101-2110, p 1-10, 2013), is brought suddenly to a speed of 20,000 ° C per minute at the
- sucrose Sigma S-1888
- PBS PBS
- Table 3 Survival rate after 1 hour (T0) and blastocysts at day 5 after one-step vitrification of zygotes on aseptic and non-aseptic media and instantaneous rewarming / rehydration in M2 medium without sucrose
- the shortest duration of exposure to the SV causes the dehydration of the cell without allowing the entry of CPs or water (Vanderzwalmen et al, 2013), which translated into previous examples (1 and 2) by superior efficiency (survival and development) compared to the longer exposures that allow the entry of CPs and water (Vanderzwalmen et al., 2013).
- the dehydration state reached by the cells is such that the cooling rate is less important for achieving and maintaining the vitreous state than during vitrification according to the state of the art.
- longer exposure to VS 150 seconds
- allowing the entry of CPs and water into the cell after the initial dehydration process Vanderzwalmen et al, Human
- Example 4 Use of zygote transfer in pseudopregnant recipient mice to confirm the competence of embryos to become suckling mice after one-step vitrification according to the invention
- Example 4a Non-aseptic vitrification: effects of the duration of exposure to SV on the% of births after transfers The birth of young after transfers of vitrified embryos in one step is the ultimate proof of the lack of toxicity of the method .
- Table 4 shows the percentages of births obtained.
- Groups of 154 and 103 zygotes were vitrified according to the one-step non-aseptic vitrification protocol and exposures at 30 and 150 seconds, respectively; after heating in 0.25M sucrose, 142 and 53 of them were transferred on the same day into pseudopregnant recipient mice.
- Table 4 Percentages of young people after transfers of vitrified zygotes according to the non-aseptic vitrification protocol in one step and exposures to respectively 30 and 150 seconds; the heating was carried out in 0.25M sucrose.
- Example 4b Percentages of births after zygote transfers that have undergone aseptic vitrification in one step (50 sec exposure to VS) and instant rehydration in M2.
- Table 5 shows the percentages of births obtained.
- a group of 52 zygotes was vitrified according to the one-step aseptic vitrification protocol with exposure to 50-second VS; warming was done directly in M2 (washing medium according to Quinn, J.Reprod.Fert 1982, Sep: 66 (1): 161-8). All embryos were transferred the same day into 2 pseudopregnant recipients.
- Table 5 Percentages of juveniles after vitreous zygote transfers according to the one-step aseptic vitrification protocol and 50-second OS exposures; the heating was done directly in M2, inducing instant rehydration.
- the percentages of births are here also comparable to those obtained routinely (20.9%, F.Ectors, data not shown) during reimplantations of vitrified embryos according to the state of the art.
- Examples 4a and 4b confirm that one-step vitrification according to the invention followed or not by direct heating in M2 medium (washing medium according to Quinn, J.Reprod.Fert, 1982). , Sept. 66 (1): 161-8), whether or not aseptically, does not affect the ability of zygotes to ensure normal gestation after transfer to a recipient.
- Example 5 Vitrification and heating in one step according to
- mESCs murine embryonic stem cells
- mESCs (Nagy RI, line 129SV agouti color) vitrified in one step according to the invention were microinjected in the blastocoele of C57BL / 6j mouse blastocysts and gave an extremely high percentage of chimerism in the 9 pups born. (near or equal to 100%!), the coat of chimeras showing a
- the shortest exposure times at the VS (30 seconds) dehydrate the cell without allowing the entry of CPs or water, which resulted in this example by a higher efficiency of vitrification (survival and development) compared to the longest exposures that allow the entry of CPs followed by water
- Example 2 (vitrification in one step and direct rewarming in M2 resulting in instant rehydration, Table 2), we reinforced the hypothesis of the (near) absence of intracellular CPs with regard to the good survival rates observed. despite direct warming in a normotonic solution (medium M2- wash medium according to Quinn, J.Reprod.Fert, 1982, sep: 66 (1): 161-8 - without sucrose).
- vitrification in one step according to the invention drastically limits or even cancels the entry of CPs into the cells is thus verified.
- this experiment 2 shows that the process of vitrification in one step / heating in one step according to the invention is at least as effective as vitrification according to the state of the art with respect to the viability and development of the cells, while ensuring a reduction or cancellation of their CP content potentially deleterious in the short, medium or long term.
- Example 3 in which aseptic and non-aseptic carriers are used, makes it possible to evaluate the effect of the cooling rate during vitrification according to the invention. The results obtained are unexpected in that they do not correspond to what is observed during vitrification according to the state of the art, where the cooling rate is critical to avoid crystallization.
- Cooling is less important for achieving and maintaining the vitreous state than during vitrification according to the state of the art.
- the relationship between the ICCP and the cooling rate to reach the vitreous state recovers, as suggested by the efficiency which tends to decrease when the aseptic support is used.
- Our conclusions regarding the efficacy and safety of vitrification according to the invention with short exposure to VS before cooling and direct dilution in M2 washing medium are further reinforced by the birth rates obtained after transfers (Tables 4 and 5). ).
- mice obtained routinely after transfers of non-cryopreserved and vitrified zygotes according to the state of the art are respectively 20.2% and 20.9% (F. Ectors, results not shown).
- Examples 4a and 4b show comparable efficiencies of the vitrification according to the invention which does not affect the competence of zygotes to ensure a normal pregnancy after transfers in a recipient.
- vitreous states There would therefore be two types of vitreous states involved in our experiments: (i) an intracellular vitreous state linked to the absence of vicinal water associated with the transformation of the cytoplasmic gel into a vitreous solid during cooling, and (ii) a vitreous state of the extracellular medium less concentrated in macromolecules and polysaccharides and where the amorphous solidification of water requires the presence of CPs at high concentrations. This demonstrates that as long as extracellular vitrification conditions are met, and cell dehydration is sufficient, the presence of intracellular CPs is not essential for cell survival during the entire vitrification process.
- the examples and validations of the vitrification according to the invention were carried out on embryos at the zygote stage and at the morula stage. Recall that the cells present in the morula stage (+/- 16 cells) have a ratio surface / volume and general biological properties (physiology) quite comparable to most other mammalian cells, which allows the extrapolation of the results. to these other types of cells. Moreover, experiments on these cells (mESCs RI) confirm that the vitrification in one step according to the invention is effective on other cells without altering the biology.
- vitrification according to the invention is based on an unprecedented approach involving cellular dehydration eliminating free water without penetration of CPs.
- it has the advantage of eliminating toxic effects, including genotoxic, known or unknown, in the short, medium or long term , related to prolonged intracellular exposure to CPs.
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020197021195A KR20190097222A (ko) | 2016-12-27 | 2017-12-15 | 신규의 단일 단계 유리화 방법 |
CN201780080896.0A CN110113940A (zh) | 2016-12-27 | 2017-12-15 | 单步玻璃化方法 |
US16/470,268 US20190307118A1 (en) | 2016-12-27 | 2017-12-15 | Single-step vitrification methods |
JP2019555062A JP2020503065A (ja) | 2016-12-27 | 2017-12-15 | 新規な一段階ガラス化方法 |
EP17832768.0A EP3562302A1 (fr) | 2016-12-27 | 2017-12-15 | Methode de vitrification en une etape |
CA3045956A CA3045956A1 (fr) | 2016-12-27 | 2017-12-15 | Novel single-step vitrification method |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BE2016/5981A BE1024850B1 (fr) | 2016-12-27 | 2016-12-27 | Nouvelle methode de vitrification en une etape |
BEBE20165981 | 2016-12-27 | ||
EP16206907.4 | 2016-12-27 | ||
EP16206907.4A EP3342288A1 (fr) | 2016-12-27 | 2016-12-27 | Nouvelle methode de vitrification en une etape |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018122011A1 true WO2018122011A1 (fr) | 2018-07-05 |
Family
ID=61017890
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2017/083123 WO2018122011A1 (fr) | 2016-12-27 | 2017-12-15 | Methode de vitrification en une etape |
Country Status (7)
Country | Link |
---|---|
US (1) | US20190307118A1 (fr) |
EP (1) | EP3562302A1 (fr) |
JP (1) | JP2020503065A (fr) |
KR (1) | KR20190097222A (fr) |
CN (1) | CN110113940A (fr) |
CA (1) | CA3045956A1 (fr) |
WO (1) | WO2018122011A1 (fr) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1326492B1 (fr) * | 2000-10-19 | 2004-05-12 | Organ Recovery Systems, Inc. | Procede de conservation par le froid de tissus ou d'organes autres que des vaisseaux sanguins, par vitrification |
WO2013096659A1 (fr) * | 2011-12-20 | 2013-06-27 | Cook General Biotechnology Llc | Procédés et compositions pour le stockage de cellules animales |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1133369C (zh) * | 2000-05-08 | 2004-01-07 | 中国农业大学 | 一步法胚胎玻璃化冷冻保存 |
-
2017
- 2017-12-15 US US16/470,268 patent/US20190307118A1/en not_active Abandoned
- 2017-12-15 CA CA3045956A patent/CA3045956A1/fr not_active Abandoned
- 2017-12-15 EP EP17832768.0A patent/EP3562302A1/fr not_active Withdrawn
- 2017-12-15 WO PCT/EP2017/083123 patent/WO2018122011A1/fr unknown
- 2017-12-15 JP JP2019555062A patent/JP2020503065A/ja active Pending
- 2017-12-15 KR KR1020197021195A patent/KR20190097222A/ko not_active Application Discontinuation
- 2017-12-15 CN CN201780080896.0A patent/CN110113940A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1326492B1 (fr) * | 2000-10-19 | 2004-05-12 | Organ Recovery Systems, Inc. | Procede de conservation par le froid de tissus ou d'organes autres que des vaisseaux sanguins, par vitrification |
WO2013096659A1 (fr) * | 2011-12-20 | 2013-06-27 | Cook General Biotechnology Llc | Procédés et compositions pour le stockage de cellules animales |
Non-Patent Citations (11)
Title |
---|
BEHRINGER ET AL.: "Manipulating the mouse embryo, a laboratory manual", 2013, CSH PRESS |
BEHRINGER ET AL.: "Manipulating the mouse embryo, a laboratory manual", 2013, CSHL PRESS |
E.M.M. ABDEL-GAWA ET AL: "Effect of Cryoprotective Solutions, Ethylene Glycol, Dimethyle-sulfoxide and Ficoll 70 with Different Combination Ratios on Vitrification of Bovine Oocytes and Embryos Produced in vitro", ASIAN JOURNAL OF ANIMAL AND VETERINARY ADVANCES, vol. 11, no. 10, 1 October 2016 (2016-10-01), pages 608 - 619, XP055349195, ISSN: 1683-9919, DOI: 10.3923/ajava.2016.608.619 * |
NAGY ET AL., PNAS, vol. 90, 1993, pages 8424 - 8428 |
QUINN, J.REPROD.FERT., vol. 66, no. 1, September 1982 (1982-09-01), pages 161 - 168 |
VANDERZWALMEN ET AL., GYNECOL. OBSTET. FERTIL., vol. 38, 2010, pages 541 - 546 |
VANDERZWALMEN ET AL., HUMAN REPRODUCTION, 2013 |
VANDERZWALMEN ET AL., HUMAN REPRODUCTION, vol. 28, 2013, pages 1 - 10 |
WHITTINGHAM, J.REPROD.FERTIL, vol. 14, 1971, pages 7 - 21 |
WHITTINGHAM, J.REPROD.FERTIL., vol. 14, June 1971 (1971-06-01), pages 7 - 21 |
YAVIN ET AL., HUM REPROD, vol. 24, no. 4, April 2009 (2009-04-01), pages 797 - 804 |
Also Published As
Publication number | Publication date |
---|---|
JP2020503065A (ja) | 2020-01-30 |
EP3562302A1 (fr) | 2019-11-06 |
CN110113940A (zh) | 2019-08-09 |
KR20190097222A (ko) | 2019-08-20 |
CA3045956A1 (fr) | 2018-07-05 |
US20190307118A1 (en) | 2019-10-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7270946B2 (en) | Method for treatment of cellular materials with sugars prior to preservation | |
FR2625073A1 (fr) | Liquide de perfusion pour la conservation d'organes | |
Lardet et al. | Effect of exogenous calcium on post-thaw growth recovery and subsequent plant regeneration of cryopreserved embryogenic calli of Hevea brasiliensis (Müll. Arg.) | |
Towill | Germplasm preservation | |
Robles et al. | Sperm cryopreservation of sex-reversed rainbow trout (Oncorhynchus mykiss): parameters that affect its ability for freezing | |
Bao et al. | Development of vitrified bovine secondary and primordial follicles in xenografts | |
Lakey et al. | NOVEL APPROACHES TO CRYOPRESERVATION OF HUMAN PANCREATIC ISLETS1 | |
Donfack et al. | In vivo and in vitro strategies to support caprine preantral follicle development after ovarian tissue vitrification | |
Cleary et al. | Effect of ovariectomy and graft position on cryopreserved common wombat (Vombatus ursinus) ovarian tissue following xenografting to nude mice | |
EP3342288A1 (fr) | Nouvelle methode de vitrification en une etape | |
EP3562302A1 (fr) | Methode de vitrification en une etape | |
BE1024850B1 (fr) | Nouvelle methode de vitrification en une etape | |
Nagano et al. | Effects of isolation method and pre-treatment with ethylene glycol or raffinose before vitrification on in vitro viability of mouse preantral follicles | |
JP4683408B2 (ja) | 新規***前培養培地 | |
Brandhorst et al. | Pancreas storage in oxygenated perfluorodecalin does not restore post‐transplant function of isolated pig islets pre‐damaged by warm ischemia | |
CN102197130A (zh) | 高通量和非侵袭性玻璃化猪胚胎的方法 | |
Hardikar et al. | Islet cryopreservation: improved recovery following taurine pretreatment | |
EP1626623B1 (fr) | Procede de clonage du rat par transfert nucleaire | |
Griveau et al. | La vitrification: principes et résultats | |
Păcală et al. | Vitrification of mice embryos in different developmental stages using four vitrification methods | |
Shaw et al. | Experimental models for ovarian tissue and immature follicles | |
Hesters et al. | Santé des enfants nés après transfert d'embryons congelés. | |
Wyns | Future of fertility preservation | |
Qiu et al. | Equilibrium vitrification of oocytes using low concentrations of cryoprotectants | |
Malter | Life Interrupted: The Nature and Consequences of Cryostasis |
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: 17832768 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 3045956 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2019555062 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 20197021195 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2017832768 Country of ref document: EP Effective date: 20190729 |