CN106665560B - Immune cell frozen stock solution for direct venous return and application thereof - Google Patents
Immune cell frozen stock solution for direct venous return and application thereof Download PDFInfo
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Classifications
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses immune cell frozen stock solution for direct venous return and application thereof. The invention adopts the raw materials of clinical medicine grade such as dimethyl sulfoxide, human serum albumin, dextran, hydroxyethyl starch, heparin sodium and the like, and finally prepares the immune cell frozen stock solution for direct intravenous infusion by optimizing the use amount of each raw material. In addition, the invention also simplifies the operation steps and improves the freezing and storing effect by optimizing the freezing and storing method. On one hand, the invention avoids the potential risk of introducing animal-derived antigen substances in the cell treatment process; on the other hand, the immune cell cryopreservation effect of the invention is better than that of the conventional cryopreservation liquid, thereby ensuring the safety and effectiveness of clinical use. The invention can store the frozen stock solution stably for a long time by storing the two components. The method is simple to operate, provides stronger operability for implementation of large-scale cryopreservation, and provides basic technical support for implementation of immune cell therapy.
Description
Technical Field
The invention relates to a cell cryopreservation solution, in particular to an immune cell cryopreservation solution for direct venous return and an application method thereof. The invention belongs to the technical field of biological medicines.
Background
Cancer is the primary problem which afflicts human health, and cellular immunotherapy provides a broad prospect for the prevention and treatment of tumors. However, in the process of clinical practical application, there are many problems that autoimmune cells of some tumor patients are difficult to collect, the cell expansion capability is poor, the time windows of the immune cells and other treatment modes are difficult to match, and the like; in addition, ex situ transport, storage conditions and survival time of fresh immune cells are limited from the standpoint of drug development and production. Therefore, the effective cryopreservation of the immune cells provides necessary basic technical guarantee for the industrial application of the immune cells as medicines.
However, most of the currently marketed immune cell frozen stocks are prepared from fetal bovine serum, high-proportion DMSO, autologous patient plasma and non-clinical application-level reagents, and the frozen stocks have the following problems: 1. the freezing effect is poor; 2. the frozen stock solution has undefined components; 3. the cryopreservation process and subsequent application programs are relatively complicated; 4. the cost is too high; 5. there is a risk in clinical use; 6. is not suitable for industrialization. For example:
the patent application with the publication number of CN104026118A discloses an immune cell freezing solution, a preparation method and an application thereof, wherein dimethyl sulfoxide, inactivated human autologous plasma and immune cell culture medium components are used for freezing immune cells by a programmed cooling method. The technical problems are that the cryopreservation liquid of the application uses autologous plasma inactivated by a patient, the quantity and the source are limited, the cryopreservation liquid is not suitable for large-scale cryopreservation after cell amplification culture, the cryopreservation process is complicated, and batch control is difficult.
Patent application with publication number CN105248413A discloses a CIK cell cryopreservation solution, which uses dimethyl sulfoxide, grape seed extract, raffinose, human serum albumin, and fetal bovine serum to cryopreserve CIK cells. The technical problem is that the frozen stock solution contains animal-derived serum, has potential risk of introducing animal-derived antigen substances, and cannot be directly returned to a human body.
Patent application publication No. CN105211051A discloses a frozen stock solution of cultured NK cells and a preparation method thereof, wherein dimethyl sulfoxide, inactivated human autologous plasma, and grape seed extract, tea polyphenol and vitamin C are added as components of the frozen stock solution. The technical problem is that the cryopreservation solution of the application uses autologous inactivated plasma of a patient, has limited quantity and source and is not suitable for large-scale cryopreservation after cell amplification culture.
Patent application with publication number CN105123671A discloses a cell cryopreservation liquid, application and an immune cell cryopreservation method, wherein dimethyl sulfoxide, propylene glycol, a cell culture medium, human serum albumin are used as components of the cryopreservation liquid, and non-essential amino acid, trehalose, lentinan and vitamin C are added. The technical problem is that the main component of the frozen stock solution is a cell culture medium, does not have the effect of a stabilizer and cannot be directly used for clinical reinfusion.
The patent application with publication number CN104862275A discloses a method for freezing and recovering cells and a cell preparation prepared by the method, wherein dimethyl sulfoxide, 1640 cell basal medium, fetal calf serum, dextran and ddH2O are used as components of a freezing solution. The technical problem is that the frozen stock solution contains animal-derived serum, has potential risk of introducing animal-derived antigen substances, and cannot be directly returned to a human body.
In conclusion, the effective components of the existing freezing medium mostly contain scientific research-grade cell culture medium, fetal bovine serum and other components, can not be directly applied to human infusion, and is expensive. Therefore, there is a need to research a frozen immune cell stock solution with better frozen effect, lower price, more convenient use and safer application.
Disclosure of Invention
The invention aims to solve the technical problems of poor freezing effect, undefined freezing solution component, relatively complicated freezing process and subsequent application program, high cost, risk in clinical application, unsuitability for large-scale production and the like in the prior art, and provides the immune cell freezing solution for direct intravenous return transfusion and the application method thereof.
In order to achieve the purpose, the invention adopts the following technical means:
the immune cell frozen stock solution directly transfused by vein of the invention is composed of solution A and solution B, wherein, the solution A contains the following components: 4-16 v/v% of dimethyl sulfoxide, 0.3-5.5 w/v% of hydroxyethyl starch, 0.3-5.5 w/v% of dextran, 0.5-5 w/v% of glucose, 1-1000U/ml of heparin sodium and the balance of medical injection; the B solution is medical injection containing 2-25 w/v% human serum albumin.
In the present invention, the content of each component in the solution a is preferably: 8.3 v/v% of dimethyl sulfoxide, 2.75 w/v% of hydroxyethyl starch, 2.75 w/v% of dextran, 2.29 w/v% of glucose, 625U/ml of heparin sodium and the balance of medical injection; the B solution is medical injection containing 20 w/v% human serum albumin.
In the present invention, preferably, the medical injection solution includes, but is not limited to: one or more of sterile water for injection, physiological saline, electrolyte injection, dextran injection or hydroxyethyl starch sodium chloride injection.
The raw materials for preparing the frozen stock solution can be raw material medicines or finished products of medicines prepared by solution. For example, the hydroxyethyl starch can be hydroxyethyl starch sodium chloride injection, the dextran and the glucose can be dextran 40 glucose injection, and the heparin sodium can be heparin sodium injection. As long as the final concentration of the active ingredient satisfies the above requirements.
The invention is prepared by optimizing the mixture ratio of dimethyl sulfoxide, human serum albumin, dextran, hydroxyethyl starch and heparin sodium, and adopting raw materials of clinical medicinal grade and medical injection. On one hand, the risk of pollution of animal-derived substances is avoided, and on the other hand, the immune cell cryopreservation effect is better than that of the conventional cryopreservation liquid. Thereby ensuring the safety and effectiveness of clinical use. In addition, the method is simple to operate, is suitable for implementation of large-scale cryopreservation, can be directly infused after cell resuscitation, and is convenient for clinical implementation.
Furthermore, the invention also provides application of the cryopreservation liquid in cryopreservation of immune cells.
Among them, preferably, the immune cells include but are not limited to: t cells, NK cells, monocytes, DC cells, γ δ T cells, CAR-T cells.
Furthermore, the invention also provides a method for freezing and storing immune cells in vitro, which comprises the following steps:
1) uniformly mixing the solution A and the solution B of the frozen stock solution according to the volume ratio of 3:2, and placing the mixture in a refrigerator for precooling at 4 ℃;
2) collecting immune cells to be frozen and washing twice;
3) re-suspending the immune cells by using a pre-cooled freezing medium, transferring the immune cells into a freezing bag, and placing the immune cells in a refrigerator at 4 ℃ for balancing or directly performing the step 4) by skipping the balancing step if the operation time of the step 3) is longer than 30 min;
4) the freezing bag with the cells is placed in a refrigerator at minus 80 ℃ overnight and then transferred to a liquid nitrogen tank for long-term storage.
In the present invention, it is preferable that the cryopreserved immune cells are used by rapidly taking out the cryopreserved bag from a liquid nitrogen tank, placing the cryopreserved bag in a water bath at 37 ℃ until the cryopreserved bag is completely thawed, and completing intravenous infusion within 1 hour after thawing.
In the present invention, it is preferable that the time for the refrigerator equilibration at 4 ℃ is 1-30 min.
In the present invention, preferably, the immune cells include, but are not limited to: t cells, NK cells, monocytes, DC cells, γ δ T cells, CAR-T cells.
In the present invention, preferably, the freezing density of the immune cells is 1 × 106/ml-1×108/ml。
Compared with the prior art, the invention has the beneficial effects that:
the invention overcomes the defects of poor freezing effect, undefined freezing medium component, relatively complicated freezing process and subsequent application program, high cost, risk in clinical application, unsuitability for large-scale production and the like in the prior art, and provides the immune cell freezing medium for direct venous return and the application method thereof.
Compared with the conventional freezing method: 1. compared with freshly cultured immune cells, the immune cells recovered by the method have no significant difference in immunobiological characteristics (cell viability, apoptosis level, immunophenotype and the like) and biological functions (killing effect on target cells, cytokine secretion level and the like); 2. the frozen stock solution has definite raw materials, no animal-derived components, clinical medicinal grade and clinical application safety; 3. patient autologous plasma is not used, the quantity and source of raw materials are not limited, the cost is low, and the method is suitable for industrialization; 4. the frozen stock solution has good storage stability through a component storage mode; the DMSO content is relatively low, and the cytotoxicity of the DMSO in the frozen stock solution is reduced by precooling the frozen stock solution; 6. the freezing and storing process of the immune cells does not need long-time programmed cooling; 7. after resuscitation, the conventional centrifugal cleaning step is not needed, and venous return transfusion can be directly carried out, thereby greatly facilitating clinical application.
In conclusion, the invention abandons the components which are not suitable for clinical use in the conventional frozen stock solution, selects the clinically common medicines to prepare the frozen stock solution through scientific proportion, simplifies the preparation operation of the frozen stock solution through a scientific storage mode, simplifies the operation steps through an optimized frozen stock method, improves the frozen stock effect and provides basic guarantee for the implementation of immune cell therapy. Compared with the prior art, the invention has high safety, does not contain animal-derived serum with potential pathogenic risk, and can be stably stored for a long time by the two components. The cryopreservation method is simple and easy to implement, can meet the requirements of clinical large-scale operation, is superior to the conventional cryopreservation method in cryopreservation effect, can be directly infused after cells are recovered, and reduces the damage to the cells caused by intermediate complicated treatment links and washing. And the raw material cost is low, the method is suitable for industrial production, and great economic benefit can be generated.
Detailed Description
The present invention is further illustrated by the following specific examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
Example 1: preparation and application of immune cell frozen stock solution for direct intravenous infusion
1. Raw materials and specifications
Hydroxyethyl starch sodium chloride injection: 500ml of hydroxyethyl starch (200/0.5)30g
Dextran 40 glucose injection: 500ml of 30g of dextran 40 and 25g of glucose
Heparin sodium injection: 2ml:1.25 ten thousand units
Human serum albumin injection: 50 ml: 10g Total protein
2. Preparation and application of immune cell frozen stock solution for direct intravenous infusion
1) Respectively preparing solution A and solution B
Solution a (100 ml): 8.3ml of dimethyl sulfoxide, 45.8ml of hydroxyethyl starch and sodium chloride injection, 45.8ml of dextran 40 and glucose injection and 0.1ml of heparin sodium injection.
The content of each component in the solution A is as follows: 8.3 v/v% of dimethyl sulfoxide, 2.75 w/v% of hydroxyethyl starch, 2.75 w/v% of dextran, 2.29 w/v% of glucose, 625U/ml of heparin sodium and the balance of medical injection.
Solution B (100 ml): 20 w/v% human serum albumin injection.
2) Mixing the solution A and the solution B in a volume ratio of 3:2, and placing in a refrigerator at 4 ℃ for later use.
3) 50ml of peripheral blood of healthy volunteers is extracted, slowly added into a centrifuge tube filled with lymphocyte separation liquid, centrifuged for 20 minutes at 500g, and leucocyte layer cells are sucked. Washed twice with PBS and counted.
4) Based on the counting result, use2) Resuspending PBMC cells in a well-prepared, pre-cooled frozen stock solution to a cell density of 1X 107Perml, transfer cells to cryopreservation bags.
5) The frozen bag with cells was placed in a refrigerator at 4 ℃ and equilibrated for 15 min.
6) The frozen bags containing the cells were placed in a-80 ℃ freezer overnight.
6) And (4) placing the cryopreservation bag containing the cells into liquid nitrogen for preservation.
7) Taking out cells from a liquid nitrogen tank after 1, 6 and 12 months, placing the cells in a water bath kettle at 37 ℃ for thawing, taking a small amount of cells, and detecting the cell viability by a trypan blue staining method.
PBMC cells treated with control cryopreservation (50% FBS + 40% RPMI1640+ 10% DMSO) were also used as controls, and the procedure was the same as above except for the cryopreservation.
3. Results
A comparison of PBMC resuscitation activity rates with different freezing media is shown in table 1:
TABLE 1 comparison of PBMC resuscitation Activity rates with different cryopreservation fluids
| The cryopreservation liquid of the invention | Control frozen stock solution | |
| Survival rate of 1 month | 98.5％ | 87.1％ |
| Survival rate of 6 months | 96.2％ | 85.4％ |
| Survival rate of 12 months | 93.7％ | 80.7％ |
Example 2 preparation and application of immune cell cryopreservation solution for direct intravenous infusion
1. Raw materials and specifications
Hydroxyethyl starch sodium chloride injection: 500ml of hydroxyethyl starch (200/0.5)30g
Dextran 40 glucose injection: 500ml of 30g of dextran 40 and 25g of glucose
Heparin sodium injection: 2ml:1.25 ten thousand units
Human serum albumin injection: 50 ml: 5g Total protein
2. Preparation and application of immune cell frozen stock solution for direct intravenous infusion
1) Respectively preparing solution A and solution B
Solution a (100 ml): 10ml of dimethyl sulfoxide, 70ml of hydroxyethyl starch and sodium chloride injection, 19.9ml of dextran 40 and glucose injection and 0.1ml of heparin sodium injection
The content of each component in the solution A is as follows: 10 v/v% of dimethyl sulfoxide, 4.2 w/v% of hydroxyethyl starch, 1.19 w/v% of dextran, 1.0 w/v% of glucose, 625U/ml of heparin sodium and the balance of medical injection;
solution B (100 ml): 10 w/v% human serum albumin injection
2) And uniformly mixing the solution A and the solution B according to the volume ratio of 3:2, and placing the mixture in a refrigerator at 4 ℃ for later use.
3) And (4) taking the cultured and amplified NK cells to a centrifuge tube, and centrifuging for 10min at 300 g. Washed twice with PBS and counted.
4) According to the counting result, the NK cells are re-suspended by using the frozen stock solution prepared and pre-cooled in the step 2) so that the cell density is 1 multiplied by 108Perml, transfer cells to cryopreservation bags.
5) The freezing bag with the cells is placed in a 4-degree refrigerator to be balanced for 15 min.
6) The frozen bags containing the cells were placed in a-80 ℃ freezer overnight.
6) And (4) placing the cryopreservation bag containing the cells into liquid nitrogen for preservation.
7) Taking out cells from a liquid nitrogen tank after 1, 6 and 12 months, placing the cells in a water bath kettle at 37 ℃ for thawing, taking a small amount of cells, and detecting the cell viability by a trypan blue staining method.
NK cells were also treated with a control cryopreservation solution (50% FBS + 40% RPMI1640+ 10% DMSO) as a control, and the procedure was the same as above except for the cryopreservation solution.
3. Results
Comparison of NK resuscitation activity with different frozen stocks is shown in table 2:
TABLE 2 comparison of NK cell resuscitative Activity rates with different cryopreservation solutions
| Example 2 | Example 2 comparison | |
| Survival rate of 1 month | 96.4％ | 82.6％ |
| Survival rate of 6 months | 92.8％ | 75.3％ |
| Survival rate of 12 months | 90.5％ | 60.2％ |
Example 3 preparation and application of immune cell cryopreservation solution for direct intravenous infusion
1. Raw materials and specifications
Hydroxyethyl starch sodium chloride injection: 500ml of hydroxyethyl starch (200/0.5)30g
Dextran 40 glucose injection: 500ml of 30g of dextran 40 and 25g of glucose
Heparin sodium injection: 2ml:1.25 ten thousand units.
Human serum albumin injection: 50 ml: 5g Total protein
2. Preparation and application of immune cell frozen stock solution for direct intravenous infusion
1) Respectively preparing solution A and solution B
Solution a (100 ml): 9ml of dimethyl sulfoxide, 33ml of hydroxyethyl starch and sodium chloride injection, 57.9ml of dextran glucose injection and 0.1ml of heparin sodium injection.
The content of each component in the solution A is as follows: 9 v/v% of dimethyl sulfoxide, 1.98 w/v% of hydroxyethyl starch, 3.47 w/v% of dextran, 2.9 w/v% of glucose, 625U/ml of heparin sodium and the balance of medical injection;
solution B (100 ml): 10 w/v% human serum albumin injection.
2) Mixing solution A and solution B at ratio of 3:2, and placing in refrigerator at 4 deg.C for use.
3) And taking the CAR-T cells after culture and amplification into a centrifuge tube, and centrifuging for 10min at 300 g. Washed twice with PBS and counted.
4) According to the counting results, the CAR-T cells were resuspended in the prepared, precooled cryopreservation solution of 2) so that the cell density was 5X 107Perml, transfer cells to cryopreservation bags.
5) The frozen bag with cells was placed in a refrigerator at 4 ℃ and equilibrated for 15 min.
6) The frozen bags containing the cells were placed in a-80 ℃ freezer overnight.
6) And (4) placing the cryopreservation bag containing the cells into liquid nitrogen for preservation.
7) Taking out cells from a liquid nitrogen tank after 1, 6 and 12 months, placing the cells in a water bath kettle at 37 ℃ for thawing, taking a small amount of cells, and detecting the cell viability by a trypan blue staining method.
CAR-T cells treated with control cryopreservation (50% FBS + 40% RPMI1640+ 10% DMSO) were also used as controls, except for the cryopreservation.
3. Results
A comparison of CAR-T resuscitation activity rates with different cryopreservation fluids is shown in Table 3:
TABLE 3 CAR-T cell Resuscitation Activity comparisons with different cryopreservatives
| Example 3 | Example 3 comparison | |
| Survival rate of 1 month | 98.1％ | 85.5％ |
| Survival rate of 6 months | 96.7％ | 80.5％ |
| Survival rate of 12 months | 93.3％ | 72.4％ |
The above examples are merely illustrative of the present invention and should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Claims (13)
1. The immune cell frozen stock solution for direct intravenous infusion is characterized by consisting of a solution A and a solution B, wherein the solution A contains the following components: 8.3-10 v/v% of dimethyl sulfoxide, 1.98-4.2 w/v% of hydroxyethyl starch, 1.19-3.47 w/v% of dextran, 1.0-2.9 w/v% of glucose, 625U/ml of heparin sodium and the balance of medical injection; the B solution is a medical injection containing 10-20 w/v% human serum albumin, the medical injection is one or two of sterile water for injection and physiological saline, and the volume ratio of the A solution to the B solution is 3: 2.
2. The cryopreservation solution of claim 1, wherein the contents of the components in the solution A are as follows: 8.3 v/v% of dimethyl sulfoxide, 2.75 w/v% of hydroxyethyl starch, 2.75 w/v% of dextran, 2.29 w/v% of glucose, 625U/ml of heparin sodium and the balance of medical injection; the B solution is medical injection containing 20 w/v% human serum albumin.
3. Use of the cryopreservation solution of claim 1 or 2 for cryopreservation of immune cells.
4. The use of claim 3, wherein said immune cells comprise: PBMC cells.
5. The use of claim 3, wherein said immune cells comprise: t cells, NK cells, DC cells.
6. The use of claim 5, wherein said T cells comprise: γ δ T cells, CAR-T cells.
7. A method for freezing immune cells in vitro is characterized by comprising the following steps:
1) uniformly mixing the solution A and the solution B of the frozen stock solution according to the volume ratio of 3:2, and placing the mixture in a refrigerator for precooling at 4 ℃;
2) collecting immune cells to be frozen and washing twice;
3) re-suspending the immune cells by using a pre-cooled freezing medium, transferring the immune cells into a freezing bag, and placing the immune cells in a refrigerator at 4 ℃ for balancing or directly performing the step 4) by skipping the balancing step if the operation time of the step 3) is longer than 30 min;
4) the freezing bag with the cells is placed in a refrigerator at minus 80 ℃ overnight and then transferred to a liquid nitrogen tank for long-term storage.
8. The method of claim 7, wherein the cryopreserved immune cells are used by rapidly removing the cryopreserved bag from a liquid nitrogen tank, placing the cryopreserved bag in a water bath at 37 ℃ until the cryopreserved bag is completely thawed, and completing the intravenous infusion within 1 hour after thawing.
9. The method of claim 7, wherein the time to equilibrate in a 4 ℃ freezer is 15 min.
10. The method of claim 7, wherein the immune cell comprises: PBMC cells.
11. The method of claim 7, wherein the immune cell comprises: t cells, NK cells, DC cells.
12. The method of claim 11, wherein said T cell comprises: γ δ T cells, CAR-T cells.
13. The method of claim 7, wherein said immune cells are cryopreserved at a density of 1 x 107/ml-1×108/ml。
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