WO2017174032A1 - 细胞冻存保护组合物、该组合物的用途以及细胞冻存的方法 - Google Patents

细胞冻存保护组合物、该组合物的用途以及细胞冻存的方法 Download PDF

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WO2017174032A1
WO2017174032A1 PCT/CN2017/079744 CN2017079744W WO2017174032A1 WO 2017174032 A1 WO2017174032 A1 WO 2017174032A1 CN 2017079744 W CN2017079744 W CN 2017079744W WO 2017174032 A1 WO2017174032 A1 WO 2017174032A1
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cell
cryopreservation
group
cells
solution
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PCT/CN2017/079744
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English (en)
French (fr)
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张雷
杨静
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天津大学
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Priority to US16/087,617 priority Critical patent/US11497206B2/en
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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
    • 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
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/22—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated the carbon skeleton being further substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/50—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the nitrogen atom of at least one of the carboxamide groups quaternised
    • 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
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0695—Stem cells; Progenitor cells; Precursor cells

Definitions

  • the present application belongs to the field of cell biology, and in particular to the use of a cell cryoprotective composition comprising an amphiphilic molecule having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 and which is non-toxic and highly efficient .
  • the application also relates to the use of the composition and to a method of cryopreservation of cells.
  • living cells extracted from animals are increasingly used in various fields of biomedicine.
  • living cells are used to examine the efficacy, dosage and toxicity of drugs, provide direction guidance for animal experiments, and reduce the amount of animals used in experiments to reduce risks.
  • the work obtained from the human body can be used for detection and diagnosis.
  • cells with therapeutic functions such as stem cells, immune cells, blood cells
  • cryopreservation at low temperatures (-80 ° C or -196 ° C). Under such ultra-low temperature conditions, cells slow down or even halt metabolism for long-term preservation. However, in the process of freezing and cooling, the formation of ice crystals will cause fatal mechanical damage and solute damage. Therefore, cryopreservation of cells often requires the addition of cryoprotectants to reduce the production of intracellular and extracellular liquid ice crystals. Dimethyl sulfoxide (DMSO) and egg yolk and glycerol are the most commonly used traditional cryoprotectants, and these cryoprotectants are suitable for different cell types due to different cell permeability and structure.
  • DMSO dimethyl sulfoxide
  • egg yolk and glycerol are the most commonly used traditional cryoprotectants, and these cryoprotectants are suitable for different cell types due to different cell permeability and structure.
  • DMSO is suitable for cryopreservation of animal somatic cells; Oil is often used as a cryopreservation agent for bacterial cells, human and animal germ cells, and blood cells; egg yolk is often used as an auxiliary additive for germ cell cryopreservation agents.
  • traditional cryoprotectants have their fatal flaws: DMSO has certain cytotoxicity. For example, DMSO inhibits cell proliferation, changes the biological function of cells, and induces stem cell differentiation (see K. Aita et al for details). .Apoptosis in murine lymphoid organs following intraperitoneal administration of dimethyl sulfoxide (DMSO). "Experimental and Molecular Pathology", 2005, 79, 265-271; R. Pal et al.
  • DMSO dimethyl sulfoxide
  • Glycerin was the first traditional cryoprotectant to be found to successfully cryopreserve chicken sperm.
  • the disadvantage of glycerol is that it has poor permeability, is slow to enter cells, and has no cryoprotective effect on various cells.
  • cells are highly susceptible to osmotic stress during deglycerolation, especially at high concentrations.
  • Glycerol cryoprotectant blood cryoprotectant usually contains 40% glycerol
  • Toxicity for human peritoneal mesothelial cells in culture comparison with glucose. International Journal of Artificial Organs, 1994, 17(5): 252-260).
  • the auxiliary additive of egg yolk as a cryoprotectant also has the following disadvantages: 1) the yolk component is complex, contains some components harmful to cells such as large particulate matter, high concentration of lipoprotein, etc.; 2) yolk is usually derived from poultry Eggs may carry some potentially infectious pathogens (viruses, bacteria, mycoplasma), increasing the risk of disease transmission; 3) the quality of egg yolk from different sources is uneven, and the effects of cryopreservation are different and difficult to standardize. Therefore, the limitations of low efficiency and risk of traditional cryoprotectants have become bottlenecks in the successful application of living cells in various fields, especially in cell therapy and regenerative medicine.
  • the cryopreservation procedure using conventional protectants is a gradient cooling, which typically takes about one day.
  • gradient cooling such as a cooling rate of 1 ° C per minute
  • the glycerol cryopreservation agent is mixed with the cells, and placed at a temperature of 4 ° C, -20 ° C, and -80 ° C for a certain period of time, and finally transferred to a liquid nitrogen tank to meet the requirements of the gradient cooling in the conventional cryopreservation procedure.
  • Patent Document 1 discloses a composition for sperm ovum transport storage of livestock, which comprises components such as glycerin 5 ml (87%) and Merk solution, and Merk solution contains EDTA. It is known to those skilled in the art that glycerol is the main substance for protecting frozen cells. Patent Document 1 uses 5 ml (87%) of glycerin to preserve animal sperm. On the one hand, glycerin is a cryoprotectant that plays a decisive role in cryopreserved sperm. For details, see the reference (Polge et al. Revival of spermatozoa after vitrification and dehydration at low temperature.
  • EDTA is highly cytotoxic. As we all know, EDTA is a Ca 2+ ion chelating agent, which will chelate the calcium ions necessary for cells to affect cells. In addition, EDTA also has significant toxic effects on the human body, which may irritate the skin and mucous membranes, causing asthma, skin rash and other symptoms. Is a substance that may cause allergies. It can cause calcium deficiency, blood pressure lowering, kidney disorders, chromosomal abnormalities and native variability through the ingestion of propylene glycol and other transdermal absorption aids (see Singh K. et al. Interaction of EDTA with tributyltin induced). Cellular toxicity. Indian J Exp Biol. 1989 Sep; 27(9): 833-4.,).
  • the invention aims to address the deficiencies of the existing cell cryopreservation technology, such as the toxicity of the cryoprotectant, the cumbersome operation, the unstable storage of cryopreservation (hereinafter referred to as "freezing"), and the provision of a structure having the general structure R 1 - An amphoteric molecule of N + (CH 3 ) 2 -(CH 2 ) n -R 2 , a cell cryopreservation protective composition assisted by a cell cryopreservation component (such as glycerol or DMSO) which is toxic to cells, and Use of the composition and a method of cryopreservation of cells using the composition.
  • a cell cryopreservation protective composition assisted by a cell cryopreservation component (such as glycerol or DMSO) which is toxic to cells
  • the cell cryopreservation composition of the present invention can be applied to a wide range of cryopreserved cell concentrations and cell types, and can achieve high survival rate after cell resuscitation.
  • the cryopreservation composition provided by the method has the steps of simple and easy to freeze, and does not need to use a plurality of large-scale cryopreservation devices, which can effectively reduce the cell cryopreservation cost and improve the cryopreservation efficiency.
  • a first aspect of the present application provides a cell cryoprotective composition comprising one or more amphiphilic molecules having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 , And cell nutrients,
  • amphiphilic molecule is 10 to 2530 parts by mass with respect to 100 parts by mass of the nutrient component for the cell;
  • the cell cryoprotective composition does not comprise: glycerin, diaminoethanetetraacetic acid or a salt thereof, dimethyl sulfoxide, or egg yolk,
  • R 1 is a linear or branched alkyl group having 1 to 10 carbon atoms, which is optionally selected from (meth)acrylamide group, (meth)acryloyloxy group, alkenyl group, hydroxyl group, hydroxyalkyl group. a substituent in the group consisting of an alkoxy group, and a halogen,
  • R 2 is selected from the group consisting of -COO - , -SO 4 - , -SO 3 - , and Any of the group consisting of one negatively charged group, said R 3 is selected from the group consisting of (meth) acryloyloxy group, an alkyl group, an alkenyl group, and the group consisting of,
  • the (CH 3 ) 2 and (CH 2 ) n may each independently be optionally selected from the group consisting of alkyl, alkenyl, hydroxy, hydroxyalkyl, alkoxy, polyalkylene oxide, and halogen. Replacement of substituents,
  • n is an integer from 1 to 10.
  • R 1 is a linear or branched alkyl group having 1 to 5 carbon atoms, which is optionally selected from the group consisting of a (meth)acrylamide group, a (meth)acryloyloxy group, a hydroxyl group, a hydroxyalkyl group, and an alkoxy group. a substituent in the group consisting of a group and a halogen,
  • R 2 is selected from the group consisting of -COO - , -SO 3 - , and Any one of the group consisting of R 3 being a (meth)acryloyloxyalkyl group,
  • the hydrogen atoms in the (CH 3 ) 2 and (CH 2 ) n may each independently be optionally selected from the group consisting of an alkyl group, a hydroxyl group, a hydroxyalkyl group, an alkoxy group, a polyalkylene oxide group, and a halogen. Substituted in the substituent,
  • n is an integer from 1 to 5.
  • the amphiphilic molecule is 420 to 1120 parts.
  • R 1 is a linear or branched alkyl group having 1 to 3 carbon atoms, which is optionally selected from a (meth)acrylamide group, a (meth)acryloyloxy group, a hydroxyl group, and a carbon number of 1 to 5. Substituted by a substituent in the group consisting of a hydroxyalkyl group and an alkoxy group having 1 to 5 carbon atoms,
  • the hydrogen atoms in the (CH 3 ) 2 and (CH 2 ) n may each independently be optionally selected from an alkyl group having 1 to 5 carbon atoms, a hydroxyl group, a hydroxyalkyl group having 1 to 5 carbon atoms, and carbon. Substituted with alkoxy groups having an atomic number of 1 to 5 and a substituent in the group consisting of polyalkylene oxide groups,
  • n is an integer from 1 to 3.
  • the 100 parts by mass of the cell nutrient component comprises the following parts by mass:
  • the amphiphilic molecule having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 is CH 3 -N + (CH 3 ) 2 - CH 2 -COO - .
  • the cell cryopreservation protection composition is for protecting human cells or animal cells during cryopreservation; preferably for protecting human cells or mammalian cells.
  • the human cell or mammalian cell includes at least one of a cancer cell, a somatic cell, and a stem cell; preferably, the somatic cell is an immune cell or a blood cell.
  • the cell cryopreservation composition is used for cryopreservation
  • the human cells are protected during the period; preferably, the human cells include at least one of lung cancer cells, cervical cancer cells, breast cells, hematopoietic stem cells, immune cells, umbilical mesenchymal stem cells, bone marrow mesenchymal stem cells, lymphatic cancer cells, and blood cells.
  • the cell nutrient component is one or more salts; preferably the salt comprises an inorganic salt; and preferably the inorganic salt is present in the form of physiological saline or a buffer; further Preferably, the buffer is a phosphate buffer.
  • the second aspect of the present application provides a cell cryopreservation solution, which is an aqueous solution of the cell cryoprotective composition, wherein the content of the amphiphilic molecule in the cell cryoprotection solution is 0.1-20% by mass, Based on the total mass of the cell cryoprotectant.
  • the cell cryopreservation protection solution wherein the content of the amphiphilic molecule in the cell cryopreservation protection solution is 4 to 10% by mass based on the total mass of the cell cryopreservation protection solution.
  • a third aspect of the present application provides a method for cryopreservation of a cell, comprising: suspending the cell in the cell cryopreservation solution, placing it in a cryopreservation container, and then performing cryopreservation.
  • the cryopreservation is ultra-fast cryopreservation.
  • cryopreserved cells are resuscitated, and then the cells are directly used or the cells are used in a diluted manner.
  • the recovery survival rate of the cells is at least 70%; preferably, the recovery survival rate of the cells is 80%-99%.
  • 1 ⁇ 10 4 to 1 ⁇ 10 9 cells are suspended in 1.0 ml to 5.0 ml of the concentration of the cell cryopreservation protective solution, and the cells are suspended in the cell cryopreservation protection.
  • 1 ⁇ 10 5 to 1 ⁇ 10 7 cells are suspended in a concentration of 1.5 mL to 1.8 mL of the cell cryopreservation solution, and the cells are suspended in the cell cryopreservation solution.
  • the cryopreservation temperature ranges from -20 ° C to -196 ° C; preferably from -80 ° C to -196 ° C.
  • a fourth aspect of the present application provides a cell cryopreservation composition as described above for cryopreservation in a cell
  • the cell is a human cell or an animal cell; preferably the cell is a human cell or a mammalian cell.
  • the human cell or mammalian cell comprises at least one of a cancer cell, a somatic cell, and a stem cell; preferably, the somatic cell is an immune cell or a blood cell.
  • the cell is a human cell; preferably the human cell comprises a lung cancer cell, a cervical cancer cell, a breast cell, a hematopoietic stem cell, an immune cell, a umbilical cord mesenchymal stem cell, a bone marrow mesenchymal stem cell, a lymphoma cell, and At least one of blood cells.
  • the present invention provides a non-toxic and high-efficiency method, and the cryopreservation method is simple, and the cell cryopreservation composition which can be directly used after resuscitating the cells can be directly used and used. Frozen method.
  • a cryoprotective composition based on an amphiphilic molecule comprising a general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 has not been reported. Therefore, the cell cryopreservation protection composition provided by the present application has urgent practical significance and application value, and has a revolutionary promotion effect on scientific research, biomedicine, clinical treatment and the like.
  • the cell cryopreservation protection composition provided by the present invention wherein the amphiphilic molecule having a general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 has a regulation of cell osmotic pressure or It reduces the effect of freezing point, so it can play a protective role in the process of cell freezing, and the survival rate of cells after resuscitation is extremely high.
  • the cryopreservation step of the cell cryopreservation protection composition provided by the invention is simple and convenient, and is preferably directly placed in a cryopreservation container in a cryopreservation device (including a liquid nitrogen or a low temperature freezer refrigerator) for ultra-free freezing without precipitation.
  • a cryopreservation device including a liquid nitrogen or a low temperature freezer refrigerator
  • the rapid cryopreservation avoids the low survival rate of cell resuscitation caused by the instability of the cooling rate in the traditional "slow-frozen" storage technology, and greatly reduces the number of frozen storage devices.
  • the cell cryopreservation composition provided by the present invention has an active ingredient having an amphiphilic molecule of the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 , and the cell cryopreservation protection combination of the present invention
  • the substance does not contain the following substances: glycerin, diaminoethanetetraacetic acid or a salt thereof, dimethyl sulfoxide, or egg yolk, and thus is different from the conventional cryoprotectant and is completely harmless to cells.
  • the amphoteric molecule having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 according to the present invention includes a compound isolated from a natural substance, and is modified on the basis of a natural compound.
  • Compounds obtained by derivatization or derivatization, and artificially synthesized compounds conforming to the above-described general structure, etc., the compounds themselves are not toxic to cells. Therefore, it is not necessary to resuspend the cells in order to remove the toxic cryoprotectant after resuscitation, which brings broad application prospects in the medical clinical field, especially in the field of cell therapy.
  • Figure 1a Schematic diagram of the principle of freezing damage of cells during routine cryopreservation.
  • Fig. 1b is a schematic diagram showing the principle of protecting the cells by the cell cryopreservation protection composition provided by the present invention when the cells are subjected to conventional cryopreservation.
  • Figure 2 Re-attachment test of different cell cryopreservation protection compositions on human lung cancer cell GCL-82 cells.
  • Figure 3 Re-attachment test of different cell cryopreservation protection compositions on human cervical cancer Hela cells.
  • Figure 4 Re-attachment test of different cell cryopreservation protection compositions on human mammary gland MCF-10 cells.
  • Fig. 5a Test chart of adherence of different cell cryopreservation protection compositions to human lung cancer cell GCL-82 cells after suspension culture for 1 day.
  • Fig. 5b Test chart of adherence of different cell cryopreservation protection compositions to human lung cancer cell GCL-82 cells after 2 days of suspension culture.
  • Fig. 5c Test chart of adherence of different cell cryopreservation protection compositions to human lung cancer cell GCL-82 cells after 3 days of suspension culture.
  • Figure 6 DSC plot of betaine at different concentrations (% by mass).
  • Figure 7 DSC curve of L-carnitine at different concentrations (% by mass).
  • Figure 8 DSC graph of sulfobetaine at different concentrations (% by mass).
  • Figure 9 DSC chart of (3-acrylamidopropyl)-(2-carboxyethyl)-dimethylammonium salt at different concentrations (% by mass).
  • Figure 10 DSC chart of 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionic acid inner salt at different concentrations (% by mass).
  • Figure 11 shows the effect of washing operation on cell survival rate during cryopreservation of sheep blood cells with different concentrations (% by mass) of betaine and different concentrations (% by mass) of glycerol and DMSO.
  • solute damage if the rate of cooling is too fast, the water in the cell has not oozing out of time and icing in the cell, forming Ice crystals can cause fatal damage to organelles, proteins, and membrane structures inside the cell, which is mechanical damage (II).
  • the cell cryopreservation protecting agent 3 (specifically, an amphiphilic molecule) contained in the cell cryopreservation composition provided by the present invention has a function of regulating osmotic pressure, alleviating stress reaction, lowering freezing point of water, and protecting protein.
  • the cell cryopreservation composition of the present application comprises a cell cryoprotectant and a cell nutrient component, wherein the cell cryoprotectant comprises one or more of the general structures R 1 -N + (CH 3 ) 2 - ( CH 2 ) an amphoteric molecule of n -R 2 , for example, a cell cryoprotectant in the cell cryoprotective composition of the present application is the amphiphilic molecule, or a combination of the amphoteric molecule and other conventional cryoprotectants.
  • the amphiphilic molecule is contained in an amount of 10 to 2530 parts by mass based on 100 parts by mass of the cell nutrient component, and the cell cryopreservation composition does not contain a harmful substance, for example, a cell
  • harmful substances are glycerin, diaminoethanetetraacetic acid or a salt thereof, dimethyl sulfoxide, or egg yolk.
  • the amphoteric molecule is preferably contained in an amount of from 420 to 1120 parts by mass based on 100 parts by mass of the cell nutrient component.
  • R 1 is a linear or branched alkyl group having 1 to 10 carbon atoms, which is optionally selected from the group consisting of a (meth)acrylamide group, a (meth)acryloyloxy group, an alkenyl group, a hydroxyl group, and a hydroxyl group.
  • substitution means that the hydrogen atom in the group is substituted by a substituent,
  • R 2 is selected from the group consisting of -COO - , -SO 4 - , -SO 3 - , and Any of the groups of the composition that are negatively charged.
  • R 3 is a group selected from the group consisting of a (meth)acryloyloxyalkyl group, an alkyl group, and an alkenyl group.
  • the (CH 3 ) 2 and (CH 2 ) n may each independently be optionally selected from the group consisting of alkyl, alkenyl, hydroxy, hydroxyalkyl, alkoxy, polyalkylene oxide, and halogen. Replaced by the base,
  • n is an integer from 1 to 10.
  • the amphoteric molecule having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 belongs to a nitrogen-containing and oxygen-containing amphiphilic molecule, and the general structure may have a plurality of substituents.
  • the cell cryoprotective composition of the present application comprises an amphoteric molecule of the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 without glycerin, diaminoethanetetraacetic acid or It is a harmful component such as salt, dimethyl sulfoxide, or egg yolk.
  • the amphoteric molecule having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 includes a compound isolated from a natural substance, modified or derived on the basis of a natural compound Compounds obtained by the synthesis, and artificially synthesized compounds conforming to the above general structure.
  • amphiphiles including CH 3 -N + (CH 3 ) 2 -CH 2 -COO - (this compound is sometimes abbreviated as "betaine” or "natural betaine"
  • phosphoric acid extracted from natural substances Choline derivatives, L-carnitine, and the like are naturally occurring amphiphiles, including CH 3 -N + (CH 3 ) 2 -CH 2 -COO - (this compound is sometimes abbreviated as "betaine” or "natural betaine"
  • the amphoteric molecule having such a general structure is preferably a betaine compound.
  • the betaine compound comprises all compounds having a structure of R 1 -N + (CH 3 ) 2 -(CH 2 ) n -COO - and has been extended to similar compounds containing sulfur and phosphorus in the anion.
  • n is an integer of 1 to 10
  • (CH 2 ) n may each independently be optionally selected from the group consisting of an alkyl group, an alkenyl group, a hydroxyl group, and a hydroxy alkane.
  • Substituents in the group consisting of a group, an alkoxy group, a polyalkylene oxide group, and a halogen are substituted.
  • R 1 is as defined above.
  • betaine and its derivatives phosphorylcholine derivatives, and L-carnitine are preferably used.
  • the structure of betaine can be expressed as (ie, CH 3 -N + (CH 3 ) 2 -CH 2 -COO - , which is also known as ammonium trimethylammonate or betaine).
  • the betaine derivative is preferably a sulfobetaine, a carboxybetaine (meth)acrylamide, a carboxybetaine (alkyl)acrylate or the like.
  • Betaine is extracted from many microorganisms, plants and animals and is an important ingredient in many foods such as wheat, spinach, beets, shellfish and fish. According to reports, the safe food intake of betaine per day for an adult is 9g ⁇ 15g, and the concentration of betaine in the blood can reach 20 ⁇ 70 ⁇ mol / L. In addition to natural betaine, synthetic betaines are equally suitable for use in the present invention.
  • the amphiphilic molecule having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 has the effect of rapidly regulating the osmotic pressure in the body, alleviating the stress reaction, and also the protein in the cell ( For example, enzymes play a protective role. It has good biocompatibility and is easily absorbed by cells. It has been widely used in many fields such as anti-tumor, anti-fatty liver, ulcer treatment and skin care products.
  • R 2 is selected from -COO - , -SO 3 - , and Any one of the group consisting of R 3 is a (meth)acryloyloxyalkyl group. It is also preferred that the hydrogen atom in (CH 2 ) n is substituted by a hydroxyl group and R 2 is -COO ⁇ .
  • amphoteric molecules having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 , most preferably CH 3 -N + (CH 3 ) 2 -CH 2 -COO - .
  • amphoteric molecule having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 in the case where R 2 is -COO - , the general structure R 1 -N + (CH 3) 2 - ( CH 2) n -R 2 of amphipathic molecules may also be wherein R 1 is a carboxy betaine (meth) acrylamide group, (meth) acrylamide, and / or in which R 1 is It is a carboxybetaine (alkyl) acrylate of a (meth) acrylate alkyl ester group.
  • amphoteric molecule having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 it can be used as the betaine (compound 1) (hereinafter sometimes Referred to as "betaine"); structure is (3-acrylamidopropyl)-(2-carboxyethyl)-dimethylammonium salt ((2-carboxyethyl)-dimethylammonium) (compound 2); 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propanoate (3-((2-(methacryloyloxy)ethyl)dimethylammonio)propanoate) (compound 3); 3-[N,N-Dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid internal salt (3-[Dimethyl-[2 -(2-methylprop-2-eno
  • alkyl group an alkenyl group, a hydroxyl group, a hydroxyalkyl group, an alkoxy group, a polyalkylene oxide group.
  • alkenyl group, the hydroxyl group, the hydroxyalkyl group or the alkoxy group each group has 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, and further preferably 1 to 5 carbon atoms. Also preferred are from 1 to 3 carbon atoms, most preferably 1 or 2 carbon atoms.
  • the polyalkylene oxide has a carbon number of 4 to 20 carbon atoms, more preferably 4 to 10 carbon atoms; and the polyalkylene oxide group is more preferably a polyethylene oxide group (ie, polyoxyethylene) . It is also preferred that all of the two methyl groups of the N + linkage in the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 are substituted by polyoxyethylene or hydroxyethyl.
  • the (meth)acryloyloxyalkyl group in R 3 is preferably a (meth)acryloyloxy group-substituted linear or branched alkyl group having 1 to 10 carbon atoms, more preferably (methyl)
  • the acryloyloxy group is substituted with a linear or branched alkyl group having 1 to 5 carbon atoms, and most preferably a (meth)acryloyloxy group-substituted methyl group or ethyl group.
  • the cell nutrient component is a substance conventionally used for culturing cells in the field of cell culture media, and the substance has, for example, a function of supplying cell nutrition and/or a function of promoting cell proliferation.
  • the cell nutrient component is preferably at least one selected from the group consisting of one or more amino acids, one or more salts, one or more sugars, one or more vitamins, And one or more proteins. More preferably, the cell nutrient component comprises one or more amino acids, one or more salts, one or more sugars, one or more vitamins, and one or more proteins.
  • the amino acid may be a common 20 natural amino acids, for example, a natural amino acid including at least one of arginine, glycine, leucine, glutamic acid, isoleucine, and glutamine; Regulating the osmotic pressure of the extracellular environment, and providing the salts necessary for the cells, preferably the inorganic salts, which may include NaCl, NaHCO 3 , KCl, Ca(NO 3 ) 2 , MgSO 4 , and KH 2 PO At least one of 4 ; the saccharide is capable of providing the necessary protection of the cells and regulating the osmotic pressure, providing a suitable environment for the cells, etc., the saccharide may include at least one of glucose and sucrose; the vitamins include vitamin B1, vitamins B6, at least one of biotin, D-calcium pantoth
  • the content of a specific component in the nutrient component of the cell may be the content of a common corresponding component in the cell culture medium.
  • the 100 parts by mass of the cell nutrient component comprises the following parts by mass: 5 to 15 parts of one or more amino acids; 45 to 75 parts of one or more salts; one or more sugars 8 to 32 parts; 0.1 to 1.0 parts of one or more vitamins; 0.5 to 10 parts of one or more proteins.
  • the 100 parts by mass of the cell nutrient component comprises the following parts by mass: 9 to 14 parts of one or more amino acids; 50 to 73 parts of one or more salts; one or more 9 to 24 parts of sugar; 0.1 to 0.5 parts of one or more vitamins; 0.9 to 9 parts of one or more proteins.
  • the composition of the nutrient component for cells is preferably maintained within the range of the osmotic pressure of the aqueous solution obtained by dissolving the cell nutrient component in water.
  • the pH of the aqueous solution is preferably about 7.4.
  • an aqueous solution obtained by mixing all components of the composition (amphiphilic molecule and cell nutrient component) and dissolving in an appropriate amount of water is used as a cell cryopreservation solution.
  • some of the components of the composition eg, cell nutrient components
  • the remaining components of the composition eg, amphiphilic molecules
  • the aqueous solution is used as a cell cryoprotectant.
  • the cell cryoprotection solution of the present application can be obtained by the above two modes of operation.
  • the amount of water used in the preparation of the cell cryoprotectant of the present application there is no particular limitation on the amount of water used in the preparation of the cell cryoprotectant of the present application, and the usual amount in the art can be used. Among them, it is preferably used in the cell cryoprotectant
  • the content of the amphiphilic molecule is from 0.1 to 20% by mass, and further preferably the content of the amphiphilic molecule is from 4 to 10% by mass based on the total mass of the cell cryoprotectant.
  • the pH is preferred to about neutral, for example, adjusting the pH of the cryopreservation solution to 7.2 with an appropriate amount of HCl/NaOH.
  • filter and sterilize the cell cryopreservation solution subjected to the treatment for example, by filtration through a 0.22 ⁇ m disposable sterile filter.
  • the main steps of cryopreservation of cells by the cryopreservation protection composition provided by the present invention are as follows: (1) preparing an amphiphilic molecule comprising a compound having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 a cell cryopreservation protection solution for cryopreservation of the composition; (2) suspending a certain number of cells in the cell cryopreservation solution and placing it in a cryopreservation container for cryopreservation, preferably by direct immersion in a cryopreservation device ( The cryopreservation device in the art, such as liquid nitrogen or cryogenic refrigerator, etc., is cryopreserved; (3) after resuscitating the cells, the frozen storage container is taken out from the cryopreservation device, and then thawed and resuscitated, and then directly or slightly diluted.
  • the cells (such as cell therapy can be injected directly into the patient).
  • the cryopreservation protection composition provided by the present invention and the cell cryopreservation solution containing the same are useful for protecting human cells or animal cells during cryopreservation; preferably for protecting human cells or mammalian cells.
  • the human cell or mammalian cell preferably comprises at least one of a cancer cell, a somatic cell, and a stem cell; more preferably the somatic cell is an immune cell or a blood cell.
  • stem cells include cells such as hematopoietic stem cells, embryonic stem cells, and pluripotent stem cells.
  • the somatic cells are human or animal cells other than non-differentiated stem cells, cancer cells, and germ cells; the immune cells therein include cells such as T cells and macrophages.
  • the cell cryopreservation protection composition and the cell cryopreservation solution containing the same are preferably used for protecting human cells during cryopreservation; more preferably, the human cells include lung cancer cells, cervical cancer cells, breast cells, hematopoietic stem cells At least one of immune cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, lymphatic cancer cells, and blood cells.
  • the reagents, raw materials, and fines used in the preparation examples and effect examples of the present application are as specified.
  • the cells and the like are commercially available or can be prepared by conventional methods in the art.
  • L-carnitine (from Dalian Meilun, article number: MB3278) - compound 6.
  • the product was washed once with diethyl ether every 15-30 min, and after collecting the product, the product was placed in a round bottom flask, vacuumed for 1.5 h (diaphragm pump), and then vacuumed for 1-2 h (vacuum pump with cold trap), dissolved in methanol.
  • Product (1:1 w/w); adding triethylamine to the solution to turbidity of the solution; adding methanol to make the solution clear, stirring for 5 h, adding anhydrous acetone, solid precipitation, static for 30 min, and all the solids were precipitated to obtain a monomer.
  • the structure of the compound was determined to be (3-acrylamidopropyl)-(2-carboxyethyl)-dimethylammonium salt, and specifically, it was found to have a carboxyl group and a propylene group in a nuclear magnetic resonance spectrum. Amido group compound.
  • Table 1 lists the respective components (ammonium molecules and cell nutrient components) of the respective parts by mass of the cell cryopreservation protective composition contained in the cell cryopreservation solution of each amphiphilic concentration obtained.
  • the cell cryopreservation solution was prepared by dissolving the mass fraction of the cells shown in Table 1 in water, and then adding the amphiphilic molecules shown in Table 1 to obtain the concentration of each amphiphilic molecule.
  • the cells are cryopreserved.
  • Preparation Examples 22 to 39 The cell cryopreservation solution was prepared by mixing each component (amphiphilic molecule and cell nutrient component) shown in Table 1 to obtain a cell cryopreservation protective composition, and freezing the cells.
  • the protective composition is dissolved in water to obtain a cell cryopreservation solution having a concentration of each amphiphilic molecule.
  • the cell nutrient components used in the respective preparation examples are composed of the respective components (mg) shown in the composition of the cell nutrient component of the following Table No. 1 to No. 5:
  • Compound name Content (mg) Compound name Content (mg) Calcium chloride 8.86 L-valine 2.15 Potassium chloride 17.72 L-serine 2.26 Potassium nitrate 0.00 L-threonine 5.10 Anhydrous magnesium sulfate 5.25 L-tryptophan 0.86 Sodium chloride 241.97 L-tyrosine 3.84 Anhydrous sodium dihydrogen phosphate 5.84 L-valine 5.05 Sodium selenite 0.00 D-glucose 241.70 L-alanine 1.34 Phenol red 0.81 L-arginine hydrochloride 4.51 HEPES 320.02 L-asparagine 1.34 Sodium pyruvate 5.91 L-aspartate 1.61 Vitamin H 0.00 L-cystine hydrochloride 4.90 Nicotinamide 0.21 L-glutamic acid 4.03 Pyridoxal hydrochloride 0.21 L-glutamine 31.37 D-calcium pantothenate 0.21 Glycine 1.61 Ribofla
  • the cells are commonly used in the laboratory to freeze the protective solution (80% basal medium + 10% DMSO + 10% fetal bovine serum, sometimes referred to as the traditional protective solution; see Situ Zhenqiang's "Cell Culture", R. Ian Freshney “Culture of Animal Cells” and other classic textbooks, as well as different concentrations of glycerol-containing cryoprotectants to preserve animal cells for use with the present invention comprising a general structure R 1 -N + (CH 3 ) 2
  • the (CH 2 ) n -R 2 amphiphilic cell cryopreservation protection composition was compared and the survival rate after cell resuscitation was compared.
  • a solution containing calcein (0.5 mmol/L)/EthD-1 (2 mmol/L) reagent mixture (100 ⁇ L) was added to a suspension containing cells (10 ⁇ L) and injected into a 96-well TCPS plate to stain the cells at room temperature. Protected from light for 30 minutes, then observe and use an inverted fluorescence microscope (brand: Nikon Eclipse, model: Ti-S) and calculate the number of viable cells and the number of dead cells in more than 3 different samples to determine the cell survival rate.
  • human lung cancer cell GCL-82 human lymphoma cell line U937 used in this application is from Tianjin Cancer Hospital; human cervical cancer cell line HELA, human breast cell MCF-10 from Tianjin University, College of Life Sciences; human immune cell lymphocyte H9 and Human hematopoietic stem cells are from Tianjin Medical University; human umbilical cord mesenchymal stem cells, human bone marrow mesenchymal stem cells from the Chinese Academy of Sciences.
  • the experimental results are shown in Table 2 and Table 3 below.
  • the experimental results show that the cell recovery survival rate of the conventional protective solution is very low in the case of directly placing in liquid nitrogen, and the present invention provides a structure having the general structure R 1 -N + (CH 3 ) 2 -(CH 2
  • the n- R 2 amphiphilic cryopreservation composition has a high recovery rate and is safe and non-toxic, and does not require repeated cleaning of the cryoprotective composition.
  • the best ratio of the betaine cryopreservation composition in the preparation example was used to freeze the cells of different species, and the survival rate after cell recovery was compared.
  • the experimental results are shown in Table 4 below.
  • the experimental results show that the cell cryopreservation protection composition provided by the present invention is suitable for various human cell lines, and the conventional cryopreservation protection solution survives the resuscitation cells preserved by various human cell lines by directly placing liquid nitrogen. The rate is very low.
  • the human lung cancer cell GCL-82 cells of different cell concentrations were cryopreserved using the cryopreservation solution of Preparation Example 1, and the survival rate after cell resuscitation was compared.
  • the experimental results are shown in Table 5 below.
  • the experimental results show that the cell cryopreservation protection composition provided by the present invention is suitable for cryopreservation of various cell concentrations, and the cryopreservation recovery survival rate is not affected by the cell concentration.
  • the human lung cancer cell GCL-82 cells were cryopreserved using the cryopreservation solution of Preparation Example 1, and the survival rate after cell recovery after different periods of frozen storage was compared.
  • the experimental results are shown in Table 6 below.
  • the experimental results show that the cell cryopreservation composition provided by the present invention can be cryopreserved for a long time, and maintains a high survival rate of cryopreservation recovery even in the case of a long cryopreservation time.
  • the human lung cancer cell GCL-82 cells were subjected to conventional gradient cooling using the cryopreservation solution of Preparation Example 1 and directly placed in liquid nitrogen cryopreservation, and the cell survival survival rates after cryopreservation of the two cooling rates were compared.
  • a sample was directly placed in liquid nitrogen, and b sample was placed in a gradient cryopreservation box for 30 min at 4 ° C, 90 min at -20 ° C, and 10 h at -80 ° C, and then transferred to liquid nitrogen for cryopreservation; (4) After cryopreservation in liquid nitrogen for 24 h, the cells were thawed at 37 ° C, and the survival rate was counted by staining with a live-staining kit.
  • the experimental results are shown in Table 7 below.
  • the experimental results show that the cell cryopreservation composition provided by the present invention can cryopreservate cells for a long time, and the storage efficiency is not drastically reduced due to a slight change in the freezing rate.
  • the different cell types of the cells were subjected to a combined cryopreservation protection composition dilution and affixing test using a plurality of cell cryopreservation solutions of the present application and a conventional DMSO protection solution.
  • the samples having the betaine concentrations of 2%, 6%, and 10% in the effects of Examples 6 and 7 were the cell cryopreservation solutions of Preparation Example 7, Preparation Example 5, and Preparation Example 3, respectively.
  • a 10% protective solution of DMSO was prepared according to the composition of the conventional protective solution shown in Table 2. See Table 8 below to obtain the DMSO-containing protective solutions in Effect Examples 6 and 7.
  • Figure 2-4 shows different cell types (human lung cancer cell GCL-82 cells, human cervical cancer cell Hela cells, and human breast cell MCF-10 cells, respectively) after cryopreservation with different cryopreservation solutions at the same cell concentration. After being diluted 5 times with the corresponding medium, the culture was directly cultured for the adherence test. As shown in the figure, the cells cultured in the cryopreservation solution containing the betaine cell cryopreservation solution of the present application can be reattached, and the morphology and proliferation function are not affected, while the cells cultured by the conventional DMSO protection solution are reduced and floated. Above the bottom surface, it is not attached to the wall. It can be seen from the morphology that the cells have died.
  • the experimental results show that the cell cryopreservation solution provided by the present invention has no toxicity, can be diluted or directly injected into the body, and the function of the cells after storage is completely normal.
  • the same human lung cancer cell GCL-82 cells were tested for toxicity using the cell cryopreservation solution of Preparation Example 7 of the present application and a conventional DMSO protection solution and a common medium (control).
  • FIG. 5 shows that human lung cancer cell GCL-82 was subjected to an adherent test after suspension culture for 1-3 days at the same cell concentration with different cryoprotective compositions, as shown in the figure, cultured with a cell cryopreservation solution containing 2% betaine.
  • the cells can be attached, the morphology and the proliferation function are the same as the suspension culture of the common medium (control), while the cells cultured by the traditional DMSO protection solution are reduced, floating above the bottom surface, and can not be attached to the wall. death.
  • the experimental results show that the use of this hair
  • the cell cryopreservation solution provided by Ming has no observable toxicity compared to the conventional culture solution (DMSO).
  • DSC Differential Scanning Calorimeter
  • DSC Differential Scanning Calorimeter
  • the DSC is equipped with two sets of compensation heating wires under the sample and the reference container.
  • the differential thermal amplifier circuit and the differential heat compensation amplifier are passed.
  • the current flowing into the compensation heating wire is changed.
  • the compensation amplifier immediately increases the current on one side of the sample; conversely, when the sample is exothermic, the current on the side of the reference object is increased until The heat balance on both sides, the temperature difference ⁇ T disappears.
  • the change in heat that occurs during the thermal reaction of the sample is compensated for by the timely input of electrical power, so the actual difference between the thermal power of the two electrothermal compensation heating wires under the sample and the reference is recorded over time t.
  • the rate of temperature rise is constant, the difference in thermal power recorded as a function of temperature T is recorded. Therefore, when the ambient temperature of the measured sample rises and the temperature reaches the melting point of the sample, there is an endothermic reaction, and the compensation amplifier immediately increases the current on one side of the sample, and the graph shows a peak.
  • Figure 6-10 shows the DSC curves of the specific compounds in the aqueous solution of the betaine compound at different concentrations, where the abscissa is the temperature (°C) and the ordinate is the power difference (W/g). The temperature point is the crystallization temperature.
  • 0% is pure water. As shown in Figure 6-10, as the concentration of the effective component increases, the freezing point of water becomes lower and lower, and it can be proved that the specific compounds contained in the betaine compound are water-free. There is a reduction.
  • Blood transfusion also known as "blood transplantation”
  • Blood transplantation is an important rescue and treatment in clinical practice. It is an important method for rescuing trauma/surgical bleeding and treating diseases such as aplastic anemia, thalassemia and hemolytic anemia.
  • diseases such as aplastic anemia, thalassemia and hemolytic anemia.
  • the blood needs to be preserved to preserve the vitality of the blood cells and maintain the function of the blood cells.
  • the most common method of preserving blood is to place whole blood or blood cells in a preservation solution and store at 4 ° C. In the refrigerator, but this method is only valid for about 42 days, which is easy to cause blood spoilage.
  • Ultra-low temperature cryopreservation can achieve long-term preservation of blood, but there are still two main problems in the cryopreservation solution currently used: 1. Its main component is 40% glycerol, high concentration of glycerol will increase the risk of osmotic damage of blood cells. In the process of deglycerolation, it is particularly serious; 2, it needs to be saved by time-consuming and gradual gradient cooling.
  • the animal cells are preserved with a cryoprotectant containing different concentrations of glycerol and DMSO for use with the present invention comprising R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 having the general structure
  • the amphiphilic cell cryopreservation protection composition was compared to compare the survival rate after cell resuscitation.
  • the cryopreservation agent used in this experiment consists of a buffer (e.g., physiological saline, Phosphate-buffered Saline (PBS)) that is balanced with the physiological osmotic pressure of a human or animal body, and a cryoprotective active ingredient (i.e., The amphoteric molecule of the general structure R 1 -N + (CH 3 ) 2 -(CH 2 ) n -R 2 of the present application; or DMSO or glycerol or the like which is conventionally used. That is, the cryopreservation agent used in this experiment does not require other cell nutrient components.
  • a buffer e.g., physiological saline, Phosphate-buffered Saline (PBS)
  • PBS Phosphate-buffered Saline
  • a cryoprotective active ingredient i.e., The amphoteric molecule of the general structure R 1 -N + (CH 3 ) 2 -(CH
  • each cell cryopreservation solution (which consists only of PBS and cryopreserved active ingredient) according to Table 9 below; (2) Take about 1 ⁇ 10 7 sheep blood cells or 20 ⁇ L rabbit blood, and add about 1.5 mL ⁇ 1.8mL of cell cryopreservation solution, frozen in liquid nitrogen directly in a suitable cryotube; (3) thawed and resuscitated cells at 37 °C after cryopreservation, using a microplate reader (brand: TECAN, model: INFINITE 200PRO The hemolysis in each sample is measured to calculate the survival rate.
  • the method for measuring hemolysis is as follows:
  • sheep blood cells and rabbit blood used in this effect example are purchased from Guangzhou Future Bioscience Department. Technology Co., Ltd.
  • the experimental results are shown in Table 9 and Table 10 below.
  • the experimental results show that under the ultra-rapid cooling process, the cell survival rate of the traditional protective solution and the glycerol protection solution is very low, and the present invention provides a structure having the general structure R 1 -N + (CH 3 ) 2 -(CH 2 )
  • the cryopreservation composition of the n- R 2 amphiphilic group has a high recovery rate and is safe and non-toxic.
  • Effect Example 10 Effect of washing operation on cell survival rate during cryopreservation of sheep blood with different concentrations (% by mass) of betaine and different concentrations (% by mass) of glycerol
  • the sheep blood cells were separately incubated with the various cell cryopreservation solutions of the present application and the traditional blood cell glycerol protection solution to test the effect of the washing process on the survival rate of the sheep blood cells.
  • FIG. 11 shows that sheep blood cells are taken out after soaking for 24 hours at the same cell concentration with different cryoprotective solutions, and diluted 1 times with PBS, then centrifuged to remove the cryopreservation agent in the sample, and the supernatant is tested with a microplate reader. Light transmittance to calculate the survival rate of blood cells.
  • the blood cells after soaking in the cryopreservation solution containing the betaine of the present application are not affected by the blood cell survival rate before and after the dilution washing; and the blood cells after the immersion in the cryopreservation solution containing glycerin and DMSO are diluted, and the glycerin permeability is poor.
  • the cells cannot be discharged in time, resulting in imbalance of intracellular and extracellular osmotic pressure, resulting in hemolysis of a large number of blood cells, and the survival rate is greatly reduced; although the permeability of DMSO is stronger than that of glycerol, it still causes hemolysis of blood cells during the washing process.
  • the experimental results show that with the cell cryopreservation solution provided by the present application, the washing process does not cause osmotic pressure damage to blood cells, which is much better than the most effective traditional glycerin and DMSO cryoprotectant.
  • the cryopreservation solution used in the above effect examples 9-10 consisted only of PBS and a cryoprotective active ingredient.

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Abstract

本申请涉及一种细胞冻存保护组合物、该组合物的用途以及细胞冻存的方法。所述细胞冻存保护组合物包含具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子，其中上述R1为碳数1至10的直链或支链的烷基，其任选被取代基取代，R2为选自由-COO-、-SO4 -、-SO3 -、和（I）组成的组中的任一种带负电荷的基团，所述R3为选自由(甲基)丙烯酰氧基烷基、烷基、和烯基组成的组中的基团；所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子优选为甜菜碱类化合物。所述细胞冻存保护组合物能够无毒高效地实施细胞冻存，细胞复苏后成活率极高且无需梯度冻存；复苏细胞之后，可直接使用或稍稍稀释后使用该细胞。

Description

细胞冻存保护组合物、该组合物的用途以及细胞冻存的方法 技术领域
本申请属于细胞生物领域，具体涉及利用一种包含具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子、且无毒高效的细胞冷冻保护组合物。本申请还涉及该组合物的用途以及对细胞进行冷冻保存的方法。
背景技术
随着生物医学技术的发展，从动物体内提取出的活细胞越来越广泛应用于生物医学中的各个领域。比如，在医药开发领域，活细胞被用于考察药物疗效、剂量和毒性，为动物实验测试提供方向引导，同时也减少实验了动物用量，降低风险；在医疗领域，一方面从人体获得的活细胞样品可以用于检测和诊断，另一方面，具有治疗功能的细胞(比如干细胞、免疫细胞、血细胞)，已被用于攻克危害人类健康的诸多疑难杂症，如癌症、糖尿病、器官损伤等。然而，活细胞离开动物体环境后，在不适宜的条件下会迅速死亡；在细胞培养环境下(37℃培养箱，CO2等)虽然可以存活，但却会不断增殖、衰老，甚至癌变。因此，细胞的长效保存是各类细胞应用的必要前提和一直以来的挑战。
目前，细胞最有效的保存手段是在低温(-80℃或-196℃)下冷冻保存。在这种超低温的条件下，细胞会减缓甚至暂停新陈代谢，从而达到长期保存的目的。然而，在冷冻降温的过程中，冰晶的形成会使细胞遭受致命性的机械损伤和溶质损伤，因此，细胞的冷冻保存往往需要加入冻存保护剂来降低细胞内外液冰晶的产生。二甲基亚砜(DMSO)和卵黄以及甘油是目前最常用的传统冻存保护剂，而由于不同种细胞渗透性以及结构的不同，这些冻存保护剂适用于不同的细胞类型。DMSO适合用作动物体细胞类的冻存保护；而甘 油则常用作细菌细胞、人畜类生殖细胞、以及血细胞的冷冻保存剂；卵黄常用作生殖细胞冻存保护剂的辅助添加剂。但是，传统的冻存保护剂都有他们致命的缺陷：DMSO具有一定的细胞毒性，例如，DMSO会抑制细胞繁殖、改变细胞的生物功能、以及引发干细胞的分化等(具体参见K.Aita et al.Apoptosis in murine lymphoid organs following intraperitoneal administration of dimethyl sulfoxide(DMSO).《Experimental and Molecular Pathology》,2005,79,265-271；R.Pal et al.Diverse effects of dimethyl sulfoxide(DMSO)on the differentiation potential of human embryonic stem cells.《Archives of Toxicology》,2012,86,651-661；B.Hegner et al.Differential regulation of smooth muscle markers in human bone marrow-derived mesenchymal stem cells.《Journal of Hypertension》2005,23,1191-1202.)。因此，如果使用DMSO作为细胞冻存保护剂，必须要尽可能的缩短它在非冻结状态下与细胞的接触时间，另外，在细胞使用之前，需要充分冲洗去除DMSO。但依然有大量研究表明，在细胞治疗领域中，使用DMSO保存过的干细胞，无论如何冲洗，注入人体仍会对人体产生致命的副作用。一方面可能是因为已进入细胞内的DMSO难以彻底清洗掉，另一方面可能是DMSO已经对细胞的生理功能和活性产生了有害的影响(具体参见B.Calmels et al.Preclinical evaluation of an automated closed fluid management device:CytomateTM,for washing out DMSO from hematopoietic stem cell grafts after thawing.Bone Marrow Transplantation.2003,31,823-828.)。
甘油作为第一种被发现的传统冻存保护剂成功冷冻保存了鸡的精子。但是甘油的缺点是，渗透性很差，进入细胞很慢，对于多种细胞没有冻存保护的效果；同时由于渗透性差，在去甘油过程中，细胞极易受到渗透压损伤，尤其是高浓度的甘油冻存保护剂(血液冻存保护剂通常含有40％甘油)；而且，也有甘油影响细胞功能的实例报道(具体参见J Witowski et al.Glycerol  toxicity for human peritoneal mesothelial cells in culture:comparison with glucose.《International Journal of Artificial Organs》,1994,17(5):252-260)。
此外，卵黄的作为冻存保护剂的辅助添加剂也具有以下不足：1)卵黄成分复杂，含有一些对细胞有害的成分如大颗粒物质，高浓度的脂蛋白等；2)卵黄通常来源于家禽的蛋，可能携带一些潜在的传染性病原体(病毒、细菌、支原体)，增加疾病传播的风险；3)不同来源的卵黄质量参差不齐，冻存保护效果存在差异，难以标准化。因此，传统冻存保护剂效率低以及风险性等局限性已成为活细胞在各个领域，尤其是细胞治疗、再生医学等临床领域成功应用的瓶颈问题。
除传统冻存保护剂本身所具有的毒性以外，使用传统保护剂的冻存程序为梯度降温，通常需要约一天的时间。这是因为DMSO或甘油进入细胞能力有限，为了使它们能够慢慢渗入细胞，减少冰晶造成的细胞内机械和溶质损伤，因此需要梯度降温(如降温速率1℃每分钟)，即必须将含有DMSO或甘油的冻存保护剂与细胞混合，在4℃、-20℃、-80℃的条件下依次分别放置一定的时间，最后转移至液氮罐，以达到传统冻存程序中梯度降温的要求。这种梯度降温程序非常繁琐耗时，不仅加剧了有毒冻存保护剂与细胞的接触时间，增加了细胞损伤的机会，还需要多种冷冻设备(如-80℃低温冰箱，-20℃冰箱、4℃冰箱、液氮罐等)，另外一旦没有达到“慢冻”的降温速率要求，冻存效率就可能会显著下降。因此，开发一种无毒、高效，并且不需要繁琐冻存步骤的冷冻保护剂对生物医学等领域具有重大意义。
此外，专利文献1(US4980277)公开了用于家畜的精子卵子运输储存的组合物，该组合物包括甘油5ml(87％)和Merk溶液等组分，而Merk溶液中含有EDTA。本领域专业人士可知，甘油是保护冻存细胞的主要物质，专利文献1用5ml(87％)的甘油保存动物精子，一方面甘油才是在冻存精子中起到了决定性作用的冻存保护剂，具体请参见参考文献(Polge et al.Revival of spermatozoa after vitrification and dehydration at low temperature.Nature  164:666(1949))；另一方面，EDTA有很大细胞毒性。众所周知，EDTA是Ca2+离子螯合剂，会螯合细胞所必需的钙离子从而影响细胞，另外，EDTA对人体也有显著的毒害作用，会刺激皮肤、黏膜，从而引起哮喘、皮肤发疹等症状，是一种可能引起过敏的物质。它通过丙二醇等透皮吸收助剂被人体摄取后会引起钙缺乏症，血压降低，肾脏障碍，染色体异常和原生变异等一系列有害作用(具体参见Singh K.et al.Interaction of EDTA with tributyltin induced cellular toxicity.Indian J Exp Biol.1989Sep；27(9):833-4.,)。
发明内容
技术问题
本发明旨在针对现有细胞冻存技术中冻存保护剂毒性大、操作繁琐、冷冻保存(以下简称为“冻存”)效率不稳定等不足，提供一种包含具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子、无需任何对细胞有毒性的细胞冻存保护组分(如甘油或DMSO)辅助的细胞冻存保护组合物，以及该组合物的用途和使用该组合物的对细胞进行冷冻保存的方法。本发明所述细胞冻存保护组合物可以适用的冻存细胞浓度和细胞种类范围较广，能够实现细胞复苏后存活率高。另外本方法提供的冻存保护组合物冻存步骤简单易行，不需要使用多种大型冻存设备，能够有效降低细胞冻存成本，提高冻存效率。
解决方案
本申请的第一方面提供一种细胞冻存保护组合物，其包含一种或多种具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子、以及细胞用养料成分，
其中相对于100质量份的所述细胞用养料成分，所述两性分子为10～2530质量份；
所述细胞冻存保护组合物不包含以下物质：甘油、二氨基乙烷四乙酸或其盐、二甲基亚砜、或卵黄，
在所述通式结构R1-N+(CH3)2-(CH2)n-R2中，
上述R1为碳数1至10的直链或支链的烷基，其任选被选自由(甲基)丙烯酰胺基、(甲基)丙烯酰氧基、烯基、羟基、羟基烷基、烷氧基、和卤素组成的组中的取代基取代，
R2为选自由-COO-、-SO4 -、-SO3 -、和
Figure PCTCN2017079744-appb-000001
组成的组中的任一种带负电荷的基团，所述R3为选自由(甲基)丙烯酰氧基烷基、烷基、和烯基组成的组中的基团，
所述(CH3)2和(CH2)n可各自独立地任选被选自由烷基、烯基、羟基、羟基烷基、烷氧基、聚环氧烷基、和卤素组成的组中的取代基取代，
n为1至10的整数。
所述细胞冻存保护组合物中，所述通式结构R1-N+(CH3)2-(CH2)n-R2中，
上述R1为碳数1至5的直链或支链的烷基，其任选被选自由(甲基)丙烯酰胺基、(甲基)丙烯酰氧基、羟基、羟基烷基、烷氧基、和卤素组成的组中的取代基取代，
上述R2为选自由-COO-、-SO3 -、和
Figure PCTCN2017079744-appb-000002
组成的组中的任一种基团，所述R3为(甲基)丙烯酰氧基烷基，
所述(CH3)2和(CH2)n中的氢原子可各自独立地任选被选自由烷基、羟基、羟基烷基、烷氧基、聚环氧烷基、和卤素组成的组中的取代基取代，
n为1至5的整数。
所述细胞冻存保护组合物中，相对于100质量份的所述细胞用养料成分， 所述两性分子为420～1120份；和
所述通式结构R1-N+(CH3)2-(CH2)n-R2中，
上述R1为碳数1至3的直链或支链的烷基，其任选被选自由(甲基)丙烯酰胺基、(甲基)丙烯酰氧基、羟基、碳原子数1至5的羟基烷基、和碳原子数1至5的烷氧基组成的组中的取代基取代，
所述(CH3)2和(CH2)n中的氢原子可各自独立地任选被选自由碳原子数1至5的烷基、羟基、碳原子数1至5的羟基烷基、碳原子数1至5的烷氧基、和聚环氧烷基组成的组中的取代基取代，
n为1至3的整数。
所述细胞冻存保护组合物中，所述100质量份的细胞用养料成分包含下述质量份数的组分：
Figure PCTCN2017079744-appb-000003
所述细胞冻存保护组合物中，所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子为CH3-N+(CH3)2-CH2-COO-。
所述细胞冻存保护组合物中，所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子中，(CH2)n中的氢原子被羟基取代且R2为-COO-。
所述细胞冻存保护组合物中，所述细胞冻存保护组合物用于在冷冻保存期间保护人类细胞或动物细胞；优选用于保护人类细胞或哺乳动物细胞。
所述细胞冻存保护组合物中，所述人类细胞或哺乳动物细胞包括癌细胞、体细胞、和干细胞的至少之一；优选所述体细胞为免疫细胞或血细胞。
所述细胞冻存保护组合物中，所述细胞冻存保护组合物用于在冷冻保存 期间保护人类细胞；优选所述人类细胞包括肺癌细胞、宫颈癌细胞、乳腺细胞、造血干细胞、免疫细胞、脐带间质干细胞、骨髓间充质干细胞、淋巴癌细胞、和血细胞的至少之一。
所述细胞冻存保护组合物中，所述细胞用养料成分为一种或多种盐；优选所述盐包括无机盐；并还优选所述无机盐以生理盐水或缓冲液的形式存在；进一步优选所述缓冲液为磷酸盐缓冲液。
本申请的第二方面提供一种细胞冻存保护液，其为上述细胞冻存保护组合物的水溶液，其中在所述细胞冻存保护液中所述两性分子的含量为0.1～20质量％，基于所述细胞冻存保护液的总质量。
所述细胞冻存保护液中，其中在所述细胞冻存保护液中所述两性分子的含量为4～10质量％，基于所述细胞冻存保护液的总质量。
本申请的第三方面提供一种对细胞进行冷冻保存的方法，其包括：将细胞悬浮于上述细胞冻存保护液中，并置于冻存容器内，然后进行冷冻保存。
所述对细胞进行冷冻保存的方法中，所述冷冻保存为超快速冷冻保存。
所述对细胞进行冷冻保存的方法中，在进行所述冷冻保存后，使经冷冻保存的细胞复苏，然后直接使用所述细胞、或以稀释方式使用所述细胞。
所述对细胞进行冷冻保存的方法中，所述细胞的复苏成活率为至少70％；优选所述细胞的复苏成活率为80％-99％。
所述对细胞进行冷冻保存的方法中，以1×104～1×109个细胞悬浮于1.0ml-5.0ml所述细胞冻存保护液的浓度，将细胞悬浮于所述细胞冻存保护液中；优选以1×105～1×107个细胞悬浮于1.5mL～1.8mL所述细胞冻存保护液的浓度，将细胞悬浮于所述细胞冻存保护液中。
所述对细胞进行冷冻保存的方法中，所述冷冻保存的温度范围为-20℃至-196℃；优选为-80℃至-196℃。
本申请的第四方面提供一种上述细胞冻存保护组合物在细胞冷冻保存 中的用途，所述细胞为人类细胞或动物细胞；优选所述细胞为人类细胞或哺乳动物细胞。
所述用途中，所述人类细胞或哺乳动物细胞包括癌细胞、体细胞、和干细胞的至少之一；优选所述体细胞为免疫细胞或血细胞。
所述用途中，所述细胞为人类细胞；优选所述人类细胞包括肺癌细胞、宫颈癌细胞、乳腺细胞、造血干细胞、免疫细胞、脐带间质干细胞、骨髓间充质干细胞、淋巴癌细胞、和血细胞的至少之一。
有益效果
本发明为了解决现有冻存保护细胞技术中的不足之处，提供一种无毒而又高效，冻存方法简便，复苏细胞之后无需清洗可直接使用的细胞冻存保护组合物及使用其进行冻存的方法。基于包含具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子的冻存保护组合物还未见报道。因而本申请提供的细胞冻存保护组合物具有迫切的现实意义和应用价值，对科学研究、生物医药、临床治疗等领域有着革命性的促进作用。
对本发明的技术效果综述如下：
1)本发明所提供的细胞冻存保护组合物，由于其组分具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子具有调节细胞渗透压或者降低冰点的作用，因此可以在细胞冷冻过程中起到保护作用，复苏后细胞成活率极高。
2)本发明所提供的细胞冻存保护组合物其冻存步骤简便易行，优选直接放入冷冻保存装置(包括，液氮或低温冷冻冰箱)中的冻存容器进行无需梯度冻存的超快速冷冻保存，避免了传统“慢冻”储存技术中因为降温速率的不稳定而造成的细胞复苏成活率低下这种情况，也极大减少了冻存设备的使用数量。
3)本发明所提供的细胞冻存保护组合物有效成份具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子，本发明的细胞冻存保护组合物不包含以 下物质：甘油、二氨基乙烷四乙酸或其盐、二甲基亚砜、或卵黄，因此不同于传统的冻存保护剂，对细胞完全无害。本发明所述的具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子包括从天然物质中分离得到的化合物、以及在天然化合物的基础上进行改性或者衍生化而得到的化合物、以及人工合成的符合上述通式结构的化合物等，化合物本身对于细胞无毒性。因此，无需复苏后为了去除有毒性的冻存保护剂而反复冲洗细胞，为医疗临床领域，尤其是为细胞治疗等领域带来了广阔的应用前景。
附图说明
图1a：细胞在进行常规低温冷冻保存时受到冷冻损伤的原理示意图。
图1b：细胞在进行常规低温冷冻保存时，本发明提供的细胞冻存保护组合物保护细胞的原理示意图。
图2：不同细胞冻存保护组合物对人肺癌细胞GCL-82细胞的再贴壁测试图。
图3：不同细胞冻存保护组合物对人宫颈癌细胞Hela细胞的再贴壁测试图。
图4：不同细胞冻存保护组合物对人乳腺细胞MCF-10细胞的再贴壁测试图。
图5a：不同细胞冻存保护组合物对人肺癌细胞GCL-82细胞悬浮培养1天后再贴壁测试图。
图5b：不同细胞冻存保护组合物对人肺癌细胞GCL-82细胞悬浮培养2天后再贴壁测试图。
图5c：不同细胞冻存保护组合物对人肺癌细胞GCL-82细胞悬浮培养3天后再贴壁测试图。
图6：不同浓度(质量％)下的甜菜碱的DSC曲线图。
图7：不同浓度(质量％)下的左旋肉碱DSC曲线图。
图8：不同浓度(质量％)下的磺基甜菜碱的DSC曲线图。
图9：不同浓度(质量％)下的(3-丙烯酰胺基丙基)-(2-羧乙基)-二甲基铵盐的DSC曲线图。
图10：不同浓度(质量％)下的3-[[2-(甲基丙烯酰氧基)乙基]二甲基铵]丙酸内盐的DSC曲线图。
图11：示出了采用不同浓度(质量％)的甜菜碱和不同浓度(质量％)的甘油和DMSO对绵羊血细胞进行冻存保护过程中，洗涤操作对细胞成活率的影响。
附图标记列表
1 细胞
2 冰晶
3 细胞冻存保护剂
具体实施方式
以下结合实施例对本发明的技术方案做进一步的详细说明，但本发明不受这些具体实施例的限制。
如图1a所示，细胞在低温冷冻保存时经常会受到冷冻损伤以致细胞死亡。1970年，Mazur提出了冷冻损伤的两种因素假设，如图1a所示。当细胞1降低到冰点以下温度时，如果降温速度过于缓慢，细胞外的冰晶2要早于细胞内先形成，于是导致细胞外渗透压骤升，由于细胞内外渗透压差，细胞内的流动水会通过细胞膜大量渗出细胞外，导致细胞脱水而亡，这就是所谓的溶质损伤(I)；如果降温速率过快，细胞内的水分还没有时间渗出细胞就在细胞内结冰，形成的冰晶对细胞内部的细胞器，蛋白，和膜结构等会产生致命的损伤，这就是机械损伤(II)。
如图1b所示，本发明提供的细胞冻存保护组合物所包含的细胞冻存保护剂3(具体为，两性分子)具有调节渗透压、缓和应激反应、降低水的冰点，以及保护蛋白等作用，在细胞冷冻保存中对避免两种冷冻损伤起到了决定作用，从而能够保护细胞成活。
本申请的细胞冻存保护组合物包含细胞冻存保护剂和细胞用养料成分，其中的细胞冻存保护剂包含一种或多种具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子，例如，本申请细胞冻存保护组合物中的细胞冻存保护剂为该两性分子、或该两性分子与其他常规冻存保护剂的组合。
本申请的细胞冻存保护组合物中，相对于100质量份的细胞用养料成分，以10-2530质量份包含上述两性分子，所述细胞冻存保护组合物不包含有害物质，如，对细胞有害的物质，举例为甘油、二氨基乙烷四乙酸或其盐、二甲基亚砜、或卵黄。
本申请的细胞冻存保护组合物中，相对于100质量份的细胞用养料成分，优选以420～1120质量份包含上述两性分子。
所述通式结构R1-N+(CH3)2-(CH2)n-R2中，氮原子带有正电荷，R2为带有负电荷的基团。其中，上述R1为碳数1至10的直链或支链的烷基，其任选被选自由(甲基)丙烯酰胺基、(甲基)丙烯酰氧基、烯基、羟基、羟基烷基、烷氧基、和卤素组成的组中的取代基取代，所述取代是指基团中的氢原子被取代基取代，
R2为选自由-COO-、-SO4 -、-SO3 -、和
Figure PCTCN2017079744-appb-000004
组成的组中的任一种带负电荷的基团。R3为选自由(甲基)丙烯酰氧基烷基、烷基、和烯基组成的组中的基团。
所述(CH3)2和(CH2)n可各自独立地任选被选自由烷基、烯基、羟基、羟基烷基、烷氧基、聚环氧烷基、和卤素组成的组中的取代基地取代，
n为1至10的整数。
所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子属于含氮及含氧的两性分子，该通式结构中可以具有多种取代基。本申请的细胞冻存保护组合物中，包含通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子，同时不包含甘油、二氨基乙烷四乙酸或其盐、二甲基亚砜、或卵黄等有害组分。
所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子，包括从天然物质中分离得到的化合物、在天然化合物的基础上进行改性或者衍生化而得到的化合物、以及人工合成的符合上述通式结构的化合物。作为天然存在的两性分子，包括从天然物质中提取出来的CH3-N+(CH3)2-CH2-COO-(该化合物有时简称为“甜菜碱”或“天然甜菜碱”)、磷酸胆碱衍生物、左旋肉碱等。
具备该通式结构的两性分子优选为甜菜碱类化合物。该甜菜碱类化合物包含具有R1-N+(CH3)2-(CH2)n-COO-结构的所有化合物，且已经扩展到负离子中含硫和含磷的类似化合物。其中，正负离子之间的连接基团(CH2)n中，n为1至10的整数，且(CH2)n可各自独立地任选被选自由烷基、烯基、羟基、羟基烷基、烷氧基、聚环氧烷基、和卤素组成的组中的取代基取代。R1的定义同上所定义。
在甜菜碱类化合物中，优选使用甜菜碱及其衍生物、磷酸胆碱衍生物、左旋肉碱。甜菜碱的结构可表示为
Figure PCTCN2017079744-appb-000005
(即，CH3-N+(CH3)2-CH2-COO-，其又称为三甲基乙酸铵、或三甲铵乙内酯)。甜菜碱衍生物优选磺基甜菜碱、羧基甜菜碱(甲基)丙烯酰胺、羧基甜菜碱(甲基)丙烯酸烷基酯等。
甜菜碱可从许多微生物、植物和动物中提取出来，是许多食物比如小麦、菠菜、甜菜、贝类和鱼类中的等重要的组成成份。据报道，一个成人每天对甜菜碱的安全摄食量是9g～15g，血液中的甜菜碱的浓度可达到20～70μmol/L。除天然甜菜碱外，人工合成的甜菜碱同样适用于本发明。
具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子具有迅速调节体内细胞渗透压，缓和应激反应的功效，而且还可以对细胞内的蛋白(比如酶)起到保护作用。它生物相容性好、容易被细胞快速吸收，已在很多如抗肿瘤、抗脂肪肝、治疗溃疡等医药以及护肤品领域广泛使用。
所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子中，优选上述R2为选自由-COO-、-SO3 -、和
Figure PCTCN2017079744-appb-000006
组成的组中的任一种基团，所述R3为(甲基)丙烯酰氧基烷基。还优选，(CH2)n中的氢原子被羟基取代且R2为-COO-。
所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子中，最优选为CH3-N+(CH3)2-CH2-COO-。
所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子中，在R2为-COO-的情况下，所述通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子还可以为其中R1为(甲基)丙烯酰胺基的羧基甜菜碱(甲基)丙烯酰胺、和/或为其中R1为(甲基)丙烯酸烷基酯基的羧基甜菜碱(甲基)丙烯酸烷基酯。
在具体技术方案中，作为具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子，可以使用为三甲铵乙内酯(化合物1)(下文中有时简称为“甜菜碱”)；结构 为
Figure PCTCN2017079744-appb-000007
的(3-丙烯酰胺基丙基)-(2-羧乙基)-二甲基铵盐((3-Acryloylaminopropyl)-(2-carboxyethyl)-dimethylammonium)(化合物2)；结构为
Figure PCTCN2017079744-appb-000008
的3-[[2-(甲基丙烯酰氧基)乙基]二甲基铵]丙酸内盐(3-((2-(methacryloyloxy)ethyl)dimethylammonio)propanoate)(化合物3)；结构为
Figure PCTCN2017079744-appb-000009
的3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐(3-[Dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]azaniumyl]propane-1-sulfonate)(化合物4)(下文中有时简称为“磺基甜菜碱”)；结构为
Figure PCTCN2017079744-appb-000010
的2-(甲基丙烯酰氧基)乙基-2-(三甲基氨基)乙基磷酸酯(2-(Methacryloyloxy)ethyl-2-(Trimethylammonio)ethyl Phosphate)(化合物5)(下文中有时简称为“磷酸胆碱衍生物”)；或，结构为
Figure PCTCN2017079744-appb-000011
的3-羧基-2-羟基-N,N,N-三甲基-1-丙酸铵内盐(2R)(1-Propanaminium,3-carboxy-2-hydroxy-N,N,N-trimethyl-,inner salt, (2R)-)(化合物6)(下文中有时简称为“左旋肉碱”)。其中，本段中表示化合物1至6的各结构式中未明确示出的各基团分别为碳原子及与其相连接的氢原子，如CH3、CH2、CH、和C等。
需要说明的是，本申请中使用的各种未经碳原子数限定的基团(该基团包括但不限于烷基、烯基、羟基、羟基烷基、烷氧基、聚环氧烷基、烯基、羟基、羟基烷基、烷氧基)中，各基团的碳数为1-20个碳原子，更优选为1-10个碳原子，并进一步优选为1-5个碳原子，还优选1-3个碳原子，最优选1或2个碳原子。其中，聚环氧烷基的碳数为4-20个碳原子，更优选为4-10个碳原子；聚环氧烷基更优选为聚环氧乙烷基(即，聚氧乙烯基)。还优选通式结构R1-N+(CH3)2-(CH2)n-R2中N+连接的两个甲基全部或部分被聚氧乙烯基或羟乙基取代。此外，R3中的(甲基)丙烯酰氧基烷基，优选为(甲基)丙烯酰氧基取代的碳数1至10的直链或支链的烷基，更优选(甲基)丙烯酰氧基取代的碳数1至5的直链或支链的烷基，最优选(甲基)丙烯酰氧基取代的甲基或乙基。
在本发明中，所述细胞用养料成分为细胞培养基领域中常规用于培养细胞的物质，该物质例如具有供给细胞营养的功能和/或促使细胞生殖增殖的功能。所述细胞用养料成分优选为选自由以下物质组成的组的至少一种物质：一种或多种氨基酸、一种或多种盐、一种或多种糖类、一种或多种维生素、和一种或多种蛋白质。所述细胞用养料成分更优选包含一种或多种氨基酸、一种或多种盐、一种或多种糖类、一种或多种维生素、和一种或多种蛋白质。
在本发明中，细胞用养料成分中的具体组分，例如氨基酸、盐、糖类、维生素、和蛋白质等采用细胞培养基中的常见相应组分即可。所述氨基酸可为常见的20种天然氨基酸，例如，包括精氨酸、甘氨酸、亮氨酸、谷氨酸、异亮氨酸、和谷氨酰胺的至少之一的天然氨基酸；所述盐能够调节细胞外环境的渗透压，以及提供细胞必须的盐，所述盐优选为无机盐，所述无机盐可 包括NaCl、NaHCO3、KCl、Ca(NO3)2、MgSO4、和KH2PO4的至少之一；所述糖类能够提供细胞必要的保护以及调节渗透压，提供细胞适宜的环境等，所述糖类可包括葡萄糖和蔗糖的至少之一；所述维生素包括维生素B1、维生素B6、生物素、D-泛酸钙、叶酸、i-肌醇、烟酰胺、氯化胆碱、盐酸吡哆醇、核黄素、盐酸硫胺素、和维生素B12的至少之一；和/或，所述蛋白包括白蛋白、和血清(其包含白蛋白)的至少之一。
在本发明中，细胞用养料成分中的具体组分含量，例如氨基酸、盐、糖类、维生素、和蛋白质等的含量采用细胞培养基中的常见相应组分的含量即可。例如，所述100质量份的细胞用养料成分包含下述质量份数的组分：一种或多种氨基酸5～15份；一种或多种盐45～75份；一种或多种糖类8～32份；一种或多种维生素0.1～1.0份；一种或多种蛋白质0.5～10份。优选地，所述100质量份的细胞用养料成分包含下述质量份数的组分：一种或多种氨基酸9～14份；一种或多种盐50～73份；一种或多种糖类9～24份；一种或多种维生素0.1～0.5份；一种或多种蛋白质0.9～9份。
在本发明中，细胞用养料成分的组成，优选在将细胞用养料成分溶于水之后得到的水溶液维持在细胞渗透压范围之内，例如，优选该水溶液的pH值在7.4左右。
本发明的细胞冻存保护组合物在使用时，可将所述组合物的全部组分(两性分子和细胞用养料成分)混合之后溶于适量水中而得到的水溶液作为细胞冻存保护液使用。可选地，可先将所述组合物的部分组分(如，细胞用养料成分)溶于水，之后在该水溶液中加入所述组合物的剩余组分(如，两性分子)而得到的水溶液作为细胞冻存保护液使用。以上两种操作方式均可得到本申请的细胞冻存保护液。
对于制备本申请细胞冻存保护液的过程中所使用的水的量没有特别的限制，本领域的常规用量即可。其中，优选采用在所述细胞冻存保护液中所 述两性分子的含量为0.1～20质量％，并进一步优选所述两性分子的含量为4～10质量％，基于所述细胞冻存保护液的总质量。
制得上述细胞冻存保护液后，优选还需要调节pH值至中性左右，例如用适量的HCl/NaOH将冻存保护液调pH至7.2。此外，还优选再将经过该处理的细胞冻存保护液过滤除菌，例如用0.22μm的一次性无菌滤膜过滤除菌。
本发明提供的冻存保护组合物冷冻保存细胞的主要步骤如下：(1)配制包含含有具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子的细胞冻存保护组合物的细胞冻存保护液；(2)将一定数量的细胞悬浮于所述细胞冻存保护液中并置于冻存容器内进行冷冻保存，优选采用直接浸入冷冻保存装置(本领域的常规冻存装置，例如液氮或低温冷冻冰箱等)的方式进行冷冻保存；(3)复苏细胞之后，从冷冻保存装置内取出冻存容器解冻复苏后即可直接或稍稍稀释后使用该细胞(比如细胞治疗可直接注入病人体内)。
本发明提供的冻存保护组合物及包含其的细胞冻存保护液用于在冷冻保存期间保护人类细胞或动物细胞；优选用于保护人类细胞或哺乳动物细胞。其中，所述人类细胞或哺乳动物细胞优选包括癌细胞、体细胞、和干细胞的至少之一；更优选所述体细胞为免疫细胞或血细胞。
其中，干细胞包含造血干细胞、胚胎干细胞、和多能干细胞等细胞。体细胞为除了非分化的干细胞、癌细胞和生殖细胞之外的人类或动物细胞；其中的免疫细胞包含T细胞、和巨噬细胞等细胞。
进一步地，所述细胞冻存保护组合物及包含其的细胞冻存保护液优选用于在冷冻保存期间保护人类细胞；更优选所述人类细胞包括肺癌细胞、宫颈癌细胞、乳腺细胞、造血干细胞、免疫细胞、脐带间质干细胞、骨髓间充质干细胞、淋巴癌细胞、和血细胞的至少之一。
制备例
除特别注明之外，本申请制备例以及效果例中所使用的试剂、原料、细 胞等均可以由市场购得，或者由本领域常规方法制备而得。
材料的来源
甜菜碱(来源于伊诺凯Acros，货号：204241000)—化合物1；
磺基甜菜碱(来源于希恩思，货号：M-58510)—化合物4；
磷酸胆碱衍生物(来源于希恩思，货号：P-013898)—化合物5；
左旋肉碱(来源于大连美仑，货号：MB3278)—化合物6。
(3-丙烯酰胺基丙基)-(2-羧乙基)-二甲基铵盐(化合物2)，其制备方法如下：用500mL三口瓶将体系用氮气吹扫15min，除去空气及其中的水蒸气；加入300mL无水丙酮，加入甲基丙烯酸二甲氨乙酯53.98g，室温搅拌半小时至完全溶解，氮气保护，之后滴加25g的β-丙内酯，反应2h，开始过滤。每隔15-30min用乙醚洗涤一次产物，收集齐产物后，将产物置于圆底烧瓶中，抽真空1.5h(隔膜泵)，再抽真空1-2h(真空泵带冷阱)，用甲醇溶解产品(1:1w/w)；向溶液中加入三乙胺至溶液浑浊；加入甲醇使溶液澄清，搅拌5h，滴加无水丙酮，有固体析出，静止30min，待固体全部析出得到单体。核磁共振波谱检测后确定该化合物的结构为(3-丙烯酰胺基丙基)-(2-羧乙基)-二甲基铵盐，具体地，在核磁共振波谱中显示得到了具有羧基和丙烯酰胺基的化合物。
3-[[2-(甲基丙烯酰氧基)乙基]二甲基铵]丙酸内盐(化合物3)，其制备方法如下：向两颈瓶内加入156g二甲胺基丙基丙烯酰胺，再加入200mL甲醇，混合体系用冰浴冷却；向冷却后的体系中滴加60g的乙酸，再加入72.06g的丙烯酸；用铝箔包裹两颈瓶做避光处理，室温下搅拌48h；缓慢滴加250mL三乙胺(约15min)；搅拌50min后，缓慢滴加1200mL的丙酮/三乙胺(摩尔比1/1)混合物(40-50min)，使羧基甜菜碱甲基丙烯酸析出；搅拌20min后，过滤，滤饼用500mL丙酮洗涤，反复3次，真空干燥，得到133g羧基甜菜碱甲基丙烯酸；利用266mL甲醇溶解上步得到固体，加入100mL三乙胺，混合液搅拌 混合1h；缓慢加入1000mL的丙酮/三乙胺(摩尔比1/1)混合物(40-50min)，得到130g产物。核磁共振波谱检测后确定该化合物的结构为3-[[2-(甲基丙烯酰氧基)乙基]二甲基铵]丙酸内盐，具体地，在核磁共振波谱中显示得到了具有羧基和甲基丙烯酸酯基的化合物。
以下表1中列出了，所得的各两性分子浓度的细胞冻存保护液中所包含的、细胞冻存保护组合物的相应质量份的各组分(两性分子和细胞用养料成分)。
其中，制备例1至21制备细胞冻存保护液的操作为，将表1所示质量份的细胞用养料成分溶于水中，然后再添加表1所示质量份的两性分子得到各两性分子浓度的细胞冻存保护液。制备例22至39制备细胞冻存保护液的操作为，将表1所示各质量份的各组分(两性分子和细胞用养料成分)混合后得到细胞冻存保护组合物，并将细胞冻存保护组合物溶于水中得到各两性分子浓度的细胞冻存保护液。
表1
Figure PCTCN2017079744-appb-000012
表1续(1)
Figure PCTCN2017079744-appb-000013
表1续(2)
Figure PCTCN2017079744-appb-000014
表1续(3)
Figure PCTCN2017079744-appb-000015
表1续(4)
Figure PCTCN2017079744-appb-000016
表1续(5)
Figure PCTCN2017079744-appb-000017
其中，各制备例中采用的细胞用养料成分由下表编号1至编号5的细胞用养料成分的组成中示出的各组分以其相应的质量(毫克)组成：
编号1的细胞用养料成分的组成
Figure PCTCN2017079744-appb-000018
编号2的细胞用养料成分的组成
Figure PCTCN2017079744-appb-000019
编号3的细胞用养料成分的组成
Figure PCTCN2017079744-appb-000020
编号4的细胞用养料成分的组成
Figure PCTCN2017079744-appb-000021
编号5的细胞用养料成分的组成
化合物名称 含量(mg) 化合物名称 含量(mg)
氯化钙 8.86 L-脯氨酸 2.15
氯化钾 17.72 L－丝氨酸 2.26
硝酸钾 0.00 L-苏氨酸 5.10
无水硫酸镁 5.25 L-色氨酸 0.86
氯化钠 241.97 L-酪氨酸 3.84
无水磷酸二氢钠 5.84 L-缬氨酸 5.05
五水亚硒酸钠 0.00 D-葡萄糖 241.70
L-丙氨酸 1.34 酚红 0.81
L-精氨酸盐酸盐 4.51 HEPES 320.02
L－天门冬酰胺 1.34 丙酮酸钠 5.91
L－天门冬氨酸 1.61 维生素H 0.00
L-胱氨酸盐酸盐 4.90 烟酰胺 0.21
L-谷氨酸 4.03 盐酸吡哆醛 0.21
L-谷氨酰胺 31.37 D-泛酸钙 0.21
甘氨酸 1.61 核黄素 0.02
L-组氨酸盐酸盐 2.26 氯化胆碱 0.21
L-异亮氨酸 5.64 盐酸硫胺 0.21
L-亮氨酸 5.64 叶酸 0.21
L－赖氨酸盐酸盐 7.84 i-肌醇 0.39
L-蛋氨酸 1.61 维生素B12 0.00
L-苯丙氨酸 3.54 人血白蛋白 53.71
效果例1、对不同冻存保护液配方的比较
1、实验设计
以实验室常用的细胞冻存保护液(80％基础培养基+10％DMSO+10％胎牛血清，后文有时简称传统保护液；见司徒振强的《细胞培养》、R.Ian Freshney的《Culture of Animal Cells》等经典教材)，以及不同浓度的含有甘油的冻存保护剂对动物细胞进行保存，用来与本发明提供的包含具有通式结 构R1-N+(CH3)2-(CH2)n-R2的两性分子的细胞冻存保护组合物进行比较，比较细胞复苏后的成活率。
2、实验方法
(1)取人肺癌细胞GCL-82进行传代培养；(2)按照下表2配制含有所述细胞冻存保护液并进行细胞计数；(3)收集约1×106个细胞悬浮于约1.5mL～1.8mL的细胞冻存保护液加入合适冻存管并直接放入液氮中冻存；(4)在液氮中冻存24h后在37℃融化复苏细胞，利用活死染色试剂盒染色计数(来源于life technology,货号：L3224)，用如下方法统计成活率：
将含有钙黄绿素(0.5mmol/L)/EthD-1(2mmol/L)试剂混合物溶液(100μL)加入到含有细胞(10μL)的悬液注入到96孔TCPS板对细胞进行染色，并在室温下避光30分钟，然后观察并使用倒置荧光显微镜(品牌：Nikon Eclipse，型号：Ti-S)并计算超过3个不同样本的活细胞数目和死细胞数目，求出细胞成活率。
此外，并对人宫颈癌细胞HELA和人乳腺细胞MCF-10进行与针对人肺癌细胞GCL-82的上述实验相同的实验。
其中，本申请采用的人肺癌细胞GCL-82，人淋巴癌细胞U937来自天津市肿瘤医院；人宫颈癌细胞HELA，人乳腺细胞MCF-10来自天津大学生命科学学院；人免疫细胞淋巴细胞H9与人造血干细胞来自天津医科大学；人脐带间质干细胞，人骨髓间充质干细胞来自中国科学院。
3、实验结果
实验结果示于下表2和表3。实验结果表明，在直接放入液氮中的情况下，传统保护液的细胞复苏成活率很低，而本发明提供的包含具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子的冻存保护组合物复苏成活率极高，且安全无毒，无需反复清洗冻存保护组合物。
表2.不同冻存保护液配方冻存人肺癌细胞GCL-82的复苏成活率
Figure PCTCN2017079744-appb-000022
表3.不同冻存保护液配方冻存人宫颈癌细胞HELA和人乳腺细胞MCF-10的复苏成活率
Figure PCTCN2017079744-appb-000023
效果例2、对不同细胞种类的冻存效果比较
1、实验设计
使用制备例中甜菜碱冻存保护组合物的较佳配比进行不同种细胞的冻存，比较细胞复苏后的成活率。
2、实验方法
(1)取人细胞系进行传代培养；(2)配制包含相应的较佳配比的甜菜碱冻存保护组合物的冻存保护液和传统保护液，并进行细胞计数；(3)收集约1×106个细胞悬浮于约1.5mL～1.8mL的细胞冻存保护液加入合适冻存管并直接放入液氮中冻存；(4)在液氮中冻存24h后在37℃融化复苏细胞，利用活死染色试剂盒染色计数统计成活率。
3、实验结果
实验结果示于下表4。实验结果表明，利用本发明提供的细胞冻存保护组合物适用于各种人类细胞系，而传统的冻存保护液利用直接放入液氮的方法对各类人细胞系进行保存的复苏细胞成活率很低。
表4.甜菜碱冻存保护组合物对不同种类细胞冻存效果比较
Figure PCTCN2017079744-appb-000024
效果例3、对不同细胞浓度的冻存效果比较
1、实验设计
使用制备例中1的冻存保护液对不同细胞浓度的人肺癌细胞GCL-82细胞进行冷冻保存，比较细胞复苏后的成活率。
2、实验方法
(1)取人肺癌细胞GCL-82细胞进行传代培养；(2)配制制备例1的冻存保护液，并进行细胞计数；(3)分别收集约1×105、5×105、1×106个细胞悬浮于约1.5mL～1.8mL的细胞冻存保护液中、加入合适冻存管并直接放入液氮中冻存；(4)在液氮中冻存24h后在37℃融化复苏细胞，利用活死染色试剂盒染色计数统计成活率。
3、实验结果
实验结果示于下表5。实验结果表明，利用本发明提供的细胞冻存保护组合物适用于各种细胞浓度的冷冻保存，其冻存复苏成活率不受细胞浓度的影响。
表5.制备例1的冻存保护液对人肺癌细胞GCL-82不同细胞浓度的冻存效果比较
Figure PCTCN2017079744-appb-000025
效果例4、冷冻储存时间对冻存复苏成活率的影响
1、实验设计
使用制备例1的冻存保护液对人肺癌细胞GCL-82细胞进行冷冻保存，比较冷冻储存不同时间后细胞复苏后的成活率。
2、实验方法
(1)取人肺癌细胞GCL-82细胞进行传代培养；(2)配制制备例1的冻存保 护液并进行细胞计数；(3)收集约1×106个细胞悬浮于约1.5mL～1.8mL的细胞冻存保护液中、加入合适冻存管并直接放入液氮中冻存；(4)在液氮中冻存指定时间后在37℃融化复苏细胞，利用活死染色试剂盒染色计数统计成活率。
3、实验结果
实验结果示于下表6。实验结果表明，利用本发明提供的细胞冻存保护组合物可长时间冷冻保存，即使在较长冻存时间的情况下也保持较高的冻存复苏成活率。
表6.制备例1的冻存保护液对人肺癌细胞GCL-82保存不同时间的冻存效果比较
Figure PCTCN2017079744-appb-000026
效果例5、降温速率对冻存复苏成活率的影响
1、实验设计
使用制备例1的冻存保护液对人肺癌细胞GCL-82细胞进分别进行传统的梯度降温和直接放入液氮冷冻保存，比较两种降温速率冻存后的细胞复苏成活率。
2、实验方法
(1)取人肺癌细胞GCL-82细胞进行传代培养；(2)配制制备例1的冻存保护液，并进行细胞计数；(3)分别收集约1×106个细胞悬浮于约1.5mL～1.8mL的细胞冻存保护液、加入两支合适的冻存管，分别形成a样和b样。将a样直接放入液氮中，将b样置于梯度冻存盒中在4℃下放置30min、-20℃下90min、-80℃下10h后转入液氮冷冻保存；(4)在液氮中冻存24h后在37℃融化复苏细胞，利用活死染色试剂盒染色计数统计成活率。
3、实验结果
实验结果示于下表7。实验结果表明，利用本发明提供的细胞冻存保护组合物可长时间冷冻保存细胞，保存效率不会因冷冻速率的些许改变而剧烈降低。
表7.制备例1的冻存保护液对人肺癌细胞GCL-82不同降温速率方法的冻存效果比较
Figure PCTCN2017079744-appb-000027
效果例6、不同种冻存保护剂冻存后的细胞联合冻存保护剂直接稀释再贴壁
1、实验设计
使用本申请的多个细胞冻存保护液与传统的DMSO保护液分别对不同种细胞进行联合冻存保护组合物稀释再贴壁测试。效果例6和7中的甜菜碱浓度为2％、6％和10％的样品分别为制备例7、制备例5和制备例3的细胞冻存保护液。
按照表2所示的传统保护液的组成制备得到DMSO为10％的保护液。参见下表8得到效果例6和7中的含DMSO的保护液。
表8.含DMSO的保护液组成
Figure PCTCN2017079744-appb-000028
2、实验方法
(1)不同细胞冻存保护液保护下的细胞在液氮中冻存24h后在37℃融化复苏细胞，并联合冻存保护组合物用相应的细胞培养基以体积比1:4进行稀释，直接注入培养瓶中放入37℃、CO2培养箱内培养进行再贴壁测试；(2)24h后， 将培养瓶取出，将上清液轻轻吸取出来，并在显微镜下观看细胞贴壁情况。
3、实验结果
实验结果示于图2-4。图2-4为不同种细胞(分别为人肺癌细胞GCL-82细胞、人宫颈癌细胞Hela细胞、和人乳腺细胞MCF-10细胞)在用不同冻存保护液，以相同细胞浓度下冷冻保存后，用相应培养基稀释5倍后，直接培养进行贴壁测试。如图所示，包含本申请甜菜碱的细胞冻存保护液的冻存保护液培养的细胞可再贴壁，形态以及增殖功能都未受到影响，而传统DMSO保护液培养的细胞缩小，漂浮于底面上方，不能贴壁，从形态中可以看出细胞已死亡。实验结果表明，利用本发明提供的细胞冻存保护液没有毒性，可稀释后或直接注入体内，并且保存后的细胞功能完全正常。
效果例7、冻存保护剂对细胞毒性测试
1、实验设计
使用本申请制备例7的细胞冻存保护液与传统的DMSO保护液以及普通培养基(对照)对同一种人肺癌细胞GCL-82细胞进行毒性测试。
2、实验方法
(1)取人肺癌细胞GCL-82细胞进行传代培养；(2)配制不同冻存保护液并进行细胞计数，用普通培养基(基础培养基与胎牛血清体积比8:1)作为对照实验组；(3)收集约1×106个细胞，悬浮于15mL离心管中浸泡1-3天；(4)将浸泡后的细胞进行再贴壁测试。
3、实验结果
实验结果示于图5。图5为人肺癌细胞GCL-82在用不同冻存保护组合物以相同细胞浓度下悬浮培养1-3天后进行贴壁测试，如图所示，含2％甜菜碱的细胞冻存保护液培养的细胞可在贴壁，形态以及增殖功能都与普通培养基(对照)悬浮培养无异，而传统DMSO保护液培养的细胞缩小，漂浮于底面上方，不能贴壁，从形态中可以看出细胞已死亡。实验结果表明，利用本发 明提供的细胞冻存保护液较传统培养液(DMSO)相比，并无可观察到的毒性。
效果例8、冻存保护剂对冰点的影响测试
DSC(Differential Scanning Calorimeter)意为“差示扫描量热法”，指的是在程序控制温度下，测量输给物质和参比物的功率差与温度关系的一种技术。DSC是在试样和参比物容器下装有两组补偿加热丝，当试样在加热过程中由于热效应与参比物之间出现温差ΔT时，通过差热放大电路和差动热量补偿放大器，使流入补偿电热丝的电流发生变化，当试样吸热时，补偿放大器使试样一边的电流立即增大；反之，当试样放热时则使参比物一边的电流增大，直到两边热量平衡，温差ΔT消失为止。换句话说，试样在热反应时发生的热量变化，由于及时输入电功率而得到补偿，所以实际记录的是试样和参比物下面两只电热补偿加热丝的热功率之差随时间t的变化关系。如果升温速率恒定，记录的也就是热功率之差随温度T的变化关系。因此，当测量样品的环境温度上升，温度到达样品熔点时，就会有吸热反应，补偿放大器使试样一边的电流立即增大，曲线图就会出现峰值。图6-10示出了不同浓度下的甜菜碱类化合物水溶液中各具体化合物的DSC曲线，其中横坐标为温度(℃)，纵坐标为功率差(W/g)，曲线中向下开始降低的温度点为结晶温度。
图6-10中0％为纯水，如图6-10可知，随着有效组分浓度的增加，水的冰点越来越低，可证明甜菜碱类化合物所包含的各具体化合物对水冰点有降低作用。
效果例9：不同冻存保护液配方冻存血细胞的复苏成活率
输血，又称“血液移植”，是临床上一种重要的抢救和治疗措施，是抢救外伤/手术出血，以及治疗再生障碍性贫血、地中海贫血、溶血性贫血等疾病的重要方法。但是，新鲜血液从供血者体内取出后，如果不能及时输入患者体内，就需要将血液保存，以保存血细胞的生命力，维持血细胞应有的功能。目前，保存血液最常用的方法是将全血或血细胞置入保存液并存于4℃ 冰箱内，但这种方法的有效期仅有大约42天，容易导致血液变质浪费。超低温冻存可以实现血液的长期保存，但是目前所使用的冻存保护液仍存在两个主要问题：1.其主要成分是40％的甘油，高浓度的甘油会加剧血细胞遭受渗透压损伤的风险，在去甘油化的过程中尤为严重；2，需要采用耗时繁琐的梯度降温的方式保存。
1、实验设计
以含有不同浓度甘油与DMSO的冻存保护剂对动物细胞进行保存，用来与本发明提供的包含具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子的细胞冻存保护组合物进行比较，比较细胞复苏后的成活率。本实验采用的冻存保护剂由与人或动物体的生理渗透压平衡的缓冲液(如，生理盐水、磷酸缓冲盐(Phosphate-buffered Saline，PBS))、和冻存保护有效成分(即，本申请的通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子；或者，常规使用的DMSO或甘油等)组成。即，本实验采用的冻存保护剂无需其他细胞用养料成分。
2、实验方法
(1)按照下表9配制各细胞冻存保护液(其仅由PBS和冻存保护有效成分组成)；(2)取约1×107个绵羊血细胞或者20μL兔血液，加入约1.5mL～1.8mL的细胞冻存保护液，在合适冻存管中直接放入液氮中冻存；(3)冻存过后在37℃融化复苏细胞，利用酶标仪(品牌：TECAN,型号：INFINITE 200PRO)，测定每个样本中的溶血情况来统计成活率，测定溶血的方法如下：
将复苏后的样本以及PBS和水的样品放入离心机离心(2000r/min，10min)后取上清液体，利用酶标仪测试其透光率，并计算血细胞成活率。将新鲜的1×107个绵羊血细胞(或者20μL兔血液)加入到不会导致溶血的PBS中作为100％成活率(正对照组)，加入会完全导致溶血的纯水中作为0％成活率(负对照组)；
其中，本效果例采用的绵羊血细胞以及兔血液均购自广州未来生物科 技有限公司。
3、实验结果
实验结果示于下表9和表10。实验结果表明，在超快速降温的程序下，传统保护液和甘油保护液的细胞复苏成活率很低，而本发明提供的包含具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子的冻存保护组合物复苏成活率极高，且安全无毒。
表9.不同冻存保护液冻存绵羊血细胞的复苏成活率
Figure PCTCN2017079744-appb-000029
表10.不同冻存保护液冻存兔血的复苏成活率
Figure PCTCN2017079744-appb-000030
效果例10：采用不同浓度(质量％)的甜菜碱和不同浓度(质量％)的甘油对绵羊血进行冻存保护过程中，洗涤操作对细胞成活率的影响
1、实验设计
使用本申请的多种细胞冻存保护液与传统的血细胞甘油保护液分别对绵羊血细胞进行孵育，测试洗去过程对绵羊血细胞的成活率影响。
2、实验方法
(1)将等量的绵羊血细胞(1×107个细胞)分别置于甜菜碱浓度为2％、4％和6％的样品(体积1ml，其由甜菜碱和PBS缓冲液组成)以及甘油浓度为2％、4％和6％的样品(体积1ml，其由甘油和PBS缓冲液组成)中，放入37℃、CO2培养箱内浸泡孵育24小时；(2)24小时后，将样品取出，并用PBS稀释1倍后，离心洗去样本中的冻存保护剂，并用酶标仪测试上清液透光率，以计算血细胞成活率。
3、实验结果
实验结果示于图11。图11为绵羊血细胞在用不同冻存保护液，以相同细胞浓度下浸泡24小时后取出，并用PBS稀释1倍后，离心洗去样本中的冻存保护剂，并用酶标仪测试上清液透光率，以计算血细胞成活率。如图11所示， 包含本申请甜菜碱的冻存保护液浸泡后的血细胞，稀释洗涤前后，血细胞成活率没受到影响；而包含甘油和DMSO的冻存保护液浸泡后的血细胞，稀释洗涤后，由于甘油渗透性差，不能及时排出细胞，造成胞内外渗透压失衡，从而导致大量血细胞发生溶血，成活率大大降低；DMSO的渗透性虽然与甘油相比较强，但依然会在洗涤过程中造成血细胞溶血。实验结果表明，利用本申请提供的细胞冻存保护液，洗涤过程不会对血细胞产生渗透压损伤，大大优于当前最有效的传统甘油与DMSO冻存保护剂。以上效果例9-10中采用的冻存保护液仅由PBS和冻存保护有效成分组成。然而可知，在将PBS替换为与人或动物体的生理渗透压平衡的缓冲液(如，生理盐水等)的情况下的技术方案，也将达到与效果例9-10公开的类似的技术效果的对比。
以上制备例和效果例均是对于本发明具体实施方案和效果的示例，而不得被视为对本发明的限制。本发明公开和提出的所有方法，本领域技术人员可通过借鉴本文内容，适当改变原料和条件等环节实现，尽管本发明的方法已通过较佳实施例进行了描述，相关技术人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或重新组合，来实现最终的制备技术。特别需要指出的是，所有相类似的替换和改动对本领域技术人员来说是显而易见的，他们都被视为包括在本发明精神、范围和内容中。

Claims (21)

  1. 一种细胞冻存保护组合物，其包含一种或多种具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子、以及细胞用养料成分，
    其中相对于100质量份的所述细胞用养料成分，所述两性分子为10～2530质量份；
    所述细胞冻存保护组合物不包含以下物质：甘油、二氨基乙烷四乙酸或其盐、二甲基亚砜、或卵黄，
    在所述通式结构R1-N+(CH3)2-(CH2)n-R2中，
    上述R1为碳数1至10的直链或支链的烷基，其任选被选自由(甲基)丙烯酰胺基、(甲基)丙烯酰氧基、烯基、羟基、羟基烷基、烷氧基、和卤素组成的组中的取代基取代，
    R2为选自由-COO-、-SO4 -、-SO3 -、和
    Figure PCTCN2017079744-appb-100001
    组成的组中的任一种带负电荷的基团，所述R3为选自由(甲基)丙烯酰氧基烷基、烷基、和烯基组成的组中的基团，
    所述(CH3)2和(CH2)n可各自独立地任选被选自由烷基、烯基、羟基、羟基烷基、烷氧基、聚环氧烷基、和卤素组成的组中的取代基取代，
    n为1至10的整数。
  2. 根据权利要求1所述的细胞冻存保护组合物，其中，所述通式结构R1-N+(CH3)2-(CH2)n-R2中，
    上述R1为碳数1至5的直链或支链的烷基，其任选被选自由(甲基)丙烯酰胺基、(甲基)丙烯酰氧基、羟基、羟基烷基、烷氧基、和卤素组成的组中的取代基取代，
    上述R2为选自由-COO-、-SO3 -、和
    Figure PCTCN2017079744-appb-100002
    组成的组中的任一种基团，所述R3为(甲基)丙烯酰氧基烷基，
    所述(CH3)2和(CH2)n中的氢原子可各自独立地任选被选自由烷基、羟基、羟基烷基、烷氧基、聚环氧烷基、和卤素组成的组中的取代基取代，
    n为1至5的整数。
  3. 根据权利要求2所述的细胞冻存保护组合物，其中，相对于100质量份的所述细胞用养料成分，所述两性分子为420～1120份；和
    所述通式结构R1-N+(CH3)2-(CH2)n-R2中，
    上述R1为碳数1至3的直链或支链的烷基，其任选被选自由(甲基)丙烯酰胺基、(甲基)丙烯酰氧基、羟基、碳原子数1至5的羟基烷基、和碳原子数1至5的烷氧基组成的组中的取代基取代，
    所述(CH3)2和(CH2)n中的氢原子可各自独立地任选被选自由碳原子数1至5的烷基、羟基、碳原子数1至5的羟基烷基、碳原子数1至5的烷氧基、和聚环氧烷基组成的组中的取代基取代，
    n为1至3的整数。
  4. 根据权利要求1至3任一项所述的细胞冻存保护组合物，其中，所述100质量份的细胞用养料成分包含下述质量份数的组分：
    Figure PCTCN2017079744-appb-100003
  5. 根据权利要求1至4任一项所述的细胞冻存保护组合物，其中，所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子为CH3-N+(CH3)2-CH2-COO-。
  6. 根据权利要求1至4任一项所述的细胞冻存保护组合物，其中，所述具有通式结构R1-N+(CH3)2-(CH2)n-R2的两性分子中，(CH2)n中的氢原子被羟基取代且R2为-COO-。
  7. 根据权利要求1至6任一项所述的细胞冻存保护组合物，其中，所述细胞冻存保护组合物用于在冷冻保存期间保护人类细胞或动物细胞；优选用于保护人类细胞或哺乳动物细胞。
  8. 根据权利要求7所述的细胞冻存保护组合物，其中，所述人类细胞或哺乳动物细胞包括癌细胞、体细胞、和干细胞的至少之一；优选所述体细胞为免疫细胞或血细胞。
  9. 根据权利要求7或8所述的细胞冻存保护组合物，其中，所述细胞冻存保护组合物用于在冷冻保存期间保护人类细胞；优选所述人类细胞包括肺癌细胞、宫颈癌细胞、乳腺细胞、造血干细胞、免疫细胞、脐带间质干细胞、骨髓间充质干细胞、淋巴癌细胞、和血细胞的至少之一。
  10. 根据权利要求1至3和5至9任一项所述的细胞冻存保护组合物，其中，所述细胞用养料成分为一种或多种盐；优选所述盐包括无机盐；并还优选所述无机盐以生理盐水或缓冲液的形式存在；进一步优选所述缓冲液为磷酸盐缓冲液。
  11. 一种细胞冻存保护液，其为根据权利要求1-10任一项所述的细胞冻存保护组合物的水溶液，其中在所述细胞冻存保护液中所述两性分子的含量为0.1～20质量％，基于所述细胞冻存保护液的总质量。
  12. 根据权利要求11所述的细胞冻存保护液，其中在所述细胞冻存保护液中所述两性分子的含量为4～10质量％，基于所述细胞冻存保护液的总质量。
  13. 一种对细胞进行冷冻保存的方法，其包括：将细胞悬浮于权利要求11或12所述的细胞冻存保护液中，并置于冻存容器内，然后进行冷冻保存。
  14. 根据权利要求13所述的方法，其中，所述冷冻保存为超快速冷冻保存。
  15. 根据权利要求13或14所述的对细胞进行冷冻保存的方法，其中，在进行所述冷冻保存后，使经冷冻保存的细胞复苏，然后直接使用所述细胞、或以稀释方式使用所述细胞。
  16. 根据权利要求13至15任一项所述的方法，其中，所述细胞的复苏成活率为至少70％；优选所述细胞的复苏成活率为80％-99％。
  17. 根据权利要求13至16任一项所述的方法，其中，以1×104～1×109个细胞悬浮于1.0ml-5.0ml所述细胞冻存保护液的浓度，将细胞悬浮于所述细胞冻存保护液中；优选以1×105～1×107个细胞悬浮于1.5mL～1.8mL所述细胞冻存保护液的浓度，将细胞悬浮于所述细胞冻存保护液中。
  18. 根据权利要求13至17任一项所述的方法，其中，所述冷冻保存的温度范围为-20℃至-196℃；优选为-80℃至-196℃。
  19. 一种根据权利要求1-10任一项所述的细胞冻存保护组合物在细胞冷冻保存中的用途，所述细胞为人类细胞或动物细胞；优选所述细胞为人类细胞或哺乳动物细胞。
  20. 根据权利要求19所述的用途，其中，所述人类细胞或哺乳动物细胞包括癌细胞、体细胞、和干细胞的至少之一；优选所述体细胞为免疫细胞或血细胞。
  21. 根据权利要求19或20所述的用途，其中，所述细胞为人类细胞；优选所述人类细胞包括肺癌细胞、宫颈癌细胞、乳腺细胞、造血干细胞、免疫细胞、脐带间质干细胞、骨髓间充质干细胞、淋巴癌细胞、和血细胞的至少之一。
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