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WO1992003046A1 - Procede ameliore de cryoconservation de cellules - Google Patents

Procede ameliore de cryoconservation de cellules Download PDF

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Publication number
WO1992003046A1
WO1992003046A1 PCT/US1991/006010 US9106010W WO9203046A1 WO 1992003046 A1 WO1992003046 A1 WO 1992003046A1 US 9106010 W US9106010 W US 9106010W WO 9203046 A1 WO9203046 A1 WO 9203046A1
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WO
WIPO (PCT)
Prior art keywords
cells
medium
concentration
thawing
freezing
Prior art date
Application number
PCT/US1991/006010
Other languages
English (en)
Inventor
Mary Anne Whyte
Paul J. Price
Original Assignee
Somatix Therapy Corporation
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 Somatix Therapy Corporation filed Critical Somatix Therapy Corporation
Publication of WO1992003046A1 publication Critical patent/WO1992003046A1/fr

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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
    • 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

Definitions

  • the present invention relates to a method for freezing and thawing cells, particularly organ digests and primary cell cultures.
  • a major obstacle encountered in organ transplantation is the need to transplant the organ shortly after removal from the body of the donor. This limited time period makes it difficult to type the tissue, ship over long distances, or detect the presence of viral contaminants.
  • a problem with cultured digests is that the cell composition can change during culture and fibroblasts may predominate over differentiated parenchymal cells. Furthermore, parenchymal cells may dedifferentiate in culture and lose their specialized function.
  • organ digest and primary cell cultures tend to have very poor viability following freezing and thawing.
  • fetal liver organ digests tend to have about 15% viable cells following a freeze/thaw process.
  • a freeze/thaw process that substantially increased the viability of organ digest and primary cells would allow time for quality control procedures and transportation of cells over long distances.
  • such methods would facilitate banking cells for later transplantation when a suitable- recipient was available.
  • the methods would also facilitate research, permitting studies requiring a portion of the cells isolated from a single organ to be performed over a period of time.
  • the present invention provides a method for cryopreservation of cells that substantially enhances the viability of organ digests and primary cell cultures.
  • the method comprises suspending the cells in a freezing medium comprising an effective amount of DMSO and Hana freezing solution at 0 to 4°C to form a cell suspension; gradually lowering the temperature of the cell suspension to at least -1 °C to produce a frozen cell suspension; and storing the frozen cell suspension, preferably in liquid nitrogen.
  • Hana freezing solution comprises potassium lactobionate at a concentration in the range of from about 60 to about 140 mM, glutathione at a concentration in the range of from about 1 to about 4 mM, raffinose at a concentration in the range of from about 15 to about 40 mM, and insulin at a* concentration in the range of from about 2.5to about 10 ⁇ g/ l.
  • the frozen cell suspension is thawed until the suspension contains both solid and liquid.
  • the partially thawed suspension is combined with at least an equal volume of a thawing medium to finish thawing the suspension while diluting the concentration of DMSO.
  • the thawed cell suspension is pelleted.
  • the supernatant medium is removed, and the cell pellet is resuspended in thawing medium and plated in a culture vessel.
  • the cells are allowed to attach in the thawing medium for not more than about four hours and the thawing medium is replaced with growth medium.
  • the method has been used successfully to cryopreserve digests of human fetal hepatocytes which, previously, did not survive in culture after freezing.
  • the present invention provides an improved method for freezing and thawing organ digests and primary cell cultures which greatly improves the viability of the cells.
  • the method comprises suspending the cells in a freezing medium comprising a freezing solution and an effective amount of DMSO at 0 to 4°C to form a cell suspension; gradually lowering the temperature of the cell suspension to at least -12°C to produce a frozen cell suspension; and storing the frozen cell suspension, preferably in liquid nitrogen.
  • the frozen cell suspension is thawed until the suspension contains both solid and liquid.
  • the partially thawed suspension is combined with at least an equal volume of a thawing medium to finish thawing the suspension while diluting the concentration of DMSO.
  • the thawed cell suspension is pelleted.
  • the supernatant medium is removed, and the cell pellet is resuspended in thawing medium and plated in a culture vessel.
  • the cells are allowed to attach in the thawing medium for not more than about four hours and the thawing medium is replaced with growth medium.
  • an organ digest refers to cells (single cells and cell aggregates) obtained from the intact tissue of an animal.
  • a primary cell culture refers to cells obtained from the intact tissue of an animal and grown on glass or plastic surfaces for the first time.
  • the cell freezing and thawing method can be used for organ digests and primary cell cultures.
  • the method is also useful for early passage cells which have poor viability on freezing and thawing.
  • the method can be used with cells of any organ from any animal species and is particularly useful with human fetal hepatocytes and human fetal islet cells. Although the method is also effective for use with established cell lines, less expensive and less time consuming methods provide suitable results with cell lines.
  • the freezing medium for the present method is Hana freezing solution with an effective amount of DMSO.
  • Hana freezing solution is a physiological solution that comprises potassium lactobionate at a concentration in the range of from about 60 to about 140 mM; glutathione at a concentration in the range of from about 1 to about 4 mM; raffinose at a concentration in the range of from about 15 to about 40 mM; and insulin at a concentration in the range of from about 2.5 to about 10 ⁇ g/ml.
  • a preferred formulation of Hana freezing solution is shown below in Table 1.
  • Hana freezing solution additionally comprises Na KH 2 P0 4 at a concentration in the range of from about 10 to about 35 mM; adenosine at a concentration in the range of from about 0.6 to about 10 mM; MgS0 4 at a concentration in the range of from about 3 to about 6 mM; allopurinol at a concentration in the range of from about 0.5 to about 1.5 mM; and bactrim at a concentration in the range of from about 0.1 to about 1.0 ml/L.
  • a preferred formulation for each of the additional components is listed below in Table 2.
  • Belzer's solution can be used as the freezing solution.
  • Belzer's solution is an organ transport solution that additionally contains hydroxyethyl starch.
  • Belzer's solution is described in D'Alessandro et al., Diabetes 38:7-9 (1989), which article is incorporated herein by reference in its entirety.
  • the formulation for Belzer's solution is shown below in Table 3.
  • Belzer's solution is available commercially from DuPont. TABLE 3 Belzer's Solution
  • Effective amounts of DMSO for freezing cells are well known and can vary from about 7.5 to 20% (v/v), and are preferably about 10%.
  • the freezing medium optionally includes an antibiotic at an effective concentration.
  • the thawing medium of this invention is the growth medium for the type of cell that is being thawed, preferably supplemented with DNase and, for use with organ digests, preferably, a substance to increase the osmolality.
  • the DNase is present at a concentration which is effective to prevent clumping due to DNA released from dead cells. A concentration of about 100 ⁇ g/ml DNase is effective.
  • the osmolality of the solution is preferably high to prevent swelling and subsequent rupture of cells during the thawing process and the first few hours thereafter.
  • a sugar preferably glucose at a concentration of from about 1.0 to about 1.5 M, more preferably about 1 M glucose, to enhance the osmolality is preferred.
  • 1 M glucose for increased osmolality is well known.
  • Other suitable substances for use in increasing osmolality in tissue culture solutions or cell processing solutions are well known and can be used in place of glucose.
  • the osmolality enhancer is preferably not present in the thawing medium.
  • the osmolality enhancer is detrimental to cells which were plated prior to freezing.
  • the thawing medium is preferably also supplemented with serum for use with cells having a serum-free growth medium.
  • a preferred serum is fetal calf serum at a concentration of at least about 10% (v/v) .
  • a growth medium for an organ digest or primary cell culture is not affected by the method of this invention.
  • a preferred growth medium for use with hepatocytes and pancreatic islet cells is H 3 H Basal medium.
  • a most preferred thawing medium for those cells is listed in Table 4.
  • EGF Extracellular Growth Factor
  • Se0 2 selenium oxide 0.035 ⁇ g/ml
  • the organ digest is prepared by a standard method for preparation of a primary cell culture for the species and type of organ.
  • the cell suspension can contain single cells and small cell aggregates.
  • the cell suspension is prepared by standard methods such as trypsinization, scraping and the like.
  • the cells are pelleted and counted. Then the cells are resuspended in a sufficient amount of freezing medium at 4°C to provide a concentration of from about 10 5 to 2X10 7 cells/ml. If there will be a delay before freezing the cells, preferably the cells are suspended in freezing medium without DMSO, since DMSO is toxic to cells at temperatures of 0°C or above. Conveniently, the cells are suspended in DMSO-free freezing medium and then diluted with an equal volume of freezing medium having twice the desired final concentration of DMSO just prior to freezing.
  • the temperature of the cell suspension in freezing medium is sequentially lowered to about -100°C prior to placing the vials in liquid nitrogen storage.
  • the temperature is slowly lowered, preferably at about 1°C per minute, to the phase change where the liquid supernatant begins to freeze.
  • the temperature at which the phase change occurs depends on the freezing medium used, particularly the DMSO concentration. For freezing media containing 10% DMSO, the phase change occurs from about -10°C to about -12°C.
  • the temperature is lowered rapidly, preferably at about 10°C per minute, more preferably at more than 10°C per minute, until the cell suspension is solid.
  • the temperature is gradually lowered, preferably at from 2 to 5°C per minute.
  • a most preferred sequential temperature lowering scheme is listed below. Departures from the scheme can be tolerated, particularly after the cells are frozen.
  • the vials are transferred to liquid nitrogen storage.
  • the cells should remain frozen for at least about a week prior to thawing.
  • the frozen cell suspension is preferably thawed until there is both liquid and solid freezing medium in the vials.
  • the vials are thawed to about a half crystalline, half liquid state.
  • the frozen cell mixture is diluted with an excess of thawing medium to dilute the concentration of DMSO in the supernatant medium to at least about one-half, preferably to about one-fifth, preferably to about one-tenth or less of the original DMSO concentration of the freezing medium.
  • the thawing medium is at room temperature.
  • the diluted cells are pelleted, and the DMSO-con ining supernatant is removed.
  • the cell pellet is resuspended in thawing medium.
  • the cells are preferably counted prior to plating.
  • the cells are plated in thawing medium for about 2 to 4 hours post-thawing to permit attachment of the cells.
  • the cells preferably do not remain in the thawing medium more than about 4 hours. Thereafter, the medium is aspirated and replaced with growth medium.
  • Freezing media having varying concentrations of FCS were compared to determine the effect of the FCS concentration on the viability of human fetal hepatocytes.
  • the hepatocytes were an organ digest prepared the day the study was performed.
  • the cell suspension, freezing vials and freezing media were kept at 4°C until just before addition to a controlled rate freezer (Cryomed freezer with a thermocouple probe) .
  • the cell suspension was pelleted and resuspended in half of the final volume of freezing medium without DMSO.
  • the cells were frozen at a final concentration of 1X10 6 in a final volume of 1 ml.
  • the freezing media used were:
  • F12K with CHO supplement 10% DMSO and 5% FCS
  • F12K with CHO supplement 10% DMSO and 0% FCS (CHO supplement is a cell culture medium supplement which is commercially available from Irvine Scientific, Irvine CA) .
  • FCS 20% FCS: 18.8% 10% FCS: 10.5% 5% FCS: 40.5% 0% FCS: 11.2%
  • PBS phosphate buffered saline
  • the cells were fed with H 3 H basal medium with 10% FCS.
  • the thawing medium used was ⁇ -MEM with 1 M glucose and 10% FCS.
  • cells in freezing medium with 10% FCS looked best.
  • Cells from 20% and 0% FCS freezing medium looked worst. Plates were discarded at 24 hours post-plating.
  • EXAMPLE 2 Effect of Thawing Medium
  • the study of Example 1 was repeated with the following changes.
  • the thermocouple probe of the freezer was placed in a "blank" vial which contained freezing medium with cells.
  • the freezing medium used was H 3 H basal medium with 5% FCS, CHO supplement and 10% DMSO.
  • Four vials of cells were used to evaluate each of the following two thawing media. 1) MEM with 1 M glucose and 10% FCS 2) H 3 H basal medium and 10% FCS
  • the cells contained numerous vacuoles at 24 hours.
  • Example 2 Evaluation of Freezing Media The study of Example 2 was repeated with the changes described below to evaluate the effects of freezing media of the cells. The freezing procedure was modified so that the rate of freezing from -4°C to -10°C was 25°C/minute.
  • the thawing media used was 125 ml H 3 H basal media; 13.8 ml FCS; 25 g glucose and 2.5 ml H3 supplement.
  • the freezing media evaluated were:
  • L-15 media was purchased from GIBCO (Grand Island, NY) .
  • Example 4 Evaluation of Freeze/Thaw Method The study of Example 4 was repeated using Belzer's solution with 10% DMSO as the freezing medium and H 3 H basal media with H3 supplement, FCS and glucose as the thawing medium for plating the cells. At two hours post-plating, the 1 ml of thawing was supplemented with an additional 1 ml to dilute residual DMSO. At three hours post-plating, the thawing medium was removed and replaced with H 3 H basal media with CHO supplement. By three hours post-plating, the cells looked healthy and appeared to be attached, but were rounded.
  • the cells were attached and looked healthy. No differences between the three formulations of freezing medium were apparent. Few viable eels were observed clumped together or to dead cells using thawing media with DNase. This was an improvement in comparison to the number of clumped, viable cells using thawing media without DNase.
  • organ digests of human fetal hepatocytes are well known for failing to survive after freezing.
  • survival rates of 70% or more were achieved.

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  • 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)

Abstract

La présente invention concerne un procédé de congélation et de dégel de cellules, ce procédé augmentant sensiblement la viabilité de digestes d'organes (cellules simples et agrégats de cellules) et des cultures de cellules primaires.
PCT/US1991/006010 1990-08-28 1991-08-22 Procede ameliore de cryoconservation de cellules WO1992003046A1 (fr)

Applications Claiming Priority (2)

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US574,168 1984-01-26
US57416890A 1990-08-28 1990-08-28

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997047192A1 (fr) * 1996-06-14 1997-12-18 Biostore New Zealand Limited Compositions et procedes de conservation de tissus vivants
US5827640A (en) * 1996-06-14 1998-10-27 Biostore New Zealand Limited Methods for the preservation of cells and tissues using trimethylamine oxide or betaine with raffinose or trehalose
EP0834252A3 (fr) * 1996-09-25 1999-03-03 W.R. Grace & Co.-Conn. Méthode de décongélation de cellules cryoconservées
US5879875A (en) * 1996-06-14 1999-03-09 Biostore New Zealand Compositions and methods for the preservation of living tissues
US5962213A (en) * 1996-06-14 1999-10-05 Biostore New Zealand Limited Compositions and methods for the preservation of living tissues
US6037116A (en) * 1996-06-14 2000-03-14 Biostore New Zealand, Ltd. Compositions comprising betaine, sodium citrate and sodium chloride and methods for the preservation of biological materials
US6114107A (en) * 1996-06-14 2000-09-05 Biostore New Zealand Limited Composition comprising raffinose, TMAO, sodium citrate and methods for the preservation of living tissues
US6361933B1 (en) 1996-06-14 2002-03-26 Biostore New Zealand Limited Solutions for the preservation of tissues
US6743575B2 (en) 1996-06-14 2004-06-01 Biostore New Zealand Ltd. Compositions and methods for the preservation of living tissues
WO2005054450A1 (fr) * 2003-12-01 2005-06-16 Vertex Pharmaceuticals Incorporated Compositions renfermant des cellules hepatiques foetales, et procedes utiles pour le traitement de l'infection par le vhc
WO2015077523A1 (fr) * 2013-11-22 2015-05-28 Le Centre Nationale De La Recherche Scientifique (Cnrs) Cellule congelée prête pour l'analyse et procédé pour réduire la variabilité de ses performances
WO2017194954A1 (fr) * 2016-05-12 2017-11-16 University Of Leeds Formulation
WO2018187439A1 (fr) * 2017-04-06 2018-10-11 The University Of North Carolina At Chapel Hill Procédé de cryoconservation

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SU935050A1 (ru) * 1978-11-10 1982-06-15 Институт проблем криобиологии и криомедицины АН УССР Способ консервировани щитовидной железы
US4879283A (en) * 1985-10-03 1989-11-07 Wisconsin Alumni Research Foundation Solution for the preservation of organs
US4965185A (en) * 1988-06-22 1990-10-23 Grischenko Valentin I Method for low-temperature preservation of embryos

Patent Citations (3)

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SU935050A1 (ru) * 1978-11-10 1982-06-15 Институт проблем криобиологии и криомедицины АН УССР Способ консервировани щитовидной железы
US4879283A (en) * 1985-10-03 1989-11-07 Wisconsin Alumni Research Foundation Solution for the preservation of organs
US4965185A (en) * 1988-06-22 1990-10-23 Grischenko Valentin I Method for low-temperature preservation of embryos

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Title
BIOSIS ABSTRACT, MOUNIB, M.S., "Cryogenic Preservation of Fish and Mammalian Spermatozoa". See Biosis No. 67031505; & J. REPROD. FERTIL., 53(1), 13-18 (1978). *
BIOSIS ABSTRACT, TANIGUCHI et al., "Non-Frozen Cold Storage is Favorable for Islet Function and Morphology". See Biosis Number: 86014057; & DIABETES RES. CLIN. PRACT., 4(4), 295-302 (1988). *
BIOSIS ABSTRACT, YU et al., "Rat Liver Preservation I. the Components of UW Solution That Are Essential to its Success", See Biosis No. 90073422; & TRANSPLANTATION (Baltimore), 49(6), 1060-1066 (1990). *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997047192A1 (fr) * 1996-06-14 1997-12-18 Biostore New Zealand Limited Compositions et procedes de conservation de tissus vivants
US5827640A (en) * 1996-06-14 1998-10-27 Biostore New Zealand Limited Methods for the preservation of cells and tissues using trimethylamine oxide or betaine with raffinose or trehalose
US5879875A (en) * 1996-06-14 1999-03-09 Biostore New Zealand Compositions and methods for the preservation of living tissues
US5962213A (en) * 1996-06-14 1999-10-05 Biostore New Zealand Limited Compositions and methods for the preservation of living tissues
US6037116A (en) * 1996-06-14 2000-03-14 Biostore New Zealand, Ltd. Compositions comprising betaine, sodium citrate and sodium chloride and methods for the preservation of biological materials
US6114107A (en) * 1996-06-14 2000-09-05 Biostore New Zealand Limited Composition comprising raffinose, TMAO, sodium citrate and methods for the preservation of living tissues
US6361933B1 (en) 1996-06-14 2002-03-26 Biostore New Zealand Limited Solutions for the preservation of tissues
US6743575B2 (en) 1996-06-14 2004-06-01 Biostore New Zealand Ltd. Compositions and methods for the preservation of living tissues
EP0834252A3 (fr) * 1996-09-25 1999-03-03 W.R. Grace & Co.-Conn. Méthode de décongélation de cellules cryoconservées
US5895745A (en) * 1996-09-25 1999-04-20 W.R. Grace & Co.-Conn. Method of thawing cryopreserved cells
WO2005054450A1 (fr) * 2003-12-01 2005-06-16 Vertex Pharmaceuticals Incorporated Compositions renfermant des cellules hepatiques foetales, et procedes utiles pour le traitement de l'infection par le vhc
JP2007516706A (ja) * 2003-12-01 2007-06-28 バーテックス　ファーマシューティカルズ　インコーポレイテッド 胎児肝臓細胞を含む組成物およびｈｃｖ感染で有用な方法
WO2015077523A1 (fr) * 2013-11-22 2015-05-28 Le Centre Nationale De La Recherche Scientifique (Cnrs) Cellule congelée prête pour l'analyse et procédé pour réduire la variabilité de ses performances
US20160302405A1 (en) * 2013-11-22 2016-10-20 Le Centre Nationale De La Recherche Scientifique (Cnrs) Assay-ready frozen cell and method for minimizing variability in the performance thereof
US10271539B2 (en) 2013-11-22 2019-04-30 Le Centre Nationale De La Recherche Scientifique (Cnrs) Assay-ready frozen cell and method for minimizing variability in the performance thereof
WO2017194954A1 (fr) * 2016-05-12 2017-11-16 University Of Leeds Formulation
CN109219347A (zh) * 2016-05-12 2019-01-15 利兹大学 制剂
JP2019515940A (ja) * 2016-05-12 2019-06-13 ユニバーシティ オブ リーズ 製剤
WO2018187439A1 (fr) * 2017-04-06 2018-10-11 The University Of North Carolina At Chapel Hill Procédé de cryoconservation
IL269602A (en) * 2017-04-06 2019-11-28 Sapienza Univ Di Roma Freezing method
CN110913691A (zh) * 2017-04-06 2020-03-24 北卡罗来纳大学教堂山分校 低温保存方法
JP2020516257A (ja) * 2017-04-06 2020-06-11 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒルＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｎｏｒｔｈ Ｃａｒｏｌｉｎａ Ａｔ Ｃｈａｐｅｌ Ｈｉｌｌ 凍結保存方法
EP3612027A4 (fr) * 2017-04-06 2021-02-24 The University of North Carolina at Chapel Hill Procédé de cryoconservation

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