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WO2013096659A1 - Procédés et compositions pour le stockage de cellules animales - Google Patents

Procédés et compositions pour le stockage de cellules animales Download PDF

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Publication number
WO2013096659A1
WO2013096659A1 PCT/US2012/071025 US2012071025W WO2013096659A1 WO 2013096659 A1 WO2013096659 A1 WO 2013096659A1 US 2012071025 W US2012071025 W US 2012071025W WO 2013096659 A1 WO2013096659 A1 WO 2013096659A1
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WO
WIPO (PCT)
Prior art keywords
cells
cryopreservation medium
medium composition
composition
cryopreservation
Prior art date
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Ceased
Application number
PCT/US2012/071025
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English (en)
Inventor
Xu Han
Erik John Woods
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Cook General Biotechnolgy LLC
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Cook General Biotechnolgy LLC
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Publication date
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Publication of WO2013096659A1 publication Critical patent/WO2013096659A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • A01N1/128—Chemically defined matrices for immobilising, holding or storing living parts, e.g. alginate gels; Chemically altering living parts, e.g. by cross-linking
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells

Definitions

  • the present disclosure relates generally to the storage of animal cells, and in certain embodiments to polymer-modified cryopreservation media with beneficial vitrification properties, and associated cellular compositions and methods.
  • Cryopreservation of embryos from animal models enables genetic resource centers to operate in a more cost-effective manner by maintaining and shipping animal lines as cryopreserved germplasm rather than live animals.
  • the associated cryopreservation procedures inevitably cause cell damage. Survival of a cell during cryopreservation depends on whether this damage can be minimized.
  • Vitrified samples are generally stored in liquid nitrogen at -196°C or in liquid nitrogen vapor at -120°C. Devitrification is a crystallization process which is detrimental to cells due to the associated mechanical damage and should be avoided during storage or shipping.
  • cryoprotectant agents such as dimethyl sulfoxide
  • cryoprotectant agent T ⁇ values are typically between -120°C and -100°C which are higher than liquid nitrogen and its vapor storage
  • aqueous cryoprotectant solution can be developed to be thermodynamically stable at higher temperatures, for example temperatures as high as -80°C, it is then practical to store samples using conventional freezers such as -80°C freezers and ship them safely on dry ice in inexpensive shipping containers, such as polyethylene boxes. The need therefore exists for improved compositions cryopreservation compositions and methods.
  • the present disclosure provides unique compositions and methods for cryopreservation of biological samples.
  • such compositions and methods are effective at maintaining sample vitrification at temperatures above (warmer than) -100 °C. Accordingly, in one embodiment, the present disclosure provides a
  • cryopreservation medium that includes at least a cryoprotectant and a polymer, wherein the cryopreservation medium has a devitrification temperature above - 100°C and in particular embodiments between about -100 °C and about -60 °C.
  • the cryoprotectant comprises dimethyl sulfoxide, glycerol, and/or lactose-egg yolk extender.
  • the polymer comprises Ficoll and/or polyvinylpyrrolidone.
  • the cryopreservation medium has a devitrification temperature greater than -80 °C.
  • the cryopreservation medium has a Ficoll concentration between about 1% to about 15%.
  • the cryopreservation medium is configured for use with mammalian cells, including for example, murine, porcine, and/or human cells. In some forms the
  • cryopreservation medium of the present disclosure is configured for use with embryonic stem cells, embryos, pluripotent stem cells, and/or spermatozoa.
  • the disclosure provides a cellular composition comprised of cells suspended in a cryopreservation medium as described above.
  • the suspended cells are mammalian cells including for example, murine, porcine, and/or human cells.
  • the suspended cells are embryonic stem cells, embryos, pluripotent stem cells, and/or spermatozoa.
  • the disclosure provides a method of cryogenically storing cells, the method comprising vitrifying cells in the presence of the cryopreservation medium described above.
  • the vitrified cells are mammalian cells including for example, murine, porcine, and/or human cells.
  • the vitrified cells may be embryonic stem cells, embryos, pluripotent stem cells, and/or spermatozoa. Additional methods involve devitrifying such cryogenically stored cells, and optionally also administering the cells to a human or other animal for example to treat the animal for a disease state or condition.
  • FIG. 1 illustrates the exothermic curves of water-Ficoll 70-DMSO solutions with differing R values.
  • cryopreservation media with preferred devitrification characteristics.
  • a cryopreservation medium may comprise a cryoprotectant and a polymer.
  • cryopreservation media of the present disclosure will have a devitrification temperature between about -100°C and about -60°C.
  • the cryopreservation medium includes a cryoprotectant.
  • the cryoprotectant of the present disclosure may include for example, dimethyl sulfoxide (DMSO), glycerol and/or lactose-egg yolk extender.
  • the cryopreservation medium of the present disclosure includes a polymer.
  • addition of a polymer may increase the vitrification temperature of the disclosed cryopreservation medium.
  • the polymer may thicken or increase the viscosity of the
  • the polymer of the present disclosure may comprise a sucrose polymer.
  • the polymer of the present disclosure may comprise for example, Ficoll, and/or polyvinylpyrrolidone.
  • Certain inventive variants may include for example, percoll, hyaluronic acid, albumin and/or glycerol.
  • the polymer component may comprise between about 1% and about 20% by weight of the cryopreservation medium.
  • the polymer will comprise between about 5% to about 15% by weight of the cryopreservation medium.
  • the polymer composition may comprise 10% by weight of the cryopreservation medium.
  • the polymer may comprise about 5% by weight of the cryopreservation medium. In some forms, the polymer concentration is less than 20% by weight of the cryopreservation medium. It is noted that the concentration should be sufficient to stabilize cryopreservation solutions between about -100°C to about -60°C, preferably greater than about -80°C by preventing devitrification, but polymer concentration should be low enough to attenuate the cell osmotic damage associated with the addition of excessive polymer.
  • the polymer of the present invention comprises Ficoll.
  • Ficoll is an uncharged, highly branched polymer formed by the co- polymerization of sucrose and epichlorohydrin.
  • the polymer comprises Ficoll or another sucrose polymer having a weight average molecular weight of less than about 500,000 Daltons.
  • the polymer of the present invention comprises Ficoll 70, in which the Ficoll molecules of the composition have an average molecular weight of about 70,000 Daltons.
  • the polymer of the present invention comprises Ficoll 400, in which the Ficoll molecules of the composition have an average a molecular weight of about 400,000 Daltons.
  • the cryopreservation medium of the present disclosure has a devitrification temperature between about -100°C and about - 60°C. In certain embodiments, the cryopreservation medium of the present disclosure has a devitrification temperature between about -90°C to about -70°C. In preferred embodiments, the cryopreservation medium of the present disclosure has a devitrification temperature greater than about -80°C.
  • cryopreservation medium of the present disclosure is configured for use with mammalian cells including, but not limited to, murine cells, porcine cells and/or human cells.
  • mammalian cells including, but not limited to, murine cells, porcine cells and/or human cells.
  • the cryopreservation medium of the present disclosure is configured for use with mammalian cells including, but not limited to, murine cells, porcine cells and/or human cells.
  • the cryopreservation medium of the present disclosure is configured for use with mammalian cells including, but not limited to, murine cells, porcine cells and/or human cells.
  • cryopreservation medium of the present disclosure is configured for use with cells, including but not limited to, embryonic stem cells, pluipotent stem cells, spermatozoa and/or embryos.
  • cells are isolated from patient or donor tissue, for example for autologous, allogenic or xenogenic use in a subject.
  • a variety of cell types may be preserved according to the present disclosure.
  • Methods of isolating, for example, embryonic stem cells, pluripotent stem cells, spermatozoa and/or embryos are known in the art.
  • the isolated cells may comprise one or more individual cells.
  • the isolated cells are suspended in the cryopreservation medium of the present disclosure. Suspension may occur, for example, in a cryovial, straw, or other suitable container.
  • the composition comprising the cells suspended in the cryopreservation medium may be initially cooled in dry ice.
  • the composition is stored for a period of time at a temperature between about -100°C and about -60°C.
  • the cells may be stored for at least 1 week. In other embodiments the cells may be stored for at least one month, for example for 1-12 months. In yet another embodiment, the cells may be stored for more than 12 months.
  • the cells may be thawed after the cryopreservation storage. In some forms, the cells are thawed by immersing the frozen container in a water bath of about 37°C. In certain embodiments the thawed cells are further cultured before administration or other use, and in other embodiments they are used without further culture and/or without expansion in cell number.
  • the thawed cells are administered to an animal or human patient, e.g. for treatment of a disease state or condition, or in the case of embryos implantation in a uterus of a subject, e.g. for reproductive purposes or in case of sperm for fertilization of an egg, e.g. for reproductive purposes.
  • the thawed cells may be utilized for research purposes.
  • Procine induced pluripotent stem cells were prepared with the procedure described in a previous study [Telugu, B., et al, Porcine Induced Pluripotent Stem Cells Analogous to naive and Primed Embryonic Stem Cells of the Mouse, Int. J. Dev. Biol, 54: 1703-1711 (2010)].
  • the solutions used for cryopreservation were made with the same compositions as shown in Table 1 of Example 1 except that cultured media for porcine induced pluripotent stem cells was different from that used for murine embryonic stem cells.
  • the cryopreservation procedures and warming procedures were the same as those used for murine embryonic stem cells as described in Example 1.
  • the storage duration was three weeks.
  • the post-thaw cell membrane integrity was also tested by Trypan blue staining and the results for cell survival rates are listed in Table 3. Table 3
  • Porcine spermatozoa were cryopreserved with solutions whose
  • compositions are listed in Table 4.
  • Tr f cryopreservation media
  • T ⁇ of the water of a water Ficoll 70 DMSO solution with different solute mass ratios (R) were measured using DSC, where R is defined as the ratio of the mass of polymers over that of DMSO.
  • R is defined as the ratio of the mass of polymers over that of DMSO.
  • the total weight percent (W t ) of both solutes (Ficoll 70 and DMSO) in the solutions was fixed as 50%, simulating the composition of the unfrozen fraction of initial cryopreservation solutions (W t ⁇ 20% before freezing), after losing approximately 70% of their freezable water from an equilibrium freezing procedure.
  • the R values were chosen as 1.0, 0.5, and 0. A volume of 8 ⁇ 1 of each solution was sealed in a ⁇ aluminum crucible.
  • Ficoll 70 increases the value of T ⁇ of DMSO solutions at about -67°C when R equals 1 and can be used as the polymer to stabilize DMSO solutions stored in a freezer at about -80°C or on dry ice.
  • Ficoll 70 was chosen as the polymer for mouse embryo cryopreservation at about -80°C.
  • Female ICR mice (6-12 weeks old) were induced to super-ovulate and morula- stage embryos were obtained using standard procedures.
  • To determine the optimal Ficoll 70 concentrations six FHM solutions labeled as A-F containing 1.5M DMSO and different concentrations of Ficoll 70 were prepared and their compositions are listed in Table 7.
  • the Ficoll 70 concentration was calculated as the mass ratio of Ficoll 70 versus the FHM base solution without DMSO. Approximately 30 embryos were suspended in each solution and given approximately 10 minutes to fully equilibrate with the DMSO. Cell suspensions were drawn into the cryo-straws with a diameter of 2mm and thermally sealed. The straws were first pre-cooled to -4°C and nucleated using a cold metal clamp. They then were cooled to -40°C at approximately 0.5°C/min in a programmable cooling device. After the termination of controlled rate cooling, the straws containing solution A (the control group) were directly plunged into liquid nitrogen for storage.
  • a Ficoll concentration of about 10% is the preferred concentration in FHM solutions with 1.5M DMSO for mouse embryo cryopreservation at about -80°C.
  • the resulting cell survival rate (92, ⁇ 7%) was higher than that of the control group (88 ⁇ 7%).
  • FIG. 1 which as stated above illustrates the exothermic curves of water-Ficoll 70-DMSO solutions with differing R values including 1 010, 0.5 020, and 0 030.
  • a higher concentration of Ficoll 70 results in a higher value of T A with T rf being higher than -80°C when R equals 1. Therefore, addition of Ficoll 70 to DMSO aqueous solutions is an efficient approach to increasing the values of T ⁇ and preventing devitrification during storage and shipping of biological samples.
  • Ficoll 400 PVP 40, PVP 360 and sucrose can also increase the values of T rf of DMSO solutions.
  • the solution containing Ficoll 400 has a T ⁇ of - 75.7°C which may also satisfy the requirement for cryopreservation media stable at -80°C.
  • solution D FHM solution containing 10% Ficoll 70 and 1.5M DMSO
  • R the value of R has to be sufficiently high
  • T ⁇ (approximately 1.0); otherwise, the value of T ⁇ will be lower than -80°C after the equilibrium freezing procedures, resulting in a thermodynamically unstable solidified cell suspension.
  • Ficoll 70 When the concentration of Ficoll 70 is higher than 10%, the lower cell survival rates may be caused by the fact that the relatively high concentration of polymers may considerably increase solute osmolalities or reduce the amount of osmotically active water during the freezing procedures. Consequently, the high concentrations of Ficoll may result in severe osmotic damage that cells will undergo during freezing. Therefore, it is likely that an optimal concentration of Ficoll 70 exists for different cell types. The concentration should be high enough to stabilize the cryopreservation media solutions at -80°C by preventing

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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  • Developmental Biology & Embryology (AREA)
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  • Wood Science & Technology (AREA)
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Abstract

Dans certains aspects, la présente invention concerne des compositions et des procédés uniques pour la cryopréservation d'échantillons biologiques. Selon certaines formes de pratique de l'invention, ces compositions et procédés sont efficaces pour maintenir la vitrification d'échantillon à des températures au-dessus de -100°C. Par conséquent, dans une forme de réalisation, la présente invention concerne un milieu de cryopréservation qui inclut au moins un cryoprotecteur et un polymère, le milieu de cryopréservation ayant une température de dévitrification entre environ -100°C et environ -60°C.
PCT/US2012/071025 2011-12-20 2012-12-20 Procédés et compositions pour le stockage de cellules animales Ceased WO2013096659A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017088025A1 (fr) * 2015-11-24 2017-06-01 The University Of Adelaide Procédés, milieux et produits pour la culture d'embryons
EP3342288A1 (fr) * 2016-12-27 2018-07-04 Universite De Liege Nouvelle methode de vitrification en une etape
WO2018122011A1 (fr) * 2016-12-27 2018-07-05 Universite De Liege Methode de vitrification en une etape
BE1024850B1 (fr) * 2016-12-27 2018-07-23 Universite De Liege Établissement Public De La Communauté Française De Belgique Nouvelle methode de vitrification en une etape
CN109414008A (zh) * 2016-05-13 2019-03-01 密苏里大学管理机构 防止重结晶的冷冻保存培养基和方法
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

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WO2000030441A1 (fr) * 1998-11-24 2000-06-02 Bresagen Limited Cryopreservation d'ovocytes et d'embryons et methodes de production d'animaux par cette technique
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WO2007077560A2 (fr) * 2006-01-04 2007-07-12 Do-Coop Technologies Ltd. Compositions cryoprotectrices et procédés d'utilisation de celles-ci
US20110206776A1 (en) * 2010-02-18 2011-08-25 Samson Tom Methods of manufacture of immunocompatible amniotic membrane products
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3380603A4 (fr) * 2015-11-24 2019-07-10 The University of Adelaide Procédés, milieux et produits pour la culture d'embryons
WO2017088025A1 (fr) * 2015-11-24 2017-06-01 The University Of Adelaide Procédés, milieux et produits pour la culture d'embryons
CN108884439A (zh) * 2015-11-24 2018-11-23 阿德莱德大学 用于培养胚胎的方法、培养基和产品
JP2019502372A (ja) * 2015-11-24 2019-01-31 ザ ユニバーシティー オブ アデレード 胚を培養するための方法、培地および製品
JP2019514442A (ja) * 2016-05-13 2019-06-06 ザ・キュレイターズ・オブ・ザ・ユニバーシティー・オブ・ミズーリＴｈｅ Ｃｕｒａｔｏｒｓ Ｏｆ Ｔｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｍｉｓｓｏｕｒｉ 凍結防止培地及び再結晶防止方法
EP3454653A4 (fr) * 2016-05-13 2020-01-22 The Curators of the University of Missouri Milieu de cryoconservation et procédé pour empêcher la recristallisation
CN109414008A (zh) * 2016-05-13 2019-03-01 密苏里大学管理机构 防止重结晶的冷冻保存培养基和方法
BE1024850B1 (fr) * 2016-12-27 2018-07-23 Universite De Liege Établissement Public De La Communauté Française De Belgique Nouvelle methode de vitrification en une etape
WO2018122011A1 (fr) * 2016-12-27 2018-07-05 Universite De Liege Methode de vitrification en une etape
CN110113940A (zh) * 2016-12-27 2019-08-09 列日大学 单步玻璃化方法
EP3342288A1 (fr) * 2016-12-27 2018-07-04 Universite De Liege Nouvelle methode de vitrification en une etape
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

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