WO2019172112A1 - Solution de cryoconservation de cellules et son utilisation - Google Patents

Solution de cryoconservation de cellules et son utilisation Download PDF

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Publication number
WO2019172112A1
WO2019172112A1 PCT/JP2019/008063 JP2019008063W WO2019172112A1 WO 2019172112 A1 WO2019172112 A1 WO 2019172112A1 JP 2019008063 W JP2019008063 W JP 2019008063W WO 2019172112 A1 WO2019172112 A1 WO 2019172112A1
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Prior art keywords
cells
cell cryopreservation
cell
solution
cryopreservation solution
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PCT/JP2019/008063
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English (en)
Japanese (ja)
Inventor
博明 小玉
小川　淳
尚登 山城
孝一 佐瀬
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ゼノアックリソース株式会社
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Priority to JP2020504979A priority Critical patent/JPWO2019172112A1/ja
Publication of WO2019172112A1 publication Critical patent/WO2019172112A1/fr
Priority to JP2023061004A priority patent/JP2023076609A/ja

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to a cell cryopreservation solution and use thereof.
  • cryopreservation has been performed in order to prevent cell deterioration due to passage of cultured cells or contamination by various bacteria and to use the cells for a long time.
  • a general method for cryopreserving cells a method is known in which cells are suspended in a solution containing dimethyl sulfoxide (DMSO) or serum, and cryopreserved (for example, in liquid nitrogen) in a cryotube or ampoule.
  • DMSO dimethyl sulfoxide
  • Patent Document 1 describes that cells were cryopreserved using a cell preservation aqueous solution containing DMSO, a thickener and glucose, and a good survival rate was achieved.
  • DMSO is considered to be the best anti-frost damage agent in terms of cell viability. However, it can affect the expression, differentiation and proliferation of various cell functions. In addition, in hematopoietic stem cell transplantation using a cell cryopreservation solution containing DMSO, side effects such as nausea, vomiting, headache, increased blood pressure, diarrhea, and abdominal cramps have been reported (Non-Patent Document 1, Non-Patent Document 2). ).
  • Non-patent Document 1 Although there is an attempt to reduce the amount of DMSO used by combining other frost damage protective agents (Non-patent Document 1), securing of both performance and safety to the living body by replacement with other frost damage protective agents is still unsuccessful. It is insufficient.
  • the present invention has been made to solve such problems, and an object of the present invention is to provide a cell cryopreservation solution that can achieve both good survival rate and safety to living bodies. .
  • the cell cryopreservation solution comprises at least one saccharide selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose, and maltose, and propylene glycol, dimethyl sulfoxide, a thickener, and natural Contains no animal-derived ingredients.
  • a method for cryopreserving cells includes a mixing step of mixing the cell cryopreservation solution and cells, and freezing in which the cells are mixed with the cell cryopreservation solution. And a process.
  • the method for producing a frozen product of cells includes a mixing step of mixing the cell cryopreservation solution and cells, and freezing the cells in a state of being mixed with the cell cryopreservation solution. Freezing step.
  • the frozen cell product according to one embodiment of the present invention is prepared using the above-described method for preparing a frozen cell material.
  • the survival rate of the cells after thawing is good, and the thawed cells can be safely applied to a living body.
  • the cell cryopreservation solution contains at least one saccharide selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose, and maltose and propylene glycol, dimethyl sulfoxide (DMSO), a thickener, and natural Contains no animal-derived ingredients.
  • a saccharide selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose, and maltose and propylene glycol, dimethyl sulfoxide (DMSO), a thickener, and natural Contains no animal-derived ingredients.
  • DMSO dimethyl sulfoxide
  • the saccharide is at least one selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose, and maltose. That is, there are one, two, three, four, five or six saccharides selected from the group.
  • Glucose is a monosaccharide
  • mannitol and sorbitol are sugar alcohols
  • trehalose, sucrose, and maltose are disaccharides.
  • the saccharide may be D-form, L-form, or a mixture of D-form and L-form, but it should be D-form from the viewpoint of production cost and safety. Is preferred.
  • the saccharide is a monosaccharide.
  • the saccharide is a disaccharide.
  • the saccharide is a sugar alcohol.
  • the saccharide is a mixture of any of monosaccharides, disaccharides, and sugar alcohols.
  • the concentration of the saccharide in the cell cryopreservation solution is not particularly limited, but in one example, it is preferably 0.5 w / v% or more, more preferably 1.0 w / v% or more in terms of monosaccharide. 1.5 w / v% or more is more preferable, 2.0 w / v% or more is particularly preferable, 10.0 w / v% or less is preferable, and 6.0 w / v%. More preferably, it is more preferably 5.0 w / v% or less, still more preferably 4.5 w / v% or less, and particularly preferably 4.0 w / v% or less.
  • the concentration based on “monosaccharide conversion” refers to the concentration calculated by weight for monosaccharides and sugar alcohols, and the concentration calculated by assuming that the weight is half of that for disaccharides. That is, with the disaccharide, the actual concentration (w / v%) is twice the above concentration.
  • the concentration of propylene glycol in the cell cryopreservation solution is not particularly limited, but in one example, it is preferably 2.5 w / v% or more, more preferably 5.0 w / v% or more, and 7.5 w / W or more is more preferable, 10.0 w / v or more is particularly preferable, and from the viewpoint of safety with respect to a living body, 18.0 w / v% or less is preferable, and 16.0 w / v is preferable. It is more preferably v% or less, further preferably 15.0 w / v% or less, and particularly preferably 13.0 w / v% or less.
  • DMSO Dimethyl sulfoxide
  • the thickener examples include carboxymethyl cellulose (hereinafter referred to as CMC), sodium carboxymethyl cellulose (hereinafter referred to as CMC-Na), organic acid polymer, propylene glycol alginate, and sodium alginate.
  • CMC carboxymethyl cellulose
  • CMC-Na sodium carboxymethyl cellulose
  • organic acid polymer propylene glycol alginate
  • propylene glycol alginate propylene glycol alginate
  • sodium alginate sodium alginate.
  • Examples of natural animal-derived components include albumin, serum, plasma, and basal medium.
  • Examples of serum include adult calf serum, calf serum, newborn calf serum, and fetal calf serum.
  • Examples of the basal medium include RPMI medium, MEM medium, HamF-12 medium, DM-160 medium and the like. Since the cell cryopreservation solution of the present invention does not contain natural animal-derived components, there is no problem of quality differences among lots of natural animal-derived components, and various cytokines and growth contained in serum. It is possible to avoid the risk of changes in cell properties due to factors and hormones that are essentially unnecessary for cell preservation, and it is also possible to avoid the effects of components that are unknown in the basal medium.
  • the cell cryopreservation solution of the present invention is very useful from the viewpoint that it can be safely applied to a living body particularly in clinical use. Moreover, as shown in the examples described later, cells can be cryopreserved with a good survival rate even if natural animal-derived components are not contained.
  • the cell cryopreservation solution may further contain other components.
  • other components include a pH adjuster.
  • the pH adjuster include phosphate buffer and carbonate buffer.
  • BSS Basic Stock Solution
  • a solution containing physiological saline can also be used.
  • BSS Basic Stock Solution
  • the pH adjuster is preferably used as appropriate in order to adjust the pH in the cell cryopreservation solution to about 6.5 to 9.0, preferably 7.0 to 8.5.
  • the phosphate buffer in the present invention is sodium chloride, monosodium phosphate (anhydrous), monopotassium phosphate (anhydrous), disodium phosphate (anhydrous), trisodium phosphate (anhydrous), potassium chloride, And potassium dihydrogen phosphate (anhydrous), and sodium chloride, monosodium phosphate (anhydrous), potassium chloride, or potassium dihydrogen phosphate (anhydrous) is particularly preferable.
  • a combination of potassium dihydrogen phosphate, sodium chloride, potassium chloride, and disodium hydrogen phosphate is also preferable.
  • the pH adjuster is preferably contained in the cell cryopreservation solution in an amount of 0.01 to 1.0 w / v%, more preferably 0.05 to 0.5 w / v%.
  • the composition of the inorganic salt solution used in the examples is also suitable.
  • the cell cryopreservation solution is preferably an aqueous solution.
  • the osmotic pressure of the cell cryopreservation solution is preferably 1000 mOsm or more, more preferably 1000 to 2700 mOsm, in order to maintain performance as a preservation solution.
  • composition of the cell cryopreservation solution may be any combination of components among the specific examples of each component listed above as long as the composition can sufficiently preserve the cells.
  • concentrations can be selected and combined.
  • components and / or concentrations in Examples described later can be selected and combined. That is, the individual components and / or concentrations disclosed herein can be selected and combined, respectively.
  • the cell cryopreservation solution is an aqueous solution containing saccharides and propylene glycol and not containing DMSO, thickener and natural animal-derived components.
  • the cell cryopreservation solution is an aqueous solution containing saccharides, propylene glycol and a pH adjuster, and free of DMSO, thickeners and natural animal-derived components.
  • the cell cryopreservation solution is an aqueous solution containing only saccharides, propylene glycol and a pH adjuster and not containing DMSO, thickener and natural animal-derived components (ie, saccharides, propylene glycol, It consists only of pH adjuster and water).
  • the cell cryopreservation solution is preferably sterilized. This is because the risk of infection of bacteria and the like is reduced, so that it can be applied to a living body more safely. Moreover, in research use, the risk of contamination by bacteria or the like is reduced.
  • Glucose, mannitol, sorbitol, trehalose, sucrose, and maltose have been used as components of intravenous injection solutions.
  • 5% glucose injection is usually intravenously injected at 500 to 1000 mL per adult due to water supply, drug / toxic poisoning, and liver disease.
  • the six saccharides are very safe for living bodies.
  • propylene glycol is sometimes used as a solubilizing agent in an injection when the active ingredient is hardly soluble in a solvent.
  • the maximum amount used in the human body is 3.2 g by intravenous injection.
  • propylene glycol is very safe for living organisms.
  • the cells to be cryopreserved are not particularly limited.
  • cell lines of various organisms lymphoid cells, spleen cells, thymocytes, fertilized eggs, hematopoietic stem cells, adult stem cells, mesenchymal stem cells, embryos Sex stem cells (ES cells) and induced pluripotent stem cells (iPS cells).
  • Examples of the living organism include humans and non-human animals, and more specifically, insects, fish, amphibians, reptiles, birds, mammals, and the like. Mammals include laboratory animals such as primates excluding mice, rats, rabbits, guinea pigs and humans; pets such as dogs and cats (pets); domestic animals such as pigs, cows, goats, sheep and horses; Can be mentioned.
  • the cell cryopreservation solution of the present invention varies depending on the cells to be stored, but can achieve a good survival rate after, for example, one week of storage or more (for example, after 10 years). .
  • the survival rate after thawing may be 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, and particularly preferably 90% or more.
  • the cell cryopreservation solution of the present invention has an advantage that a good survival rate can be achieved, it can be used as an excellent cell cryopreservation solution even when it is not applied to a living body (for example, for research use). it can.
  • the cell cryopreservation solution of the present invention may be combined with, for example, a cold-resistant container, a temperature-controllable freezer or a slow-freezing container, or an instruction manual.
  • the instructions for use of the kit include, for example, the contents of the cryopreservation method according to the present invention described in the section of [Method for cryopreserving cells] described below, and / or [Method for preparing a frozen product of cells] described below. At least a part of the content of the manufacturing method according to the present invention described in the column is recorded.
  • the method of cryopreserving cells according to the present invention includes a mixing step of mixing the above-described cell cryopreservation solution and cells, and a freezing step of freezing the cells in a state of being mixed with the cell cryopreservation solution; ,including.
  • the number of cells per 1 mL of the cell cryopreservation solution is not particularly limited, but it is preferably 10 3 to 10 9 cells / mL, more preferably 10 4 to 10 8 cells / mL. Is preferred.
  • the cells Before the mixing step, the cells may be washed with a washing solution such as PBS (phosphate buffered saline). Thereby, for example, mixing of components such as a culture medium can be further reduced.
  • a washing solution such as PBS (phosphate buffered saline).
  • the mixture may be transferred to a cold resistant container such as an ampoule or cryotube.
  • the cold-resistant container is preferably sterilized inside.
  • the cooling rate is not particularly limited, but from the viewpoint of further increasing the survival rate, it is preferably cooled at a slow rate, more preferably at a cooling rate of about -1 ° C / min. Therefore, the freezing step is preferably performed using a temperature-controllable freezer or slow freezing container.
  • slow freezing containers include Corning's CoolCell (registered trademark), Nalgene's Mr. Frosty, and Nihon Freezer Corporation's Bicell.
  • the final freezing temperature is not particularly limited, but is preferably ⁇ 80 ° C. or lower, more preferably ⁇ 150 ° C. or lower, and further preferably ⁇ 196.5 ° C. or lower. Further, after storing at around ⁇ 80 ° C., it may be transferred to ⁇ 180 ° C. to ⁇ 200 ° C. (for example, in liquid nitrogen) for storage.
  • cells cryopreserved using the cryopreservation method according to the present invention can exhibit a good survival rate after thawing (see also examples described later).
  • Thawing is preferably performed quickly, for example, by immersing in a water bath at 37 ° C. ⁇ 1 ° C.
  • the cell cryopreservation solution may or may not be removed.
  • the thawed mixture can be applied to a living body without removing the cell cryopreservation solution. Since the cell cryopreservation solution of the present invention can be composed of components with extremely high safety to living organisms, it can be directly applied to living organisms. Therefore, it can be possible to perform cell transplantation easily and quickly at the site of cell transplantation (operating room).
  • the thawed mixture may be added to a culture medium or the like, and used after the cell cryopreservation solution is removed by centrifugation or washing. The liquid used for washing may be appropriately selected according to the content of subsequent treatment and the intended use of the cells. For example, culture medium, physiological saline, PBS, or the like may be used.
  • the cell cryopreservation solution in a research scene where it is desired to strictly control the components during cell culture.
  • the thawed mixture can be used for experiments such as cell culture without removing the cell cryopreservation solution. Since the cell cryopreservation solution of the present invention can be composed of components with extremely high safety against cells, cell culture can be suitably performed by diluting with a medium or the like without washing.
  • the method for producing a frozen product of cells according to the present invention includes a mixing step of mixing the above-described cell cryopreservation solution and cells, and freezing in which the cells are frozen in a state mixed with the cell cryopreservation solution. And a process.
  • a frozen product of cells produced using the production method according to the present invention can exhibit a good survival rate after thawing (see also examples described later).
  • the survival rate after thawing may be 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, and particularly preferably 90% or more.
  • the frozen material can be, for example, a cell preparation, a normal tissue or a cancer tissue having a size within several cm.
  • the cells contained in the cell preparation for example, mesenchymal stem cells, hematopoietic stem cells, lymphocyte cells, dendritic cells, nervous system cells, keratinocytes, and fibroblasts are preferable.
  • the cell cryopreservation solution comprises at least one saccharide selected from the group consisting of glucose, mannitol, sorbitol, trehalose, sucrose, and maltose, and propylene glycol, dimethyl sulfoxide, a thickener, and natural Contains no animal-derived ingredients.
  • the propylene glycol concentration is preferably 5.0 w / v% or more.
  • the propylene glycol concentration is more preferably 10.0 w / v% or more and 15.0 w / v% or less.
  • the concentration of the saccharide is preferably 1.0 w / v% or more and 5.0 w / v% or less in terms of monosaccharide.
  • the concentration of the saccharide is preferably 2.0 w / v% or more and 4.0 w / v% or less in terms of monosaccharide.
  • the cell cryopreservation solution preferably further contains a pH adjuster.
  • a method for cryopreserving cells includes a mixing step of mixing the cell cryopreservation solution and cells, and freezing in which the cells are mixed with the cell cryopreservation solution. And a process.
  • the method for producing a frozen product of cells includes a mixing step of mixing the cell cryopreservation solution and cells, and freezing the cells in a state of being mixed with the cell cryopreservation solution. Freezing step.
  • the freezing step is preferably performed using a temperature-controllable freezer or a slow freezing container.
  • the frozen cell product according to one embodiment of the present invention is prepared using the above-described method for preparing a frozen cell material.
  • ⁇ Used cells> Jurkat: Human T cell line leukemia cell line P3U1: Mouse myeloma cell line Mesenchymal stem cells: MSenchymal stem cells (MSC) (Source: National Institute of Biomedical Innovation, Health and Nutrition JCRB Cell Bank) iPS cells (induced pluripotent stem cells): 201B7 strain (source: iPS Academia Japan)
  • ⁇ Test method> [Freeze storage] 1. Various cells were collected from the culture flask, and viable cells were counted by trypan blue staining. 2. A required amount of cell suspension was dispensed into a 15 mL falcon tube, centrifuged at 1,200 rpm ⁇ 5 minutes (4 ° C.), and the supernatant was removed. 3. 3 mL (iPS cells: 0.6 mL) of each cell cryopreservation solution was added to suspend the pellet. 4). 1 mL (iPS cells: 0.2 mL) of cell suspension was dispensed into 1.5 mL cryotubes.
  • the number of cells was 3 ⁇ 10 6 cells / tube (Jurkat), 1 ⁇ 10 6 cells / tube (MSC and P3U1), or 2 ⁇ 10 5 cells / tube (iPS). 5.
  • Cryotubes were placed in Mr. Frosty (Nalgene), which had been ice-cooled in advance, and stored frozen in a ⁇ 80 ° C. freezer overnight. 6). The next day, the cryotube was transferred to a -152 ° C freezer and stored frozen for over 1 week.
  • Solution 1-A 3.0 w / v% glucose
  • Solution 1-B 3.0 w / v% glucose + 10.0 w / v% DMSO (MW 78.13)
  • Solution 1-C 3.0 w / v% glucose + 10.0 w / v% glycerol (MW 92.09)
  • Solution 1-D 3.0 w / v% glucose + 10.0 w / v% propylene glycol (MW 76.09)
  • Solution 2-A 2.5 w / v% propylene glycol + 3 w / v% glucose
  • 2-B 5.0 w / v% propylene glycol + 3 w / v% glucose
  • 2-C 7.5 w / v% propylene glycol + 3 w / V% glucose solution
  • 2-D 10.0 w / v% propylene glycol + 3 w / v% glucose solution
  • 2-E 12.5 w / v% propylene glycol + 3 w / v% glucose solution
  • 2-F 15.0 w / v % Propylene glycol + 3 w / v% glucose
  • Solution 3-A 10.0 w / v% propylene glycol Solution 3-B: 3.0 w / v% glucose (MW 180.2) + 10.0 w / v% propylene glycol Solution 3-C: 3.0 w / v% mannitol ( MW 182.2) +10.0 w / v% propylene glycol solution 3-D: 3.0 w / v% sorbitol (MW 182.2) +10.0 w / v% propylene glycol solution 3-E: 6.0 w / v% trehalose (MW 342.3 ) + 10.0 w / v% propylene glycol solution 3-F: 6.0 w / v% sucrose (MW 342.3) + 10.0 w / v% propylene glycol solution 3-G: 6.0 w / v% maltose (MW342.3) + 10.0w / v% propylene glycol solution 3-G: 6.0 w / v% maltose
  • Solution 4-A 10.0 w / v% propylene glycol Solution 4-B: 1.0 w / v% glucose + 10.0 w / v% propylene glycol Solution 4-C: 2.0 w / v% glucose + 10.0 w / v % Propylene glycol solution 4-D: 3.0 w / v% glucose + 10.0 w / v% propylene glycol solution 4-E: 4.0 w / v% glucose + 10.0 w / v% propylene glycol solution 4-F: 5. 0 w / v% glucose + 10.0 w / v% propylene glycol solution 4-G: 6.0 w / v% glucose + 10.0 w / v% propylene glycol
  • Solution 5-A 10.0 w / v% propylene glycol + 3.0 w / v% glucose (MW 180.2)
  • Solution 5-B 3.0 w / v% glucose (MW 180.2)
  • Solution 5-C 3.0 w / v% mannitol (MW 182.2)
  • Solution 5-D 3.0 w / v% sorbitol (MW 182.2)
  • Solution 5-E 6.0 w / v% trehalose (MW 342.3)
  • Solution 5-F 6.0 w / v% sucrose (MW 342.3)
  • Solution 5-G 6.0 w / v% maltose (MW 342.3)
  • the present invention can be used for cryopreservation of cells in medicine and research.

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Abstract

L'invention concerne une solution de cryoconservation de cellules qui peut atteindre à la fois une bonne viabilité et une bonne sécurité par rapport à un corps vivant. Cette solution de cryoconservation de cellules comprend : au moins un sucre choisi dans le groupe constitué du glucose, du mannitol, du sorbitol, du tréhalose, du saccharose et du maltose ; et du propylène glycol, et ne comprend pas de diméthylsulfoxyde, d'épaississant et de composant dérivé d'un animal naturel.
PCT/JP2019/008063 2018-03-06 2019-03-01 Solution de cryoconservation de cellules et son utilisation WO2019172112A1 (fr)

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JP2020504979A JPWO2019172112A1 (ja) 2018-03-06 2019-03-01 細胞凍結保存用溶液およびその利用
JP2023061004A JP2023076609A (ja) 2018-03-06 2023-04-04 細胞凍結保存用溶液およびその利用

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Publication number Priority date Publication date Assignee Title
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