CN114667999A - Immune cell cryopreservation stabilizer, cryopreservation liquid and cryopreservation method - Google Patents
Immune cell cryopreservation stabilizer, cryopreservation liquid and cryopreservation method Download PDFInfo
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- CN114667999A CN114667999A CN202210585624.9A CN202210585624A CN114667999A CN 114667999 A CN114667999 A CN 114667999A CN 202210585624 A CN202210585624 A CN 202210585624A CN 114667999 A CN114667999 A CN 114667999A
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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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
Abstract
The invention discloses an immune cell cryopreservation stabilizer, a cryopreservation solution and a cryopreservation method, wherein the immune cell cryopreservation stabilizer comprises, by mass, 2-10% of glycine, 0.1-2% of gelatin, 5-10% of mannitol, 0.1-5% of lactose, 10-20% of human serum albumin, 1-10% of dextran, and the balance of a basic cell culture solution. The cryopreservation stabilizer can effectively protect cell phenotype, and can remarkably improve the activity of immune cells under cryopreservation conditions, so that the physiological function and the biological function of the recovered cells are maintained; and the stabilizer adopts serum-free culture solution, no exogenous animal protein is introduced, the possibility of animal source pollution is reduced, and adverse reaction and toxic and side effects which occur when the cells are returned to the patient after recovery are avoided.
Description
Technical Field
The invention relates to the technical field of human cell cryopreservation, in particular to an immune cell cryopreservation stabilizer, a cryopreservation solution and a cryopreservation method.
Background
Immune cell therapy, which is an emerging technology for clinical tumor therapy in recent years, is also considered to be one of the most promising technologies. The immune cell therapy is to induce self-antiviral immune response by in vitro culture, proliferation and activation and to be infused back into the body, and once the human antiviral immunity is activated, antiviral substances are continuously generated to kill viruses.
Immune cell therapy, also known as autologous cell immunotherapy, currently mainly includes three immune cell therapies — DC, CIK, DC-CIK cell therapy. The specific method is that immature immune cells in a patient body are extracted (blood is collected), activated and cultured in a laboratory to enable the immature immune cells to have high-efficiency cell recognition and cell capacity, and then the immature immune cells are transfused into the patient body.
Therefore, the freezing storage of the immune cells also becomes an indispensable important link for immunotherapy. Cell cryopreservation is a technology for storing cells in a low-temperature environment to slow down cell metabolism and achieve long-term storage. The principle is that the cells are placed in liquid nitrogen at the temperature of 196 ℃ below zero for ultralow temperature preservation, so that the cells can be temporarily separated from the growth state to preserve the characteristics of the cells, and the cells can be used by reviving the cells when needed.
At present, the cryopreservation of cells is mainly performed under the ultralow temperature condition of liquid nitrogen at the temperature of-196 ℃, the metabolic function of the cells is arrested under the ultralow temperature condition, the cells can be preserved for a long time, and when the cells are needed, the cryopreserved cells are taken out and quickly thawed in water bath at the temperature of 37 ℃, so that the activity and the function of the cells can be recovered. However, the existing immune cell cryopreservation method only solves the cryopreservation of immune cells and tumor cells, but the immune cell cryopreservation method cannot be well solved. Due to the cell structure and the cell characteristics of immune cells, when the conventional cell cryopreservation solution and the conventional cell cryopreservation method are used for cryopreserving the immune cells, the immune cells are easily damaged by ice crystals, the survival rate of the recovered cells is low, the cell proliferation quantity is insufficient, the cells are easy to age, the efficiency is low, the cryopreservation quality is low, the activity and the survival rate of the recovered cells are poor, and the like.
Disclosure of Invention
In view of the above problems, the present invention provides a cryopreservation agent for immune cells, a cryopreservation solution, and a cryopreservation method for immune cells, which can stably protect the biological activity, morphological structure, cell survival rate, and the like of cells.
The first technical scheme of the invention is as follows:
an immune cell cryopreservation stabilizer comprises, by mass, 2-10% of glycine, 0.1-2% of gelatin, 5-10% of mannitol, 0.1-5% of lactose, 10-20% of human serum albumin, 1-10% of dextran, and the balance of basic cell culture solution.
In one embodiment, the immune cell cryopreservation stabilizer comprises, by mass, 5% of glycine, 1% of gelatin, 7% of mannitol, 3% of lactose, 14% of human serum albumin, 6% of dextran, and the balance of basic cell culture solution.
In one embodiment, the immune cell cryopreservation stabilizer is a CORNING serum-free culture solution, a KBM581 serum-free culture solution or an RPMI-1640 serum-free culture solution.
The second technical scheme provided by the invention is as follows:
an immune cell freezing solution comprises an immune cell freezing nutrient, a protective agent and the immune cell freezing stabilizer.
In one embodiment, the mass percentages of the nutrient, the protective agent and the stabilizer in the immune cell cryopreservation solution are 60-90%, 5-30% and 1-15%, respectively.
In one embodiment, the mass percentages of the nutrient, the protective agent and the stabilizer are 70%, 25% and 5%, respectively.
In another embodiment, the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 60%, 30% and 10%.
In one embodiment, the nutrient in the immune cell cryopreservation solution comprises a cell culture medium and 15-20 w/w% human serum albumin; the cell culture solution is a CORNING serum-free culture solution, a KBM581 serum-free culture solution or an RPMI-1640 serum-free culture solution, and the concentration of the human serum albumin is 15-35 g/l.
In one embodiment, the immune cell cryopreservation solution further comprises 50% -60% of an alpha-MEM basal medium.
In one embodiment, in the immune cell cryopreservation solution, the protective agent comprises at least one of DMSO, sulfosalicylic acid, and acetamide; preferably, the protective agent is a mixture of DMSO, sulfosalicylic acid and acetamide, and the mass ratio of the sulfosalicylic acid to the acetamide is 1-3: 1-5.
The third technical scheme provided by the invention is as follows:
a method for freezing cells by using the freezing medium comprises the following steps:
at normal temperature, resuspending immune cells by using a freezing medium to form a suspension, and then subpackaging the suspension in a freezing tube;
and (3) pre-cooling the freezing tube for 10-15 hours at the temperature of 2-8 ℃, and finally transferring the tube into liquid nitrogen for long-term freezing.
In one embodiment, in the method for freezing immune cell freezing medium, when the freezing medium re-suspends immune cells to form a suspension, the density of the immune cells is controlled to be 1-2 × 107One per ml.
Compared with the prior art, the invention has the following advantages:
1. the immune cell cryopreservation stabilizer can effectively protect cell phenotype and remarkably improve the activity of immune cells under cryopreservation conditions, so that the physiological function and the biological function of the recovered cells are maintained;
2. the stabilizing agent and the nutritional agent both adopt serum-free culture solution, and no exogenous animal protein is introduced, so that the possibility of animal pollution is reduced, and adverse reaction and toxic and side effects caused by the transfusion of the patient after cell recovery are avoided.
The protective agent in the frozen stock solution can prevent immune cells from being damaged by ice crystals, and improve the survival rate of the recovered cells and the cell amplification quantity; the survival rate of the cells after cryopreservation and recovery is up to more than 97%, and the cell expansion multiple is up to 289 times after the cells are cultured for 30 days after recovery;
4. after the frozen stock solution is used for resuspending immune cells at normal temperature, the frozen stock solution can be transferred into liquid nitrogen for long-term freezing only by precooling for 10-15 hours at the temperature of 2-8 ℃, so that the intermediate procedure cooling link of-20 ℃ and/or-80 ℃ is reduced, the freezing cost is reduced, and the freezing efficiency is improved;
5. the stabilizer and the nutrient agent both adopt serum-free culture solution, and no exogenous animal protein is introduced, so that the possibility of animal pollution is reduced, and adverse reaction and toxic and side effects generated when the cells are recovered and returned to a patient are avoided.
Drawings
FIG. 1 is a graph showing the growth of CIK cells cultured for 18 days before and after the CIK cells are frozen for 30 days in examples 1, 2, 3 and 1;
FIG. 2 is a graph showing the proliferation of NK cells in culture before cryopreservation and after further culturing for 18 days after cryopreservation of NK cells in example 4, example 5, example 6 and comparative example 2;
FIG. 3 is a graph showing the cell recovery viability rate of CIK cells in example 1, example 2, example 3 and comparative example 1 after the CIK cells are frozen for 30 days;
FIG. 4 is a graph showing the cell recovery survival rate of NK cells in example 4, example 5, example 6 and comparative example 2 after cryopreservation for 30 days;
FIGS. 5, 6, 7, 8 and 9 are flow charts of phenotypic assay of CD3+ CD56+ after incubation for 7 days before and after freezing of CIK cells in example 1, example 2, example 3 and comparative example 1, respectively;
FIGS. 10, 11, 12, 13 and 14 are the CD3-CD56+ phenotype detection flow charts of NK cells cultured for another 7 days after being frozen for 30 days in example 4, example 5, example 6 and comparative example 2 before being frozen and stored respectively;
FIG. 15 shows the killing rate of CIK cells before cryopreservation on, against K562 after culturing for 7 days after 30 days of cryopreservation of CIK cells in examples 1, 2, 3 and 1.
FIG. 16 shows the killing rate of NK cells on K562 cells after 30 days of cryopreservation and 7 days of culture of CIK cells in example 4, example 5, example 6 and comparative example 2 before cryopreservation.
Detailed Description
In the present invention, the immunocyte may be any of DC, CIK, DC-CIK, NK, NKT, and the like.
Aiming at the immune cells, the invention provides a corresponding immune cell cryopreservation stabilizer, which comprises 2-10% of glycine, 0.1-2% of gelatin, 5-10% of mannitol, 0.1-5% of lactose, 10-20% of human serum albumin, 1-10% of dextran and the balance of basic cell culture solution by mass percentage; preferably, 4-8% of glycine, 1-2% of gelatin, 6-8% of mannitol, 1-3% of lactose, 12-16% of human serum albumin, 3-8% of dextran and the balance of basic cell culture solution.
In one embodiment, the immune cell cryopreservation stabilizer comprises, by mass, 5% of glycine, 1% of gelatin, 7% of mannitol, 3% of lactose, 14% of human serum albumin, 6% of dextran, and the balance of basic cell culture solution; or the immune cell cryopreservation stabilizer comprises 2% of glycine, 2% of gelatin, 5% of mannitol, 0.1% of lactose, 20% of human serum albumin, 1% of dextran and the balance of basic cell culture solution by mass percentage; or the immune cell cryopreservation stabilizer comprises, by mass, 10% of glycine, 0.1% of gelatin, 10% of mannitol, 5% of lactose, 10% of human serum albumin, 10% of dextran, and the balance of basic cell culture solution.
In one embodiment, the immune cell cryopreservation stabilizer is a Corning serum-free culture solution, a serum-free culture solution of KBM581, or an RPMI-1640 serum-free culture solution.
The stabilizer can be used for preparing immune cell cryopreservation liquid, and the cryopreservation liquid also comprises an immune cell cryopreservation nutrient and a protective agent; wherein the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 60-90%, 5-30% and 1-15%; preferably, the mass percentages of the nutrient, the protective agent and the stabilizer are 65-80%, 10-28% and 2-12% respectively; or the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 70%, 25% and 5%; or the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 60%, 30% and 10%; or the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 70%, 20% and 10%; or the mass percentages of the nutrient, the protective agent and the stabilizer are 65%, 25% and 10% respectively; or the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 80%, 10% and 10%; or the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 75%, 20% and 5%; or the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 90%, 5% and the like.
Wherein the nutrient comprises a serum-free culture solution and 15-20 w/w% of human serum albumin (the concentration of the human serum albumin in the initial solution before the human serum albumin is added into the culture solution is 15-20 w/w%); preferably, the cell culture solution is a CORNING serum-free culture solution, a KBM581 serum-free culture solution or an RPMI-1640 serum-free culture solution, and the addition amount of the human serum albumin in the culture solution is 15-35 g/l; or the addition amount of human serum albumin in the cell culture solution is 20 g/l; or the addition amount of human serum albumin in the cell culture solution is 35 g/l; alternatively, the amount of human serum albumin added to the cell culture medium was 15 g/l.
In one embodiment, the cell culture solution further comprises 50% -60% of an alpha-MEM basal medium; preferably, the cell culture solution comprises 54% of alpha-MEM basic culture medium; or, the cell culture solution comprises 50% of alpha-MEM basal medium; alternatively, the cell culture solution may contain 60% of an α -MEM basal medium or the like.
The protective agent comprises at least one of DMSO, sulfosalicylic acid and acetamide; preferably, the protective agent is a mixture of DMSO, sulfosalicylic acid and acetamide, and the mass ratio of the sulfosalicylic acid to the acetamide is 1-3: 1-5; or the protective agent is any one of DMSO, sulfosalicylic acid and acetamide; alternatively, the protectant is a mixture of DMSO in 1:1 combination with sulfosalicylic acid; alternatively, the protective agent is a mixture of DMSO and acetamide in a ratio of 1: 2; alternatively, the protectant is a mixture of sulfosalicylic acid and acetamide in a 1:5 ratio; or the protective agent is a mixture of DMSO, sulfosalicylic acid and acetamide according to a ratio of 1:1: 5; or the protective agent is a mixture of DMSO, sulfosalicylic acid and acetamide according to a ratio of 1:3: 1; or the protective agent is a mixture of DMSO, sulfosalicylic acid and acetamide according to a ratio of 1:1: 1; or the protective agent is a mixture of DMSO, sulfosalicylic acid and acetamide according to a ratio of 1:2: 2; alternatively, the protectant is a mixture of DMSO, sulfosalicylic acid, and acetamide in a 1:3:5 ratio.
In the frozen stock solution, the stabilizer is used for effectively protecting cell phenotype and can remarkably improve the activity of immune cells under the frozen stock condition, so that the physiological function and the biological function of the cells after recovery are kept; the nutrient adopts serum-free culture solution, so that the possibility of pollution of immune cells to animal pathogens can be reduced, and the method is safe and reliable; the protective agent can prevent immune cells from being damaged by ice crystals, and improve the cell survival rate and the cell amplification quantity after recovery.
The frozen stock solution can be applied to frozen stock of immune cells, and the frozen stock process comprises the following steps:
s1, resuspending the immune cells with the frozen stock solution at normal temperature to form a suspension, and then subpackaging the suspension in frozen stock tubes;
s2, pre-cooling the cryopreservation tube for 12 hours at 4 ℃, and then placing the cryopreservation tube at-20 ℃ to cryopreservation for 30 minutes; then transferring to-80 deg.C to freeze-store for 4 hr, finally transferring to liquid nitrogen to freeze-store for a long time.
When the immune cells are frozen and frozen, and the frozen stock solution is used for re-suspending the immune cells to form a suspension, the density of the immune cells is controlled to be 1-2 multiplied by 107One per ml.
The present invention will be further described in detail below with reference to the attached drawings, which take immune cells CIK and NK as examples. In the following examples, human albumin is 15-20 w/w% human albumin.
Preparation of freezing medium and cell freezing and recovery
(I) CIK immune cells as an example
Example 1
1. 1000g of frozen stock solution is prepared, and the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 70%, 25% and 5%, namely the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 700g, 250g and 50 g.
The concrete preparation process is as follows
1.1, preparing 50g of freezing storage stabilizer
According to the mass percentage, 5% of glycine, 1% of gelatin, 7% of mannitol, 3% of lactose, 14% of human serum albumin, 6% of dextran, and the balance of basic cell culture solution; weighing 2.5g of glycine, 0.5g of gelatin, 3.5g of mannitol, 1.5g of lactose, 7g of human serum albumin, 3g of dextran and 32g of CORNING serum-free culture solution, and uniformly mixing to prepare a cell cryopreservation stabilizer;
1.2, preparing 250g of freezing protective agent
Weighing 50g of DMSO, 100g of sulfosalicylic acid and 100g of acetamide according to the ratio of 1:2:2, and uniformly mixing to obtain a protective agent;
1.3, preparing 750g of frozen nutrient
750g of serum-free CORNING culture solution is weighed, and the culture solution contains an alpha-MEM basic culture medium with the concentration of 54% and human albumin with the concentration of 20 g/l.
1.4 preparing the frozen stock solution
And (4) mixing the stabilizing agent, the protective agent and the nutritional agent prepared in the steps 1.1, 1.2 and 1.3 to prepare the frozen stock solution.
2. CIK immune cell cryopreservation
2.1, measuring 100ml of the frozen stock solution prepared in the step 1, adding CIK immune cells and controlling the cell density to be 1.5 multiplied by 107Resuspending into suspension at each ml; then the mixture is divided into 5ml freezing tubes;
2.2, pre-cooling the freezing tube at 4 ℃ for 4 hours, and then freezing the freezing tube at-20 ℃ to 30 minutes; then transferring to-80 deg.C for freezing for 4 hr, then transferring to liquid nitrogen for long-term freezing.
3. CIK immune cell resuscitation
Taking out the frozen tube from the liquid nitrogen, and putting the tube at the temperature of 40 ℃ for 2 minutes to recover the CIK immune cells for later culture.
Example 2
1. 1000g of frozen stock solution is prepared, and the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 60%, 30% and 10%, namely the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 600g, 300g and 100 g.
The concrete preparation process is as follows
1.1, preparing 100g of freezing storage stabilizer
Calculated according to the mass percentage, 2 percent of glycine, 2 percent of gelatin, 5 percent of mannitol, 0.1 percent of lactose, 20 percent of human serum albumin, 1 percent of dextran, and the balance of basic cell culture solution; weighing 2g of glycine, 2g of gelatin, 5g of mannitol, 0.1g of lactose, 20g of human serum albumin, 1g of dextran and 69.9g of serum-free culture solution of KBM581, and uniformly mixing to prepare a cell cryopreservation stabilizer;
1.2, preparing 300g of freezing protective agent
Weighing 75g of DMSO and 225g of sulfosalicylic acid according to the proportion of 1:3, and uniformly mixing to obtain a protective agent;
1.3, preparing 600g of frozen nutrient
600g of KBM581 serum-free culture medium containing 60% alpha-MEM basal medium and 35g/l human serum albumin was weighed.
1.4 preparation of frozen stock solution
And (3) mixing the stabilizer, the protective agent and the nutrient prepared in the steps 1.1, 1.2 and 1.3 to prepare the frozen stock solution.
2. CIK immune cell cryopreservation
2.1, measuring 100ml of the frozen stock solution prepared in the step 1, adding CIK immune cells and controlling the cell density to be 2 multiplied by 107Resuspending into suspension at each ml; then the mixture is divided into 5ml freezing tubes;
2.2, pre-cooling the freezing tube at 4 ℃ for 4 hours, and then freezing the freezing tube at-20 ℃ to 30 minutes; then transferring the mixture to be frozen for 4 hours at the temperature of minus 80 ℃, and then transferring the mixture to be frozen in liquid nitrogen for a long time.
3. CIK immune cell resuscitation
Taking out the frozen tube from the liquid nitrogen, and putting the tube at the temperature of 37 ℃ for 3 minutes to recover the CIK immune cells for later culture.
Example 3
1. 1000g of frozen stock solution is prepared, and the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 90%, 5% and 5%, namely the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 900g, 50g and 50 g.
The concrete preparation process is as follows
1.1, preparing 50g of freezing storage stabilizer
According to the mass percentage, 10 percent of glycine, 0.1 percent of gelatin, 10 percent of mannitol, 5 percent of lactose, 10 percent of human serum albumin, 10 percent of dextran and the balance of basic cell culture solution; weighing 5g of glycine, 0.05g of gelatin, 5g of mannitol, 2.5g of lactose, 5g of human serum albumin, 5g of dextran and 27.45g of RPMI-1640 serum-free culture solution, and uniformly mixing to prepare a cell cryopreservation stabilizer;
1.2, preparing 50g of freezing protective agent
DMSO is selected as a protective agent, and 75g of DMSO is weighed.
1.3, preparing 900g of frozen nutrient
900g of RPMI-1640 serum-free culture solution containing 50% alpha-MEM basal medium and 15g/l human albumin was weighed.
1.4 preparation of frozen stock solution
And (3) mixing the stabilizer, the protective agent and the nutrient prepared in the steps 1.1, 1.2 and 1.3 to prepare the frozen stock solution.
2. CIK immune cell cryopreservation
2.1, measuring 100ml of the frozen stock solution prepared in the step 1, adding CIK immune cells and controlling the cell density to be 1 multiplied by 107Resuspending into suspension at each ml; then the mixture is divided into 5ml freezing tubes;
2.2, pre-cooling the freezing tube at 4 ℃ for 4 hours, and then freezing the freezing tube at-20 ℃ to 30 minutes; then transferring to-80 deg.C for freezing for 4 hr, then transferring to liquid nitrogen for long-term freezing.
3. CIK immune cell resuscitation
Taking out the frozen tube from the liquid nitrogen, and putting the tube at the temperature of 43 ℃ for 2 minutes to recover the CIK immune cells for later culture.
Comparative example 1
Comparative example 1 contained no stabilizer.
1. 1000g of frozen stock solution is prepared, and the mass percentages of the nutrient and the protective agent are respectively 70 percent and 30 percent, namely the mass percentages of the nutrient and the protective agent are respectively 700g and 300 g.
The concrete preparation process is as follows
1.1, preparing 300g of freezing protective agent
Weighing 60g of DMSO, 120g of sulfosalicylic acid and 120g of acetamide according to the ratio of 1:2:2, and uniformly mixing to obtain a protective agent;
1.2, preparing 700g of frozen nutrient
700g of serum-free CORNING culture solution is weighed, and the culture solution contains an alpha-MEM basic culture medium with the concentration of 54% and human albumin with the concentration of 20 g/l.
1.3 preparation of frozen stock solution
And (3) mixing the protective agent and the nutrient prepared in the steps 1.1 and 1.2 to prepare the frozen stock solution.
2. CIK immune cell cryopreservation
2.1, measuring 100ml of the frozen stock solution prepared in the step 1, adding CIK immune cells and controlling the cell density to be 1.5 multiplied by 107Resuspending into suspension at each ml; then the mixture is divided into 5ml freezing tubes;
2.2, pre-cooling the freezing tube at 4 ℃ for 4 hours, and then freezing the freezing tube at-20 ℃ to 30 minutes; then transferring to-80 deg.C for freezing for 4 hr, then transferring to liquid nitrogen for long-term freezing.
3. CIK immune cell resuscitation
Taking out the frozen tube from the liquid nitrogen, and putting the tube at the temperature of 40 ℃ for 2 minutes to recover the CIK immune cells for later culture.
(II), NK immune cells for example
Example 4
1. 1000g of frozen stock solution is prepared, and the mass percentages of the nutrient, the protective agent and the stabilizer are 65%, 25% and 10% respectively, namely the mass percentages of the nutrient, the protective agent and the stabilizer are 650g, 250g and 100g respectively.
The concrete preparation process is as follows
1.1, preparing 100g of freezing storage stabilizer
According to the mass percentage, 6% of glycine, 1.5% of gelatin, 8% of mannitol, 4% of lactose, 16% of human serum albumin, 8% of dextran, and the balance of basic cell culture solution; weighing 6g of glycine, 1.5g of gelatin, 8g of mannitol, 4g of lactose, 16g of human serum albumin, 8g of dextran and 56.5g of CORNING serum-free culture solution, and uniformly mixing to prepare a cell cryopreservation stabilizer;
1.2, preparing 250g of freezing protective agent
Weighing 83.4g of DMSO, 83.3g of sulfosalicylic acid and 83.3g of acetamide according to the proportion of 1:1:1, and uniformly mixing to obtain a protective agent;
1.3, preparing 650g of frozen nutrient
650g of serum-free CORNING culture medium containing 54% alpha-MEM basal medium and 14g/l human albumin was weighed.
1.4 preparation of frozen stock solution
And (3) mixing the stabilizer, the protective agent and the nutrient prepared in the steps 1.1, 1.2 and 1.3 to prepare the frozen stock solution.
2. NK immune cell cryopreservation
2.1, measuring 100ml of the frozen stock solution prepared in the step 1, adding NK immune cells and controlling the cell density to be 1.5 multiplied by 107Resuspending into suspension at each ml; then the mixture is divided into 5ml freezing tubes;
2.2, pre-cooling the freezing tube at 4 ℃ for 4 hours, and then freezing the freezing tube at-20 ℃ to 30 minutes; then transferring to-80 deg.C for freezing for 4 hr, then transferring to liquid nitrogen for long-term freezing.
3. NK immune cell resuscitation
Taking out the frozen tube from the liquid nitrogen, and putting the tube at the temperature of 40 ℃ for 2 minutes to recover NK immune cells for later culture.
Example 5
1. 1000g of frozen stock solution is prepared, and the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 80%, 10% and 10%, namely the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 800g, 100g and 100 g.
The concrete preparation process is as follows
1.1, preparing 100g of freezing storage stabilizer
According to the mass percentage, 8 percent of glycine, 0.5 percent of gelatin, 7 percent of mannitol, 2.5 percent of lactose, 14 percent of human serum albumin, 8 percent of dextran and the balance of basic cell culture solution; weighing 8g of glycine, 0.5g of gelatin, 7g of mannitol, 2.5g of lactose, 14g of human serum albumin, 8g of dextran and 60g of serum-free culture solution of KBM581, and uniformly mixing to prepare a cell cryopreservation stabilizer;
1.2, preparing 100g of freezing protective agent
Weighing 20g of DMSO, 60g of sulfosalicylic acid and 20g of acetamide according to the ratio of 1:3:1, and uniformly mixing to obtain a protective agent;
1.3, preparing 800g of frozen nutrient
800g of KBM581 serum-free culture medium containing 52% alpha-MEM basal medium and 28g/l human serum albumin was weighed.
1.4 preparation of frozen stock solution
And (3) mixing the stabilizer, the protective agent and the nutrient prepared in the steps 1.1, 1.2 and 1.3 to prepare the frozen stock solution.
2. NK immune cell cryopreservation
2.1, measuring 100ml of the frozen stock solution prepared in the step 1, adding NK immune cells and controlling the cell density to be 2 multiplied by 107Resuspending into suspension at each ml; then the mixture is respectively packed in a freezing tube of 5mlPerforming the following steps;
2.2, pre-cooling the freezing tube at 4 ℃ for 4 hours, and then freezing the freezing tube at-20 ℃ to 30 minutes; then transferring to-80 deg.C for freezing for 4 hr, then transferring to liquid nitrogen for long-term freezing.
3. NK immune cell resuscitation
Taking out the frozen tube from the liquid nitrogen, and putting the tube at the temperature of 37 ℃ for 3 minutes to recover NK immune cells for later culture.
Example 6
1. 1000g of frozen stock solution is prepared, and the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 70%, 20% and 10%, namely the mass percentages of the nutrient, the protective agent and the stabilizer are respectively 700g, 200g and 100 g.
The concrete preparation process is as follows
1.1, preparing 100g of freezing storage stabilizer
Calculated according to the mass percentage, 3 percent of glycine, 1 percent of gelatin, 6 percent of mannitol, 2 percent of lactose, 12 percent of human serum albumin, 5 percent of dextran, and the balance of basic cell culture solution; weighing 3g of glycine, 1g of gelatin, 6g of mannitol, 2g of lactose, 12g of human serum albumin, 5g of dextran and 61g of serum-free culture solution of KBM581, and uniformly mixing to prepare a cell cryopreservation stabilizer;
1.2, preparing 200g of freezing protective agent
Weighing 33.33g of sulfosalicylic acid and 166.67g of acetamide according to the ratio of 1:5, and uniformly mixing to obtain a protective agent;
1.3, preparing 700g of frozen nutrient
700g of KBM581 serum-free culture medium was weighed, and the culture medium contained a 54% alpha-MEM basal medium and 35g/l human albumin.
1.4 preparation of frozen stock solution
And (3) mixing the stabilizer, the protective agent and the nutrient prepared in the steps 1.1, 1.2 and 1.3 to prepare the frozen stock solution.
2. NK immune cell cryopreservation
2.1, measuring 100ml of the frozen stock solution prepared in the step 1, adding NK immune cells and controlling the cell density to be 2 multiplied by 107Resuspending to form a suspension; then subpackaging in 5ml of frozen stockIn the tube;
2.2, pre-cooling the freezing tube at 4 ℃ for 4 hours, and then freezing the freezing tube at-20 ℃ to 30 minutes; then transferring to-80 deg.C for freezing for 4 hr, then transferring to liquid nitrogen for long-term freezing.
3. NK immune cell resuscitation
Taking out the frozen tube from the liquid nitrogen, and putting the tube at the temperature of 37 ℃ for 3 minutes to recover NK immune cells for later culture.
Comparative example 2
Comparative example 2 contained no stabilizer.
1. 1000g of frozen stock solution is prepared, and the mass percentages of the nutrient and the protective agent are respectively 70 percent and 30 percent, namely the mass percentages of the nutrient and the protective agent are respectively 700g and 300 g.
The concrete preparation process is as follows
1.1, preparing 300g of freezing protective agent
Weighing 83.4g of DMSO, 83.3g of sulfosalicylic acid and 83.3g of acetamide according to the proportion of 1:1:1, and uniformly mixing to obtain a protective agent;
1.2, preparing 700g of frozen nutrient
700g of serum-free CORNING culture solution is weighed, and the culture solution contains an alpha-MEM basic culture medium with the concentration of 54% and human albumin with the concentration of 14 g/l.
1.3 preparation of frozen stock solution
And (3) mixing the stabilizer, the protective agent and the nutrient prepared in the steps 1.1 and 1.2 to prepare the frozen stock solution.
2. NK immune cell cryopreservation
2.1, measuring 100ml of the frozen stock solution prepared in the step 1, adding NK immune cells and controlling the cell density to be 1.5 multiplied by 107Resuspending into suspension at each ml; then the mixture is divided into 5ml freezing tubes;
2.2, pre-cooling the freezing tube at 4 ℃ for 4 hours, and then freezing the freezing tube at-20 ℃ to 30 minutes; then transferring to-80 deg.C for freezing for 4 hr, then transferring to liquid nitrogen for long-term freezing.
3. NK immune cell resuscitation
Taking out the frozen tube from the liquid nitrogen, and putting the tube at the temperature of 40 ℃ for 2 minutes to recover NK immune cells for later culture.
Second, analysis of test results
1. Proliferation of cells
FIG. 1 is a CIK cell culture growth graph, FIG. 2 is a NK cell culture growth graph; in FIGS. 1 and 2, "non-cryopreserved cells" indicate that non-cryopreserved CIK cells and NK cells were directly cultured, whereas the cells of examples 1 to 6 and comparative examples 1 to 2 were recovered by freezing for 30 days and then cultured for 18 days. Table 1 corresponds to fig. 1 and fig. 2, respectively.
As is clear from FIG. 1, FIG. 2 and Table 1, CIK cells and NK cells were 1X 10 on day 0, respectively6Each/ml, after 18 days of respective culture; after the cryopreservation recovery by using the cryopreservation agent, the recovery growth number of the frozen CIK cells and NK cells is close to the growth number of the cells without cryopreservation, for example, the recovery culture growth number of the CIK cells after cryopreservation reaches 278.79 multiplied by 10 in example 16Per ml, CIK cell culture growth number of 281.39X 10 without frozen storage6Perml, in examples 2 and 3 and comparative example 1, the number of CIK cells grown in culture was 260.22X 106/ml、252.78×106/ml、180.64×106/ml。
Similarly, in example 4, the number of the NK cells recovered and cultured after cryopreservation reaches 285.82X 106The cultured number of unfrozen NK cells was 289.41X 106Per ml; in examples 5 and 6 and comparative example 2, the culture growth numbers of NK cells were 271.22X 10, respectively6/ml、268.78×106/ml、184.37×106/ml。
According to the growth quantity of the cells, firstly, the immune cells cryopreserved by using the cryopreservation agent disclosed by the invention, for example, after CIK (cytokine induced killer) cryopreservation is recovered, the continuous culture growth amplification multiple is between 252 and 279 and is close to the direct culture growth amplification multiple of the non-cryopreserved CIK cells, which is about 281; after the NK immune cells are frozen and recovered, the expansion times of continuous culture growth are 268-286 times, and are approximate to 289 times of the expansion times of direct culture growth of the NK cells which are not frozen and stored; in comparative examples 1 and 2, the cryopreservation stabilizer provided by the invention is not added in the cryopreservation solution, so that the expansion multiple of CIK cells in recovery from cryopreservation is about 180 times, and the expansion multiple of NK cells in growth is about 182 times. That is, the cryopreservation stabilizer provided by the invention can effectively stabilize cell phenotype, prevent cell membrane from breaking, maintain the activity and physiological function of cells after recovery, and ensure that the activity, growth and amplification and physiological function of immune cells are basically consistent with those of unfrozen cells.
TABLE 1 immune cell culture growth number counting table,. times.106Per ml
2. CIK cell cryopreservation recovery survival rate
Respectively staining the CIK and NK cells of examples 1 to 6 and comparative examples 1 and 2 with trypan blue, storing at 18-25 ℃ for 4 hours, counting the cells by using a cell counting plate, and calculating the cell number and the cell viability, as shown in FIG. 3, FIG. 4, tables 2 and 3; wherein the data of table 2 is consistent with fig. 3; the data in table 3 correspond to fig. 4.
TABLE 2 CIK cell number and Activity tables before and after cryopreservation
TABLE 3 NK cell number and Activity Table before and after cryopreservation
As can be seen from fig. 3, fig. 4, tables 2 and 3, the number of CIK cells and the cell viability rate were higher in examples 1 to 3 than in comparative example 1; the number of NK cells and the cell survival rate were higher in examples 4 to 6 than in comparative example 2. Therefore, the cryopreservation stabilizer provided by the invention can effectively stabilize cell phenotype, prevent cell membrane from breaking, maintain the activity and physiological function of cells after recovery, and ensure that the activity of immune cells in cryopreservation is basically consistent with that of immune cells not in cryopreservation.
3. Detection of CD3+ CD56+ phenotype of CIK cells and detection of CD3-CD56+ phenotype of NK cells
FIGS. 5, 6, 7, 8 and 9 are the graphs showing the phenotype of CD3+ CD56+ after 7 days of CIK cell culture, respectively, wherein FIG. 5 shows the direct culture of CIK cells without freezing; FIGS. 6, 7, 8 and 9 show the recovery and re-culture of CIK cells in examples 1 to 3 and comparative example 1 after 30 days of cryopreservation.
FIGS. 10, 11, 12, 13, 14 are graphs of the CD3-CD56+ phenotype after 7 days of NK cell culture, respectively, wherein FIG. 10 is a direct culture of unfrozen NK cells; FIGS. 11, 12, 13 and 14 show recovery and re-culture of NK cells in examples 4 to 6 and comparative example 2 after cryopreservation for 30 days.
CIK cells are cultured to 7 days, and the CIK cells which are not frozen and cultured in examples 1 to 3 and comparative example 1 are respectively taken to prepare cell suspensions and the final concentration of the cells is adjusted to be 1 multiplied by 106Adding labeled CD3+ CD56+ monoclonal antibody into the cells per ml, incubating the cells in the dark at room temperature for 15 minutes, washing off excessive antibody, detecting the excessive antibody by using an up-flow cytometer, and testing the results to obtain a CIK cell phenotype analysis chart shown in figures 5, 6, 7, 8 and 9. As shown in fig. 5, 6, 7, 8 and 9, the CD3+ CD56+ content of the CIK cells cultured after cryopreservation recovery in examples 1 to 3 was 46.03%, 43.28% and 38.08%, respectively, and was 73.40%, 71.72% and 67.86% higher than the CD3+ CD56+ content (12.24%) of the CIK cells of comparative example 1; but the ratio of the CD3+ CD56+ in the direct culture of the CIK cells which are not frozen is not much different from 49.40%.
Culturing NK cells to day 7, preparing cell suspensions from unfrozen NK cells and NK cells cultured in examples 4 to 6 and comparative example 2, and adjusting the final cell concentration to 1 × 106Adding labeled CD3-CD56+ monoclonal antibody into the mixture per ml, incubating the mixture for 15 minutes at room temperature in the dark, washing off excessive antibody, and detecting the excessive antibody by using an up-flow cytometer, wherein the test results are shown in NK cell phenotype analysis graphs of figures 10, 11, 12, 13 and 14. As shown in FIGS. 10, 11, 12, 13 and 14, the proportions of CD3-CD56+ cells in NK cells cultured after cryopreservation and recovery in examples 4 to 6 were 82.88%, 81.04% and 77.35%, respectively, and were 39.02%, 37.64% and 34.66% higher than the content (50.54%) of CD3-CD56+ in NK cells of comparative example 2; but the proportion of CD3-CD56+ in the direct culture of unfrozen NK cells is not much different from 85.94%.
Therefore, after the frozen immune cells of the freezing storage agent provided by the invention are recovered, the freezing storage stabilizer has a better protective effect on the phenotype of the immune cells, the stabilizer has a smaller influence on surface markers of the immune cells, no external pollution is caused, and the immune cells can be directly used without being washed after the freezing storage recovery.
4. Killing rate of K562 by immune cells.
The CIK cells which are not frozen and stored in examples 1 to 3 and comparative example 1 are frozen and stored for 30 days, and the number of the CIK cells is the same after the CIK cells are recovered and cultured for 7 days. Transferred to 5 groups of centrifuge tubes (4 centrifuge tubes per group, each group corresponding to non-cryopreserved CIK cells and CIK cells cultured in examples 1 to 3 and comparative example 1), and then the 5 groups of centrifuge tubes were individually adjusted to a final concentration of about 1.0X 10 using the respective culture liquids6/ml、1.5×106/ml、2.0×106/ml、3.0×106/ml、4.0×106Cell suspension in ml, as 5 groups of effector cells.
And (3) paving holes for the target cells K562 and the effector cells CIK according to an effective target ratio of 1:1, 5:1, 10:1 and 20:1 by using the K562 cells as the target cells, wherein each group is provided with 3 multiple holes. Each well of CIK cells and K562 target cells was 500. mu.l. Mu.l of the respective media of examples 6 to 10 and comparative example 1 were added to each well.
Effector cell CIK cell natural release group: 1.0X 106/ml、1.5×106/ml、2.0×106/ml、3.0×106/ml、4.0×106CIK cells/ml, 500. mu.l per well, 3 replicates per group, 500. mu.l per well.
K562 target cell maximal release group: 1.0X 105Perml of target cells, 500. mu.l per well, 3 replicates per group, 500. mu.l per well of medium, 5% CO at 37 ℃2Incubate in incubator for 40 minutes.
K562 target cell spontaneous release group: 1.0X 105Perml of target cells, 500. mu.l per well, 3 replicates per group, 500. mu.l per well.
Culture solution blank control test group: mu.l per well, 3 replicates per group, 500. mu.l per well.
After plating, the plates were centrifuged at 250g for 4 minutes and placedAt 37 ℃ with 5% CO2The culture was carried out in an incubator for 4 hours. 45 minutes before the end of the culture, the culture plate was removed, the thawing solution was added to the maximum release group of the target cells, the mixture was centrifuged at 250g for 4 minutes, the cells were gently mixed, the mixture was centrifuged at 300g for 5 minutes, the supernatant was collected, and the absorbance at 492nm was measured using LDH (lactate dehydrogenase).
The killing activity was calculated according to the following formula:
killing activity = (A-B-C)/(D-C). times.100%;
wherein:
a-represents the corrected value of the absorbance value of the test group;
b-represents effector cell natural release group;
c-represents a target cell spontaneous release group;
d-represents the maximum release group of target cells.
Similarly, the NK cells also employ CIK cells in a manner similar to that described above and will not be described further herein.
The killing effect target ratio of CIK and NK to K562 cells is detailed in figure 15, figure 16 and table 4; the data in table 4 corresponds to the data in fig. 15 and 16, respectively.
TABLE 4 tables of immune cell (CIK, NK) and K562 killing test results
As can be seen from table 4, fig. 15 and fig. 16, when killing K562 cells, the frozen immune cells (e.g., CIK, NK) of the frozen stock solution containing the stabilizer provided by the present invention are recovered and cultured, as in examples 1 to 6, and have better killing effect and obvious difference than the immune cells (e.g., CIK, NK) recovered and cultured in frozen stock solutions of comparative examples 1 and 2; and the killing effect of the direct culture of the unfrozen immune cells is not greatly different from that of the recovery culture of the unfrozen immune cells in examples 1 to 6, and particularly the killing effect of the corresponding immune cells in examples 1 and 4 is better. In examples 1 to 6, after 7 days of immune cells (e.g., CIK, NK) culture, the cell killing rate is 86% to 95% under the condition of an effective target ratio of 20:1 when killing K562 cells, which is far higher than the clinical application standard, i.e., greater than or equal to 40%.
It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. The immune cell cryopreservation stabilizer is characterized by comprising, by mass, 2-10% of glycine, 0.1-2% of gelatin, 5-10% of mannitol, 0.1-5% of lactose, 10-20% of human serum albumin, 1-10% of dextran, and the balance of a basic cell culture solution.
2. The immune cell cryopreservation stabilizer of claim 1, comprising 5% of glycine, 1% of gelatin, 7% of mannitol, 3% of lactose, 14% of human serum albumin, 6% of dextran, and the balance of basic cell culture solution by mass percentage.
3. The immune cell cryopreservation stabilizer of claim 1 or 2, wherein the basal cell culture solution is a CORNING serum-free culture solution, a KBM581 serum-free culture solution or an RPMI-1640 serum-free culture solution.
4. An immune cell cryopreservation liquid comprises an immune cell cryopreservation nutrient and a protective agent; the cryopreservation solution further comprising an immune cell cryopreservation stabilizer according to any one of claims 1 to 3.
5. The frozen stock solution of immune cells according to claim 4, wherein the mass percentages of the nutrient, the protective agent and the stabilizer in the frozen stock solution of immune cells are 60-90%, 5-30% and 1-15%, respectively.
6. The immune cell cryopreservation solution of claim 4, wherein the nutritional agents comprise a cell culture solution and 15-20 w/w% human serum albumin; the cell culture solution is a CORNING serum-free culture solution, a KBM581 serum-free culture solution or an RPMI-1640 serum-free culture solution, and the addition amount of the human serum albumin is 15-35 g/l.
7. The immune cell cryopreservation liquid of claim 6, wherein the cell culture liquid further comprises 50-60% of an alpha-MEM basal medium.
8. The immune cell cryopreservation solution of claim 4 wherein the protectant comprises at least one of DMSO, sulfosalicylic acid, acetamide.
9. The immune cell cryopreservation solution of claim 8, wherein the protectant is a mixture of DMSO, sulfosalicylic acid and acetamide, and the mass ratio of sulfosalicylic acid to acetamide is 1-3: 1-5.
10. A method of cryopreserving immune cells using the cryopreserving fluid of any one of claims 4 to 8, comprising the steps of:
at normal temperature, resuspending immune cells by using a freezing medium to form a suspension, and then subpackaging the suspension in a freezing tube;
and (3) pre-cooling the freezing tube for 10-15 hours at the temperature of 2-8 ℃, and finally transferring the tube into liquid nitrogen for long-term freezing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115777686A (en) * | 2022-09-06 | 2023-03-14 | 北京唐颐惠康生物医学技术有限公司 | Cell cryopreservation solution and preparation method and application thereof |
| CN117178980A (en) * | 2023-09-08 | 2023-12-08 | 苏州依科赛生物科技股份有限公司 | Immune cell low-temperature frozen stock solution and application thereof |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1124617A (en) * | 1994-07-02 | 1996-06-19 | 武汉中科康益生物制药厂 | Lyophilized bougie and producing process thereof |
| CN102657870A (en) * | 2012-06-04 | 2012-09-12 | 长春祈健生物制品有限公司 | Vaccine cryoprotectant without composition of gelatin and human albumin |
| CN105638642A (en) * | 2016-01-27 | 2016-06-08 | 上海润泉生物技术有限公司 | Immune cell cryopreservation solution and application thereof |
| CN106635984A (en) * | 2016-11-16 | 2017-05-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Human umbilical cord blood immune cell bank establishing method |
| CN108184819A (en) * | 2018-01-24 | 2018-06-22 | 北京臻溪谷医学研究中心（有限合伙） | A kind of people's umbilical cord China Tong Shi glue tissues freeze protection liquid and its preparation and application |
| CN109843052A (en) * | 2016-10-04 | 2019-06-04 | 全崴生技股份有限公司 | The composition and method saved for cell freezing |
| CN109864064A (en) * | 2019-02-19 | 2019-06-11 | 武汉普诺赛生命科技有限公司 | A kind of immunocyte frozen stock solution and immunocyte cryopreservation methods |
| CN110292038A (en) * | 2018-03-23 | 2019-10-01 | 北京世纪劲得生物技术有限公司 | Frozen solution of mescenchymal stem cell and preparation method thereof |
| CN111345282A (en) * | 2020-03-17 | 2020-06-30 | 广东万海细胞生物科技有限公司 | Cell cryopreservation liquid and cryopreservation method |
| CN111387174A (en) * | 2020-01-17 | 2020-07-10 | 广东先康达生物科技有限公司 | Immune cell cryopreservation liquid and immune cell cryopreservation method |
| CN112806354A (en) * | 2021-01-18 | 2021-05-18 | 圣至同合(北京)生物科技有限公司 | Immune cell cryopreservation liquid as well as preparation method and application thereof |
| CN113261557A (en) * | 2021-05-28 | 2021-08-17 | 广东先康达生物科技有限公司 | Stem cell cryopreservation liquid and stem cell cryopreservation method |
| CN113444685A (en) * | 2021-07-05 | 2021-09-28 | 上海南滨江细胞生物科技有限公司 | Culture method for culturing immune cells by autologous peripheral blood |
| CN114041455A (en) * | 2021-12-01 | 2022-02-15 | 东莞再立健生物科技有限公司 | Stem cell cryopreservation solution, and preparation method and application thereof |
-
2022
- 2022-05-27 CN CN202210585624.9A patent/CN114667999A/en active Pending
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1124617A (en) * | 1994-07-02 | 1996-06-19 | 武汉中科康益生物制药厂 | Lyophilized bougie and producing process thereof |
| CN102657870A (en) * | 2012-06-04 | 2012-09-12 | 长春祈健生物制品有限公司 | Vaccine cryoprotectant without composition of gelatin and human albumin |
| CN105638642A (en) * | 2016-01-27 | 2016-06-08 | 上海润泉生物技术有限公司 | Immune cell cryopreservation solution and application thereof |
| CN109843052A (en) * | 2016-10-04 | 2019-06-04 | 全崴生技股份有限公司 | The composition and method saved for cell freezing |
| CN106635984A (en) * | 2016-11-16 | 2017-05-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Human umbilical cord blood immune cell bank establishing method |
| CN108184819A (en) * | 2018-01-24 | 2018-06-22 | 北京臻溪谷医学研究中心（有限合伙） | A kind of people's umbilical cord China Tong Shi glue tissues freeze protection liquid and its preparation and application |
| CN110292038A (en) * | 2018-03-23 | 2019-10-01 | 北京世纪劲得生物技术有限公司 | Frozen solution of mescenchymal stem cell and preparation method thereof |
| CN109864064A (en) * | 2019-02-19 | 2019-06-11 | 武汉普诺赛生命科技有限公司 | A kind of immunocyte frozen stock solution and immunocyte cryopreservation methods |
| CN111387174A (en) * | 2020-01-17 | 2020-07-10 | 广东先康达生物科技有限公司 | Immune cell cryopreservation liquid and immune cell cryopreservation method |
| CN111345282A (en) * | 2020-03-17 | 2020-06-30 | 广东万海细胞生物科技有限公司 | Cell cryopreservation liquid and cryopreservation method |
| CN112806354A (en) * | 2021-01-18 | 2021-05-18 | 圣至同合(北京)生物科技有限公司 | Immune cell cryopreservation liquid as well as preparation method and application thereof |
| CN113261557A (en) * | 2021-05-28 | 2021-08-17 | 广东先康达生物科技有限公司 | Stem cell cryopreservation liquid and stem cell cryopreservation method |
| CN114304134A (en) * | 2021-05-28 | 2022-04-12 | 广东先康达生物科技有限公司 | Stem cell cryopreservation liquid and stem cell cryopreservation method |
| CN113444685A (en) * | 2021-07-05 | 2021-09-28 | 上海南滨江细胞生物科技有限公司 | Culture method for culturing immune cells by autologous peripheral blood |
| CN114041455A (en) * | 2021-12-01 | 2022-02-15 | 东莞再立健生物科技有限公司 | Stem cell cryopreservation solution, and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 罗满林: "《兽医生物制品学》", 30 April 2019, 中国农业大学出版社 * |
| 赵满仓等: "自制细胞冻存液对树突状细胞存活率及活性的影响", 《中国肿瘤生物治疗杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115777686A (en) * | 2022-09-06 | 2023-03-14 | 北京唐颐惠康生物医学技术有限公司 | Cell cryopreservation solution and preparation method and application thereof |
| CN117178980A (en) * | 2023-09-08 | 2023-12-08 | 苏州依科赛生物科技股份有限公司 | Immune cell low-temperature frozen stock solution and application thereof |
| CN117178980B (en) * | 2023-09-08 | 2024-04-16 | 苏州依科赛生物科技股份有限公司 | Immune cell low-temperature frozen stock solution and application thereof |
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