CN114258910A - Cell cryopreservation solution capable of being infused and application thereof - Google Patents
Cell cryopreservation solution capable of being infused and application thereof Download PDFInfo
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Abstract
The application provides an infusible cell cryopreservation solution, which comprises the following components in percentage by volume: 10% of glycerol; 20% of human serum albumin injection; and the balance of hydroxyethyl starch 40 sodium chloride injection; wherein the human serum albumin injection comprises 20% of human serum albumin by mass; the hydroxyethyl starch 40 sodium chloride injection comprises 60g/L of hydroxyethyl starch 40 and 9g/L of sodium chloride. The infusible cell cryopreservation solution does not contain dimethyl sulfoxide, so that clinical adverse reactions caused by dimethyl sulfoxide in the traditional cell cryopreservation solution are avoided, and efficient, safe and stable cell cryopreservation is realized; moreover, the infusible cell cryopreservation solution can be used for CAR-NK cell clinical grade cryopreservation and can be directly infused after resuscitation.
Description
Technical Field
The application relates to the technical field of cell and cell engineering, in particular to an infusible cell cryopreservation solution for CAR-NK cell clinical grade cryopreservation.
Background
The immune cell therapy is a therapeutic method in which immature immune cells of a patient are activated, cultured and proliferated in vitro and then returned to the body to induce an autoimmune response. The cells currently applied to immune cell therapy mainly comprise T cells, NK cells, CIK cells and the like, wherein the NK cells are more likely to become the products in stock because the NK cells do not need strict HLA match and do not carry the risk of graft-versus-host disease, and can be frozen for standby use for a long time.
The CAR-NK cell technology refers to that an scFv antibody fragment capable of recognizing a specific tumor antigen is subjected to genetic engineering method to transform NK cells into CAR-NK cells capable of specifically attacking tumors, so that the tumors are ablated. However, the CAR-NK cells are low in cryopreservation efficiency at present, and are low in cell survival rate and weak in tumor killing capacity after recovery, so that clinical application is greatly limited.
Dimethyl sulfoxide and serum are usually added into cell freezing medium commonly used in laboratories or cell freezing medium sold in markets. In the cryopreservation of cells, dimethyl sulfoxide (DMSO) can effectively prevent cell damage caused by the formation of ice crystals, changes in osmotic pressure, cell structural disorder, and the like in cells.
Although it is generally believed that the toxic effects of dmso are minimal at concentrations less than 10%, dmso concentrations at different concentrations still produce many side effects during disease treatment. Clinically, the use of cells cryopreserved with dmso causes extensive physical discomfort, most commonly diarrhea, and severe damage and even death to the nervous system and respiratory system of the patient.
Serum, a substance whose composition is not yet known, has a certain lot-to-lot variation, and the effect of a cell frozen stock solution is unstable, and clinical use may cause allergy.
Therefore, there is a need to provide a novel cell cryopreservation solution to overcome the above-mentioned drawbacks and achieve efficient, safe and stable cell cryopreservation.
Disclosure of Invention
The application aims to provide the cell cryopreservation solution capable of being infused, the cell cryopreservation solution capable of being infused does not contain dimethyl sulfoxide, so that clinical adverse reactions caused by dimethyl sulfoxide in the traditional cell cryopreservation solution are avoided, and efficient, safe and stable cell cryopreservation is realized; moreover, the infusible cell cryopreservation solution can be used for CAR-NK cell clinical grade cryopreservation and can be directly infused after resuscitation.
To achieve the above objects, according to one aspect of the present application, there is provided an infusible cell cryopreservation liquid comprising by volume percent: 10% of glycerol, 20% of human serum albumin injection and the balance of hydroxyethyl starch 40 sodium chloride injection; the human serum albumin injection comprises human serum albumin with the mass ratio of 20%, and the hydroxyethyl starch 40 sodium chloride injection comprises 60g/L of hydroxyethyl starch 40 and 9g/L of sodium chloride.
In some embodiments, the infusible cell cryopreservation solution further comprises 1-10% by volume of bcg polysaccharide nucleic acid injection.
In some embodiments, the bcg polysaccharide nucleic acid injection comprises the following components: 0.35mg/ml BCG polysaccharide and 30ug/ml nucleic acid.
As will be understood by those skilled in the art, the BCG polysaccharide nucleic acid injection solution can be a conventional commercial BCG polysaccharide nucleic acid injection solution, and the main components of the BCG polysaccharide nucleic acid injection solution are BCG polysaccharide and nucleic acid. The BCG polysaccharide nucleic acid injection is prepared by extracting polysaccharide and nucleic acid from BCG and preparing the polysaccharide and nucleic acid with sterilized normal saline solution. In addition, the BCG polysaccharide nucleic acid injection can also contain medicinal sodium chloride as an auxiliary material.
In some embodiments, the infusible cell cryopreservation solution further comprises 3 to 50 percent by volume of dextran 40 amino acid injection.
In some embodiments, the dextran 40 amino acid injection comprises the components: dextran 40, leucine, isoleucine, phenylalanine, threonine, valine, tryptophan, methionine, glycine, lysine, arginine, and histidine; wherein the mass concentration of the dextran 40 is 60 g/L.
In some embodiments, the mass concentration of each component of the dextran 40 amino acid injection is: the mass concentration of the dextran 40 is 60 g/L; the mass concentration of leucine was 4.1g/L, the mass concentration of isoleucine was 1.8g/L, the mass concentration of phenylalanine was 2.9g/L, the mass concentration of threonine was 1.8g/L, the mass concentration of valine was 2.0g/L, the mass concentration of tryptophan was 0.6g/L, the mass concentration of methionine was 2.4g/L, the mass concentration of glycine was 3.4g/L, the mass concentration of lysine was 5.0g/L, the mass concentration of arginine was 2.2g/L, and the mass concentration of histidine was 1.0 g/L.
It will be understood by those skilled in the art that the dextran 40 amino acid injection solution described in the present application may be a conventional commercially available dextran 40 amino acid injection solution (also referred to as low molecular dextran amino acid injection solution), the main component of which is low molecular dextran (average molecular weight 40, also referred to as dextran 40). The dextran 40 amino acid injection is colorless or yellowish slightly viscous clear sterile solution which is prepared from dextran and 11 amino acids (leucine, isoleucine, phenylalanine, threonine, valine, tryptophan, methionine, glycine, lysine, arginine and histidine), wherein the total amino acid content of the solution is 2.72%, the dextran content of each 1ml is 60mg, the total amino acid content is 27.2mg, and the effective nitrogen content is 4.2 mg.
In some embodiments, the infusible cell cryopreservation solution further comprises 1-50% of a compound electrolyte injection by volume percentage.
In some embodiments, the compound electrolyte injection comprises the components: sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.
In some embodiments, the mass concentration of each component of the compound electrolyte injection is as follows: the mass concentration of sodium chloride is 5.26g/L, the mass concentration of sodium gluconate is 5.02g/L, the mass concentration of sodium acetate is 3.68g/L, the mass concentration of potassium chloride is 0.37g/L, and the mass concentration of magnesium chloride is 0.30 g/L.
As can be understood by those skilled in the art, the compound electrolyte injection can be a conventional commercially available compound electrolyte injection, and is a compound preparation mainly comprising sodium chloride, sodium gluconate and sodium acetate (C)2H3NaO2·3H2O), potassium chloride and magnesium chloride (MgCl)2·6H2O), and is prepared by using water for injection. In addition, the compound electrolyte injection can also contain a proper amount of sodium hydroxide for adjusting the pH value to 7.0-7.8.
In some embodiments, the infusible cell lysate does not comprise dimethyl sulfoxide and serum.
According to another aspect of the application, the application of the above-mentioned infusible cell cryopreservation liquid in CAR-NK cell cryopreservation is also provided.
In the application, the cell cryopreservation liquid which does not contain dimethyl sulfoxide and can be infused is provided, so that clinical adverse reactions caused by the traditional cell cryopreservation liquid are avoided, and efficient, safe and stable cell cryopreservation is realized. The infusible cell cryopreservation solution can be used for CAR-NK cell clinical grade cryopreservation and can be directly infused after resuscitation through reasonable formula selection. In addition, human serum albumin in the human serum albumin injection is used in the infusible cell frozen stock solution to replace components such as serum and plasma contained in the traditional cell frozen stock solution, so that the infusible cell frozen stock solution has higher clinical safety and can be used for replacing the traditional frozen solution containing dimethyl sulfoxide.
Therefore, compared with the traditional cell cryopreservation liquid capable of being infused, the cell cryopreservation liquid capable of being infused has the following advantages:
1) the cell cryopreservation solution capable of being infused can be prepared by directly using a commercially available injection, and is simple and convenient to operate and low in cost;
2) the infusible cell frozen stock solution has clear addition components and is convenient for scientific research and analysis;
3) the cell cryopreservation solution capable of being infused is high in safety and small in damage to cells;
4) the cells cryopreserved by the cell cryopreserving liquid capable of being infused have good survival rate and killing capacity after recovery.
Detailed Description
Hereinafter, the technology of the present application will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for assisting those skilled in the art in understanding the present application, and is not intended to limit the present application.
EXAMPLE 1 infusible cell cryopreservation solution
The embodiment provides an infusible cell cryopreservation solution, which comprises the following components in percentage by volume: 10 v/v% of glycerol, 20 v/v% of human serum albumin injection, 62.2 v/v% of hydroxyethyl starch 40 sodium chloride injection, 3.6 v/v% of dextran 40 amino acid injection, 0.5 v/v% of BCG polysaccharide nucleic acid injection and 3.7 v/v% of compound electrolyte injection.
Example 2 infusible cell cryopreservation solution
The embodiment provides an infusible cell cryopreservation solution, which comprises the following components in percentage by volume: 10 v/v% of glycerol, 20 v/v% of human serum albumin injection and 70 v/v% of hydroxyethyl starch 40 sodium chloride injection.
Example 3 infusible cell cryopreservation solution
The embodiment provides an infusible cell cryopreservation solution, which comprises the following components in percentage by volume: 10 v/v% of glycerol, 20 v/v% of human serum albumin injection, 60 v/v% of hydroxyethyl starch 40 sodium chloride injection and 10 v/v% of BCG polysaccharide nucleic acid injection.
Example 4 infusible cell cryopreservation solution
The embodiment provides an infusible cell cryopreservation solution, which comprises the following components in percentage by volume: 10 v/v% of glycerol, 20 v/v% of human serum albumin injection, 21.7 v/v% of hydroxyethyl starch 40 sodium chloride injection, 5 v/v% of BCG polysaccharide nucleic acid injection, 21.7 v/v% of dextran 40 amino acid injection and 21.6 v/v% of compound electrolyte injection.
Example 5 infusible cell cryopreservation solution
The embodiment provides an infusible cell cryopreservation solution, which comprises the following components in percentage by volume: 10 v/v% of glycerol, 20 v/v% of human serum albumin injection, 10.8 v/v% of hydroxyethyl starch 40 sodium chloride injection, 2.5 v/v% of BCG polysaccharide nucleic acid injection, 10.8 v/v% of dextran 40 amino acid injection and 45.9 v/v% of compound electrolyte injection.
Example 6 infusible cell cryopreservation
The embodiment provides an infusible cell cryopreservation solution, which comprises the following components in percentage by volume: 10 v/v% of glycerol, 20 v/v% of human serum albumin injection, 10.8 v/v% of hydroxyethyl starch 40 sodium chloride injection, 2.5 v/v% of BCG polysaccharide nucleic acid injection, 45.8 v/v% of dextran 40 amino acid injection and 10.9 v/v% of compound electrolyte injection.
Comparative example 1 conventional cell cryopreservation solution 1
In this example, for validation purposes, a conventional cell cryopreservation solution 1 is provided, which comprises the following components: fetal bovine serum and dimethyl sulfoxide; wherein the volume ratio of the fetal calf serum to the dimethyl sulfoxide is 9: 1.
Comparative example 2 conventional cell cryopreservation solution 2
In this example, for the purpose of verification, a conventional cell freezing medium 2 was provided, which is a dimethylsulfoxide-free and serum-free cell freezing medium commercially available from Irvine Scientific.
Effect verification examples
In order to verify the effects of the infusible cell cryopreservation solutions described in the present application, the infusible cell cryopreservation solutions prepared in examples 1 to 6 were subjected to cell cryopreservation and recovery experiments using the following methods, respectively, as compared with the conventional cell cryopreservation solution 1 of comparative example 1 and the conventional cell cryopreservation solution 2 of comparative example 2.
The experimental reagent comprises: serum-free medium T009 (sold by Shanghai Bitsu Biotech Co., Ltd.), and fetal bovine serum (Biological Industries).
CAR-NK cells cryopreserved
CAR-NK cells were cultured in T009 serum-free medium, and at the logarithmic growth phase, an appropriate amount of the cells were removed and centrifuged at 1000rpm for 5 minutes in a centrifuge, and the supernatant was discarded. Then, the conventional cell culture solutions of examples 1 to 6 and comparative examples 1 to 2 were added, respectively, and mixed well.
At 1 × 107cell/ml, 0.5ml system put into 1.5ml freezing tube; the vial was stored overnight in a freezer at-80 ℃ and then transferred to liquid nitrogen.
Survival rates of CAR-NK cells were measured after 7 days and 30 days of cryopreservation, respectively, and the results are shown in tables 1 and 2.
TABLE 1 survival of CAR-NK cells after 7 days of cryopreservation
| Cell cryopreservation liquid | CAR-NK cell survival (%) |
| Before freezing | 95.00％ |
| EXAMPLE 1 infusible cell cryopreservation solution | 85.40％ |
| Example 2 infusible cell cryopreservation solution | 78.82％ |
| Example 3 infusible cell cryopreservation solution | 68.50％ |
| Example 4 infusible cell cryopreservation solution | 72.27％ |
| Example 5 infusible cell cryopreservation solution | 79.92％ |
| Example 6 infusible cell cryopreservation | 75.00％ |
| Comparative example 1 conventional cell cryopreservation solution 1 | 67.70％ |
| Comparative example 2 conventional cell cryopreservation solution 2 | 83.70％ |
TABLE 2 survival of CAR-NK cells after 30 days of cryopreservation
| Cell cryopreservation liquid | CAR-NK cell survival (%) |
| Before freezing | 95.00％ |
| EXAMPLE 1 infusible cell cryopreservation solution | 82.75％ |
| Example 2 infusible cell cryopreservation solution | 70.77％ |
| Comparative example 1 conventional cell cryopreservation solution 1 | 66.40％ |
| Comparative example 2 conventional cell cryopreservation solution 2 | 82.14％ |
As can be seen from the data in tables 1 and 2, compared with the conventional cell cryopreservation solution 1 containing fetal bovine serum and dimethyl sulfoxide, the survival rate of CAR-NK cells is obviously improved after the cell cryopreservation solution capable of being infused is used for cryopreservation. Compared with the traditional cell frozen stock solution 2 without dimethyl sulfoxide and serum cells, the survival rate of CAR-NK cells subjected to freezing by using the cell frozen stock solution capable of being infused is close to the survival rate of the traditional cell frozen stock solution 2.
CAR-NK cell resuscitation
Freezing the cellsThe tube was taken out of the liquid nitrogen and quickly placed in a 37 ℃ water bath for rotary thawing. The cells were transferred to a six-well plate containing 5ml of fresh medium, settled in a cell incubator, then 4ml of supernatant was aspirated, and the supernatant was replaced with 5X 105cells/ml density was re-inoculated with fresh medium and 1000IU/ml IL-2 was added.
Among them, for the cells using the conventional cell cryopreservation solution of comparative example 1, that is, for the cells using the conventional cell cryopreservation solution containing dimethyl sulfoxide, the thawed cells were transferred to a centrifuge tube containing 10ml of fresh medium, centrifuged at 1000rpm for 5 minutes to remove dimethyl sulfoxide, and then inoculated into a T25 square flask.
CAR-NK cell killing ability assay
In this assay, the killing ability of CAR-NK cells to K562 cells was tested 30 days after cryopreservation.
Take 1X 105The CAR-NK cells obtained by re-inoculation after the recovery and 2 x 104Individual K562 cells were co-incubated in 96-well plates at 37 ℃ for 4 hours in an incubator. Then, 10 ul/well of CCK-8 reagent was added and mixed well, and after standing for 2 hours, OD at 450nm of each group was measured using a microplate reader.
The killing ability of CAR-NK cells against K562 cells after 30 days of cryopreservation is shown in table 3.
TABLE 3 killing ability of CAR-NK cells on K562 cells after 30 days of cryopreservation
| Cell cryopreservation liquid | Cell killing ability (%) |
| Before freezing | 75.98％ |
| EXAMPLE 1 infusible cell cryopreservation solution | 45.90％ |
| Example 2 infusible cell cryopreservation solution | 30.05％ |
| Comparative example 1 conventional cell cryopreservation solution 1 | 29.03％ |
| Comparative example 2 conventional cell cryopreservation solution 2 | 18.77％ |
CAR-NK cell phenotype detection
In this assay, the phenotypic ratio of CAR-NK cells and the ratio of secreted toxic particles were examined 30 days after cryopreservation.
Take 2X 105And (3) inoculating the obtained CAR-NK cells again after the recovery into a 1.5ml EP tube, centrifuging at 1000rpm for 5 minutes, reserving about 50ul of supernatant in each tube, adding flow antibodies such as CD3, CD56, CD16 and CAR markers, flicking, mixing uniformly, and incubating in a refrigerator at 4 ℃ for 30 minutes. Then, washed and stored in the dark, and detected as soon as possible by flow cytometry analysis.
The phenotypic ratio and the ratio of secretory toxic particles of CAR-NK cells after 30 days of cryopreservation are shown in table 4.
TABLE 4 phenotypic ratio of CAR-NK cells and ratio of secretory toxic particles after 30 days of cryopreservation
As can be seen from the experimental data in tables 1 to 4, compared with the conventional cell frozen stock solution 1 containing fetal bovine serum and dimethyl sulfoxide and the commercially available conventional cell frozen stock solution 2 without dimethyl sulfoxide and serum cells, the CAR-NK cell frozen stock solution capable of being infused has the advantages of high survival rate of the CAR-NK cell, good killing capability and capability of better maintaining the specific immunophenotype of the CAR-NK cell.
The infusible cell cryopreservation liquid does not contain dimethyl sulfoxide, has the characteristics of safety and no toxicity, and does not bring harm to the health and the environment of experimenters. Meanwhile, the infusible cell cryopreservation solution does not contain components such as serum and the like which cannot be directly clinically infused, so that the infusible cell cryopreservation solution can be used for CAR-NK cell clinical grade cryopreservation and can be directly prepared into clinical injection after resuscitation, and the method is very convenient. Moreover, the infusible cell cryopreservation liquid has simple and definite components and low manufacturing cost, so the infusible cell cryopreservation liquid can be widely applied to the technical field of cell cryopreservation and has better application prospect.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are included in the scope of the present invention.
Claims (11)
1. An infusible cell cryopreservation fluid comprising by volume percent:
10% of glycerol;
20% of human serum albumin injection; and
the balance is hydroxyethyl starch 40 sodium chloride injection; wherein the content of the first and second substances,
the human serum albumin injection comprises 20% of human serum albumin by mass;
the hydroxyethyl starch 40 sodium chloride injection comprises 60g/L of hydroxyethyl starch 40 and 9g/L of sodium chloride.
2. The infusible cell cryopreservation solution of claim 1 further comprising 1% to 10% by volume bcg polysaccharide nucleic acid injection.
3. The infusible cell cryopreservation solution of claim 2 wherein the BCG polysaccharide nucleic acid injection comprises the components: 0.35mg/ml BCG polysaccharide and 30ug/ml nucleic acid.
4. The infusible cell cryopreservation solution of claim 1 or 2 further comprising 3% to 50% dextran 40 amino acid injection by volume percent.
5. The infusible cell cryopreservation solution of claim 4 wherein the dextran 40 amino acid injection comprises the components: dextran 40, leucine, isoleucine, phenylalanine, threonine, valine, tryptophan, methionine, glycine, lysine, arginine, and histidine; wherein the mass concentration of the dextran 40 is 60 g/L.
6. The infusible cell cryopreservation solution of claim 5 wherein the mass concentrations of the components of the dextran 40 amino acid injection are:
7. the infusible cell cryopreservation liquid of claim 4 further comprising 1% to 50% by volume of a compound electrolyte injection.
8. The infusible cell cryopreservation solution of claim 7 wherein the complex electrolyte injection comprises the components: sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.
9. The infusible cell cryopreservation solution of claim 8 wherein the mass concentrations of the components of the compound electrolyte injection are as follows:
10. the infusible cell cryopreservation fluid of claim 1 wherein the infusible cell cryopreservation fluid does not comprise dimethyl sulfoxide and serum.
11. Use of an infusible cell cryopreservation solution of claim 1 in CAR-NK cell cryopreservation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108552159A (en) * | 2018-05-04 | 2018-09-21 | 武汉波睿达生物科技有限公司 | A kind of frozen stock solution for being used for CAR-T clinical grades Cord blood and being directly injected intravenously feedback |
| CN108739798A (en) * | 2018-08-22 | 2018-11-06 | 北京常箐藤生物科技有限公司 | A kind of cells frozen storing liquid |
| CN109792985A (en) * | 2019-02-20 | 2019-05-24 | 华东理工大学 | Cells frozen storing liquid |
| CN113424820A (en) * | 2021-08-04 | 2021-09-24 | 北京全式金生物技术有限公司 | Serum-free, protein-free and DMSO-free cell cryopreservation liquid and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108552159A (en) * | 2018-05-04 | 2018-09-21 | 武汉波睿达生物科技有限公司 | A kind of frozen stock solution for being used for CAR-T clinical grades Cord blood and being directly injected intravenously feedback |
| CN108739798A (en) * | 2018-08-22 | 2018-11-06 | 北京常箐藤生物科技有限公司 | A kind of cells frozen storing liquid |
| CN109792985A (en) * | 2019-02-20 | 2019-05-24 | 华东理工大学 | Cells frozen storing liquid |
| CN113424820A (en) * | 2021-08-04 | 2021-09-24 | 北京全式金生物技术有限公司 | Serum-free, protein-free and DMSO-free cell cryopreservation liquid and application thereof |
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