WO2018113796A1 - 一种可临床使用的细胞冻存液 - Google Patents

一种可临床使用的细胞冻存液 Download PDF

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WO2018113796A1
WO2018113796A1 PCT/CN2017/118381 CN2017118381W WO2018113796A1 WO 2018113796 A1 WO2018113796 A1 WO 2018113796A1 CN 2017118381 W CN2017118381 W CN 2017118381W WO 2018113796 A1 WO2018113796 A1 WO 2018113796A1
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cell
cells
vitamin
concentration
cryopreservation solution
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English (en)
French (fr)
Inventor
王飞
何佳平
赵荻骏
刘维克多
杨丁柱
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Cellular Biomedicine Group Wuxi Ltd
Cellular Biomedicine Group Shanghai Ltd
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Cellular Biomedicine Group Wuxi Ltd
Cellular Biomedicine Group Shanghai Ltd
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Priority to EP17882332.4A priority Critical patent/EP3560334A4/en
Priority to US16/472,962 priority patent/US11363812B2/en
Priority to JP2019555541A priority patent/JP6850360B2/ja
Publication of WO2018113796A1 publication Critical patent/WO2018113796A1/zh
Anticipated expiration legal-status Critical
Priority to US17/749,260 priority patent/US12356981B2/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
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/0011—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
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    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/16—Magnesium; Mg chelators
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/35—Polyols, e.g. glycerin, inositol
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • C12N2500/62—DMSO

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a cryopreservation solution for long-term storage of cells and its clinical use.
  • the cultured cells After the cell culture reaches the demand or target, the cultured cells need to be collected and stored. However, conditions at room temperature and 4 ° C are not sufficient for long-term preservation of cells, and cryopreservation of cells is required, which involves the use of cryopreservation solutions.
  • cryopreservation fluids are composed of serum or animal-derived proteins, or common chemical reagents that do not meet clinical use.
  • Such cryopreservation solutions have a poor effect on cell viability or potency on the one hand. Because the ingredients cannot be unified, it is difficult to meet the standards for clinical use.
  • a first aspect of the invention provides a clinically usable cell cryopreservation solution comprising the following components:
  • cryoprotectant includes a combination of one or more of dimethyl sulfoxide, glycerin, and ethylene glycol;
  • the salt buffer is a solution containing Na + , K + , Mg + , Cl - and CH 3 COO - ions;
  • the human albumin concentration is 1% to 20% (w/v).
  • the human albumin concentration is from 3 to 15% (w/v), more preferably from 4 to 12% (w/v).
  • cryoprotectant concentration is from 0.4 mol/L to 2.2 mol/L.
  • cryoprotectant is selected from the group consisting of dimethyl sulfoxide, glycerin, ethylene glycol, or a combination thereof.
  • cryoprotectant is dimethyl sulfoxide.
  • the cell cryopreservation solution comprises one or more characteristics selected from the group consisting of:
  • the concentration of Na + ions is 0.1 to 0.2 mol / L;
  • the concentration of K + ions is 5-10 mmol/L
  • the concentration of Mg 2+ ions is 1.5 to 5 mmol/L;
  • the concentration of Cl - ions is 0.1 to 0.3 mol / L;
  • the concentration of CH 3 COO - ions is 40 to 60 mmol/L.
  • the salt buffer is a compound electrolyte injection.
  • the vitamin concentration is from 50 mmol/L to 150 mmol/L; and/or the amino acid concentration is from 0.1 mol/L to 0.5 mol/L.
  • the human albumin is selected from the group consisting of plasma extracted human albumin, recombinant human albumin, or a combination thereof.
  • the vitamin is selected from the group consisting of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, or a combination thereof.
  • the vitamin is selected from the group consisting of vitamin A, vitamin B, vitamin C, vitamin E, or a combination thereof.
  • the vitamin is selected from the group consisting of vitamin C, vitamin E, or a combination thereof.
  • the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or a combination thereof; preferably Lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, valine, or a combination thereof.
  • the amino acid is selected from the group consisting of glycine, arginine, leucine, phenylalanine, or a combination thereof.
  • a second aspect of the invention provides a cell mixture comprising the following components:
  • the cell is a Car-T cell, a mesenchymal stem cell, or a combination thereof.
  • the cell mixture is a mixture of cells after cryopreservation.
  • the cell mixture is used to preserve cells for long periods of time and/or to establish a cell bank.
  • a third aspect of the invention provides a cryopreservation method for a cell, comprising the steps of:
  • the cells to be cryopreserved in the step (i) are prepared by the following steps:
  • step (ii) is cooled to -80 degrees using a program cooling method gradient.
  • a fourth aspect of the invention provides a kit for cryopreservation of a cell, the kit comprising the cell cryopreservation solution according to the first aspect of the invention.
  • Figure 1 is a graph showing the relationship between Car-T cell viability and cryopreservation time in Example 1.
  • Figure 2 is a graph showing the relationship between Car-T function expression ability and cryopreservation time in Example 1.
  • Figure 3 is a graph showing the relationship between the activity of the mesenchymal stem cells and the cryopreservation time of Example 2.
  • Fig. 4 is a comparison diagram of differentiation of mesenchymal stem cells and fresh frozen pre-existing cells after cryopreservation in Example 2 for 1 month and 3 months.
  • Figure 5 is a graph showing the relationship between the activity of the mesenchymal stem cells and the cryopreservation time of Example 3.
  • Figure 6 is a graph showing the chondrogenic differentiation of the mesenchymal stem cells of Example 3.
  • Figure 7 is a graph showing the relationship between the activity of the mesenchymal stem cells and the cryopreservation time of Example 4.
  • Figure 8 is a graph showing the chondrogenic differentiation of the mesenchymal stem cells of Example 4.
  • Figure 9 is a graph showing the relationship between the activity of the mesenchymal stem cells and the cryopreservation time of Comparative Example 1.
  • Fig. 10 is a graph showing the comparison of chondrogenic differentiation of the mesenchymal stem cells of Comparative Example 1.
  • the inventors have extensively and intensively studied and found for the first time that cells are preserved using a cryopreservation solution containing human albumin, vitamins, and amino acids, such as mesenchymal stem cells, and the cells still have good viability and function after long-term storage. expression ability. On the basis of this, the present invention has been completed.
  • human albumin as used herein includes plasma extracted human albumin and recombinant human albumin, while “human albumin” as used in the specific examples and comparative examples of the present invention refers to plasma extracted human albumin, "recombinant” Human albumin refers to recombinant human albumin.
  • Mesenchymal stem cells refers to pluripotent stem cells derived from the early developmental mesoderm with high self-renewal ability and multi-directional differentiation potential, which are widely present in various tissues of the whole body and can be cultured and expanded in vitro. It can be differentiated into nerve cells, osteoblasts, muscle cells, fat cells, etc. under the control of specific conditions.
  • CAR-T cell which is a chimeric antigen receptor T cell, is an in vitro coupling of an antigen binding portion of an antibody capable of recognizing a certain tumor antigen to an intracellular portion of a CD3- ⁇ chain or Fc ⁇ RI ⁇ .
  • Cell cryopreservation technology is an important means of biological preservation of species. If cells are directly frozen without any conditions, water in the cells inside and outside the environment will form ice crystals, resulting in mechanical damage, electrolyte increase, osmotic pressure change, dehydration, pH change, protein denaturation, etc. Causes cell death. If a protective agent is added to the culture solution, the freezing point can be lowered. Under slow freezing conditions, the intracellular water can be oozing out of the cells before freezing, and storage at low temperatures can reduce the formation of ice crystals.
  • cell cryopreservation The most important part of cell cryopreservation is cell cryopreservation.
  • Traditional cell cryopreservation solutions contain animal serum, primarily fetal calf serum and/or calf serum.
  • Serum is an extremely complex mixture of fibrin removed from plasma. Some of its components are unclear, and serum composition and content often vary with the gender, age, physiological conditions, and nutritional conditions of the blood donor.
  • the serum contains various plasma proteins, peptides, fats, carbohydrates, hormones, inorganic substances and the like.
  • the term "cell resuscitation” refers to the process of reactivation of dormant cells.
  • the cryotube is rapidly transferred from liquid nitrogen to a warm water bath by a program well known to those skilled in the art, preferably at 37 ° C to 40 ° C, and the thawing is accelerated by occasional stirring; after the cells are completely thawed, The cryotubes are sterilized; the cells are washed after thawing and resuspended, transferred to a cell culture flask, and cultured in a CO 2 incubator; cell viability and viability are examined.
  • cryopreservation and resuscitation steps of Car-T cells are identical to each other.
  • cryopreservation and resuscitation steps of mesenchymal stem cells are:
  • the environment in which the cells are maintained needs to be an isotonic solution.
  • a salt buffer such as physiological saline, a compound electrolyte injection or the like can maintain cell osmotic pressure.
  • cryopreservation the cells need to replace the water in the cells with a cryoprotectant to prevent the formation of ice crystals in the cryopreservation process to damage the organelles, and the metabolism of the cells after cryopreservation needs to consume vitamins and amino acids.
  • the present inventors have unexpectedly discovered that a salt buffer solution, human albumin, vitamins, amino acids and a cryoprotectant are added to physiological saline to prepare a cell cryopreservation solution, which can freeze cells for a long time and can maintain a high cell recovery. Live rate and strong functional expression and differentiation ability.
  • the cell cryopreservation solution of the present invention comprises the following components:
  • the salt buffer includes, but is not limited to, a combination of one or more of physiological saline, compound electrolyte injection, and phosphate buffer.
  • the cryoprotectant includes, but is not limited to, a combination of one or more of dimethyl sulfoxide, glycerin, and ethylene glycol.
  • the cell cryopreservation solution of the invention can be used for:
  • the cell mixture of the invention comprises the following components:
  • the salt buffer includes, but is not limited to, a combination of one or more of physiological saline, compound electrolyte injection, and phosphate buffer.
  • the cryoprotectant includes, but is not limited to, a combination of one or more of dimethyl sulfoxide, glycerin, and ethylene glycol.
  • cells can be frozen for a long time, and the cells after resuscitation still have a high survival rate
  • the O ⁇ N group is a control group and a negative group, and there is almost no expression, and the P group still has a strong expression ability, so only the P group can be seen.
  • the results showed that the cells still had strong functional expression ability after 1 to 6 weeks of cryopreservation, and the decrease was not significant compared with that before cryopreservation.
  • Fig. 3 The relationship between the survival rate of mesenchymal stem cells and cryopreservation time is shown in Fig. 3. The results show that after 1 to 3 months of cryopreservation, the viability of cells decreased within 5%, reaching 95% or more, which is in line with the needs of clinical use.
  • the results of the potency test for differentiation of mesenchymal stem cells after cryopreservation are shown in FIG. The results showed that after 1 month and 3 months of cryopreservation, the cells were differentiated from the fresh frozen cells, and the cell-forming, osteogenic and cartilage-forming properties were not affected.
  • the viability analysis is shown in Fig. 5.
  • the results show that the survival rate of cells with recombinant human albumin after 1 to 3 months of storage decreased within 7%, reaching more than 93%, which is in line with the needs of clinical use.
  • Fig. 6 The results of the differentiation ability test are shown in Fig. 6. As can be seen from the figure, in the differentiation experiment, the recombinant human albumin cryopreservation solution was frozen for one month, and the differentiation ability was basically the same as that of the fresh frozen pre-existing cells.
  • Fig. 7 The relationship between the viability of cryopreserved mesenchymal stem cells and cryopreservation time is shown in Fig. 7. The results showed that the survival rate of cells containing recombinant human albumin after 1 to 3 months of freezing decreased within 5%, reaching more than 95%, in line with the needs of clinical use.
  • Fig. 8 The results of the differentiation ability test are shown in Fig. 8. It can be seen from the figure that in the differentiation experiment, after 1 month of cryopreservation with other vitamins and amino acid cryopreservation solution, the differentiation ability is basically the same as that of the fresh frozen pre-existing cells.
  • the formulation of the cryopreservation solution used in this comparative example is as follows:
  • Fig. 10 The results of the differentiation ability test are shown in Fig. 10. As can be seen from the figure, in the differentiation experiment, after freezing for 1 month with the high dimethyl sulfoxide cryopreservation solution, the cartilage-reducing ability was significantly decreased compared with the fresh frozen pre-existing cells.

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Abstract

本发明提供了一种可临床使用的细胞冻存液。具体地，本发明的细胞冻存液包括以下组分：（1）人白蛋白；（2）冷冻保护剂；所述的冷冻保护剂包括二甲基亚砜、甘油、乙二醇的一种或多种的组合；（3）盐缓冲液；所述的盐缓冲液为包含Na +、K +、Mg +、Cl -和CH 3COO -离子的溶液；（4）维生素；和（5）氨基酸；其中，所述人白蛋白浓度为1％～20％（w/v）。经本发明冻存液长期冻存后的细胞，其活率高，且细胞效能保持一致性高。本发明的冻存液纯度级别为药品级或USP级，临床使用安全可靠，可用于常规的贴壁和悬浮细胞。

Description

一种可临床使用的细胞冻存液 技术领域
本发明涉及生物技术领域，具体地，涉及一种用于细胞长期存储的及其临床使用的冻存液。
背景技术
在细胞培养达到需求量或目标后，需要对培养得到的细胞进行收集和保存。然而，常温和4℃的条件都不足以长期保存细胞，需要对细胞进行超低温冻存保存，这涉及到冻存液的使用。
目前，常规的冻存液都由血清、或动物来源蛋白、或不满足临床使用的普通化学试剂组成，这样的冻存液一方面对细胞的活率或者效能保持的效果较差，另一方面，由于其成分无法统一，因此难以满足临床使用的标准。
综上所述，本领域尚缺乏一种质量统一，适合临床使用的细胞冻存液。
发明内容
本发明的目的是提供一种质量统一，适合临床使用的细胞冻存液。
本发明的第一方面提供了一种可临床使用的细胞冻存液，所述的细胞冻存液包括以下组分：
(1)人白蛋白；
(2)冷冻保护剂：所述的冷冻保护剂包括二甲基亚砜、甘油、乙二醇的一种或多种的组合；
(3)盐缓冲液；所述的盐缓冲液为包含Na +、K +、Mg +、Cl -和CH 3COO -离子的溶液；
(4)维生素；和
(5)氨基酸；
其中，所述人白蛋白浓度为1％～20％(w/v)。
在另一优选例中，所述的人白蛋白浓度为3～15％(w/v)，更优选为4～12％(w/v)。
在另一优选例中，所述的冷冻保护剂浓度为0.4mol/L～2.2mol/L。
在另一优选例中，所述的冷冻保护剂选自下组：二甲基亚砜、甘油、乙二醇、或其组合。
在另一优选例中，所述的冷冻保护剂为二甲基亚砜。
在另一优选例中，所述的细胞冻存液包括选自下组的一个或多个特征：
Na +离子的浓度为0.1～0.2mol/L；
K +离子的浓度为5～10mmol/L；
Mg 2+离子的浓度为1.5～5mmol/L；
Cl -离子的浓度为0.1～0.3mol/L；
CH 3COO -离子的浓度为40～60mmol/L。
在另一优选例中，所述盐缓冲液为复方电解质注射液。
在另一优选例中，所述的维生素浓度为50mmol/L～150mmol/L；和/或所述氨基酸浓度为0.1mol/L～0.5mol/L。
在另一优选例中，所述的人白蛋白选自下组：血浆提取人白蛋白、基因重组人白蛋白，或其组合。
在另一优选例中，所述的维生素选自下组：维生素A、维生素B、维生素C、维生素D、维生素E、维生素K，或其组合。
在另一优选例中，所述的维生素选自下组：维生素A、维生素B、维生素C、维生素E、或其组合。
在另一优选例中，所述的维生素选自下组：维生素C、维生素E、或其组合。
在另一优选例中，所述的氨基酸选自下组：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸，或其组合；优选为赖氨酸、色氨酸、苯丙氨酸、甲硫氨酸、苏氨酸、异亮氨酸、亮氨酸、缬氨酸，或其组合。
在另一优选例中，所述的氨基酸选自下组：甘氨酸、精氨酸、亮氨酸、苯丙氨酸、或其组合。
本发明的第二方面提供了一种细胞混合物，所述混合物包括以下组分：
细胞，和
如本发明第一方面所述的细胞冻存液；
优选地，所述的细胞是Car-T细胞、间充质干细胞，或其组合。
在另一优选例中，所述的细胞混合物是冻存后的细胞混合物。
在另一优选例中，所述的细胞混合物被用于长期保存细胞和/或建立细胞库。
本发明的第三方面提供了一种细胞的冻存方法，包括步骤：
(i)将待冻存细胞与本发明第一方面所述的细胞冻存液进行混合，得到细胞 混合物；
(ii)将步骤(i)得到的细胞混合物降温后，于液氮中保存。
在另一优选例中，所述步骤(i)中的待冻存细胞是通过以下步骤制备的：
(i-1)提供一种活细胞，并计算活率和细胞密度；
(i-2)将步骤(i-1)的细胞离心后去除上清液，弹开细胞沉淀。
在另一优选例中，所述步骤(ii)中采用程序降温法梯度降温至-80度。
本发明的第四方面提供了一种试剂盒，所述的试剂盒用于细胞冻存，所述的试剂盒中包括如本发明第一方面所述的细胞冻存液。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为实施例1中Car-T细胞活率与冻存时间关系图。
图2为实施例1中Car-T功能表达能力与冻存时间关系图。
图3为实施例2中间充质干细胞活率与冻存时间关系图。
图4为实施例2中冻存1月和3个月后间充质干细胞和新鲜冻存前细胞分化对照图。
图5为实施例3中间充质干细胞活率与冻存时间关系图。
图6为实施例3中间充质干细胞的成软骨分化情况对比图。
图7为实施例4中间充质干细胞活率与冻存时间关系图。
图8为实施例4中间充质干细胞的成软骨分化情况对比图。
图9为对比例1中间充质干细胞活率与冻存时间关系图。
图10为对比例1中间充质干细胞的成软骨分化情况对比图。
具体实施方式
本发明人经过广泛而深入地研究，首次发现，采用含有人白蛋白、维生素、氨基酸的冻存液保存细胞，如间充质干细胞，经过长时间保存后，细胞仍具有良好的活率和功能表达能力。在此基础上，完成了本发明。
术语
人白蛋白
如本文所用术语“人白蛋白”包括血浆提取人白蛋白和基因重组人白蛋白，而在本发明的具体实施例和对比例中使用的“人白蛋白”指血浆提取人白蛋白，“重组人白蛋白”指基因重组人白蛋白。
间充质干细胞
如本文所用“间充质干细胞”是指来源于发育早期中胚层的具有高度自我更新能力和多向分化潜能的多能干细胞，广泛存在于全身多种组织中，可在体外培养扩增，并能在特定条件控制下分化为神经细胞、成骨细胞、肌肉细胞、脂肪细胞等。
CAR-T细胞
如本文所用“CAR-T细胞”，即为嵌合抗原受体T细胞，是将能识别某种肿瘤抗原的抗体的抗原结合部与CD3-ζ链或FcεRIγ的胞内部分在体外偶联为一个嵌合蛋白，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体(CAR)。
细胞冻存
细胞冻存技术是生物学保存物种的重要手段。如果在不加任何条件下直接冻存细胞时，细胞内和外环境中的水都会形成冰晶，导致细胞内发生机械损伤、电解质升高、渗透压改变、脱水、PH改变、蛋白变性等，严重时引起细胞死亡。如向培养液加入保护剂，可使冰点降低。在缓慢的冻结条件下，能使细胞内水分在冻结前透出细胞，贮存在低温下能减少冰晶的形成。
细胞冻存中最重要的部分是细胞冻存液。传统的细胞冻存液含有动物血清，主要为胎牛血清和/或小牛血清。血清是由血浆去除纤维蛋白而形成的一种极其复杂的混合物，其一部分组成成份尚不清楚，且血清组成及含量常随供血动物的性别、年龄、生理条件和营养条件的不同而不同。血清中含有各种血浆蛋白、多肽、脂肪、碳水化合物、激素、无机物等。
细胞复苏
如本发明所用，“细胞复苏”一词是指休眠的细胞重新活化的过程。一般采 用本领域技术人员所熟知的程序即快速复苏法，将冷冻管迅速由液氮转入到温水浴中，较佳地37℃-40℃，不定时搅拌加速解冻；细胞完全解冻后，对冷冻管进行消毒；将解冻后清洗细胞并重悬，转移到细胞培养瓶，CO 2孵箱中培养；检查细胞存活率及活力。
细胞冻存方法
在本发明具体实施方式中，Car-T细胞的冻存与复苏步骤：
1.取一袋悬浮培养至14天的Car-T细胞，计算活率和细胞密度，取活细胞量4×10 8离心收集；
2.离心后去上清，弹开细胞沉淀，缓缓加入20mL冻存液；
3.混匀，分装至2个20mL冻存袋，放入程序降温仪降温至-80度；
4.于液氮中保存，并于1周、6周分别取样进行细胞活度与功能表达能力的检测分析。
在本发明具体实施方式中，间充质干细胞的冻存与复苏步骤：
1.取数盘贴壁培养的间充质干细胞，观察融合度为80％～90％,通过胰酶消化收集细胞，并计算活率和细胞密度，取活细胞量4×10 7离心收集；
2.离心后去上清，弹开细胞沉淀，缓缓加入3mL冻存液；
3.混匀，分装至2个2mL冻存袋，放入程序降温仪降温至-80度；
4.于液氮中保存，并于1月、3月分别取样进行细胞活率与分化能力的检测分析。
细胞冻存液
细胞的维持环境需要是一个等渗的溶液。盐缓冲液如生理盐水、复方电解质注射液等可以维持细胞渗透压。细胞在冻存时需要用冷冻保护剂替换出细胞内的水，防止在冻存过程中水形成冰晶损害细胞器，而细胞在冻存复苏后的代谢需要消耗维生素和氨基酸。
本发明人意外发现，在生理盐水中添加了盐缓冲液、人白蛋白、维生素、氨基酸和冷冻保护剂制成细胞冻存液，可以长时间冻存细胞，并且可以维持细胞复苏后较高的活率和较强的功能表达与分化能力。
本发明的细胞冻存液，包括以下组分：
(1)盐缓冲液；
(2)人白蛋白；
(3)维生素；
(4)氨基酸；
(5)冷冻保护剂。
所述的盐缓冲液包括(但并不限于)：生理盐水、复方电解质注射液、磷酸盐缓冲液的一种或多种的组合。所述的冷冻保护剂包括(但并不限于)：二甲基亚砜、甘油、乙二醇的一种或多种的组合。
本发明的细胞冻存液，可以用于：
(a)保存细胞；
(b)建立细胞库；或
(c)保持细胞的活力。
细胞混合物
本发明的细胞混合物，包含以下组分：
(a)细胞；以及
(b)细胞冻存液，包括以下组分：
(1)盐缓冲液；
(2)人白蛋白；
(3)维生素；
(4)氨基酸；
(5)冷冻保护剂。
所述的盐缓冲液包括(但并不限于)生理盐水、复方电解质注射液、磷酸盐缓冲液的一种或多种的组合。所述的冷冻保护剂包括(但并不限于)二甲基亚砜、甘油、乙二醇的一种或多种的组合。
本发明的细胞冻存液主要优点包括：
(1)可以长时间冻存细胞，复苏后的细胞仍具有较高的活率；
(2)冻存后复苏的细胞仍然具有较好的功能表达能力和分化能力。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法， 通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1
本实施例中采用的冻存液配方如下：
序号 所用试剂 试剂配制方案
1 Na + 140mmol/L
2 K + 6mmol/L
3 Mg 2+ 2mmol/L
4 Cl - 110mmol/L
5 CH 3COO - 40mmol/L
6 人白蛋白 8％(W/V)
7 二甲基亚砜 1.4mol/L
8 维生素C 50mmol/L
9 甘氨酸 0.1mol/L
10 精氨酸 0.1mol/L
具体冻存和复苏操作步骤如下：
1.取一袋悬浮培养至14天的Car-T细胞，计算活率和细胞密度，取活细胞量4×10 8离心收集
2.离心后去上清，弹开细胞沉淀，缓缓加入20ml冻存液
3.混匀，分装至2个20ml冻存袋，放入程序降温仪降温至-80度
4.于液氮中保存，并于1周、6周分别取样进行检测。
经冻存后的Car-T细胞活率结果如图1所示。据实验统计，冻存1～6周后活率下降都在10％内，且1～6周的结果稳定，说明冻存液的效果符合需求。
Car-T功能表达能力与冻存时间关系的检测结果如图2所示，图中，O\N\P各组分别表示：
O:CarT cells only
N:CarT cells+CD19negative tumor cells
P:CarT cells+CD19positive tumor cells
从图中可以看出，O\N组为对照组和阴性组，几乎无表达，P组仍具有较强的 表达能力，所以只能看到P组。该结果表明，经冻存1～6周后细胞仍然就有较强的功能表达能力，与冻存前相比降幅不明显。
实施例2
本实施例中采用的冻存液配方如下：
序号 所用试剂 试剂配制方案
1 Na + 140mmol/L
2 K + 6mmol/L
3 Mg 2+ 2mmol/L
4 Cl - 110mmol/L
5 CH 3COO - 40mmol/L
6 人白蛋白 5％(W/V)
7 二甲基亚砜 0.7mol/L
8 维生素C 60mmol/L
9 甘氨酸 0.2mol/L
10 精氨酸 0.2mol/L
具体冻存和复苏操作步骤如下：
1.取数盘贴壁培养的间充质干细胞，观察融合度为80％～90％，通过胰酶消化收集细胞，并计算活率和细胞密度，取活细胞量4×10 7离心收集；
2.离心后去上清，弹开细胞沉淀，缓缓加入3ml冻存液；
3.混匀，分装至2个2ml冻存袋，放入程序降温仪降温至-80度；
4.于液氮中保存，并于1月、3月分别取样进行检测。
间充质干细胞活率与冻存时间关系如图3所示，结果表明，细胞在冻存1～3月后活率下降在5％以内，达到95％以上，符合临床使用的需求。
经冻存后的间充质干细胞分化能力效能检测结果如图4中所示。结果表明，冻存1月和3个月后分别和新鲜冻存前细胞对照做分化实验，细胞成脂、成骨、成软骨能力未受影响。
实施例3
本实施例中采用的冻存液配方如下：
序号 所用试剂 试剂配制方案
1 Na + 140mmol/L
2 K + 6mmol/L
3 Mg 2+ 2mmol/L
4 Cl - 110mmol/L
5 CH 3COO - 40mmol/L
6 重组人白蛋白 5％(W/V)
7 二甲基亚砜 0.7mol/L
8 维生素C 60mmol/L
9 甘氨酸 0.2mol/L
10 精氨酸 0.2mol/L
具体冻存和复苏操作步骤如下：
1.取数盘贴壁培养的间充质干细胞，观察融合度为80％～90％，通过胰酶消化收集细胞，并计算活率和细胞密度，取活细胞量4×10 7离心收集；
2.离心后去上清，弹开细胞沉淀，缓缓加入3ml冻存液；
3.混匀，分装至2个2ml冻存袋，放入程序降温仪降温至-80度；
4.于液氮中保存，并于1月、3月分别取样进行检测。
活率分析如图5中所示，结果显示，用细胞在含重组人白蛋白冻存1～3月后活率下降在7％以内，达到93％以上，符合临床使用的需求。
分化能力效能检测结果如图6所示。从图中可以看出，在分化实验中，用重组人白蛋白冻存液冻存1月后，和新鲜冻存前细胞相比，分化能力基本一致。
实施例4
本实施例中采用的冻存液配方如下：
序号 所用试剂 试剂配制方案
1 Na + 140mmol/L
2 K + 6mmol/L
3 Mg 2+ 2mmol/L
4 Cl - 110mmol/L
5 CH 3COO - 40mmol/L
6 人白蛋白 5％(W/V)
7 二甲基亚砜 0.7mol/L
8 维生素E 60mmol/L
9 亮氨酸 0.1mol/L
10 苯丙氨酸 0.2mol/L
具体冻存和复苏操作步骤如下：
1.取数盘贴壁培养的间充质干细胞，观察融合度为80％～90％，通过胰酶消化收集细胞，并计算活率和细胞密度，取活细胞量4×10 7离心收集；
2.离心后去上清，弹开细胞沉淀，缓缓加入3ml冻存液；
3.混匀，分装至2个2ml冻存袋，放入程序降温仪降温至-80度；
4.于液氮中保存，并于1月、3月分别取样进行检测。
经冻存后的间充质干细胞活率与冻存时间关系如图7所示。结果表明，用细胞在含重组人白蛋白冻存1～3月后活率下降在5％以内，达到95％以上，符合临床使用的需求。
分化能力效能检测结果如图8所示。从图中可以看出，在分化实验中，用含其他维生素和氨基酸冻存液冻存1月后，和新鲜冻存前细胞相比，分化能力基本一致。
对比例1
本对比例中采用的冻存液配方如下：
序号 所用试剂 试剂配制方案
1 Na + 140mmol/L
2 K + 6mmol/L
3 Mg 2+ 2mmol/L
4 Cl - 110mmol/L
5 CH 3COO - 40mmol/L
6 人白蛋白 40％(W/V)
7 二甲基亚砜 5mol/L
具体的冻存和复苏操作步骤如下：
1.取数盘贴壁培养的间充质干细胞，观察融合度为80％～90％,通过胰酶消化收集细胞，并计算活率和细胞密度，取活细胞量4×10 7离心收集
2.离心后去上清，弹开细胞沉淀，缓缓加入3ml冻存液
3.混匀，分装至2个2ml冻存袋，放入程序降温仪降温至-80度
4.于液氮中保存，并于1月、3月分别取样进行检测
经本对比例所述的冻存液冻存后，间充质干细胞活率分析结果如图9所示。从图中可以看出，在人白蛋白和二甲基亚砜浓度过高时，细胞在冻存1～3月内出现大幅度下降，尤其在一个月内，说明冻存过程中此冻存液未能保护细胞活率。
分化能力效能检测结果如图10所示。从图中可以看出，在分化实验中，用高二甲基亚砜冻存液冻存1月后，和新鲜冻存前细胞相比，成软骨能力明显下降。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种可临床使用的细胞冻存液，其特征在于，所述的细胞冻存液包括以下组分：
    (1)人白蛋白；
    (2)冷冻保护剂，所述的冷冻保护剂包括二甲基亚砜、甘油、乙二醇的一种或多种的组合；
    (3)盐缓冲液，所述的盐缓冲液为包含Na +、K +、Mg +、Cl -和CH 3COO -离子的溶液；
    (4)维生素；和
    (5)氨基酸；
    其中，所述人白蛋白浓度为1％～20％(w/v)。
  2. 如权利要求1所述的细胞冻存液，其特征在于，所述的冷冻保护剂浓度为0.4mol/L～2.2mol/L。
  3. 如权利要求1所述的细胞冻存液，其特征在于，所述的细胞冻存液包括选自下组的一个或多个特征：
    Na +离子的浓度为0.1～0.2mol/L；
    K +离子的浓度为5～10mmol/L；
    Mg 2+离子的浓度为1.5～5mmol/L；
    Cl -离子的浓度为0.1～0.3mol/L；
    CH 3COO -离子的浓度为40～60mmol/L。
  4. 如权利要求1所述的细胞冻存液，其特征在于，所述的维生素浓度为50mmol/L～150mmol/L；和/或所述氨基酸浓度为0.1mol/L～0.5mol/L。
  5. 如权利要求1所述的细胞冻存液，其特征在于，所述的人白蛋白选自下组：血浆提取人白蛋白、基因重组人白蛋白、或其组合。
  6. 如权利要求1所述的细胞冻存液，其特征在于，所述的维生素选自下组：维生素A、维生素B、维生素C、维生素D、维生素E、维生素K、或其组合。
  7. 如权利要求1所述的细胞冻存液，其特征在于，所述的氨基酸选自下组：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸，或其组合；优选为赖氨酸、色氨酸、苯丙氨酸、甲硫氨酸、苏氨酸、异亮氨酸、亮氨酸、缬氨酸、或其组合。
  8. 一种细胞混合物，其特征在于，所述混合物包括以下组分：
    细胞，和
    如权利要求1～7任一项所述的细胞冻存液；
    优选地，所述的细胞是Car-T细胞、间充质干细胞、或其组合。
  9. 一种细胞的冻存方法，其特征在于，包括步骤：
    (i)将待冻存细胞与如权利要求1～7任一项所述的细胞冻存液进行混合，得到细胞混合物；
    (ii)将步骤(i)得到的细胞混合物降温后，于液氮中保存。
  10. 一种试剂盒，所述的试剂盒用于细胞冻存，其特征在于，所述的试剂盒中包括如权利要求1～7任一所述的细胞冻存液。
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