WO2012067240A1 - 細胞のガラス化保存液 - Google Patents
細胞のガラス化保存液 Download PDFInfo
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- WO2012067240A1 WO2012067240A1 PCT/JP2011/076711 JP2011076711W WO2012067240A1 WO 2012067240 A1 WO2012067240 A1 WO 2012067240A1 JP 2011076711 W JP2011076711 W JP 2011076711W WO 2012067240 A1 WO2012067240 A1 WO 2012067240A1
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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
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
Definitions
- the present invention relates to a preservation solution for vitrifying cells for cryopreservation. Specifically, the present invention relates to a cell vitrification preservation solution that is less likely to cause a decrease in survival rate due to operation time and can be suitably used for primate ES cells or primate iPS cells.
- BACKGROUND ART Conventionally, in order to prevent deterioration due to passage of cultured cells and contamination by various bacteria and to use the cells for a long period of time, the cells are routinely stored frozen. When cells are frozen as they are, ice crystals form inside and outside the cells, and the cells are physically damaged and die. In particular, there are many structures essential for life activity in the cell, and ice crystal formation in the cell is fatal to the cell. In order to prevent this damage, it is necessary to store using a storage solution containing a cell membrane-permeable cryoprotectant such as dimethyl sulfoxide (DMSO), and the development of a storage solution having excellent cell storage efficiency is eagerly desired.
- DMSO dimethyl sulfoxide
- cell freezing preservation methods are roughly classified into a slow freezing method and a vitrification method.
- the “slow freezing method” is a method in which cells are suspended in a preservation solution in which about 5 to 20% by volume of a cryoprotectant is added to a medium or serum, and after the permeation of the protectant into cells, the temperature is controlled gradually. This is a method of lowering and finally storing in liquid nitrogen ( ⁇ 196 ° C.).
- both the intracellular fluid and the extracellular fluid are supercooled.
- the supercooled state is released when a certain temperature is reached, and ice crystals are formed. This temperature is determined by the concentration of the solution and the type of substance serving as a nucleus, and is generally higher in the extracellular fluid than in the intracellular fluid.
- the cooling rate is moderate, ice crystals are first formed outside the cells. Since only water freezes, the extracellular fluid is gradually concentrated and becomes hypertonic. Along with this, the intracellular water moves out of the cell due to the osmotic pressure difference with the intracellular fluid. This is a so-called “dehydration” phenomenon. Due to this dehydration, the concentration of the intracellular fluid containing the cryoprotectant is increased, and the intracellular fluid is vitrified.
- vitrification will be described.
- a liquid is understood as a state in which thermal vibration exceeds intermolecular forces and particles in the phase can move freely.
- thermal vibration falls below intermolecular force at a certain temperature, and particles in the phase lose fluidity.
- the particles are rearranged into an energetically stable crystal structure.
- water crystallizes into ice usually at a low temperature of 0 ° C. or lower.
- the viscosity of the liquid increases without being crystallized even when the temperature is lowered, and may become solid as it is. This phenomenon is called vitrification.
- glass mainly composed of silicon dioxide is known as a typical glass material.
- Vitrification is greatly affected by the cooling rate. Generally, the larger the cooling rate, the easier it is to vitrify.
- the temperature at which vitrification occurs is called the glass transition point, and varies depending on the type of substance and the cooling rate. It is known that water may be vitrified depending on conditions. For example, it is known that vitrification tends to occur when a material that easily vitrifies such as glycerin is mixed.
- the viability of the cells is maintained even after thawing by vitrifying the intracellular fluid and suppressing the formation of ice crystals.
- cells that can be efficiently stored by the slow freezing method are limited to some cells such as cell lines, and efficient storage is possible among primary cells, normal cells, germ cells, and embryonic stem cells (ES cells). It is known that there are difficult cells.
- ES cells embryonic stem cells
- the “vitrification method” refers to the vigorous application of vitrification to the preservation of cells, and is often used for the preservation of cells that cannot be efficiently preserved by the slow freezing method, such as eggs, fertilized eggs, and embryos. ing.
- the vitrification method cells are suspended in a vitrification preservation solution containing about 30 to 50% by volume of a cryoprotectant, and the cells are placed in liquid nitrogen and rapidly cooled to below the glass transition point. This is a method of storing the inside and outside of the glass by vitrification. In the sense of comparison with the slow freezing method described above, it may be referred to as “rapid freezing method”.
- save liquid used for the vitrification method may be called VS (vitrification solution).
- the intracellular solution is concentrated as a result of dehydration, and is thought to eventually become vitrified.
- the slow freezing method it is considered that cells are damaged by minute ice crystals formed outside the cells.
- the inside of the cell may not be sufficiently dehydrated, and ice crystals may be formed in the cell.
- the extracellular fluid that is, the preservation solution contains a high concentration of cryoprotectant, it vitrifies without forming ice crystals. Also, the intracellular fluid is immediately dehydrated with a high osmotic pressure preservation solution, replaced with a cryoprotectant, and vitrified.
- the vitrification method was developed for cells and the like that are difficult to store efficiently by the slow freezing method, and is industrially used for the preservation of in vitro fertilized embryos in cattle.
- improvements have been made using mouse and rat embryos as materials.
- “DAP213” containing dimethyl sulfoxide, acetamide, and propylene glycol at concentrations of 2M, 1M, and 3M, respectively, is known as a typical vitrification preservation solution used for embryo vitrification.
- iPS cells induced pluripotent stem cells having almost the same properties as ES cells have been developed, and it is common to preserve human iPS cells by vitrification.
- DAP213 is also widely used as a vitrification preservation solution for primate ES / iPS cells.
- RIKEN CDB RIKEN Research Center for Developmental Sciences
- the vitrification method using DAP213 has become a standard preservation method for primate ES / iPS cells in Japan as a simple preservation method with high survival rate.
- RIKEN CDB and RIKEN The BioResource Center recommends this as a method for preserving primate ES / iPS cells.
- DAP213 includes acetamide, which is a substance suspected to be carcinogenic. Acetamide is not necessarily suitable for use as a component of a preservation solution for preserving cells for regenerative medicine.
- WO2007 / 058308 pamphlet includes a thickener, a frost damage preventive agent, a saccharide, a natural animal-derived component, preferably a phosphate buffer, and preferably an osmotic pressure of 1000 mOsm or more.
- An aqueous solution for storage is described.
- the aqueous solution for cell preservation here is a preservation solution by a so-called slow freezing method and is different from a vitrification preservation solution.
- the osmotic pressure referred to here relates to the total osmotic pressure.
- JP-T-2001-502664 International Publication WO98 / 09514 pamphlet
- JP-T-2001-517204 International Publication WO97 / 45010 pamphlet
- a preservation solution comprising a cryoprotectant, an impermeable cryoprotectant, and an impermeable auxiliary solute is disclosed.
- cell shrinkage is detrimental, so that it should be dehydrated by a method that does not reduce the cell volume, and cell shrinkage should be avoided as much as possible.
- Japanese Patent No. 3694730 discloses at least one cell membrane permeable substance selected from methanol or ethanol, dimethyl sulfoxide, ethylene glycol, propylene glycol, 1,3-butanediol, 2,3-butanediol, and glycerol. Containing at least one substance capable of inhibiting the formation of ice crystals selected from the group consisting of: and at least one cell membrane impermeable dehydration promoting substance selected from the group consisting of sucrose, trehalose, raffinose, lactose, and fructose
- a tissue cryopreservation solution is disclosed (claim 1 of the same publication). However, this preservation solution is mainly intended for tissues and organs, particularly reproductive organs, and contains methanol or ethanol as an essential component.
- the present inventor has generally used a vitrification preservation solution for cells comprising a cell membrane permeable substance and a cell membrane impermeable substance, and the total permeation generated in the cell membrane of the cells when the cells are suspended in the vitrification preservation solution.
- a vitrification preservation solution for cells comprising a cell membrane permeable substance and a cell membrane impermeable substance, and the total permeation generated in the cell membrane of the cells when the cells are suspended in the vitrification preservation solution.
- an object of the present invention is to provide a vitrification preservation solution for cells excellent in operability and safety.
- the cell vitrification preservation solution according to the present invention is a cell vitrification preservation solution comprising a cell membrane permeable substance and a cell membrane non-permeability substance,
- the content of the cell membrane permeable substance is 30-50% by volume,
- the osmotic pressure generated due to the cell membrane impermeable substance is 280 mOsm or more.
- the cell membrane permeable substance contains ethylene glycol (EG), propylene glycol (PG), 1,3-propanediol (1,3-PD). ), Butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol (DPG), glycerin, and dimethyl sulfoxide (DMSO).
- EG ethylene glycol
- PG propylene glycol
- BG Butylene glycol
- IPG isoprene glycol
- DPG dipropylene glycol
- DMSO dimethyl sulfoxide
- the cell membrane permeable substance is selected from the group consisting of ethylene glycol (EG), propylene glycol (PG), and dimethyl sulfoxide (DMSO). 1 type or 2 types.
- the cell membrane impermeable substance is sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, a monosaccharide, One or more selected from the group consisting of disaccharides, polysaccharides, trisaccharides, sugar alcohols, ficoll, polyethylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone.
- the cell membrane impermeable substance comprises sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate.
- the cell membrane impermeable substance comprises 0.2M to 1M trehalose as a concentration in the preservation solution.
- the vitrification cell preservation solution according to the present invention further comprises an ice crystal growth inhibitor selected from the group consisting of antifreeze protein and sericin.
- the vitrification medium of the present invention is used for vitrification preservation of pluripotent stem cells, more preferably primate ES cells or primate iPS cells (primate ES cells). / IPS cells).
- the method includes suspending the cells in the vitrification solution for cells according to the present invention, and then rapidly cooling the vitrification solution containing the cells with liquid nitrogen to vitrify the cells.
- a cell vitrification method comprising: A method is provided in which, of the total osmotic pressure generated in the cell membrane of the cells when the cells are suspended in the vitrification preservation solution, the osmotic pressure generated due to the cell membrane impermeable substance is 280 mOsm or more.
- a vitrification preservation solution that is excellent in operability and safety and can efficiently preserve cells, particularly primate ES / iPS cells.
- an engraftment rate of about 60% could be obtained even after 60 seconds had passed and then put into liquid nitrogen (described later).
- “engraftment rate” is used instead of “survival rate.”
- the engraftment rate is determined after 3 or 4 days after seeding the cells without vitrification. The number of undifferentiated colonies engrafted is calculated as 100).
- the vitrification preservation solution of the present invention does not require the speed of work as with DAP213, variations due to the technical level of the operator are less likely to occur, and a stable result can be desired. Furthermore, according to the present invention, since acetamide suspected of carcinogenicity is not included, it is excellent in safety. Since the vitrification preservation solution of the present invention is useful for preserving primate ES cells and primate iPS cells, it is extremely important that the use of acetamide suspected of carcinogenicity can be avoided in consideration of application to the medical field. It is.
- FIG. 10 is a graph showing the results of Example 3. It is a graph which shows the result which the trehalose and osmotic pressure of Example 5 exert on the survival rate after vitrification.
- Example 7 It is a graph which shows the result which acts on the survival rate after vitrification of the osmotic pressure with respect to DAP of Example 6. It is a graph which shows the CMES engraftment rate of Example 7. It is a graph which shows the result which the trehalose and osmotic pressure of Example 7 have on the survival rate after vitrification. It is a graph which shows the result of the performance evaluation of the vitrification preservation solution for CMES cells of Example 7. 10 is a graph showing the results of examination of an ice crystal growth inhibitor in Example 8.
- the osmotic pressure is proportional to the molar concentration.
- the solute is ionized in a solvent like a salt, the volume molarity is considered from the number of particles after ionization.
- the amount of a solute substance that gives an osmotic pressure equal to a 1 M solution of an ideal solute that is not ionized is defined as 1 Osm, and generally Osm is also used as a unit of osmotic pressure.
- the osmotic pressure value (unit: Osm / kg) obtained from the freezing point depression degree is true as the solution concentration increases. Move away from the osmotic pressure value.
- the vitrification preservation solution according to the present invention is a relatively concentrated aqueous solution containing 30 to 50% by volume of a cell membrane permeable substance (particularly, a cryoprotectant) on a volume basis
- the unit of osmotic pressure in this specification is It means Osm / L. For simplicity, it may be expressed as Osm.
- the cell membrane freely permeates water but does not permeate macromolecules and ions, and thus exhibits a property close to that of a semipermeable membrane. Therefore, when a difference in concentration occurs between the solution inside and outside the cell, water moves due to osmotic pressure.
- the solution when a cell is immersed in a solution, when the apparent water movement does not occur and the volume of the cell is not changed, the solution is said to be “isotonic”.
- the solution is said to be “hypertonic”
- the solution is said to be “hypotonic”.
- a low solution is called a “hypotonic solution”.
- an isotonic solution is isotonic
- a hypotonic solution is hypotonic
- a hypertonic solution is hypertonic.
- a cell membrane permeable substance is contained in the solute, these relationships may be disrupted.
- the difference in the osmotic pressure inside and outside the cell due to the cell membrane permeable substance becomes smaller as the cell membrane permeable substance penetrates into the cell, and no difference occurs at equilibrium. For this reason, the volume of cells at equilibrium is determined by the concentration of the non-permeable substance in the extracellular fluid.
- an isotonic solution refers to a solution of about 270 to 300 mOsm, and is generally prepared using salts or sugars as the main solute.
- salts or sugars For example, in the case of physiological saline using sodium chloride as a solute, it is about 0.9% by weight aqueous solution.
- Other isotonic solutions used when handling biomaterials in vitro include phosphate-buffered saline (PBS), Ringer's solution, and many other salts, including sugars, sugars, amino acids, or proteins.
- PBS phosphate-buffered saline
- Ringer's solution Ringer's solution
- Tension solutions are known. The osmotic pressure of the medium used for cell culture is adjusted to be isotonic with the cells.
- the osmotic pressure is defined using an ideal semipermeable membrane (a membrane that only allows the solvent to permeate but not the solute).
- a cell membrane in addition to water as a solvent, low molecules can permeate.
- water pores called aquaporins exist for permeation of water on the cell membrane. Therefore, the water permeability is high compared to other molecules.
- Whether or not a molecule can permeate the cell membrane is determined by the size of the molecule and the presence or absence of charge or polarity. Charged ions and the like cannot pass through the cell membrane by passive diffusion, whereas small molecules without charge are likely to pass through the cell membrane. For this reason, these cell membrane permeable substances do not affect the volume at equilibrium, although there are differences in the diffusion rate due to differences in molecular weight and polarity.
- dimethyl sulfoxide, ethylene glycol, propylene glycol, and the like used as a cryoprotectant are known as cell membrane permeable substances, and are generally considered not to affect the cell volume during equilibrium.
- freeze may mean ice crystal formation
- vitrification method may be defined not as a cryopreservation method but as an ultra-low temperature storage method, but in this specification “freezing” means , Meaning that the substance changes from liquid to solid upon cooling.
- melting here means that a substance changes from solid to liquid by heating.
- total osmotic pressure refers to an osmotic pressure generated based on the entire vitrified preservative solution between cells inside and outside the cell membrane when the cells are suspended in the vitrification preservative solution.
- the osmotic pressure inside the cell increases, and the cell increases in volume (hereinafter this stage may be referred to as the “diffusion phase”). ).
- the volume change of the cells stops (hereinafter, this may be referred to as “equilibrium phase by preservation solution” or simply “equilibrium phase”). .
- the volume of cells at equilibrium depends on the concentration of the cell membrane impermeable substance in the extracellular fluid (preservation solution). If the osmotic pressure of the cell membrane impermeable substance in the extracellular fluid is “isotonic”, the cell is physiological.
- the amount of the cell membrane permeable substance present in the cells was examined.
- the amount of the cell membrane permeable substance in the cell increases with the passage of time from suspending the cell in the preservation solution to vitrification, and is maximized at the time of equilibrium (see FIG. 2).
- the amount of the cell membrane permeable substance at equilibrium is the product of the concentration of the cell membrane permeable substance and the cell volume, and the concentration of the cell membrane permeable substance is equal to the concentration of the cell membrane permeable substance in the preservation solution.
- the cell volume at equilibrium depends on the osmotic pressure of the cell membrane impermeable substance in the preservation solution (“A”, “B”, and “C” in FIG. 2 are the alphabets A in FIG. 1). , B, C).
- the volume change of the cells over time when the vitrified cells were thawed was as shown in FIG.
- Thawing is performed by adding a warmed diluent (referring to a solution for dilution, usually a medium used for culturing after thawing).
- the vitrified intracellular fluid contains a cell membrane permeable substance and has a high osmotic pressure. Therefore, at the initial stage of thawing, movement of water into the cell and diffusion of the cell membrane permeable substance to the outside of the cell occur simultaneously. Again, since the speed of water movement is higher, the cells temporarily expand (hereinafter sometimes referred to as the “water absorption period”).
- the amount of the cell membrane permeable substance present in the cell is considered to be determined by the time until vitrification, the concentration of the cell membrane permeable substance in the vitrification preservation solution, and the osmotic pressure of the cell membrane impermeable substance as described above. It was.
- A, B, and C in FIG. 3 indicate changes in volume at the time of thawing of vitrified cells at timings corresponding to the alphabets in FIG. That is, A: When the cells are vitrified within 15 seconds after being suspended in the vitrification preservation solution, the amount of the cell membrane permeable substance in the cells is small, so the volume change of the cells is small. B: When cells are equilibrated after being equilibrated with a hypotonic vitrification preservation solution, since the cells are expanded, there are many cell membrane permeable substances in the cells, and the cell volume is expanded by water absorption. Here, when the cell volume exceeds the limit volume, the cell bursts. C: When cells are equilibrated after equilibration with a hypertonic vitrification preservation solution, the amount of the cell membrane permeable substance in the cells is suppressed because the cells contract, and the cells do not expand so much.
- the osmotic pressure inside the cell decreases, and the cell decreases in volume (hereinafter sometimes referred to as “diffusion phase”).
- the concentration of the cell membrane permeable substance inside and outside the cells becomes equal, the change in the volume of the cells stops (sometimes referred to as “equilibrium phase with diluent” or simply “equilibrium phase”).
- the cryoprotectants included in DAP213 are 2M dimethyl sulfoxide, 1M acetamide, and 3M propylene glycol, but none of these ionize, so the contribution to osmotic pressure is 6000 mOsm. Become.
- DAP213 is composed of about 60% by volume of a medium, and if the osmotic pressure of the medium is estimated to be 300 mOsm for convenience, the contribution of the solute molecules in the medium to the osmotic pressure of the entire DAP213 is about 180 mOsm. When these are added together, the osmotic pressure of the entire DAP 213 can be estimated to be about 6180 mOsm.
- DAP213 behaves as a hypotonic solution for cells and expands cells at equilibrium.
- the decrease in the survival rate with the passage of time seen in the preservation of ES / iPS cells using DAP213 is due to the fact that the cells approached an equilibrium state before being put into liquid nitrogen, and the permeability of the cell membrane into the cell is increased. I thought it might be due to the increased amount of material. That is, if the time after suspending the cells in the vitrification preservation solution is 15 seconds or less, the amount of the cell membrane permeable substance in the cells is small at the time of vitrification, and the water into the cells due to the osmotic pressure difference at the time of thawing. Movement and accompanying volume change can be tolerated.
- DAP213 when the basal medium of DAP213 was replaced with PBS so that the osmotic pressure (hypotonicity) was almost the same as that of DAP213 (DAP-A), the final concentration of the salt was taken into account when the solution was diluted with a protective agent. Alive when prepared to be equal (isotonic) to the physiological environment (DAP-B) and prepared to have a final salt concentration twice that of the physiological environment (hypertonic) (DAP-C). The rate was reduced by 15-20%. From this, it was shown that the viability of the cells was lowered upon dilution even when the solution was not vitrified with a hypotonic preservation solution. Moreover, almost no dead cells that were positive for trypan blue were observed.
- the present inventors have also examined protection from crystallization associated with temperature rise during vitrification. That is, the vitrification state is stable at temperatures below the glass transition point, but when the temperature rises above the glass transition point, it easily escapes from the glass state and transitions to the crystalline state. This is thought to be because the molecules gain kinetic energy and rearrange (crystallize) spontaneously to a stable state due to an increase in temperature, but the ice crystals generated at this time can damage the cells. is there. When the phase transition from the glass state to the crystal state, a phenomenon called “devitrification” is observed in which the appearance changes from transparent to opaque due to light being refracted and scattered at many crystal interfaces.
- the glass transition point of a vitrification preservation solution is generally lower than -80 ° C.
- vitrified cells when vitrified cells are transferred to -80 ° C and stored, they are devitrified, and almost all cells are killed by ice crystal damage. To do. Since the devitrification phenomenon is observed when the vitrified cells are thawed, the cells are considered to have been damaged by ice crystals for a short time.
- the present inventors have also found that by adding sericin or antifreeze protein as an ice crystal growth inhibiting substance, ice crystals can be prevented from growing greatly during crystal transition and damage to cells can be reduced. It was. The present invention is further based on such findings.
- the cell vitrification preservation solution according to the present invention is a cell vitrification preservation solution comprising a cell membrane permeable substance and a cell membrane impermeable substance,
- the content of the cell membrane permeable substance is 30-50% by volume,
- the osmotic pressure generated due to the cell membrane impermeable substance is 280 mOsm or more.
- the vitrification preservation solution for cells can be paraphrased as a composition for preservation of vitrification of cells, particularly a liquid composition for preservation of vitrification of cells.
- the cells to be preserved are not particularly limited as long as they can be preserved using the vitrification preservation solution of the present invention.
- cell lines, primary cells, normal cells, germ cells, ES cells, iPS A cell etc. can be mentioned.
- biological tissues such as islets can be stored.
- biological species include microorganisms, plants, and animals, and examples of animal species include mammals, birds, fish, and insects.
- rodents such as mice, primates such as monkeys and humans.
- ES / iPS cells that have been difficult to efficiently preserve by slow freezing or vitrification using DAP213 are preferred, and primate ES / iPS cells (primate ES cells or primate iPS cells) are preferred. More preferred.
- Cell membrane permeable substance in the present invention, a cell membrane permeable substance is used for the purpose of vitrifying a preservation solution and an intracellular solution as a cryoprotectant.
- cell membrane permeable substances include ethylene glycol (EG), propylene glycol (PG), 1,3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), and dipropylene glycol.
- diols such as (DPG), triols such as glycerin, dimethyl sulfoxide (DMSO), etc., which are substances that achieve the above-mentioned purpose by permeating the cell membrane, and are toxic to cells and living bodies.
- the cell membrane permeable substance is ethylene glycol (EG), propylene glycol (PG), 1,3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol.
- EG ethylene glycol
- PG propylene glycol
- BG butylene glycol
- IPG isoprene glycol
- DPG dimethyl sulfoxide
- EG, PG, glycerin, and DMSO are preferably used as the cell membrane permeable substance for reasons such as glass forming ability, cell membrane permeability, protective effect on cells, and use results in preservation solutions. More preferably, PG and DMSO are used, and EG and PG are more preferable. These can be used in combination of two or more. As a combination, a combination of DMSO and PG, DMSO and EG, or EG and PG is preferable. EG alone is preferred.
- the content of the cell membrane permeable substance is typically 30 to 50% by volume, preferably 33 to 47% by volume, more preferably 35 to 45% by volume.
- the preservation solution does not vitrify, and ice crystals may be formed to damage the cells.
- the content exceeds 50% by volume, the cell structure is destroyed by excessive dehydration before vitrification, or the physiological response requiring water is stagnated, resulting in a decrease in cell viability. there's a possibility that.
- an excessive osmotic pressure difference is generated at the time of melting, and the cell membrane may be destroyed.
- the volume ratio is preferably 1: 5 to 2: 1, and 1: 3 to 1: 1. Is more preferable.
- the volume ratio is preferably 1: 5 to 2: 1, more preferably 1: 3 to 1: 1.
- the volume ratio is preferably 1: 100 to 100: 1, and more preferably 1:10 to 10: 1.
- the contribution to the osmotic pressure when the cell membrane permeable substance is contained at 30 to 50% by volume greatly differs depending on the molecular weight of the cell membrane permeable substance, and thus cannot be defined unconditionally.
- osmosis when EG is used alone The contribution to pressure is usually 5000 to 9000 mOsm, and the contribution to osmotic pressure when PG is used alone is usually 4000 to 7000 mOsm.
- DMSO and PG are used in a volume ratio of 1: 5 to 2: 1
- the contribution to the osmotic pressure is usually 4000 to 7000 mOsm
- DMSO and EG have a volume of 1: 5 to 2: 1.
- the contribution to osmotic pressure when used in a ratio is usually 4000 to 9000, and the contribution to osmotic pressure when EG and PG are used in a volume ratio of 1: 100 to 100: 1 is usually 4000 to 9000. 9000.
- the cell membrane impermeable substance is used for the purpose of adjusting osmotic pressure, adjusting to a physiological environment, and promoting vitrification. Therefore, the cell membrane impermeable substance can include a substance for adjusting to a physiological environment (physiological environment adjusting substance) and a substance for promoting vitrification (vitrification promoting substance).
- substances for adjusting to a physiological environment include salts such as sodium salt, potassium salt and phosphate, sugars such as glucose and sucrose, proteins such as albumin, minerals such as magnesium and calcium, and the like. Can do. Of these, sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate are preferably used.
- substances for promoting vitrification include monosaccharides such as glucose, galactose, and fructose, disaccharides such as trehalose, sucrose, and maltose, trisaccharides such as raffinose, polysaccharides such as cellulose and starch, xylitol, and sorbitol. And saccharides including sugar alcohols such as erythritol and mannitol, and hydrophilic polymers such as polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and ficoll.
- Glucose, sucrose, maltose, trehalose and polyethylene glycol are preferably used, and glucose, sucrose, maltose, trehalose and polyethylene glycol are more preferably used. Of these, trehalose and sucrose are more preferable, and trehalose is particularly preferable.
- the cell membrane impermeable substance is sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, monosaccharide, disaccharide, trisaccharide, polysaccharide, sugar alcohol. , One or more selected from the group consisting of polyethylene glycol, polyvinyl alcohol, and polyvinyl pyrrolidone. More preferably, the cell membrane impermeable substance is a physiological environment adjusting substance selected from the group consisting of sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, and a group consisting of trehalose and sucrose. A vitrification promoting substance selected.
- a substance for promoting vitrification is not an essential component, but for example, the vitrification ability of the vitrification preservation solution is lowered by the remaining medium, etc., and ice crystals can be formed to damage cells. Therefore, it is preferable to add it.
- Tg glass transition point
- the content of the cell membrane impermeable substance is such that the osmotic pressure caused by the cell membrane impermeable substance is 280 mOsm or more of the total osmotic pressure generated in the cell membrane of the cell when the cells are suspended in the vitrification preservation solution. Such amount is required.
- the osmotic pressure is less than 280 mOsm, the cells after being suspended in the preservation solution expand as they reach equilibrium, and the possibility that the cells burst when water moves into the cells at the time of thawing increases.
- the vitrification preservative preferably contains a cell membrane impermeable substance so that the osmotic pressure due to the cell membrane impermeable substance is preferably 600 mOsm or more, more preferably 800 mOsm or more.
- the osmotic pressure is usually 1500 mOsm or less.
- a high concentration of salt can be harmful to cells, or because of the solubility limit of saccharides, or a decrease in solution stability due to a decrease in temperature.
- the vitrification preservation solution according to the present invention preferably contains 0.2M to 1M trehalose as a concentration in the preservation solution as a cell membrane impermeable substance. .
- preferred combinations include 30-50% by weight, preferably 40% by weight, of a cell membrane permeable substance, and a 10-fold concentration phosphate buffered saline as a physiological environment adjusting substance that is a cell membrane impermeable substance.
- 10 ⁇ PBS in an amount of 5 to 20% by volume, or an osmotic pressure by 10 ⁇ PBS of 140 to 600 mOsm
- a vitrification promoting substance that is a cell membrane impermeable substance is 0 to 1.5 M, preferably It contains 0.2 to 1M or an amount by which the osmotic pressure by vitrification promoting substance is 0 to 1500 mOsm, preferably 200 to 1000 mOsm.
- the osmotic pressure by the cell impermeable substance can be adjusted to be 280 mOsm or more, preferably 600 mOsm or more, more preferably 800 mOsm or more.
- the cell membrane permeable substances include ethylene glycol (EG), propylene glycol (PG), 1,3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol. (DPG), glycerin, and dimethyl sulfoxide (DMSO) can be used singly or in combination, preferably EG or PG alone, or DMSO to PG in a ratio of 1: 3 to 1: 1, or DMSO And EG are used in a ratio of 1: 3 to 1: 1, or EG and PG are combined in a ratio of 1:10 to 10: 1.
- composition of 10-fold phosphate buffered saline used as a physiological environment adjusting substance include a solution consisting of sodium chloride 1370 mM, potassium chloride 27 mM, disodium hydrogen phosphate 100 mM, and potassium dihydrogen phosphate 17.6 mM.
- a plurality of compositions such as those containing magnesium chloride or calcium chloride are known, and any of them may be used. In the present invention, the above composition is preferably used.
- AFP antifreeze protein
- AFP suppresses crystal growth by adsorbing to the crystal plane of ice or attracting water molecules around itself.
- examples of AFP include those derived from animals, plants, insects, and the like, and commercially available products can be used as appropriate.
- AFP is difficult to obtain in large quantities at once and is generally expensive.
- sericin can be used as an alternative to AFP.
- sericin was previously known to protect cells and proteins from freezing stress, but this time it was found to newly inhibit ice crystal growth. That is, the present inventors have unexpectedly found that sericin has an effect of suppressing ice crystal growth when sericin is used in a vitrification preservation solution for cells.
- the vitrification cell preservation solution according to the present invention further comprises an ice crystal growth inhibitor selected from the group consisting of antifreeze protein and sericin. More preferably, the ice crystal growth inhibitor is sericin.
- the sericin used in the present invention may be derived from a natural product or synthesized by a conventional chemical and / or genetic engineering technique.
- the sericin may be a hydrolyzate of sericin hydrolyzed with acid, alkali, enzyme or the like.
- Naturally derived sericin can be obtained by extraction from straw or raw silk by a conventional extraction method.
- sericin can be obtained by using, for example, silkworm, raw silk, or silk fabric as a raw material, a method of partially hydrolyzing and extracting it with hot water, acid, alkali, enzyme, etc.
- a product purified to a high purity is preferable for obtaining a stable culture and storage state with a constant quality.
- sericin may be a powder or granular solid, or a liquid melted or suspended in water or a buffer solution.
- the average molecular weight of sericin is not particularly limited, but is preferably 5,000 to 100,000, and more preferably 10,000 to 50,000.
- a preferable addition amount of sericin is 0.1 to 10%, more preferably 1 to 5%, and particularly preferably 3 to 5% in terms of weight with respect to the total amount of the vitrification preservation solution.
- the vitrification solution for cells according to the present invention comprises the components described above, but preferably does not contain any animal-derived components. This is because animal-derived components such as serum may cause contamination of viruses and the like. Moreover, the vitrification preservation solution for cells according to the present invention can contain other components within a range not impairing the effects of the present invention. Examples of other components include amino acids, hormones, cytokines, antioxidants, pH buffering agents, pH adjusting agents and the like.
- the vitrification preservation solution of the present invention is prepared by using a plurality of known protocols used when vitrifying and preserving cells, such as the RIKEN CDB protocol (for example, the human pluripotent stem cell culture training protocol 2010 (http: // (available from www.cdb.riken.go.jp/)).
- RIKEN CDB protocol for example, the human pluripotent stem cell culture training protocol 2010 (http: // (available from www.cdb.riken.go.jp/)
- the vitrification preservation solution containing the cells is rapidly cooled with liquid nitrogen to be vitrified.
- a method for vitrifying a cell comprising: A method is provided in which, of the total osmotic pressure generated in the cell membrane of the cells when the cells are suspended in the vitrification preservation solution, the osmotic pressure generated due to the cell membrane impermeable substance is 280 mOsm or more.
- cells to be stored are collected, the medium is removed by centrifugation, and then suspended in the vitrification storage solution according to the present invention. Etc. are rapidly cooled and vitrified. Storage is performed at a liquid nitrogen temperature ( ⁇ 190 to ⁇ 196 ° C.) in a gas phase or a liquid phase.
- Thawing is performed by adding a warmed diluted solution directly to the enclosing tube, then further diluting and washing with a medium or the like, removing the diluted solution by centrifugation, and then suspending and culturing in the medium.
- a medium can be used as the diluent.
- the concentration of the cell membrane impermeable substance in the preservation solution cannot be increased sufficiently, or if the cells rupture due to a difference in osmotic pressure during melting because the cell membrane is brittle, etc. It is preferable to use a material with an increased height.
- the solute for adjusting the osmotic pressure may be a cell membrane permeable substance or a cell membrane non-permeable substance.
- saccharides particularly glucose and sucrose are used, but they are harmful to cells. Anything can be used as long as the osmotic pressure can be adjusted.
- Example 1 material: Cell membrane permeable substances : ⁇ Dimethyl sulfoxide (DMSO) ⁇ Ethylene glycol (EG) ⁇ Propylene glycol (PG) ⁇ Butylene glycol (BG) ⁇ Isoprene glycol (IPG) ⁇ Dipropylene glycol (DPG) -Polyethylene glycol # 400 (PEG)
- DMSO Dimethyl sulfoxide
- EG Ethylene glycol
- PG Propylene glycol
- BG Butylene glycol
- IPG Isoprene glycol
- DPG Dipropylene glycol
- PEG Polyethylene glycol # 400
- Control group of vitrification stock solution (DAP213): -0.59 g of acetamide was dissolved in 6 ml of KnockOut DMEM and then sterilized by filter; Added DMSO 1.42 ml, PG 2.2 ml; ⁇ Measured up to 10 ml with Knockout DMEM.
- Test section for vitrification stock solution ⁇ Milli-Q 400-800 ⁇ l ⁇ 10 ⁇ PBS 100 ⁇ l ⁇ 100-500 ⁇ l of cell membrane permeable substance 1000 ⁇ l in total
- CMES cells are established of novel embryonic stem cell lines derived from the common marmoset (Sasaki E, Hanazawa K, Kurita R, Akatsuka A, Yoshizaki T, Ishii H, Tanioka Y, Ohnishi Y, Suemizu H, Sugawara A, Tamaoki N, Incubated with reference to Izawa K, Nakazaki Y, Hamada H, Suemori H, Asano S, Nakatsuji N, Okano H, Tani K. Stem Cells.
- CMES cells were seeded on an adhered ⁇ 35 mm dish, and a cell membrane permeable substance corresponding to 2% of the medium amount was added. -After 3 days, microscopic observation was performed to examine the presence or absence of proliferation suppression and differentiation promotion on CMES cells.
- colony morphology was good in the control (not added) and DAP213, DMSO, EG, PG, and PEG (not shown).
- mice ES cells were cultured according to the standard method. ⁇ The collected mouse ES cells were counted and dispensed into 1.5 ml tubes so as to be 1 million cells / tube, and centrifuged at 1500 rpm for 5 minutes. Add 200 ⁇ l of vitrification stock solution and suspend. After suspension, DAP213 is put into liquid nitrogen after 15 seconds and test solution is put into liquid nitrogen after 60 seconds. ⁇ Take out the tube from liquid nitrogen, add 1800 ⁇ l of medium pre-warmed to 37 ° C., quickly suspend and thaw ⁇ Sampling part of the suspension and counting the number of cells ⁇ Live cells at the time of counting The survival rate was calculated using the selective excretion ability of trypan blue as an indicator. The remaining cells were seeded on a 24-well plate, and the state after 3 days was confirmed with a microscope. did
- DMSO + PG showed a particularly high survival rate (FIG. 4).
- EG alone was the best, but the survival rate was greatly reduced compared to DMSO + PG.
- Example 2 material: Cell membrane permeable substances: ⁇ Dimethyl sulfoxide (DMSO) ⁇ Propylene glycol (PG)
- Mouse ES cell maintenance medium ⁇ KnockOut DMEM (manufactured by GIBCO) ⁇ 20% Knockout Serum Replacement (manufactured by GIBCO) ⁇ 0.1 mM non-essential amino acid (GIBCO) ⁇ 0.1 mM 2-mercaptoethanol (manufactured by GIBCO) ⁇ 2 mM L-glutamine (manufactured by GIBCO) ⁇ 1000 U / ml LIF (Wako)
- Vitrification preservation solution ⁇ Milli Q 500 ⁇ l ⁇ 10 ⁇ PBS 100 ⁇ l ⁇ DMSO 80-200 ⁇ l ⁇ PG 200 ⁇ 320 ⁇ l 1000 ⁇ l total
- Test solution DMSO and PG contents are prepared according to the table below so that the total concentration is 40%.
- DAP213 -0.59 g of acetamide was dissolved in 6 ml of KnockOut DMEM and then sterilized by filter; -DMSO 1.42 ml, PG 2.2 ml were added; ⁇ Measured up to 10 ml with Knockout DMEM.
- mice ES cells were cultured according to the standard method. ⁇ The collected mouse ES cells were counted and dispensed into 1.5 ml tubes so as to be 1 million cells / tube, and centrifuged at 1500 rpm for 5 minutes. Add 200 ⁇ l of vitrification stock solution and suspend. After suspension, DAP213 is put into liquid nitrogen after 15 seconds and test solution is put into liquid nitrogen after 60 seconds. ⁇ Remove the tube from liquid nitrogen, add 1800 ⁇ l of medium pre-warmed to 37 ° C., quickly suspend and thaw ⁇ Sampling part of the suspension and counting the number of cells ⁇ Live cells at the time of counting The survival rate was calculated using the selective excretion ability of trypan blue as an indicator. The remaining cells were seeded on a 24-well plate, and the state after 3 days was confirmed with a microscope. The
- Example 3 material: Cell membrane permeable substances: ⁇ Dimethyl sulfoxide (DMSO) ⁇ Propylene glycol (PG) ⁇ Acetamide
- Mouse ES cell maintenance medium ⁇ KnockOut DMEM (manufactured by GIBCO) ⁇ 20% Knockout Serum Replacement (manufactured by GIBCO) ⁇ 0.1 mM non-essential amino acid (GIBCO) ⁇ 0.1 mM 2-mercaptoethanol (manufactured by GIBCO) ⁇ 2 mM L-glutamine (manufactured by GIBCO) ⁇ 1000 U / ml LIF (Wako)
- DAP213 -0.59 g of acetamide was dissolved in 6 ml of KnockOut DMEM and then sterilized by filter; Added DMSO 1.42 ml, PG 2.2 ml; ⁇ Measured up to 10 ml with Knockout DMEM.
- DP23 -DMSO 1.42ml and PG 2.2ml were added to Knockout DMEM to make up to 10ml.
- mice ES cells were cultured according to the standard method. ⁇ The collected mouse ES cells were counted and dispensed into 1.5 ml tubes so as to be 1 million cells / tube, and centrifuged at 1500 rpm for 5 minutes. Add 200 ⁇ l of vitrification stock solution and suspend.
- DAP213 is put into liquid nitrogen after 15 seconds and test solution is put into liquid nitrogen after 60 seconds. ⁇ Remove the tube from liquid nitrogen, add 1800 ⁇ l of medium pre-warmed to 37 ° C., quickly suspend and thaw ⁇ Sampling part of the suspension and counting the number of cells ⁇ Live cells at the time of counting The survival rate was calculated by discriminating between life and death based on the selective excretion of trypan blue
- Example 4 material: Vitrification promoting substances: D-(+)-glucose sucrose D-(+)-maltose monohydrate D-(-)-fructose D-(+)-trehalose dihydrate D-(+) -Raffinose, pentahydrate, erythritol, xylitol, D-sorbitol, D-(-)-mannitol
- DMSO Dimethyl sulfoxide
- Vitrification preservation solution ⁇ Vitrification accelerator solution 300-500 ⁇ l ⁇ DMSO 300 ⁇ l ⁇ PBS 200-400 ⁇ l 1000 ⁇ l in total
- the experiment was performed as follows: The following saccharides were investigated as vitrification promoters: glucose, sucrose, maltose, fructose, trehalose, raffinose, erythritol, xylitol, sorbitol, and mannitol. -30% DMSO / PBS was prepared so that the concentration of the vitrification promoting substance was 0.3M and 0.6M. -200 ⁇ l of the prepared solution was dispensed into a cryotube and immersed in liquid nitrogen for rapid cooling. -After immersing in liquid nitrogen for 5 minutes or more to stabilize the temperature, one piece was taken out and observed.
- Example 5 material: Cell membrane impermeable substances: ⁇ Dimethyl sulfoxide (DMSO) ⁇ Propylene glycol (PG)
- Mouse ES cell maintenance medium ⁇ KnockOut DMEM (manufactured by GIBCO) ⁇ 20% Knockout Serum Replacement (manufactured by GIBCO) ⁇ 0.1 mM non-essential amino acid (GIBCO) ⁇ 0.1 mM 2-mercaptoethanol (manufactured by GIBCO) ⁇ 2 mM L-glutamine (manufactured by GIBCO) ⁇ 1000 U / ml LIF (Wako)
- mice ES cells were cultured according to the standard method. ⁇ The collected mouse ES cells were counted and dispensed into 1.5 ml tubes so as to be 1 million cells / tube, and centrifuged at 1500 rpm for 5 minutes. Add 200 ⁇ l of vitrification stock solution and suspend.
- DAP213 is put into liquid nitrogen after 15 seconds and test solution is put into liquid nitrogen after 60 seconds. ⁇ Take out the tube from liquid nitrogen, add 1800 ⁇ l of medium pre-warmed to 37 ° C., quickly suspend and thaw ⁇ Sampling part of the suspension and counting the number of cells ⁇ Live cells at the time of counting The survival rate was calculated by discriminating between life and death based on the selective excretion of trypan blue
- Example 6 material: Cell membrane impermeable substances: ⁇ Dimethyl sulfoxide (DMSO) ⁇ Propylene glycol (PG) ⁇ Acetamide
- Mouse ES cell maintenance medium ⁇ KnockOut DMEM (manufactured by GIBCO) ⁇ 20% Knockout Serum Replacement (manufactured by GIBCO) ⁇ 0.1 mM non-essential amino acid (GIBCO) ⁇ 0.1 mM 2-mercaptoethanol (manufactured by GIBCO) ⁇ 2 mM L-glutamine (manufactured by GIBCO) ⁇ 1000 U / ml LIF (Wako)
- DAP-A Effect of osmotic pressure on DAP213 It was confirmed at which time point the osmotic pressure affects viability of cells.
- DAP-B three kinds of preservation solutions (DAP-A, DAP-B, DAP-C) having different osmotic pressures were prepared.
- DAP-A was prepared by replacing the basal medium of DAP213 with PBS so that the osmotic pressure was almost equal to that of DAP213.
- DAP-B was prepared so that the final concentration of salt was equal to the physiological environment in consideration of dilution of the solution with a cell membrane permeable substance.
- DAP-C was prepared such that the final salt concentration was twice that of the physiological environment.
- Mouse ES cells were suspended with the above-mentioned DAP-A to C, and vitrification was performed, and after 90 seconds, the cells were diluted with PBS, and the survival rate was measured.
- mice ES cells were cultured according to the standard method. ⁇ The collected mouse ES cells were counted and dispensed into 1.5 ml tubes so as to be 1 million cells / tube, and centrifuged at 1500 rpm for 5 minutes. Suspended with 200 ⁇ l of PBS and 3 types of DAP ⁇ Standed on ice for 90 seconds. ⁇ Suspended by adding 1800 ⁇ l of PBS preheated to 37 ° C. ⁇ Measured the number of viable cells by trypan blue staining. The survival rate was calculated by distinguishing between live and dead using the selective excretion of live cells for trypan blue as an index.
- DMSO, EG, and PG used in vitrification preservatives can all permeate cell membranes, so the volume of cells at equilibrium is solutes other than these cell membrane permeable substances (salts, sugars, amino acids, proteins, etc.) ).
- the time required for this equilibration is said to be about 1 to 5 minutes, although it depends on the cell size and the diffusion coefficient of the permeable substance.
- the cells were suspended in a preservation solution and allowed to stand for 60 seconds before vitrification, and it is considered that equilibration of mouse ES cells, which are relatively small cells, has been completed at this point.
- the osmotic pressure of the vitrification storage solution affects the storage efficiency, and the relationship between the storage efficiency and the cell volume was shown.
- the viability is improved when the vitrification stock solution is hypertonic because the amount of the cell membrane permeable substance in the cell can be suppressed by reducing the volume of the cell at the time of vitrification, and into the cell that occurs at the time of thawing and dilution. It is thought that it can withstand the inflow of water.
- the viability of cells vitrified after standing exceeds 90%, so that the cytotoxicity of the cell membrane permeable substance, which has been conventionally known, is hardly affected. It has been suggested.
- the storage efficiency of the vitrification preservation solution was greatly influenced by the osmotic pressure, and the survival rate was greatly improved by making the preservation solution hypertonic.
- Example 7 material: Cell membrane impermeable substances: ⁇ Dimethyl sulfoxide (DMSO) ⁇ Ethylene glycol (EG) ⁇ Propylene glycol (PG)
- CMES Common Marmoset ES
- cell maintenance medium ⁇ KnockOut DMEM (manufactured by GIBCO) ⁇ 20% Knockout Serum Replacement (manufactured by GIBCO) ⁇ 0.1 mM non-essential amino acid (GIBCO) ⁇ 0.1 mM 2-mercaptoethanol (manufactured by GIBCO) ⁇ 2 mM L-glutamine (manufactured by GIBCO) ⁇ 4 ng / ml bFGF (Wako)
- Alkaline phosphatase (ALP) staining ⁇ Leukocyte Alkaline phosphatase Kit (sigma 86R) ⁇ Acetone ⁇ 37% formaldehyde, milli Q
- the experiment was performed as follows: -CMES cells were cultured according to a standard method using STO cells as a feeder.-On the day before the test, STO was seeded on a 35 mm dish according to a standard method to 1 million cells / plate. ⁇ CES cells were collected according to a standard method and dispensed into 6-9 1.5 ml microtubes. ⁇ Centrifuged at 1500 rpm for 5 minutes and allowed to stand on ice. The control group (unfrozen) was removed from the supernatant and suspended in 1 ml of the medium, and then 450 ⁇ l was seeded on a petri dish to which STO was adhered.
- the test group was removed from the supernatant and suspended by adding 200 ⁇ l of a vitrification stock solution. ⁇ After suspension, DAP213 was put into liquid nitrogen after 15 seconds and 60 seconds, and test solution was put into liquid nitrogen after 60 seconds. ⁇ More than 5 minutes The tube was removed from the liquid nitrogen, and 1800 ⁇ l of the medium pre-warmed to 37 ° C. was added to quickly suspend and thaw. After thawing, 900 ⁇ l was dispensed into a 1.5 ml microtube. Centrifugation at 1500 rpm for 5 minutes, removing the supernatant, suspending in 450 ⁇ l of the medium, and inoculating the entire amount in the above-mentioned ⁇ 35 mm dish. On day 4, the cells were stained with ALP, and the number of positive colonies was counted. The ratio of the positive colonies in the test group to the control group was calculated as the survival rate.
- the osmotic pressure was adjusted, and the influence on the storage efficiency was examined.
- the result was as shown in FIG.
- the osmotic pressure was adjusted to 800 mOsm or higher by combining sugar and salt, the engraftment rate was excellent. However, even if the osmotic pressure was higher than 800 mOsm, no further improvement in storage efficiency was observed.
- Example 8 Examination of ice crystal growth inhibitory substance The ice crystal growth inhibitory substance was examined by comparing the melting time when the vitrified storage solution was crystallized. That is, even when a crystal lump having the same volume is melted, a polycrystalline body having a larger total surface area melts faster. If the growth of ice crystals is suppressed during the phase transition to the crystalline state, it is considered that a lot of minute ice crystals are formed, and therefore the melting time is considered to be shortened.
- Cell membrane permeable substances ⁇ Ethylene glycol (EG) Ice crystal growth inhibitor: ⁇ Sericin (comparative example) ⁇ Bovine serum albumin (BSA) ⁇ Trehalose ⁇ Sucrose
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Abstract
Description
本発明は、細胞をガラス化して凍結保存するための保存液に関する。詳しくは本発明は、操作時間による生存率の低下が起こりにくく、霊長類ES細胞または霊長類iPS細胞に対して好適に使用できる細胞ガラス化保存液に関する。
従来、培養細胞の継代による変質や、雑菌による汚染を防止し、細胞を長期的に利用するために、細胞を凍結保存することが日常的に行われている。細胞をそのまま凍結すると、細胞内外に氷晶が形成され、細胞は物理的な損傷を受けて死に至る。特に細胞内には生命活動に不可欠な構造が多数存在しており、細胞内の氷晶形成は細胞にとって致命的である。この損傷を防ぐためには、ジメチルスルホキシド(DMSO)など細胞膜透過性の凍結保護剤を含む保存液を用いて保存する必要があり、細胞の保存効率に優れた保存液の開発が切望されている。
液体とは、熱振動が分子間力を上回り、相内の粒子が自由に移動できる状態と理解される。液体状態の物質を冷却していくと、ある温度で熱振動が分子間力を下回り、相内の粒子が流動性を失う。このとき、物質によっては粒子がエネルギー的に安定な結晶構造に再配列する。例えば、水は通常0℃以下の低温で結晶化し氷となる。ところが、物質の種類や、圧力、冷却速度などの外部要因によっては、温度が低下しても結晶化せずに液体の粘性が高くなっていき、そのまま固体となることがある。この現象をガラス化という。ガラス化する物質として、例えば、二酸化ケイ素を主成分とするガラスは代表的なガラス物質として知られている。
しかしながら、緩慢凍結法で効率的に保存可能な細胞は、株化細胞など一部の細胞に限られ、初代細胞や正常細胞、生殖細胞、胚性幹細胞(ES細胞)のなかには効率的な保存が難しい細胞があることが知られている。また、生物種による違いも大きく、例えば、マウスなどげっ歯類のES細胞では比較的高い生存率を示しても、ヒトなど霊長類のES細胞では、十分な生存率を示さないという問題があった。
(1)低濃度の保護剤を含む溶液により胚を段階的に平衡化させる;
(2)液体窒素によって急速冷却してガラス化させる;
(3)加温した希釈液を加えて融解する;
(4)洗浄後、培養に移す。
ここでいう「平衡化」とは、細胞膜、または、膜上のチャネルや細孔を通して、細胞の内外で水やその他の細胞膜透過性物質の見かけ上の移動がなくなる(平衡化する)まで、細胞等を静置することをいう。「平衡化」に要する時間は、細胞種等によって異なるが、例えば、マウス胚では、通常1~5分程度である。
例えば、Fujiokaらの文献(A simple and efficient cryopreservation method for primate embryonic stem cells, Fujioka T, Yasuchika K, Nakamura Y, Nakatsuji N, Suemori H. Int J Dev Biol 48:1149-54, 2004)では、サルES細胞を用いた実験において、DAP213に懸濁してから液体窒素に投入するまでに要する時間が15秒以内である場合の生存率を100%としたとき、同所要時間が30秒である場合の生存率は30%以下、60秒では10%以下にまで低下することが報告されている。
細胞膜透過性物質の含有量が30~50体積%であり、
細胞をガラス化保存液に懸濁したときに細胞の細胞膜に生ずる全浸透圧のうち、細胞膜非透過性物質に起因して生じる浸透圧が280mOsm以上となるものである。
細胞をガラス化保存液に懸濁したときに細胞の細胞膜に生ずる全浸透圧のうち、細胞膜非透過性物質に起因して生じる浸透圧が280mOsm以上とする方法が提供される。
まず、物理化学における浸透圧と物質量の関係について簡単に説明する。
浸透圧(π)とは、理想的な半透膜(溶媒のみを透過させ、溶質を透過させない膜)を挟んで膜の両側に溶液と純溶媒とを置いたとき、この半透膜にかかる圧力のことであり、van't Hoffの式によって以下のように求められる:
π=MRT
[ここで、Mは溶液中の溶質分子またはイオンの体積モル濃度、Rは気体定数、および、Tは絶対温度を表す]。
細胞膜は水を自由に透過させるが高分子やイオンを透過させないため、半透膜に近い性質を示す。そのため細胞内外の溶液に濃度差が生じると、浸透圧により水の移動が起こる。
本明細書においては、細胞を溶液に浸したとき、見かけ上の水の移動が起こらず、細胞の体積を変化させない場合に、その溶液を「等張」であるといい、水の移動により細胞の体積を収縮させる場合、その溶液を「高張」であるといい、水の移動により細胞の体積を膨張させる場合、その溶液を「低張」であるということとする。
細胞を「高張液」に浸した場合、細胞外液の浸透圧が高いため、細胞内から細胞外へ水が移動し、細胞は収縮していく。細胞の収縮は細胞内液が濃縮されて細胞外液と浸透圧が等しくなるか、細胞の構造的な限界に達するまで続く。
細胞を「低張液」に浸した場合、細胞外液の浸透圧が低いため、細胞外から細胞内へ水が移動し、細胞は膨張する。収縮の場合とは逆に、細胞内液が希釈されて細胞外液と浸透圧が等しくなるか、構造的な限界(例えば細胞壁による支えなど)に達するまで続く。構造的な限界を超えて細胞内への水の移動が続いた場合には、細胞膜は破壊される。一般に、動物細胞は細胞壁を持たないため、低張な環境では細胞膜が損傷を受け易い。
電荷を持つイオンなどは受動的な拡散により細胞膜を透過できないのに対し、電荷を持たない低分子は細胞膜を透過しやすい。このため、これら細胞膜透過性物質は、分子量や極性の違いにより拡散速度の差はあるものの、平衡時の体積に影響を与えない。
一方、ここで「融解」とは、加温により物質が固体から液体に変化することを意味するものとする。
以上を踏まえ、本発明者は、細胞をガラス化させる場合に、細胞の体積がどのように変化するのかを検討した。なお以下の説明は理論であって、本発明を限定するためのものではない。
すなわち、細胞が全浸透圧の高いガラス化保存液(細胞膜透過性物質と、細胞膜非透過性物質とを含むものである)に接触すると、細胞外への水の移動と細胞内への細胞膜透過性物質の拡散が同時に起こる。細胞とガラス化保存液が接触した直後は、通常、細胞膜透過性物質が細胞内へ拡散する速度と比較して、水が細胞外へ移動する速度の方が大きいため、一時的に細胞は収縮していくと言える(図1において「A」で示した個所を参照)。ここでは、これを「脱水期」と呼ぶことがある。
なおここで「全浸透圧」とはガラス化保存液に細胞を懸濁した際に、細胞の細胞膜を挟んで細胞内と細胞外との間で、ガラス化保存液全体に基づいて生ずる浸透圧をいう。
細胞内の細胞膜透過性物質の量は、細胞を保存液に懸濁してからガラス化させるまでの時間の経過とともに増大し、平衡時に最大となる(図2参照)。平衡時の細胞膜透過性物質の量は、細胞膜透過性物質の濃度と細胞体積の積であり、細胞膜透過性物質の濃度は、保存液の細胞膜透過性物質の濃度と等しくなる。一方、平衡時の細胞体積は、前述のように、保存液の細胞膜非透過性物質の浸透圧に依存する(図2の「A」、「B」、「C」は図1の各アルファベットA、B、Cに対応)。
ガラス化された細胞内液には細胞膜透過性物質が含まれており、浸透圧が高くなっている。このため融解の初期には、細胞内への水の移動と細胞外への細胞膜透過性物質の拡散が同時に起こる。ここでも水の移動速度の方が大きいため、一時的に細胞は膨張する(以下「吸水期」と呼ぶことがある)。そして、細胞が膨張できる限界体積を超えて吸水すると、細胞は破裂する。
細胞がどの程度膨張するかは細胞内に存在する細胞膜透過性物質の量に依存し、量が多いほど細胞は膨張する。細胞内に存在する細胞膜透過性物質の量は、前述のようにガラス化するまでの時間とガラス化保存液の細胞膜透過性物質濃度、および細胞膜非透過性物質の浸透圧により決定されると考えられた。
A: 細胞をガラス化保存液に懸濁してから15秒以内にガラス化させた場合、細胞内の細胞膜透過性物質の量が少ないため、細胞の体積変化は少ない。
B: 低張なガラス化保存液で細胞を平衡化後にガラス化した場合、細胞が膨張しているため細胞内には多くの細胞膜透過性物質が存在し、細胞体積は吸水によって膨張する。
ここで、細胞体積が限界体積を超えた場合、細胞は破裂する。
C: 高張なガラス化保存液で細胞を平衡化後にガラス化した場合、細胞が収縮しているため細胞内の細胞膜透過性物質の量は抑えられており、細胞はあまり膨張しない。
なお、細胞を高張液で融解することで、細胞内外の浸透圧差を小さくし、体積の膨張を抑えることができると考えられるが、希釈液を特別に調製する必要があるという点では必ずしも有利とは言えない。
霊長類ES/iPS細胞のガラス化保存液として、従来、一般的に用いられてきたDAP213について検討した。ここでは、理研CDB(独立行政法人理化学研究所発生・再生科学総合研究センター)のホームページで公開されている方法(ヒト多能性幹細胞の維持培養プロトコール(2010))に従い、霊長類ES/iPS細胞を、DAP213を用いてガラス化保存する場合について検討した。
DAP213はこの他、約60体積%の培地で構成されており、簡便のため培地の浸透圧を300mOsmと見積もると、培地中の溶質分子によるDAP213全体の浸透圧への寄与は約180mOsmとなる。これらを合計すると、DAP213全体の浸透圧は、約6180mOsmと見積もることができる。
従って、DAP213は細胞にとって低張な溶液として振舞い、平衡時には細胞を膨張させると考えられた。
従って、細胞によって多少の差異はあるにしても、DAP213に懸濁後、所定の時間を超えた場合、細胞は平衡に達し、限界体積付近まで膨張すると考えられる。このとき、細胞内には多量の保護剤が浸透した状態となり、この状態でガラス化し、次いで融解すると、前述のように細胞は吸水期に破裂することとなると考えられた。
卵子や受精卵、胚の場合、一般的に、平衡化のためにガラス化保存液中で細胞等を1~5分静置するが、ES/iPS細胞では細胞の体積が小さいため、平衡化に要する時間が短いと言える。
すなわち、細胞をガラス化保存液に懸濁してからの時間が15秒以内であれば、ガラス化時に細胞内の細胞膜透過性物質の量が少なく、融解時の浸透圧差による細胞内への水の移動とそれに伴う体積変化を許容できる。これに対し、細胞をガラス化保存液に懸濁してからの時間が15秒を超えると、時間の経過とともに細胞内の細胞膜透過性物質の量が増加し、融解時の体積変化を許容できずに、破裂する細胞が増加する。このような仮説を考えた。
本発明はこのような知見にも基づくものである。
さらに本発明者らは、ガラス化における温度上昇に伴う結晶化からの保護についても検討を行っている。
すなわち、ガラス化状態は、ガラス転移点以下の温度では安定だが、ガラス転移点以上に昇温した場合は容易にガラス状態を脱して結晶状態へ相転移する。これは温度の上昇により分子が運動エネルギーを獲得し自発的に安定な状態へ再配列(結晶化)するためであると考えられるが、このときに生じる氷晶によって、細胞は傷害を受けることがある。ガラス状態から結晶状態へ相転移すると多数の結晶界面で、光が屈折・散乱するために外観が透明から不透明へと変化する「失透」と呼ばれる現象が見られる。
ガラス化した細胞を融解する際に失透現象が観察されることから、短時間ではあるが細胞は氷晶による傷害を受けていると考えられる。
本発明者らは、氷晶成長抑制物質として、セリシンや、不凍タンパク質などを加えることにより、結晶転移時に氷晶が大きく成長するのを抑え、細胞への傷害を低減することができることも見出した。本発明はさらに、このような知見にも基づく。
前記したように、本発明による細胞のガラス化保存液は、細胞膜透過性物質と、細胞膜非透過性物質とを含んでなる、細胞のガラス化保存液であって、
細胞膜透過性物質の含有量が30~50体積%であり、
細胞をガラス化保存液に懸濁したときに細胞の細胞膜に生ずる全浸透圧のうち、細胞膜非透過性物質に起因して生じる浸透圧が280mOsm以上となるものである。なおここで、細胞のガラス化保存液は、細胞のガラス化保存用組成物、特に細胞のガラス化保存用液体組成物と言い換えることができる。
本発明において、細胞膜透過性物質は、凍結保護剤として保存液および細胞内液をガラス化させる目的で用いられる。
細胞膜透過性物質としては、例えば、エチレングリコール(EG)、プロピレングリコール(PG)、1,3-プロパンジオール(1,3-PD)、ブチレングリコール(BG)、イソプレングリコール(IPG)、ジプロピレングリコール(DPG)等のジオール類、グリセリン等のトリオール類、ジメチルスルホキシド(DMSO)などを挙げることができるが、細胞膜を透過して前記目的を達成する物質であって、しかも、細胞や生体への毒性が低い物質であればこれらに限定されない。好ましくは、細胞膜透過性物質は、エチレングリコール(EG)、プロピレングリコール(PG)、1,3-プロパンジオール(1,3-PD)、ブチレングリコール(BG)、イソプレングリコール(IPG)、ジプロピレングリコール(DPG)、グリセリン、およびジメチルスルホキシド(DMSO)からなる群より選択される1種または2種以上のものである。
本発明において、細胞膜非透過性物質は、浸透圧を調整するという目的の他、生理的環境に調整する、ガラス化を促進する、という目的で用いられる。したがって、細胞膜非透過性物質としては、生理的環境に調整するための物質(生理環境調整物質)と、ガラス化を促進するための物質(ガラス化促進物質)とを含むことができる。
氷晶成長抑制物質としては、例えば、不凍タンパク質(Antifreeze protein, AFP)として知られる一群のタンパク質などを用いることができる。AFPは氷の結晶面に吸着したり、自らの周囲に水分子を引きつけたりすることで結晶の成長を抑制する。AFPとしては、例えば、動物、植物、昆虫等由来のものが存在し、市販品を適宜利用することができる。しかしながら、AFPは一度に大量に得ることが難しく、一般的に、高価である。このため、氷晶成長抑制物質としては、AFPに代わるものとして、例えば、セリシン、を用いることができる。
天然物由来のセリシンは、慣用の抽出方法により繭又は生糸等から抽出して得ることができる。具体的には、例えば、セリシンは、繭、生糸、又は絹織物を原料として、これを熱水、又は酸、アルカリ、酵素等によって部分的に加水分解し抽出する方法等によって得ることができ、さらに高純度に精製されたものが品質が一定で安定した培養や保存状態を得るためには好ましい。
更にセリシンは、粉体や顆粒状の固体であっても、水や緩衝液に融解または懸濁させた液体であってもよい。
セリシンの好ましい添加量は、ガラス化保存液の全量に対して重量換算で、0.1~10%、より好ましくは1~5%、とくに好ましくは3~5%である。
本発明による細胞のガラス化保存液は、以上に説明した成分を含んでなるものであるが、動物由来成分は含まないことが望ましい。血清等の動物由来成分は、ウイルス等の混入の原因となる可能性があるからである。
また本発明による細胞のガラス化保存液は、本発明の作用効果を損なわない範囲内で、他の成分を含んでなることができる。他の成分としては、例えば、アミノ酸、ホルモン、サイトカイン、抗酸化剤、pH緩衝剤、pH調整剤などを挙げることができる。
前記したように、本発明によれば、本発明の細胞のガラス化保存液に、細胞を懸濁した後、細胞を含むガラス化保存液を、液体窒素によって急速冷却しガラス化することを含んでなる、細胞のガラス化方法であって、
細胞をガラス化保存液に懸濁したときに細胞の細胞膜に生ずる全浸透圧のうち、細胞膜非透過性物質に起因して生じる浸透圧が280mOsm以上とする方法が提供される。
希釈液としては、培地を用いることができる。ただし、保存液の細胞膜非透過性物質濃度を十分に高くすることができない場合や、細胞膜が脆いなどの理由で融解時の浸透圧差により細胞が破裂してしまう場合には、希釈液として浸透圧を高めたものを用いることが好ましい。このとき、浸透圧を調整するための溶質は細胞膜透過性物質であっても細胞膜非透過性物質であってもよく、一般的には糖類、特にグルコースやスクロースが用いられるが、細胞にとって有害とならず、浸透圧を調整することのできるものであれば、どのようなものでも用いることができる。
材料:
細胞膜透過性物質:
・ジメチルスルホキシド(DMSO)
・エチレングリコール(EG)
・プロピレングリコール(PG)
・ブチレングリコール(BG)
・イソプレングリコール(IPG)
・ジプロピレングリコール(DPG)
・ポリエチレングリコール#400(PEG)
・マウスES細胞 D3株(ATCCより入手)
・コモンマーモセットES細胞 CMES40株(理研セルバンクより入手)
*コモンマーモセットES細胞は実験動物中央研究所で佐々木らが樹立した細胞を理研セルバンクを通して入手、使用した。
・フィーダー細胞 STO株(理研セルバンクより入手)
1) マウスES細胞維持培地
・KnockOut DMEM(GIBCO社製)
・20% Knockout Serum Replacement(GIBCO社製)
・0.1 mM 非必須アミノ酸(GIBCO社製)
・0.1 mM 2-メルカプトエタノール(GIBCO社製)
・2 mM L-グルタミン(GIBCO社製)
・1000 U/ml LIF(和光社製)
・KnockOut DMEM(GIBCO社製)
・20% Knockout Serum Replacement(GIBCO社製)
・0.1 mM 非必須アミノ酸(GIBCO社製)
・0.1 mM 2-メルカプトエタノール(GIBCO社製)
・2 mM L-グルタミン(GIBCO社製)
・4 ng/ml bFGF(和光社製)
・アセトアミド0.59gをKnockOut DMEM 6mlに溶解後、フィルター滅菌した;
・DMSO 1.42ml、PG 2.2mlを添加した;
・Knockout DMEMで10mlにメスアップした。
・ミリQ 400~800μl
・10×PBS 100μl
・細胞膜透過性物質 100~500μl
計 1000μl
ガラス化保存液の毒性を大きく左右する細胞膜透過性物質の種類を検討した。ガラス化を促進する物質は多く知られているが毒性や変異原性を持たず細胞内へ浸透することが好ましい。これらの観点から化粧品原料として用いられている低分子量のジオール類に着目した。EGやPGなどいくつかのジオールは凍結保護剤としての使用実績があり、またこれらは肌に対する安全性が確認されており、細胞毒性が低いことが期待された。そこで、DMSOの他にEG、PG、BG、IPG、DPG、PEGの6種のジオールについてガラス化保存液としての性能を検討した。
陰性対照であるPBSはガラス化せず、多結晶体として凍結した。多結晶体であることは結晶界面で光が散乱し、外観が不透明となることで確認した(図省略)。一方、陽性対照であるDAP213は全体が透明色を示し、ガラス状態であることが確認できた。また、温度上昇により不透明な結晶性固体へと相転移する様子が観察された。
試験した全てのジオールとDMSOはいずれも40%以上添加することでガラス化することが示された。なかでもPG、IPG、BG、DPGは30%溶液でもガラス化することが確認された。
(1-2-1) コモンマーモセット(CM)ES細胞を用いた細胞毒性、分化影響調査
細胞膜透過性物質のCMES細胞に対する影響を調べるためCMESの培地に各細胞膜透過性物質を2%添加して培養し、コロニーの状態を観察した。
具体的には、実験は下記の通りに行った:
・CMES細胞はEstablishment of novel embryonic stem cell lines derived from the common marmoset (Sasaki E, Hanazawa K, Kurita R, Akatsuka A, Yoshizaki T, Ishii H, Tanioka Y, Ohnishi Y, Suemizu H, Sugawara A, Tamaoki N, Izawa K, Nakazaki Y, Hamada H, Suemori H, Asano S, Nakatsuji N, Okano H, Tani K. Stem Cells. 2005 Oct;23(9):1304-13)を参考にして培養した
・予めフィーダー細胞を接着させたφ35mmディッシュにCMES細胞を播種し、培地量の2%に相当する細胞膜透過性物質を添加した。
・3日後に顕微鏡観察を行い、CMES細胞に対する増殖抑制、分化促進の有無を調べた。
試験した7種の細胞膜透過性物質のうち、CMES細胞に対して分化促進効果の見られなかった4種(DMSO、EG、PG、PEG)を単独または1:1で組み合わせて細胞膜透過性物質の合計が40%となるよう使用し各細胞膜透過性物質のガラス化保護作用を確認した。CMES細胞は細胞数の計数が困難であるため、試験にはマウスES細胞を用いた。
ガラス化・融解後のマウスES細胞を播種し、培養後の細胞の状態を顕微鏡観察により確認した。
・マウスES細胞は定法に従って培養した
・回収したマウスES細胞は細胞数をカウントし、100万cells/tubeとなるよう1.5mlチューブに分注し、1500rpm、5分遠心した
・上清を除き、ガラス化保存液を200μl加えて懸濁した
・懸濁後、DAP213は15秒、試験液は60秒経過後に液体窒素に投入し、ガラス化させた
・5分以上静置し、温度を安定化させた
・液体窒素からチューブを取り出し、予め37℃に加温した培地1800μlを加えて素早く懸濁して融解した
・懸濁液を一部サンプリングして細胞数をカウントした
・カウント時、生細胞のトリパンブルーに対する選択的排出能を指標に生死を判別し生存率を算出した
・残りの細胞を24wellプレートに播種し、3日後の状態を顕微鏡にて確認した
形態観察でコロニーが多数観察されたEG、DMSO+EG、DMSO+PG、DMSO+PEG、EG+PEGの5種について、ガラス化後の生存率を計測した。各細胞膜透過性物質の40%溶液をPBSで調製し、マウスES細胞を懸濁して60秒静置後に液体窒素に投入してガラス化させた。
細胞膜透過性物質としてDMSOを使用しないものの中ではEG単独が最も優れていたがDMSO+PGに比べて生存率は大きく低下していた。
材料:
細胞膜透過性物質:
・ジメチルスルホキシド(DMSO)
・プロピレングリコール(PG)
・マウスES細胞 D3株(ATCCより入手可)
マウスES細胞維持培地:
・KnockOut DMEM(GIBCO社製)
・20% Knockout Serum Replacement(GIBCO社製)
・0.1 mM 非必須アミノ酸(GIBCO社製)
・0.1 mM 2-メルカプトエタノール(GIBCO社製)
・2 mM L-グルタミン(GIBCO社製)
・1000 U/ml LIF(和光社製)
・ミリQ 500μl
・10×PBS 100μl
・DMSO 80~200μl
・PG 200~320μl
計 1000 μl
DMSOとPGの含量は下表に従い合計濃度が40%となるように調製
・アセトアミド0.59gをKnockOut DMEM 6mlに溶解後、フィルター滅菌した;
・DMSO1.42ml、PG2.2mlを添加した;
・Knockout DMEMで10mlにメスアップした。
DMSOとPGの添加比率を変えた保存液を用いてマウスES細胞をガラス化し、融解後の生存率を比較した。
試験区では細胞を懸濁してからガラス化するまでの時間を60秒とし、対照区はDAP213を用いて懸濁から15秒以内にガラス化した。
・マウスES細胞は定法に従って培養した
・回収したマウスES細胞は細胞数をカウントし、100万cells/tubeとなるよう1.5mlチューブに分注し、1500rpm、5分遠心した
・上清を除き、ガラス化保存液を200μl加えて懸濁した
・懸濁後、DAP213は15秒、試験液は60秒経過後に液体窒素に投入し、ガラス化させた
・5分以上静置し、温度を安定化させた
・液体窒素からチューブを取り出し、予め37℃に加温した培地1800μlを加えて素早懸濁して融解した
・懸濁液を一部サンプリングして細胞数をカウントした
・カウント時、生細胞のトリパンブルーに対する選択的排出能を指標に生死を判別し生存率を算出した
・残りの細胞を24wellプレートに播種し、3日後の状態を顕微鏡にて確認した
DMSO含量を10%まで減らしても生存率に影響しないが、8%まで減らすと生存率の低下が見られた。従って現行品であるDAP213のDMSO含量(14.2%)よりDMSO含量を低減できることが示された。
材料:
細胞膜透過性物質:
・ジメチルスルホキシド(DMSO)
・プロピレングリコール(PG)
・アセトアミド
・マウスES細胞 D3株(ATCCより入手可)
マウスES細胞維持培地:
・KnockOut DMEM(GIBCO社製)
・20% Knockout Serum Replacement(GIBCO社製)
・0.1 mM 非必須アミノ酸(GIBCO社製)
・0.1 mM 2-メルカプトエタノール(GIBCO社製)
・2 mM L-グルタミン(GIBCO社製)
・1000 U/ml LIF(和光社製)
・アセトアミド0.59gをKnockOut DMEM 6mlに溶解後、フィルター滅菌した;
・DMSO 1.42ml、PG 2.2mlを添加した;
・Knockout DMEMで10mlにメスアップした。
・Knockout DMEMにDMSO1.42ml、PG 2.2mlを添加して10mlにメスアップした
アセトアミドを含むDAP213と、そこからアセトアミドのみを抜いたもの(DP23)を用いて懸濁から15秒以内および60秒後にガラス化したマウスES細胞の生存率を測定した。
・マウスES細胞は定法に従って培養した
・回収したマウスES細胞は細胞数をカウントし、100万cells/tubeとなるよう1.5mlチューブに分注し、1500rpm、5分遠心した
・上清を除き、ガラス化保存液を200μl加えて懸濁した
・懸濁後、DAP213は15秒、試験液は60秒経過後に液体窒素に投入し、ガラス化させた
・5分以上静置し、温度を安定化させた
・液体窒素からチューブを取り出し、予め37℃に加温した培地1800μlを加えて素早懸濁して融解した
・懸濁液を一部サンプリングして細胞数をカウントした
・カウント時、生細胞のトリパンブルーに対する選択的排出能を指標に生死を判別し生存率を算出した
15秒以内にガラス化した場合にはアセトアミドによる生存率の向上は認められず、60秒後においてはアセトアミドにより生存率が低下していた。
材料:
ガラス化促進物質:
・D-(+)-グルコース
・スクロース
・D-(+)-マルトース・一水和物
・D-(-)-フルクトース
・D-(+)-トレハロース・二水和物
・D-(+)-ラフィノース・五水和物
・エリスリトール
・キシリトール
・D-ソルビトール
・D-(-)-マンニトール
・ジメチルスルホキシド(DMSO)
・ガラス化促進物質溶液 300~500μl
・DMSO 300μl
・PBS 200~400μl
計 1000μl
細胞に対する毒性が低くガラス形成能に優れる糖および糖アルコール類10種についてガラス化促進物質としての性能を比較検討した。
・ガラス化促進物質として以下の糖類を検討した: グルコース、スクロース、マルトース、フルクトース、トレハロース、ラフィノース、エリスリトール、キシリトール、ソルビトール、およびマンニトール。
・30%DMSO/PBSにガラス化促進物質の濃度が0.3M、0.6Mとなるよう調製した。
・調製後の溶液をクライオチューブに200μlずつ分注し、液体窒素に浸して急冷した。
・液体窒素中に5分以上浸し、温度を安定化させた後に1本ずつ取り出して観察した。
材料:
細胞膜非透過性物質:
・ジメチルスルホキシド(DMSO)
・プロピレングリコール(PG)
・D-(+)-トレハロース・二水和物
・マウスES細胞 D3株(ATCCより入手可)
マウスES細胞維持培地:
・KnockOut DMEM(GIBCO社製)
・20% Knockout Serum Replacement(GIBCO社製)
・0.1 mM 非必須アミノ酸(GIBCO社製)
・0.1 mM 2-メルカプトエタノール(GIBCO社製)
・2 mM L-グルタミン(GIBCO社製)
・1000 U/ml LIF(和光社製)
下表に従って調整した。
試作した保存液でマウスES細胞を懸濁、60秒静置後にガラス化し、融解後の生存率を測定した。
・マウスES細胞は定法に従って培養した
・回収したマウスES細胞は細胞数をカウントし、100万cells/tubeとなるよう1.5mlチューブに分注し、1500rpm、5分遠心した
・上清を除き、ガラス化保存液を200μl加えて懸濁した
・懸濁後、DAP213は15秒、試験液は60秒経過後に液体窒素に投入し、ガラス化させた
・5分以上静置し、温度を安定化させた
・液体窒素からチューブを取り出し、予め37℃に加温した培地1800μlを加えて素早く懸濁して融解した
・懸濁液を一部サンプリングして細胞数をカウントした
・カウント時、生細胞のトリパンブルーに対する選択的排出能を指標に生死を判別し生存率を算出した
トレハロースを添加した保存液でガラス化した細胞は約90%の生存率を示した。ところが、トレハロースではなく塩(PBS)で浸透圧を調節した場合にもマウスES細胞は同様の高い生存率を示しており、ガラス化保存液の保存効率には浸透圧が大きく影響することが示された。しかし、塩をまったく添加せず、糖のみで浸透圧を調製した場合にはマウスES細胞の生存率は改善されず、ナトリウムイオンなどのように細胞内のイオンバランスを保つ環境も必要であることが示唆された。
材料:
細胞膜非透過性物質:
・ジメチルスルホキシド(DMSO)
・プロピレングリコール(PG)
・アセトアミド
・マウスES細胞 D3株(ATCCより入手可)
マウスES細胞維持培地:
・KnockOut DMEM(GIBCO社製)
・20% Knockout Serum Replacement(GIBCO社製)
・0.1 mM 非必須アミノ酸(GIBCO社製)
・0.1 mM 2-メルカプトエタノール(GIBCO社製)
・2 mM L-グルタミン(GIBCO社製)
・1000 U/ml LIF(和光社製)
・DAP213:
浸透圧がガラス化のどの時点で細胞の生存率に影響を与えているのかを確認した。
まず、上記の組成に従い、浸透圧の異なる3種の保存液(DAP-A、DAP-B、DAP-C)を調製した。ここで、DAP-AはDAP213とほぼ同等の浸透圧となるようDAP213の基礎培地をPBSに置き換えて調製した。DAP-Bは細胞膜透過性物質による溶液の希釈を考慮し、塩の終濃度が生理的環境と等しくなるよう調製した。DAP-Cは塩の終濃度が生理的環境の2倍となるよう調製した。上記のDAP-A~CでマウスES細胞を懸濁し、ガラス化は行わずに90秒後にPBSで希釈して生存率を測定した。
・マウスES細胞は定法に従って培養した
・回収したマウスES細胞は細胞数をカウントし、100万cells/tubeとなるよう1.5mlチューブに分注し、1500rpm、5分遠心した
・上清を除き、PBSおよび3種のDAP200μlで懸濁した
・氷上で90秒間静置した
・予め37℃に加温したPBS1800μlを添加して懸濁した
・トリパンブルー染色により生細胞数を測定した
・カウント時、生細胞のトリパンブルーに対する選択的排出能を指標に生死を判別し生存率を算出した
このことから、低浸透圧の保存液ではガラス化後の融解・希釈時に細胞の生存率が低下することが示された。この時、DAP-Aでは生細胞が減少していたにも関わらず、トリパンブルー陽性となる死細胞はほとんど観察されなかった。このことから、死細胞は細胞膜が著しく損傷していることが示唆された。
材料:
細胞膜非透過性物質:
・ジメチルスルホキシド(DMSO)
・エチレングリコール(EG)
・プロピレングリコール(PG)
・コモンマーモセットES細胞 CMES40株(理研セルバンクより入手可)
・フィーダー細胞 STO株(理研セルバンクより入手可)
コモンマーモセットES(CMES)細胞維持培地:
・KnockOut DMEM(GIBCO社製)
・20% Knockout Serum Replacement(GIBCO社製)
・0.1 mM 非必須アミノ酸(GIBCO社製)
・0.1 mM 2-メルカプトエタノール(GIBCO社製)
・2 mM L-グルタミン(GIBCO社製)
・4 ng/ml bFGF(和光社製)
・Leukocyte Alkaline phosphatase Kit(sigma 86R)
・Acetone
・37%ホルムアルデヒド
・ミリQ
細胞膜透過性物質としてDMSO+PGまたはEG、ガラス化促進物質としてトレハロースを用いた保存液でCMES細胞をガラス化し、融解後の生存率を調べた。マウスES細胞を用いた評価では、細胞膜透過性物質としてDMSOとPGの組み合わせが最も優れていたが、DMSOはES細胞に対する分化誘導能が報告されていることから、マウスES細胞で2番目に生存率の高かったEGについても同時に評価を行った。
・CMES細胞はSTO細胞をフィーダーとし、定法に従って培養した
・試験前日に、定法に従ってφ35mmディッシュに100万cells/枚となるようSTOを播種した
・試験当日、STOの培地を1.5mlのCMES細胞維持培地に交換してインキュベートした
・CMES細胞は定法に従って回収し、φ90mmシャーレ1枚から6~9本の1.5mlマイクロチューブに分注した
・1500rpm、5分遠心し、氷上に静置した
・対照区(未凍結)は上清を除き、1mlの培地で懸濁後、STOを接着させたシャーレに450μl播種した
・試験区は上清を除き、ガラス化保存液を200μl加えて懸濁した
・懸濁後、DAP213は15秒および60秒、試験液は60秒経過後に液体窒素に投入してガラス化させた
・5分以上静置し、温度を安定化させた
・液体窒素からチューブを取り出し、予め37℃に加温した培地1800μlを加えて素早懸濁して融解した
・融解後、900μlを1.5mlマイクロチューブに分注して1500rpm、5分遠心し、上清を除いた
・450μlの培地に懸濁し、前述のφ35mmディッシュに全量を播種した
・播種2日後から毎日培地を交換し、コロニーの状態を見て3日ないし4日目にALPで染色し、陽性コロニーを計数した
・対照区に対する試験区の陽性コロニーの比を生着率として算出した。
CMES細胞に対しては細胞膜透過性物質としてEGを単独で用いた場合に高い保存効率を示すことが明らかとなった。
結果は図10に示されるとおりであった。
糖と塩を合わせて浸透圧を800mOsm以上に調製した時に生着率が優れていたが、浸透圧を800mOsmより高くしてもそれ以上の保存効率向上は認められなかった。
ET45はCMES細胞を懸濁後15秒以内にガラス化したとき、または60秒後にガラス化したときのいずれの場合においてもDAP213と比較して高い生着率を示した。
氷晶成長抑制物質の検討
氷晶成長抑制物質の検討はガラス化した保存液を結晶化させたときの融解時間を比較することによって行った。すなわち、同じ体積の結晶塊を融解させた場合であっても総表面積が大きい多結晶体の方が早く融解する。結晶状態への相転移時に氷晶の成長が抑制されていた場合、微小な氷晶が多数形成されていると考えられるため、融解時間が短くなると考えられる。
細胞膜透過性物質:
・エチレングリコール(EG)
氷晶成長抑制物質:
・セリシン
(比較例)
・ウシ血清アルブミン(BSA)
・トレハロース
・スクロース
・EG 400 μl
・10重量% 各種氷晶成長抑制物質 100~500 μl
・ミリQ 100~500 μl
計 1000 μl
・調製した試験液を1.5mlマイクロチューブに200μlずつ分注した。
・デュワー瓶に液体窒素を入れ、タイマーを準備した。
・ピンセットでマイクロチューブを掴み、液体窒素に40秒浸してガラス化した。
・液体窒素から引き上げ、チューブ内の氷が完全に消滅するまでの時間を計測した。
これらのことからセリシンによって氷晶の成長が抑制されていることが示唆された。
Claims (10)
- 細胞膜透過性物質と、細胞膜非透過性物質とを含んでなる、細胞のガラス化保存液であって、
細胞膜透過性物質の含有量が30~50体積%であり、
細胞をガラス化保存液に懸濁したときに細胞の細胞膜に生ずる全浸透圧のうち、細胞膜非透過性物質に起因して生じる浸透圧が280mOsm以上となるものである、細胞のガラス化保存液。 - 細胞膜透過性物質が、エチレングリコール(EG)、プロピレングリコール(PG)、1,3-プロパンジオール(1,3-PD)、ブチレングリコール(BG)、イソプレングリコール(IPG)、ジプロピレングリコール(DPG)、グリセリン、およびジメチルスルホキシド(DMSO)からなる群より選択される1種または2種以上のものである、請求項1に記載の細胞のガラス化保存液。
- 細胞膜透過性物質が、エチレングリコール(EG)、プロピレングリコール(PG)、およびジメチルスルホキシド(DMSO)からなる群より選択される1種または2種のものである、請求項1または2に記載の細胞のガラス化保存液。
- 細胞膜非透過性物質が、塩化ナトリウム、塩化カリウム、リン酸水素二ナトリウム、リン酸二水素カリウム、単糖類、二糖類、三糖類、多糖類、糖アルコール、フィコール、ポリエチレングリコール、ポリビニルアルコール、およびポリビニルピロリドンからなる群より選択される1種または2種以上のものである、請求項1~3のいずれか一項に記載の細胞のガラス化保存液。
- 細胞膜非透過性物質が、塩化ナトリウム、塩化カリウム、リン酸水素二ナトリウム、およびリン酸二水素カリウムからなる群より選択される生理環境調整物質と、トレハロース、およびスクロースからなる群より選択されるガラス化促進物質とを含んでなる、請求項1~4のいずれか一項に記載の細胞のガラス化保存液。
- 細胞膜非透過性物質が、保存液における濃度として0.2M~1Mのトレハロースを含んでなる、請求項1~5のいずれか一項に記載の細胞のガラス化保存液。
- 不凍タンパク質、およびセリシンからなる群より選択される氷晶成長抑制物質をさらに含んでなる、請求項1~6のいずれか一項に記載の細胞のガラス化保存液。
- 多能性幹細胞のガラス化保存に用いられる、請求項1~7のいずれか一項に記載の細胞のガラス化保存液。
- 霊長類ES細胞または霊長類iPS細胞のガラス化保存に用いられる、請求項1~8のいずれか一項に記載の細胞のガラス化保存液。
- 請求項1~9のいずれか一項に記載の細胞のガラス化保存液に、細胞を懸濁した後、細胞を含むガラス化保存液を、液体窒素によって急速冷却しガラス化することを含んでなる、細胞のガラス化方法であって、
細胞をガラス化保存液に懸濁したときに細胞の細胞膜に生ずる全浸透圧のうち、細胞膜非透過性物質に起因して生じる浸透圧が280mOsm以上とする、方法。
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EP (1) | EP2641966A4 (ja) |
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Cited By (7)
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JP2015149905A (ja) * | 2014-02-10 | 2015-08-24 | 国立大学法人北陸先端科学技術大学院大学 | 細胞シート及び三次元細胞培養体の凍結保存のための緩慢ガラス化方法 |
JP2016073207A (ja) * | 2014-10-02 | 2016-05-12 | 株式会社リプロセル | 細胞凍結保存方法、細胞解凍方法、及び細胞 |
JP2016522221A (ja) * | 2013-06-13 | 2016-07-28 | バイオマトリカ,インク. | 細胞安定化 |
JP2019187320A (ja) * | 2018-04-26 | 2019-10-31 | 三菱製紙株式会社 | 細胞又は組織の凍結保存液 |
US10568317B2 (en) | 2015-12-08 | 2020-02-25 | Biomatrica, Inc. | Reduction of erythrocyte sedimentation rate |
US10772319B2 (en) | 2014-06-10 | 2020-09-15 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
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MX2016013026A (es) | 2016-09-19 | 2018-03-19 | Centro De Investig Cientifica Y De Educacion Superior De Ensenada Baja California Cicese | Protocolo para la criopreservación de muestras biológicas de alta viscocidad. |
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WO2021177344A1 (ja) * | 2020-03-05 | 2021-09-10 | 積水メディカル株式会社 | 細胞含有液用保存容器及び保存液 |
CN112167245A (zh) * | 2020-10-27 | 2021-01-05 | 深圳华大基因细胞科技有限责任公司 | 用于细胞保存的保护剂 |
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JP2016522221A (ja) * | 2013-06-13 | 2016-07-28 | バイオマトリカ,インク. | 細胞安定化 |
JP2015149905A (ja) * | 2014-02-10 | 2015-08-24 | 国立大学法人北陸先端科学技術大学院大学 | 細胞シート及び三次元細胞培養体の凍結保存のための緩慢ガラス化方法 |
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US11116205B2 (en) | 2015-12-08 | 2021-09-14 | Biomatrica, Inc. | Reduction of erythrocyte sedimentation rate |
JP2019187320A (ja) * | 2018-04-26 | 2019-10-31 | 三菱製紙株式会社 | 細胞又は組織の凍結保存液 |
WO2019208120A1 (ja) * | 2018-04-26 | 2019-10-31 | 三菱製紙株式会社 | 凍結保存液及び凍結保存方法 |
WO2021095741A1 (ja) * | 2019-11-11 | 2021-05-20 | 国立大学法人九州大学 | 細胞の凍結に有用な新規糖誘導体 |
Also Published As
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US20130236960A1 (en) | 2013-09-12 |
EP2641966A1 (en) | 2013-09-25 |
EP2641966A4 (en) | 2014-04-30 |
JP6333513B2 (ja) | 2018-05-30 |
JPWO2012067240A1 (ja) | 2014-05-19 |
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