CN114190368B - Serum-free immune cell cryopreservation liquid, preparation method and immune cell cryopreservation method - Google Patents
Serum-free immune cell cryopreservation liquid, preparation method and immune cell cryopreservation method Download PDFInfo
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- CN114190368B CN114190368B CN202210007695.0A CN202210007695A CN114190368B CN 114190368 B CN114190368 B CN 114190368B CN 202210007695 A CN202210007695 A CN 202210007695A CN 114190368 B CN114190368 B CN 114190368B
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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
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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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- 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
Abstract
The invention provides serum-free immune cell cryopreservation liquid, which comprises the following components: the antifreeze comprises methyl glycerol, dextran and polyethylene glycol, the preservation solution comprises glucose sodium chloride solution, human serum albumin solution, compound electrolyte solution and EDTA-Na2, the volume ratio of each solution in the antifreeze and the preservation solution is 5% of methyl glycerol, 10% of dextran, 5% of polyethylene glycol, 30% of glucose sodium chloride solution, 20% of human serum albumin solution, 30% of compound electrolyte solution, and the final concentration of EDTA-Na2 is 2mM. The invention adopts human serum albumin without animal-derived protein as a nutrient protecting agent, and solves the problem that animal-derived substances activate immune cells to generate immune response. And dimethyl sulfoxide (DMSO) which is commonly used in the traditional frozen stock solution is abandoned, so that the cytotoxicity of the frozen stock solution is reduced, and the cell viability is improved.
Description
Technical Field
The invention relates to the technical field of cell cryopreservation, in particular to serum-free immune cell cryopreservation liquid, a preparation method and an immune cell cryopreservation method.
Background
Cell cryopreservation refers to placing cells in a low-temperature environment to achieve the effect of reducing cell metabolism, so as to achieve the purpose of preserving the cells for long-term storage for subsequent experiments or clinical use. And subpackaging the cells added with the frozen stock solution into frozen stock ampoule bottles, reducing the temperature to below-100 ℃ at a certain cooling rate, and putting into liquid nitrogen for frozen stock, so that the cells are in an extremely low metabolic state, stop growing, and achieve the aim of long-term storage.
The most commonly used frozen stock solution is usually pure fetal bovine serum added with a certain proportion of dimethyl sulfoxide (DMSO) or other inorganic salt culture solutions, and the fetal bovine serum and a certain proportion of dimethyl sulfoxide (DMSO) are added. Dimethyl sulfoxide (DMSO) can reduce ice crystal formation, relieve free radical damage to cells, change the permeability of biomembrane to electrolytes, drugs, poisons and metabolites, and protect cells during cell freezing.
Serum is added into the frozen stock solution, so that nutrition can be provided for cells. Meanwhile, macromolecular substances such as protein and the like which are rich in serum can have better protection effect on cells.
The conventional cell freezing method generally adopts program freezing, the conventional method generally comprises standing at-4deg.C for 30 min, standing at 20deg.C for 30 min, standing at-80deg.C overnight, and storing with liquid nitrogen. Or using a program cooling box, and directly placing in a refrigerator at-80 ℃ overnight and then preserving in liquid nitrogen.
Immune cells refer to cells involved in or associated with an immune response. Including lymphocytes, dendritic cells, monocytes/macrophages, and the like. Immune cells can be divided into a plurality of types, and various immune cells play an important role in human bodies, and are a relatively complex and fragile cell. The DMSO can protect cells at low temperature, but is harmful to the cells at normal temperature, and if the DMSO is not cleaned, the DMSO has a large influence on the state of the cells after recovery. The normal freezing solution containing DMSO is used for freezing cells, the activity of the cells is obviously reduced after resuscitating, and part of cell surface antigens are lost. Fetal bovine serum, which is an animal-derived protein and a heterologous immunogen, may activate immune cells in their original state to produce an immune response.
The optimal cooling rates of different cell types during cryopreservation vary greatly depending on whether the moisture permeation process is compatible with the cooling rate or not, depending on the cell surface area to volume ratio. The immune cells are various in cell types and large in size difference, and when the immune cells are frozen by the traditional program cooling method, the cooling is inappropriate in the freezing process and has great influence on the cell survival rate because the program is not optimized.
Disclosure of Invention
The invention solves the technical problems by providing serum-free immune cell cryopreservation liquid, a preparation method and an immune cell cryopreservation method, so as to solve the problems in the background technology.
The technical problem solved by the invention is to adopt the following technical scheme to realize serum-free immune cell frozen stock solution, which comprises the following steps: the antifreeze comprises methyl glycerol, dextran and polyethylene glycol, the preservation solution comprises glucose sodium chloride solution, human serum albumin solution, compound electrolyte solution and EDTA-Na2, the volume ratio of each solution in the antifreeze and the preservation solution is 5% of methyl glycerol, 10% of dextran, 5% of polyethylene glycol, 30% of glucose sodium chloride solution, 20% of human serum albumin solution, 30% of compound electrolyte solution, and the final concentration of EDTA-Na2 is 2mM.
Preferably, the human serum albumin solution is a 20% human serum albumin solution.
The preparation method of the serum-free immune cell frozen stock solution specifically comprises the following steps:
step 1, according to the volume ratio of the formula of serum-free immune cell cryopreservation liquid, taking quantitative glucose sodium chloride solution and compound electrolyte solution, putting the quantitative EDTA-Na2 into a liquid storage bottle, putting the quantitative EDTA-Na2 into the liquid storage bottle, and fully and uniformly mixing by using a vortex mixer to fully dissolve the EDTA-Na 2;
step 2, adding quantitative methyl glycerol, dextran and polyethylene glycol into a liquid storage bottle, and filtering and sterilizing by using a filter after fully and uniformly mixing;
and 3, after filtering and sterilizing, adding a certain amount of human serum albumin solution into the frozen stock solution, fully and uniformly mixing, and then preserving at 4 ℃.
Preferably, the filter in step 2 is a 0.22um filter.
The immune cell cryopreservation method specifically comprises the following steps:
1) Counting immune cells, centrifuging and removing cell supernatant;
2) Immune cells were isolated at less than 2 x 10 7 The density of/mL is resuspended by the prepared frozen stock solution, and the frozen stock solution is added into a frozen stock tube after being fully and uniformly mixed;
3) And (5) putting the frozen products into a program cooling instrument for freezing.
Preferably, the temperature reducing device in (3) is configured to: when the temperature is not lower than 4 ℃,1 ℃/min; when the temperature reaches 4 ℃, reducing the temperature at 20 ℃/min; when the temperature reaches-15 ℃, cooling at 2 ℃/min; when the temperature reaches minus 35 ℃, reducing the temperature at 10 ℃/min; when the temperature reaches-80 ℃, the mixture is transferred into a liquid nitrogen tank for preservation.
Compared with the prior art, the invention has the following advantages: the invention adopts human serum albumin without animal-derived protein as a nutrient protecting agent, and solves the problem that animal-derived substances activate immune cells to generate immune response. And dimethyl sulfoxide which is commonly used in the traditional frozen stock solution is abandoned, so that the cytotoxicity of the frozen stock solution is reduced, and the cell activity is improved.
The invention adopts the high molecular cryopreservation protective agent dextran to reduce the concentration of low molecular solutes in the cryopreservation liquid, lightens salt damage, and the osmotic antifreeze agent propylene glycol and methyl glycerol permeate into cells to change the intracellular supercooling state, so that the intracellular extracellular pressure is close to that of the cells, and the concentration and the speed of cell dehydration shrinkage are reduced. And a plurality of different cryopreserved cell protectants are comprehensively added, so that cells are protected to a greater extent. And the cells are in a continuous and stable state by adjusting the proportion and the formula of other hypertonic and hypotonic preservation solutions in the frozen stock solution.
EDTA-Na2 is additionally added into the frozen stock solution to be used as a calcium ion binding agent, so that the adhesion condition of immunocyte after resuscitating can be effectively reduced, and the recovery rate of cells after resuscitating is increased.
According to the invention, the cooling rate in the process of freezing immune cells is optimized through experiments, the cooling rate and time in the process of program cooling are adjusted, the formation of ice crystals in the cooling process is reduced, the exposure time of cells in a high-concentration solution is reduced, and the damage of intracellular ice and the damage of solution in the process of freezing are reduced, so that the survival rate reaches a higher level.
Drawings
FIG. 1 is a schematic diagram showing the proportion of T cell activation in the examples;
FIG. 2 is a schematic representation of T cell activation numbers in examples;
FIG. 3 shows the proportion of markers on the cell surface of unfrozen PBMC in the examples;
FIG. 4 shows the proportion of PBMC cell surface markers after the cryopreservation method of the invention is used in the examples;
FIG. 5 shows the proportion of PBMC cell surface markers after cryopreservation of the control group in the examples.
Detailed Description
In order to make the technical means, creation features, workflow, and usage method of the present invention achieve the objects and effects of the present invention easy to understand, the technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples
1. Preparing serum-free immune cell frozen stock solution: weighing 30mL of glucose sodium chloride solution and 30mL of compound electrolyte solution, putting the glucose sodium chloride solution and the 30mL of compound electrolyte solution into a liquid storage bottle, weighing 67.2412mg of EDTA-Na2, putting the liquid storage bottle, and fully and uniformly mixing the glucose sodium chloride solution and the 30mL of compound electrolyte solution by using a vortex mixer to fully dissolve the EDTA-Na 2; 5mL of methyl glycerol, 10mL of dextran and 5mL of polyethylene glycol are added into a liquid storage bottle, and after being fully and uniformly mixed, the mixture is filtered and sterilized by a 0.22um filter; after filtration and sterilization, 20mL of 20% human serum albumin solution is added into the frozen stock solution, and the mixture is fully and uniformly mixed and then is preserved at the temperature of 4 ℃.
Observing the non-frozen immune cells under a microscope, counting the immune cells, and detecting the cell number activity by using a cell counter; dividing the immune cells into two parts, centrifuging one part, removing cell supernatant to less than 2×10 7 The density of/mL is resuspended in the prepared frozen stock solution, and the mixture is added into a frozen stock tube after being fully and evenly mixed. Putting the program cooling instrument into the program cooling instrument for freezing, and setting the program cooling instrument as follows: cooling at 1 deg.C/min when the temperature is not lower than 4deg.C; when the temperature reaches 4 ℃, reducing the temperature at 20 ℃/min; when the temperature reaches-15 ℃, cooling at 2 ℃/min; when the temperature reaches minus 35 ℃, reducing the temperature at 10 ℃/min; when the temperature reaches-80 ℃, the sample is transferred into a liquid nitrogen tank for preservation and is used as an experimental group. The other part was frozen using a DMSO frozen stock solution using a conventional method as a control group. After the same time of cryopreservation, the immune cells of the experimental group and the control group are subjected to cryopreservation and resuscitated, and the cell number activities after resuscitation are compared, and the comparison results are shown in Table one, wherein the immune cells comprise PBMC cells, T cells, B cells, NK cells and monocytes.
List one
As can be seen from the comparison result in Table I, the activity of the immune cells after the freezing of the experimental group is generally higher than that of the immune cells after the freezing of the control group.
2. Comparing the proliferation efficiency of T cells after resuscitating the immune cells frozen by using serum-free immune cell cryopreservation liquid and immune cell cryopreservation method with that of T cells not frozen, using the T cells frozen by using DMSO-containing cryopreservation liquid and traditional program cooling method as a control group:
after cell resuscitation, the concentration was adjusted to 1 x 10 using RPMI-1640 complete medium 6 cells/mL and staining cells with CFDA-SE staining solution; adding CD3/CD28 antibody and IL-2, culturing in a 37 deg.C incubator for 14 days, changing the culture medium every 3 days and supplementing cell factor; detecting T cell activation proportion by using a flow cytometer on the fifth day, wherein the detection result is shown in figure 1; cell counts were performed every three days to detect the number of activated T cells, and the detection results are shown in fig. 2.
3. PBMC cells were resuspended to 1 x 10 7 cells/mL, stained with fluorescent-labeled CD3, CD4, CD8, CD14, CD19, CD56 antibodies, washed after staining, and detected using a flow cytometer. Comparing the ratio of PBMC cells recovered by using serum-free immune cell cryopreservation solution and immune cell cryopreservation method provided by the patent application of the invention to PBMC cell surface markers not frozen, using PBMC cells frozen by using DMSO-containing cryopreservation solution and conventional program cooling method as a control group, and comparing the results as shown in table two, fig. 3, fig. 4 and fig. 5.
Watch II
As can be seen from the comparison results of Table two, the ratio of the PBMC cell surface markers after resuscitating the immune cells in the serum-free immune cell cryopreservation solution and the immune cell cryopreservation method is higher than that of the PBMC cell surface markers in the control group.
The foregoing has shown and described the basic principles, main features and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. Serum-free immune cell frozen stock solution is characterized in that: comprising the following steps: the antifreeze comprises methyl glycerol, dextran and polyethylene glycol, the preservation solution comprises glucose sodium chloride solution, human serum albumin solution, compound electrolyte solution and EDTA-Na2, the volume ratio of each solution in the antifreeze and the preservation solution is 5% of methyl glycerol, 10% of dextran, 5% of polyethylene glycol, 30% of glucose sodium chloride solution, 20% of human serum albumin solution, 30% of compound electrolyte solution, and the final concentration of EDTA-Na2 is 2mM.
2. The serum-free immunocyte cryopreservation solution according to claim 1, wherein: the human serum albumin solution is 20% human serum albumin solution.
3. The method for preparing serum-free immune cell cryopreservation solution according to claim 1, which is characterized by comprising the following steps:
step 1, according to the volume ratio of the formula of serum-free immune cell cryopreservation liquid, taking quantitative glucose sodium chloride solution and compound electrolyte solution, putting the quantitative EDTA-Na2 into a liquid storage bottle, putting the quantitative EDTA-Na2 into the liquid storage bottle, and fully and uniformly mixing by using a vortex mixer to fully dissolve the EDTA-Na 2;
step 2, adding quantitative methyl glycerol, dextran and polyethylene glycol into a liquid storage bottle, and filtering and sterilizing by using a filter after fully and uniformly mixing;
and 3, after filtering and sterilizing, adding a certain amount of human serum albumin solution into the frozen stock solution, fully and uniformly mixing, and then preserving at 4 ℃.
4. The method for preparing serum-free immunocyte cryopreservation liquid according to claim 3, wherein the method comprises the following steps: the filter in the step 2 is a 0.22um filter.
5. The method for performing immune cell cryopreservation by using serum-free immune cell cryopreservation solution according to claim 1, which is characterized by comprising the following specific contents:
1) Counting immune cells, centrifuging and removing cell supernatant;
2) Immune cells were isolated at less than 2 x 10 7 The density of/mL is resuspended by the prepared frozen stock solution, and the frozen stock solution is added into a frozen stock tube after being fully and uniformly mixed;
3) The program cooling instrument is put into the program cooling instrument for freezing, and the program cooling instrument is set as follows: when the temperature is not lower than 4 ℃,1 ℃/min; when the temperature reaches 4 ℃, reducing the temperature at 20 ℃/min; when the temperature reaches-15 ℃, cooling at 2 ℃/min; when the temperature reaches minus 35 ℃, reducing the temperature at 10 ℃/min; when the temperature reaches-80 ℃, the mixture is transferred into a liquid nitrogen tank for preservation.
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