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WO2004010780A2 - Glace intracellulaire inoffensive - Google Patents

Glace intracellulaire inoffensive Download PDF

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Publication number
WO2004010780A2
WO2004010780A2 PCT/US2003/024147 US0324147W WO2004010780A2 WO 2004010780 A2 WO2004010780 A2 WO 2004010780A2 US 0324147 W US0324147 W US 0324147W WO 2004010780 A2 WO2004010780 A2 WO 2004010780A2
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Prior art keywords
cells
intracellular ice
nucleation
ice formation
cell
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Ceased
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PCT/US2003/024147
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WO2004010780A3 (fr
Inventor
Jason Paul Acker
Locksley Earl Mcgann
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General Biotechnology LLC
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General Biotechnology LLC
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Publication date
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Priority to AU2003258001A priority Critical patent/AU2003258001A1/en
Publication of WO2004010780A2 publication Critical patent/WO2004010780A2/fr
Publication of WO2004010780A3 publication Critical patent/WO2004010780A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/16—Physical preservation processes
    • A01N1/162—Temperature processes, e.g. following predefined temperature changes over time
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts

Definitions

  • the invention relates to a method for cryopreserving cells while maintaining cell viability. More particularly, cells are cryopreserved while maintaining cell viability by controlling innocuous intracellular ice formation.
  • the conventional approach to cryopreservation has therefore been to balance the toxic effects of high concentrations of solutes using chemical cryoprotectants with cooling rates slow enough to avoid ILF, but rapid enough to minimize cryoprotectant exposure.
  • the application of classic freezing techniques to the cryopreservation of cells, tissues and tissue models has assumed that intracellular ice is lethal. As it has been shown that intracellular ice formation in cells in suspension occurs during rapid freezing and that rapid freezing causes cell death, it has been assumed that IIF causes cell death. However, there is no direct evidence in the literature to lead one to conclude that the mere presence of ice within a cell is lethal. Early studies with rabbit corneal tissue, skin, tumors and plant tissue could not correlate the presence of intracellular ice with cell death.
  • a method for cryopreserving cells and maintaining cell viability comprises the steps of predetermining the nucleation temperature for extracellular and intracellular ice formation for the cells, cooling the cells to the predetermined nucleation temperature, nucleating intracellular ice formation, and cooling the cells to a temperature lower than the predetermined nucleation temperature.
  • the predetermined nucleation temperature can be from about -3°C to about -40°C, from about -4°C to about -40°C, from about -5°C to about -40°C, from about -5°C to about -30°C, from about -5°C to about -20°C, or from about -5°C to about -10°C.
  • other temperature ranges disclosed herein can be used.
  • the cells are cooled to about -30°C to about -200°C, about -30°C to about -100°C, or to about -40°C following nucleation of intracellular ice formation.
  • intracellular ice formation is nucleated by a mechanism selected from the group consisting of osmotic cycling, thermal shock, chemical permeabilization, electroporation, and surface-catalyzed nucleation.
  • a method is provided for cryopreserving cells and maintaining cell viability without the use of cryoprotective compounds. The method comprises the step of cooling the cells under conditions to optimize controlled innocuous intracellular ice formation.
  • Fig. 3 shows the effect of intracellular ice formation on the post-thaw survival of V-79W and MDCK confluent cell monolayers.
  • the incidence of intracellular ice (left column in each group), membrane integrity (second column from left in each group), metabolic activity (third column from the left in each group), and clonogenic function (right column in each group) is plotted as a function of nucleation temperature. Mean ⁇ SEM (n - 6).
  • Fig. 4 shows the metabolic activity of V-79W and MDCK single attached cells and confluent monolayers and confluent monolayers following freezing using three different cryopreservation protocols.
  • the survival of cells frozen using a standard freezing protocol left column in each group
  • a standard freezing protocol + DMSO middle column in each group
  • a modified freezing protocol involving intracellular ice formation right column in each group
  • Figs. 6 A and B show the responses of V-79W fibroblasts (panels A and B) and MDCK epithelial cells (panels C and D) to graded freezing. The samples were assessed for membrane integrity (panels A and C) and metabolic activity (panels B and D) following nucleation of extracellular ice.
  • a method for cryopreserving cells and maintaining cell viability comprises the steps of predetermining the nucleation temperature for extracellular and intracellular ice formation for the cells, cooling the cells to the predetermined nucleation temperature, nucleating intracellular ice formation, and cooling the cells to a temperature lower than the predetermined nucleation temperature.
  • a method for cryopreserving cells and maintaining cell viability without the use of cryoprotective compounds. The method comprises the step of cooling the cells under conditions to optimize controlled innocuous intracellular ice formation.
  • the cell types or cell lines for use in the presently described method include, but are not limited to, animal cells, plant cells, insect cells, mammalian cells, and human cells.
  • Transgenic cells, genetically engineered cells, or transformed cells can be used and the cells can be nucleated or non-nucleated.
  • the cell types or cell lines can comprise anchorage- dependent cells or anchorage-independent cells (e.g., cells grown in suspension) and the cells can be differentiated or undifferentiated.
  • the cells can be cultured cell types or cell lines or the cells can be derived from native tissues, organs, whole organisms, or the cells can be derived from engineered cells, tissues, organs, or whole organisms. If cultured, the cell types or cell lines can be grown in suspension or as monolayers and the cell types or cell lines can be subconfluent or confluent.
  • Cells cultured as either anchorage-dependent or anchorage- independent cell types or cell lines are known to those skilled in the art to include, but are not limited to, tumor cells, hematopoietic cells, fibroblasts, hepatocytes, epithelial cells, keratinocytes, stem cells, smooth muscle cells, dendritic cells or other immune cells such as macrophages, T-cells, B-cells, or mast cells, stromal cells, chondrocytes, endothelial cells, mesenchymal cells, or any other cell type or cell line known in the art that can be cultured.
  • the cells can be any cell type from any tissue or organ source including, but not limited to, blood, lung, heart, kidney, stroma, brain, liver, muscle, skin, ovaries, and testes.
  • Cells useful in the disclosed method can be comprised of a single cell type or cell line or of multiple cell types or cell lines.
  • the nucleation temperature is determined for extracellular and or intracellular ice formation for the specific cell type or cell line to be cryopreserved.
  • the nucleation temperature for extracellular and/or intracellular ice formation can be determined using any technique known to those skilled in the art such as the standard flash method disclosed herein where the presence of intracellular ice is denoted by a visible darkening of the cytoplasm due to the scattering of light by intracellular ice crystals.
  • any fluorometric technique can be used, such as a technique using a nucleic acid-binding fluorescent stain (e.g., SYTO 13; Molecular Probes, Eugene, OR), as described herein, to directly assess the incidence of intracellular ice formation.
  • the predetermined nucleation temperature can be from about 0 °C to about -40°C, from about -2°C to about -40°C, from about -3°C to about -40°C, from about -4°C to about -40°C, from about -5°C to about -40°C, from about -5°C to about -30°C, from about -5°C to about -20°C, from about -5°C to about -15°C, from about -5°C to about -13°C, from about -5°C to about -12°C, or from about -5°C to about -10°C.
  • the cell type or cell line to be cryopreserved is cooled at any rate to the predetermined nucleation temperature, and intracellular ice formation is induced.
  • the cell type or cell line to be cryopreserved is cooled to a temperature higher than the predetermined nucleation temperature (e.g., 0°C) prior to cooling the cell type or cell line to the predetermined nucleation temperature.
  • Intracelluar ice formation is induced when the cell type or cell line is cooled to the predetermined nucleation temperature.
  • nucleation of intracellular ice formation means that an active step is performed to induce intracellular ice formation.
  • intracellular ice formation can be induced (i.e., nucleated) by a method selected from the group consisting of osmotic cycling, thermal shock, chemical permeabilization, electroporation, and surface-catalyzed nucleation (e.g., using forceps to induce intracellular ice formation).
  • a method selected from the group consisting of osmotic cycling, thermal shock, chemical permeabilization, electroporation, and surface-catalyzed nucleation e.g., using forceps to induce intracellular ice formation.
  • any other method known to those skilled in the art for inducing intracellular ice formation can be used.
  • cryoprotectants that are known in the art are small molecular weight compounds, such as dimethylsulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, and the like.
  • intracellular ice Upon nucleation of intracellular ice formation, intracellular ice is formed within the cells, and the percentage of cells wherein intracellular ice is formed can be greater than 20%o of the cells, greater than 30% of the cells, greater than 40% of the cells, greater than 45% of the cells, greater than 50% of the cells, greater than 55% ⁇ of the cells, greater than 60% of the cells, greater than 65% of the cells, greater than 70%) of the cells, greater than 75% of the cells, greater than 80% of the cells, greater than 85% of the cells, greater than 90% of the cells, or greater than 95% of the cells.
  • conditions to optimize controlled innocuous intracellular ice formation include those conditions described herein such as cryopreserving cell types, including cell types of tissues, organs, or whole organisms, or cell lines, that are confluent, and/or inducing intracellular ice formation at a temperature that causes intracellular ice to be formed in a high percentage of the cells (e.g., greater than 60% of the cells, greater than 65% of the cells, greater than 70% of the cells, greater than 75% of the cells, greater than 80% of the cells, greater than 85%> of the cells, greater than 90% of the cells, or greater than 95% of the cells).
  • cryopreserving cell types including cell types of tissues, organs, or whole organisms, or cell lines, that are confluent, and/or inducing intracellular ice formation at a temperature that causes intracellular ice to be formed in a high percentage of the cells (e.g., greater than 60% of the cells, greater than 65% of the cells, greater than 70% of the cells, greater than 75% of the cells, greater than 80%
  • the cell type or cell line to be cryopreserved can be cooled to a predetermined storage temperature, lower than the predetermined nucleation temperature.
  • the cell type or cell line can be cooled for storage to about -30 °C to about -200 °C, about -30°C to about -150°C, about -30°C to about -100°C, about -30°C to about -80°C, about -30°C to about -60°C, about -30°C to about -40°C, or to about -40°C following nucleation of intracellular ice formation.
  • the rate of cooling can be from about l°C/min to about 500°C/min (i.e., slow or rapid cooling).
  • the cell type or cell line can be thawed, when needed, and the rate of thawing can be from about l°C/min to about 500°C/min (i.e., slow or rapid thawing).
  • Cell types or cell lines that are cryopreserved using the method disclosed herein maintain cell viability.
  • the percentage of cells that maintain viability can be greater than 20%> of the cells, greater than 30% of the cells, greater than 40%) of the cells, greater than 45% of the cells, greater than 50% of the cells, greater than 55%> of the cells, greater than 60% of the cells, greater than 65%> of the cells, greater than 70% of the cells, greater than 75% of the cells, greater than 80%) of the cells, greater than 85% of the cells, greater than 90% of the cells, or greater than 95%> of the cells.
  • Cell viability can be assessed as described herein. For example, cell viability can be assessed by examining membrane integrity, metabolic activity, or clonogenic function as described below.
  • MDCK Madin Darby Canine Kidney
  • CCL34 ATCC Madin Darby Canine Kidney epithelial cell line. These cells were incubated at 37 °C in an atmosphere of 95% air + 5%> carbon dioxide in minimum essential media (MEM) supplemented with 10% v/v fetal bovine serum (all components from GLBCO Laboratories, Grand Island, NY). Cells were grown in tissue culture flasks (25 cm 2 ; Corning Glass Works, Corning, NY) and harvested by exposure to a 0.25% trypsin- EDTA solution (GLBCO) for 10 min at 37 °C. The MDCK cells were resuspended in supplemented MEM to obtain cell suspensions and then plated on sterilized cover
  • the second cell line was the V-79W line of Chinese hamster fibroblasts.
  • Cells were incubated at 37 °C in an atmosphere of 95% air + 5% carbon dioxide in minimum essential medium (MEM) with Hanks' salts, 16 mmol L sodium bicarbonate, 2 mmol/L L-glutamine and 10%> fetal bovine serum supplemented with antibiotics (penicillin G (50 units/mL), streptomycin (50 ⁇ g/mL)) (all components from GLBCO Laboratories, Grand Island, NY).
  • MEM minimum essential medium
  • antibiotics penicillin G (50 units/mL), streptomycin (50 ⁇ g/mL)
  • GLBCO Laboratories Grand Island, NY
  • Cells were grown in tissue culture flasks (25 cm 2 ; Corning Glass Works) and harvested by exposure to a 0.25% trypsin solution (GD3CO) for 10 min at 37°C.
  • GD3CO trypsin solution
  • fibroblasts were resuspended in supplemented MEM.
  • Sterilized cover slips (12 mm circle, FISHER Brand) were placed in a petri dish (FISHER Brand, 100 x 15 mm) and covered with 15 mL of
  • the cryomicroscope and video system used for this study is known in the art. Briefly, it consisted of a Zeiss fluorescent microscope (Carl Zeiss, Germany), a CCD video camera (ZVS-47DEC, Carl Zeiss), a video recorder (GX4, Panasonic, Japan) and a convection cryostage.
  • the cryostage was connected to a computer-controlled interface (Great Canadian Computer Company, Spruce Grove, Canada). The computer monitored the temperature by analyzing the voltage from a thermocouple on the stage and via a proportional controller circuit; heat was added as necessary to allow the stage to follow a user defined thermal protocol.
  • SYTO 13 a permeant live cell nucleic acid dye (1.25 ⁇ M) and ethidium bromide (EB; Sigma Chemical
  • Percent survival based on membrane integrity was calculated as the number of SYTO positive cells over the total number of cells (SYTO and EB positive) using the following equation:
  • % survival x 100 total SYTO positive + total EB positive cells
  • AlamarBlueTM (Biosource International, CA) was used to assess the overall metabolic activity of the confluent monolayers and single attached cells post- thaw. AlamarBlue was added to tissue culture media (5-10% v/v) and confluent monolayers (MDCK and V-79W) were incubated in this solution for 12 hours at 37°C. Single attached cells (MDCK and V-79W) were incubated for 24 hours at 37°C in 10%) alamarBlue. An aliquot (100 ⁇ L) of the media was removed and measured on a spectrophotometer (570-600 nm; UVmax, Molecular Dynamics, CA). Percent survival based on metabolic activity was calculated as the mean percent difference in reduction between the experimental samples and the controls using the following equations:
  • a LW is the absorbance value of the sample minus the absorbance of the media only at 570 nm
  • a HW is the absorbance value of the sample minus the absorbance of the media only at 600 nm.
  • AO T L ,W- is the absorbance of alamarBlue in media minus the absorbance of media only at 570 and AO w is the absorbance of alamarBlue in media minus the absorbance of media only at 600 nm.
  • the cells were collected from the coverslips by exposure to a 0.25% ⁇ trypsin-EDTA solution and diluted to 750 cells/mL. Tissue culture flasks were seeded with 150 cells/flask and incubated at 37 °C for 5 days. The tissue culture media was removed and the colonies were fixed with 70% isopropanol, stained with trypan blue, and rinsed with distilled water before being counted. Percent survival based on clonogenic function was calculated as the mean of the experimental colony counts expressed as a percentage of the mean unfrozen controls.
  • intracellular ice was detected using the standard flash method known in the art where the presence of intracellular ice is denoted by a visible darkening of the cytoplasm due to the scattering of light by intracellular ice crystals.
  • a fluorometric technique was used to assist in the identification of ILF in the MDCK and V-79W confluent monolayers.
  • a nucleic acid-binding fluorescent stain (SYTO 13; Molecular Probes, Eugene, OR) was used to directly assess the incidence of ILF in the V-79W and MDCK single attached cells and confluent monolayers following extracellular ice nucleation.
  • SYTO 13 Molecular Probes, Eugene, OR
  • Prior studies have shown that the formation of intracellular ice disrupts the structures stained by fluorescent dyes resulting in a distinctive "honeycomb" pattern that can be used to directly quantify the incidence of ILF. Samples were stained with 12.5 ⁇ M SYTO prior to being placed into a pre-cooled alcohol bath.
  • Madin-Darby canine kidney (MDCK) epithelial cells and V-79W hamster fibroblasts were either attached individually or grown to confluency on glass coverslips, then stained with SYTO and EB prior to freezing. The samples were then supercooled to a defined subzero experimental temperature on a convection cryostage by cooling at 25 °C/min. The subzero experimental temperatures were carefully chosen to ensure that approximately 100% of the cells would form intracellular ice (see Fig. 1). Under video surveillance, ice was nucleated using a cold copper probe at the constant temperature and the incidence of intracellular ice formation was observed after holding for 5 min. Cells were then warmed at 25°C/min and the integrity of the cell plasma membrane was quantitatively assessed.
  • MDCK Madin-Darby canine kidney
  • the samples were then transferred from the cryomicroscope and assayed for metabolic function using the reduction-oxidation indicator alamarBlue.
  • the cells were trypsinized, plated, incubated for 5 days, and the number of colony forming units was determined.
  • the cumulative incidence of cells with intracellular ice formation was determined as a function of nucleation temperature and correlated with post-thaw survival.
  • Madin-Darby canine kidney epithelial cells and V-79W hamster fibroblasts were either used as single attached cells or grown to confluency on glass coverslips.
  • the cover slips containing a confluent monolayer were placed in 15 x 45 mm glass tubes (Kimble Glass Inc.) and placed on ice for 5 min. Following this incubation at 0°C, the samples were immersed in an alcohol bath at -5 °C and allowed to cool for 5 min. Extracellular ice formation was induced in the samples using cold forceps. The bath was then cooled at 1 °C/min to -40°C where the samples were held for 5 min. All samples were then rapidly warmed in a 37°C water bath and incubated in a 10%> alamarBlue solution at 37°C. Metabolic activity was then assessed.
  • MDCK and V-79W single attached cells and confluent monolayers were subjected to the standard freezing protocol with addition of 10% v/v DMSO (Cryoserv, Tera Pharmaceuticals Inc., Buena Park, CA).
  • the cover slips containing the cells were placed in glass tubes incubated for 5 min in an ice bath and then 500 ⁇ L of a 10%) v/v DMSO solution (in supplemented tissue culture media) was added to the glass tubes.
  • the single attached cells and confluent monolayers were allowed to incubate with the DMSO for 20 min and then 450 ⁇ L of the solution was removed prior to freezing.
  • the samples were immersed in an alcohol bath and the above freezing protocol was used.
  • MDCK and V-79W cells were either used as single attached cells or grown to confluency on glass coverslips.
  • the cover slips containing single attached cells and confluent monolayers were placed in 15 x 45 mm glass tubes and placed on ice for 5 min. Following this incubation at 0°C, the samples were immersed in an alcohol bath at -10°C and allowed to cool for 5 min. Extracellular ice formation was induced at -10°C in the samples using cold forceps. At -10°C, 100% intracellular ice formation has been shown to occur in all of the V-79W and MDCK single attached cells and confluent monolayers (see Fig. 1).
  • the bath was then cooled at l°C/min to -40°C where the samples were held for 5 min. All samples were then rapidly warmed in a 37°C water bath, incubated in a 10%> alamarBlue solution at 37°C and assessed.
  • Another controlled-rate freezing/thawing protocol (graded freezing) was used to progressively to simulate cell injury during freezing (used only in the assay shown in Figs. 6 A and B).
  • the bath was then cooled at l°C/min, and at different subzero temperatures (-5, -10, -15, -20, -30, and -40°C), samples were thawed directly in a circulating water bath at 37°C and assessed.
  • extracellular ice nucleation was initiated at -10°C by placing samples into the -5°C alcohol bath for 5 min before being cooled at l°C/min to -10°C. Nucleation was induced using cold forceps, and the samples were allowed to equilibrate at -10°C for 5 min before continuing with the graded freezing protocol. Control samples cooled to and thawed from the two nucleation temperatures (-5 and -10°C) followed the above thermal profile but did not undergo extracellular ice nucleation.
  • V-79W and MDCK confluent cell monolayers were subjected to low temperature conditions where ILF was shown to occur and then evaluated using the three different assessment techniques.
  • the effect of ILF on the post-thaw survival of V-79W and MDCK confluent monolayers is shown in Fig. 3.
  • MDCK monolayers have been shown to form intracellular ice at high subzero temperatures (see Fig. 1), it was possible to extend the range of nucleation temperatures examined for MDCK cells.
  • intracellular ice formation in confluent monolayers provides protection against the damaging effects of freezing and thawing, and inducing intracellular ice formation is an effective method for the cryopreservation of confluent cell monolayers. Ln the absence of any chemical cryoprotectant, a significant degree of cell recovery was obtained following the formation of intracellular ice.
  • Fig. 5 The results presented in Fig. 5 further demonstrate that ILF prevents damage against freezing and thawing.
  • the temperature of extracellular ice nucleation was decreased from -5 to -10°C.
  • Nucleation at -10°C resulted in a significant increase in the formation of intracellular ice in V-79W and MDCK single attached cells and V-79W confluent monolayers compared to nucleation at -5°C (Fig. 5).
  • the post-thaw metabolic activity and membrane integrity of V-79W and MDCK single attached cells and confluent monolayers following nucleation (-5 and -10 °C) and graded freezing is shown in Fig. 6 A and B.

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Abstract

La présente invention concerne une méthode de cryopréservation de cellules et de maintien de la viabilité cellulaire. Ladite méthode consiste à prédéterminer la température de nucléation pour la formation de glace extracellulaire et intracellulaire destinée aux cellules, refroidir les cellules à une température de nucléation prédéterminée, nucléer la formation de glace intracellulaire et refroidir les cellules à une température inférieure à la température de nucléation prédéterminée. Dans un autre mode de réalisation, une méthode permet de cryopréserver des cellules et de maintenir une viabilité cellulaire sans utilisation de composés cryoprotecteurs. Ladite méthode consiste à refroidir les cellules dans certaines conditions de manière à optimiser la formation régulée de glace intracellulaire inoffensive.
PCT/US2003/024147 2002-07-30 2003-07-30 Glace intracellulaire inoffensive Ceased WO2004010780A2 (fr)

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AU2003258001A AU2003258001A1 (en) 2002-07-30 2003-07-30 Innocuous intracellular ice

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005118785A1 (fr) * 2004-06-02 2005-12-15 Es Cell International Pte Ltd Methode de conservation cellulaire
AU2005250053B2 (en) * 2004-06-02 2009-08-06 Es Cell International Pte Ltd Cell preservation method
WO2012154324A1 (fr) * 2011-04-29 2012-11-15 Praxair Technology, Inc. Procédé et système de régulation de la nucléation dans le cadre de la cryopréservation de substances biologiques
CN113615681A (zh) * 2021-08-27 2021-11-09 郑州源创吉因实业有限公司 一种免疫细胞的冻存液及冻存方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3842831A (en) * 1972-10-27 1974-10-22 Genetic Labor Inc Cellular skin patch
US5891617A (en) * 1993-09-15 1999-04-06 Organogenesis Inc. Cryopreservation of harvested skin and cultured skin or cornea equivalents by slow freezing
MX9705540A (es) * 1996-01-30 1997-10-31 Organogenesis Inc Metodo y diseño de empaque para la crioconservacion y almacenamiento de equivalentes de tejido cultivado.
US5689961A (en) * 1996-01-30 1997-11-25 Organogenesis Inc. Ice seeding apparatus for cryopreservation systems
US6140123A (en) * 1998-10-07 2000-10-31 Cedars-Sinai Medical Center Method for conditioning and cryopreserving cells

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005118785A1 (fr) * 2004-06-02 2005-12-15 Es Cell International Pte Ltd Methode de conservation cellulaire
GB2429717A (en) * 2004-06-02 2007-03-07 Es Cell Int Pte Ltd Cell preservation method
JP2008501320A (ja) * 2004-06-02 2008-01-24 イーエス・セル・インターナショナル・プライヴェート・リミテッド 細胞保存方法
GB2429717B (en) * 2004-06-02 2009-04-08 Es Cell Int Pte Ltd Cell preservation method
AU2005250053B2 (en) * 2004-06-02 2009-08-06 Es Cell International Pte Ltd Cell preservation method
US9714412B2 (en) 2004-06-02 2017-07-25 Es Cell International Pte Ltd. Cell preservation method for pluripotent stem cells
US10472606B2 (en) 2004-06-02 2019-11-12 Es Cell International Pte Ltd Cell preservation method for pluripotent stem cells
US8794013B2 (en) 2006-02-10 2014-08-05 Praxair Technology, Inc. Method and system for nucleation control in a controlled rate freezer (CRF)
WO2012154324A1 (fr) * 2011-04-29 2012-11-15 Praxair Technology, Inc. Procédé et système de régulation de la nucléation dans le cadre de la cryopréservation de substances biologiques
CN113615681A (zh) * 2021-08-27 2021-11-09 郑州源创吉因实业有限公司 一种免疫细胞的冻存液及冻存方法

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WO2004010780A3 (fr) 2004-05-13
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