CN116058364B - NK cell cryopreservation liquid and cryopreservation method and application thereof - Google Patents
NK cell cryopreservation liquid and cryopreservation method 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
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses NK cell cryopreservation liquid, and a cryopreservation method and application thereof, and relates to biomedicineTechnical field. The NK cell frozen stock solution is prepared fromCS10 and human serum albumin; the said processThe volume ratio of CS10 to human serum albumin is (1-5): 1. the invention also provides a freezing and storing method of NK cells, which comprises the following steps: (1) Will beCS10 and human serum albumin are mixed according to the volume ratio to obtain NK cell frozen stock solution; (2) Adding NK cell frozen stock solution into NK cells to obtain cell suspension; (3) And (5) sub-packaging the cell suspension in a freezing tube, and cooling in sequence. The freezing solution disclosed by the invention has the advantages of simple components and good stability, the survival rate of cells after resuscitating is improved, and the damage to NK in the freezing process is reduced.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to NK cell cryopreservation liquid, and a cryopreservation method and application thereof.
Background
Chimeric antigen receptors (chimeric antigen receptor, CAR) are receptor proteins based on genetic engineering and expressed on the cell surface, consisting of an antigen recognition region, a hinge region, a transmembrane region and an intracellular signaling region. Natural killer cells (Natural killer cells, NK cells) are human innate immune lymphocytes that contain a range of activating and inhibitory receptors on the cell surface that can rapidly recognize and kill diseased cells. CAR-NK therapy is an adoptive cell therapy based on genetically engineered NK cells. The therapy can avoid a plurality of defects of autologous CAR-T, such as cytokine release syndrome, immune-related neurotoxicity syndrome, poor accessibility and the like. The selection of the auxiliary materials of the CAR-NK cell preparation is particularly important, compared with the freezing storage of NK cells, the freezing storage of the NK cells has larger damage generally, and a proper preparation prescription needs to be screened to ensure better activity.
CS10 is a serum-free and animal-component-free frozen stock solution, and the DMSO content is 10%. The frozen stock solution is widely applied to immune cell frozen stock and has better frozen stock protection effect. FDA approved version 3 CD19 directed CAR-T cell therapy Breyanzi, formulation with a prescription of +.>CS10, 24% compound electrolyte, 1% hsa.
DMSO Free GMP grade is GMP-grade completely frozen stock solution, which is colorless transparent clear liquid, does not contain DMSO, serum and animal-derived components, and has limited and safe chemical components. And has acquired U.S. FDA accession number BMF (Biological Master File) (MF#: 19364). The frozen stock solution has shown better frozen stock effect.
Chinese patent CN202211407542.1 discloses an NK cell cryopreservation liquid and a cryopreservation method, wherein the NK cell cryopreservation liquid consists of a cryopreservation liquid A and a cryopreservation liquid B; the frozen stock solution A comprises the following components in percentage by volume: 30-60% of human serum albumin, 10-30% of dextran and 25-45% of trehalose; the frozen stock solution B comprises the following components: 5-10% of honeysuckle extract, 45-50% of 551-H3 culture medium, 4-6% of culture supernatant concentrate, 18-20% of hydroxyethyl starch and 21-25% of glycerol. The NK cell cryopreservation liquid is added with the culture supernatant concentrated solution, so that the cell activity in cell cryopreservation and after resuscitation can be maintained, heterologous proteins are not added, pollution to cells caused by the exogenous proteins is avoided, and meanwhile, biological waste is applied, so that the cryopreservation cost is greatly saved. However, the NK cell cryopreservation solution is complex in composition.
Chinese patent CN202010240894.7 discloses an NK cell frozen stock solution which is prepared by adding glucose and polylysine CPLL into a culture medium. The NK cell frozen stock solution does not comprise animal and human serum, so that the potential transmission of zoonotic infectious diseases or the risk of causing alloreaction is prevented; the added glucan and CPL not only can reduce the damage of ice crystals to cells in the processes of freezing and resuscitation, but also can effectively preserve the killing activity of the cells. The frozen stock solution disclosed by the invention is simple and safe to prepare, has a good application market, and is large in reduction amplitude of the cell viability after resuscitating.
Disclosure of Invention
The invention aims to provide an NK cell cryopreservation solution, which can effectively protect cells from damage and maintain the activity of the cells in the cryopreservation and resuscitation processes by screening the types and the contents of each component in the NK cell cryopreservation solution.
Term interpretation:
as described herein, the term NK cells, also known as natural killer cells, are human innate immune lymphocytes. NK cells have antiviral effects, and can produce cytokines, interleukins, interferons, etc. without antigen stimulation when the body is infected by viruses, thus resisting tumor occurrence and virus infection. NK cells play a very good role in monitoring tumors, and can monitor the immune life of a human body, the damage condition of tumor cells and the like. NK cells also mediate human immune function and prevent autoimmune diseases and other immune dysfunction.
As described herein, the termCS10 is a serum-free and animal-component-free frozen stock solution, and the DMSO content is 10%.
In order to achieve the above object, the present invention has the following technical scheme:
in one aspect, the invention provides an NK cell cryopreservation solution, which is prepared fromCS10 and Human Serum Albumin (HSA);
the said processThe volume ratio of CS10 to human serum albumin is (2-4): 1.
preferably, the saidThe volume ratio of CS10 to human serum albumin is 3:1.
preferably, the NK cells are CAR-NK cells.
In still another aspect, the present invention provides a method for cryopreserving NK cells, wherein the NK cells are cryopreserved using the NK cell cryopreservation solution described above.
Preferably, the freezing method comprises the following steps:
(1) Will beCS10 and human serum albumin are mixed according to the volume ratio to obtain NK cell frozen stock solution;
(2) Adding NK cell frozen stock solution into NK cells to obtain cell suspension;
(3) And (5) sub-packaging the cell suspension in a freezing tube, and cooling in sequence.
Preferably, the NK cells in step (2) have a density of 3.00×10 7 cells/ml-1.00×10 8 cells/ml。
Further preferably, the NK cells in step (2) have a density of 4.00×10 7 cells/ml-6.00×10 8 cells/ml。
Most preferably, the NK cells in step (2) have a density of 5.00X 10 7 cells/ml。
Preferably, the NK cells in step (2) are treated cells by the following procedure: the NK cell suspension was centrifuged and the supernatant was discarded to obtain desired NK cells.
Further preferably, the rotational speed of the centrifugation is 400-800g, the centrifugation time is 8-15min, and the centrifugation temperature is 2-8 ℃;
still further preferably, the rotational speed of the centrifugation is 500g, the centrifugation time is 10min, and the centrifugation temperature is 4 ℃.
Preferably, the program cooling in step (3) is as follows (User 1):
a. waiting at 4.0 ℃ until the sample is put in;
b. the sample temperature was reduced to-4.0 ℃ at a rate of 1.0 ℃ per minute;
c. the temperature of the cavity is reduced to-10.0 ℃ at a rate of 10.0 ℃ per minute;
d. the temperature of the cavity is reduced to-20.0 ℃ at a rate of 0.5 ℃ per minute;
e. the sample temperature was reduced to-80.0 ℃ at a rate of 1.0 ℃ per minute;
f. and (5) finishing cooling.
Specifically, after the procedure described in the step (3) is cooled, the cell suspension is transferred to liquid nitrogen for preservation.
Preferably, the NK cell resuscitation after cryopreservation using the above NK cell cryopreservation solution comprises the following steps:
(1) Preparing an NK cell culture medium;
(2) Adding the culture medium obtained in the step (1) into a T-25 bottle;
(3) And taking out the CAR-NK cells from the liquid nitrogen gas phase, putting the CAR-NK cells into dry ice, quickly putting the cells in a water bath at 37 ℃ after leaving the dry ice for resuscitating, slightly shaking the cells, controlling the resuscitating time within 2-3min, and stopping the water bath when a small amount of ice crystals exist in the freezing storage tube.
In yet another aspect, the invention provides the use of the above NK cell cryopreservation solution for cryopreserving NK cells.
The beneficial effects of the invention are as follows:
the NK cell cryopreservation solution comprisesCS10 and human serum albumin do not contain animal source components except human body, and have the advantages of simple components and good stability. The frozen stock solution passes +.>The combined action of CS10 and human serum albumin improves the survival rate of cells after resuscitation, and reduces the damage to NK in the freezing process.
Drawings
FIG. 1 is a line graph of cell viability.
FIG. 2 is a graph of cell growth.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purpose and the effect of the present invention easy to understand, the present invention will be further elucidated with reference to the specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all of them. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. In the following examples, unless otherwise specified, the methods of operation used were conventional, the equipment used was conventional, and the materials used in the examples were the same.
Experimental raw material purchasing manufacturer and goods number:
FBS is purchased from Beijing full gold biotechnology Co., ltd, with the product number being FS301-02;
DMSO is available from WAK-Chemie Medical GmbH under the trade designation WAK-DMSO-10;
CS10 is purchased from Biolife solutions under the accession number 210102;
HSA was purchased from jetty bellin biologicals limited, switzerland under the code national standard SJ20170005;
STEM CELL BANKER DMSO Free available from ZENOAQ under the trade designation 11890F;
the CAR-NK cells of the examples and comparative examples were prepared as follows:
PBMCs were isolated from apheresis of different healthy donors, followed by isolation and purification of CD3-cd56+ NK cells using magnetic beads of methoprene against CD3, CD 56. The isolated NK cells were purified according to 1:1 and mbIL-21 modified K562 trophoblasts that lost proliferative capacity after irradiation were incubated, 5 days later, when NK cells killed trophoblasts, and when activation proliferation began, NK cells were infected (1000 g centrifugation, 30 min) with lentivirus carrying the CAR gene, and after two days of infection, NK cells were continued to be purified according to 1:1 and incubating the trophoblasts, activating and amplifying the CAR-NK cells, culturing for 7 days, after the NK cells are activated and amplified and the trophoblasts are killed completely, harvesting the CAR-NK cells, identifying the positive rate of the CAR and the purity of the NK cells, and carrying out experiments.
Example 1
An NK cell cryopreservation solution comprisingCS10 and human serum albumin, and the NK cell frozen stock solution is +.>The volume ratio of CS10 to human serum albumin is 3:1.
A freezing method of NK cells comprises mixing NK cells to be frozen with above NK cell freezing solution to obtain cell suspension, wherein the concentration of NK cells in the cell suspension is 5.00×10 7 Placing the cell suspension in a sterile cryopreservation tube for program cooling, wherein the program cooling is as follows (User 1):
a. waiting at 4.0 ℃ until the sample is put in;
b. the sample temperature was reduced to-4.0 ℃ at a rate of 1.0 ℃ per minute;
c. the temperature of the cavity is reduced to-10.0 ℃ at a rate of 10.0 ℃ per minute;
d. the temperature of the cavity is reduced to-20.0 ℃ at a rate of 0.5 ℃ per minute;
e. the sample temperature was reduced to-80.0 ℃ at a rate of 1.0 ℃ per minute;
f. and (5) finishing cooling.
Then put into liquid nitrogen for freezing.
Comparative example 1
Comparative example 1 provides an NK cell cryopreservation solution comprising 90% fbs+10% dmso.
Comparative example 2
Comparative example 2 provides an NK cell cryopreservation solution, which isCS10。
Comparative example 3
Comparative example 3 provides an NK cell cryopreservation solution comprisingCS10, 24%STEM CELL BANKER DMSO Free and 1% hsa.
Comparative example 4
Comparative example 4 provides an NK cell cryopreservation solution, which is STEM CELL BANKER DMSO Free.
Comparative example 5
Comparative example 5 provides an NK cell cryopreservation solution comprisingCS10, 75%STEM CELL BANKER DMSO Free and 1% hsa.
Test examples cell viability, viable cell density, total number of viable cells, fold expansion, PDL and PDT data statistics.
The experimental process comprises the following steps:
1. cryopreserved CAR-NK cells were resuscitated (24 h after cryopreservation, resuscitation when the diary was Day 1)
(1) Preparing a CAR-NK cell culture medium: 1%NK MACS Supplement,NK MACS Basal Medium,200IU/mL IL-2,5% CTS TM The total volume of the immune cell serum replacement was formulated to be 60mL.
(2) 5ml of the medium was added to a total of 12 flasks of T-25.
(3) And recovering the-CAR-NK cells 24h after freezing, wherein each group has 2 branches. And taking out the CAR-NK cells from the liquid nitrogen gas phase, putting the CAR-NK cells into dry ice, conveying the cells to a cell preparation laboratory, quickly putting the cells into a 37 ℃ water bath for resuscitating after leaving the dry ice, slightly shaking the cells, controlling the resuscitating time within 2-3min, and stopping the water bath when a small amount of ice crystals are still in the freezing tube.
(4) Taking 100ul of each cell, adding into T-25 bottle with culture medium, observing cell state under microscope, and adding CO 2 The incubator continues to cultivate.
(5) The remaining cells were mixed and diluted 20-fold with the corresponding cryopreservation solution, and the two samples were counted in parallel in AP/PI DMSO mode.
2. Day 2 sampling counting and equal volume fluid infusion
(1) Cell removal from CO 2 The cells were taken out of the incubator and observed for each group.
(2) The flask surface was rubbed with 75% sterile alcohol and transferred into a biosafety cabinet, each group was sampled with 1ml of cell suspension, diluted 2-fold, and AO/PI counted cell density and viability were counted in duplicate.
(3) After sampling, each T-25 bottle is supplemented with 5ml NK cell culture medium, and mixed uniformly and put back into CO 2 Culturing is continued in the incubator.
3. Day 3 sample count
(1) Cell removal from CO 2 And taking out the culture box.
(2) The surface of the flask was rubbed with 75% sterile alcohol and transferred into a biosafety cabinet, each group was sampled with 1ml of cell suspension, diluted 2-fold, and AO/PI was used to count cell density and viability, and the two counts were performed in parallel.
(3) After sampling, each T-25 bottle is supplemented with 5ml NK cell culture medium, and mixed uniformly and put back into CO 2 Culturing is continued in the incubator.
4. Day 4 sample count
(1) Cell removal from CO 2 And taking out the culture box.
(2) The surface of the flask was rubbed with 75% sterile alcohol and transferred into a biosafety cabinet, each group was sampled with 1ml of cell suspension, diluted 2-fold, and AO/PI was used to count cell density and viability, and the two counts were performed in parallel.
(3) After the completion of the culture, the remaining cells were treated as medical waste.
Experimental results and analysis:
the statistical results of the viable cell density, the viable rate and the total number of viable cells are shown in the following tables 1-2 and FIG. 1:
TABLE 1 statistical results of viable cell density, viable Rate, and viable cell count of example 1
TABLE 2 statistical results of viable cell densities, viable rates, and viable cell totals of comparative examples 1-5
The growth curve of the cells is shown in FIG. 2, and it can be seen from FIG. 2 that the cells frozen using the frozen stock solution of example 1 grew faster than comparative examples 1 to 5. Wherein, the growth rate of each group of cells is specifically: example 1 group > comparative example 3 group > comparative example 2 group > comparative example 4 group > comparative example 5 group.
Analysis of experimental results:
before freezing: example 1 frozen stock (CS 10: hsa=4:1), comparative example 1 frozen stock (90% fbs+10% dmso), comparative example 2 frozen stock (CS 10), comparative example 3 frozen stock (75%CS10+Stem cell banker DMSO Free+1%HSA), comparative example 4 frozen stock (STEM CELL BANKER DMSO Free), comparative example 5 frozen stock (24%CS10+75%STEM CELL BANKER DMSO Free+1%HSA), average cell viability was 79.12%,76.72%,79.23%,78.68%,82.77%,79.24%, respectively. Statistical analysis using GraphPad Prism 7.00 software found no differences between the experimental groups.
D1 resuscitation: example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5, and average cell viability were respectively: 77.71%,75.50%,76.57%,80.60%,75.73% and 79.75%. Statistical analysis using GraphPad Prism 7.00 software found no differences between the experimental groups.
D2: example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5, and average cell viability were respectively: 44.44%,47.74%,34.24%,35.17%,17.86% and 15.08%. The cell viability of each experimental group was reduced compared with that of D1 when the experimental groups were resuscitated after culturing to D2, and the reduction of the freezing solution of example 1 was 33.27% compared with that of D1, and the reduction of the freezing solution of comparative example 1 and D1 was 27.77% in terms of the comprehensive experimental groups. The frozen stock solution of comparative example 4 and the frozen stock solution of comparative example 5 have the largest descending amplitude of 57.87 percent and 64.68 percent respectively.
D3, time: example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5, and average cell viability were respectively: 60.10%,60.26%,48.83%,49.01%,23.30% and 21.53%. Compared with the D2 cell viability which is improved, the frozen stock solution of the comparative example 1 is up to 60.26 percent, and the frozen stock solution of the example 3 is 60.01 percent. The average activity rate of the frozen stock solution of the comparative example 4 is 23.30 percent, and the worst frozen stock solution of the comparative example 5 is 21.53 percent.
D4, at: example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5, and average cell viability were respectively: 70.40%,73.86%,61.05%,65.52%,38.12%,43.27% higher than D3 cell viability, up to 73.86% of the comparative example 1 frozen stock solution and 70.04% of the example 1 frozen stock solution. The cell viability of the comparative example 5 frozen stock solution was 43.27%, and the comparative example 4 frozen stock solution was worst compared to the other groups.
The application detects 6 frozen stock solutions altogether: example 1 (CS 10: hsa=3:1), comparative example 1 (90% fbs+10% dmso), comparative example 2 (CS 10), comparative example 3 (75%CS10+Stem cell banker DMSO Free+1%HSA), comparative example 4 (STEM CELL BANKER DMSO Free), comparative example 5 (24%CS10+75%STEM CELL BANKER DMSO Free+1%HSA); according to 5X 10 7 The cells are frozen at the density of cells/ml, and the cells are frozen in a user1 program control cooling mode. The cell viability can be maintained at more than 75% by the six frozen solutions before and after freezing. When culturing to D2, the cell viability of the frozen stock solution of example 1 was reduced by 33.27% as compared with other frozen stock solutions, and the cell viability was reduced to D4 by the frozen stock solution of example 1The cell viability was recovered to 70% or more, while the comparative example 2 frozen stock, the comparative example 3 frozen stock, the comparative example 4 frozen stock, and the comparative example 5 frozen stock were all 70.00% or less. This shows that the components and the content of the NK cell cryopreservation liquid are adjusted, so that the prepared cell cryopreservation liquid can better maintain the cell viability.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. The NK cell cryopreservation liquid is characterized by comprising a Cryostor ® CS10 and human serum albumin;
the Cryptor is described as ® The volume ratio of CS10 to human serum albumin is 3:1, a step of;
the human serum albumin is purchased from JieTeberlin biological products, inc. of Switzerland, and the product number is the national medicine standard SJ20170005.
2. The NK cell cryopreservation solution of claim 1, wherein the NK cells are CAR-NK cells.
3. A method for cryopreserving NK cells, characterized in that NK cells are cryopreserved using the NK cell cryopreservation solution according to any one of claims 1-2.
4. A method of cryopreservation according to claim 3, comprising the steps of:
(1) Cryptor is provided with ® CS10 and human serum albumin are mixed according to the volume ratio to obtain NK cell frozen stock solution;
(2) Adding NK cell frozen stock solution into NK cells to obtain cell suspension;
(3) And (5) sub-packaging the cell suspension in a freezing tube, and cooling in sequence.
5. The method according to claim 4, wherein the NK cells in the step (2) have a density of 3.00X 10 7 cells/ml-1.00×10 8 cells/ml。
6. The method according to claim 5, wherein the density of NK cells in the step (2) is 4.00X 10 7 cells/ml-6.00×10 7 cells/ml。
7. The method according to claim 6, wherein the density of NK cells in the step (2) is 5.00X 10 7 cells/ml。
8. Use of the NK cell cryopreservation solution of any one of claims 1-2 for cryopreserving NK cells.
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| CN115039763A (en) * | 2022-07-15 | 2022-09-13 | 北京大学第三医院（北京大学第三临床医学院） | Immune cell cryopreservation liquid |
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