CN112998009A - NK cell cryopreservation liquid and preparation method and application thereof - Google Patents
NK cell cryopreservation liquid and preparation method and application thereof Download PDFInfo
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- CN112998009A CN112998009A CN202110345949.5A CN202110345949A CN112998009A CN 112998009 A CN112998009 A CN 112998009A CN 202110345949 A CN202110345949 A CN 202110345949A CN 112998009 A CN112998009 A CN 112998009A
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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
- A01N1/0205—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
Abstract
The invention relates to an NK cell frozen stock solution and a preparation method and application thereof, wherein the NK cell frozen stock solution consists of 0.9% sodium chloride injection, human serum albumin, glycerol, vitamin C and trehalose. The combined action of the components can promote the proliferation of NK cells, improve the activity of the NK cells and greatly improve the survival rate of the recovered NK cells; the NK cell frozen stock solution does not contain dimethyl sulfoxide and animal-derived components except for human bodies, and has the advantages of high recovery rate, simple components, good stability and the like. NK cell frozen stock solution is used for freezing and deposits NK cell, and after freezing and depositing, the survival rate of NK cell can not change significantly, and uses NK cell after NK cell frozen stock solution freezes and deposits can directly input human, convenient and fast high-efficient, no safe risk after melting.
Description
Technical Field
The invention relates to the technical field of cell cryopreservation, in particular to an NK cell cryopreservation solution as well as a preparation method and application thereof.
Background
NK cells (Natural killer cells) are the first line of defense of human body against tumors, have important function in the innate immune system, and can directly kill tumor cells without being sensitized in advance. Although NK cells have been used for cell therapy for tumor immunity and have a good effect in clinical treatment of tumors (such as leukemia, lymphoma and melanoma); however, the long-term efficient storage of NK cells is always a problem, both for third party cell banks and for delivery and application clinical management.
The cryopreservation technology is to freeze and preserve living cells at ultralow temperature (liquid nitrogen at-198 ℃), reduce the metabolic rate of the cells, and inhibit the activity of various enzymes for regulating and controlling the growth and metabolism of the cells, so that the inherent biochemical reaction of the cells is very slow, even the cells stop growing, thereby maintaining the activity of the cells and prolonging the preservation life of the cells. The existing cell freezing medium is basically prepared from a cell basic culture medium (such as DMEM, RPMI1640 and the like), Fetal Bovine Serum (FBS) and dimethyl sulfoxide (DMSO). Wherein dimethyl sulfoxide (DMSO) (dimethyl sulfoxide) is used as cryoprotectant to protect cells from damage during freezing and resuscitation. DMSO belongs to a protective osmotic agent that rapidly penetrates into cells, reducing the time that the cells are exposed to harmful environments. However, DMSO has a toxic effect on cells at room temperature. Even if most of them are removed during resuscitation, the remainder of the micro-scale can cause side effects to the human body (neurotoxicity, cardiovascular failure, respiratory arrest and fatal heart failure). There are reports in the literature that NK cells cryopreserved using DMSO have degraded activity and functional killing activity after resuscitation. In addition, fetal bovine serum or calf serum is generally used as a protective agent in the cell freezing medium to protect cell membranes and improve the cell survival rate in the freezing recovery process. However, bovine serum may contain mad cow disease virus, endotoxin, exotoxin and potential unknown virus risks. Fetal bovine serum FBS increases the chances of pathogenic contamination of the animal, which all add uncertainty to the future use of the cells.
Disclosure of Invention
The invention mainly aims to provide an NK cell frozen stock solution as well as a preparation method and application thereof, and aims to solve the technical problem of how to provide the NK cell frozen stock solution, so that NK cells have high cell survival rate, are easy to prepare and are low in cost.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme. The NK cell cryopreservation solution provided by the invention comprises 0.9% sodium chloride injection, human serum albumin, glycerol, vitamin C and trehalose.
The object of the present invention and the technical problems solved thereby can be further achieved by the following technical measures.
Preferably, the NK cell cryopreservation solution comprises, per 100 parts by volume of the NK cell cryopreservation solution: 10-50 parts of 0.9% sodium chloride injection, 40-88 parts of human serum albumin, 1-20 parts of glycerol and 1-20 parts of vitamin C;
the NK cell cryopreservation solution further comprises: 10-60mg/mL of trehalose.
Preferably, the NK cell cryopreservation solution comprises, per 100 parts by volume of the NK cell cryopreservation solution: 30-50 parts of 0.9% sodium chloride injection, 40-60 parts of human serum albumin, 1-10 parts of glycerol and 1-10 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 30-50 mg/mL.
Preferably, the NK cell cryopreservation solution comprises, per 100 parts by volume of the NK cell cryopreservation solution: 50 parts of 0.9% sodium chloride injection, 40 parts of human serum albumin, 6 parts of glycerol and 4 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 50 mg/mL.
Preferably, the NK cell cryopreservation solution comprises, per 100 parts by volume of the NK cell cryopreservation solution: 40 parts of 0.9% sodium chloride injection, 50 parts of human serum albumin, 4 parts of glycerol and 6 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 20 mg/mL.
The object of the present invention and the technical problem to be solved are also achieved by the following technical means. The preparation method of the NK cell frozen stock solution provided by the invention comprises the following steps:
dissolving trehalose in 0.9% sodium chloride injection, adding human serum albumin, glycerol and vitamin C into the solution obtained by dissolving, and uniformly stirring to obtain a mixed solution;
sterilizing the mixed solution to obtain an NK cell frozen stock solution;
every 100 parts of the NK cell frozen stock solution comprises the following components in parts by volume: 10-50 parts of 0.9% sodium chloride injection, 40-88 parts of human serum albumin, 1-20 parts of glycerol and 1-20 parts of vitamin C;
the NK cell cryopreservation solution further comprises: 10-60mg/mL of trehalose.
The object of the present invention and the technical problems solved thereby can be further achieved by the following technical measures.
Preferably, the method for preparing the NK cell frozen stock solution comprises the step of sterilizing by using a bacterial filter, wherein the filtering size of the bacterial filter is 0.2-0.5 mu m.
The object of the present invention and the technical problem to be solved are also achieved by the following technical means. The invention provides application of the NK cell freezing solution in freezing and storing NK cells.
The object of the present invention and the technical problems solved thereby can be further achieved by the following technical measures.
Preferably, the aforementioned use, wherein the method for cryopreserving NK cells using said NK cell cryopreserving solution, comprises the steps of:
adding 0.9% sodium chloride injection into NK cells to prepare cell suspension;
centrifuging the cell suspension, removing supernatant, adding the NK cell frozen stock solution, and adjusting cell concentration to make the concentration of NK cells in the frozen stock solution be 2 × 107-4×107Precooling each cell/mL, and transferring the cells into a freezing tube;
cooling the freezing tube to-90 ℃ for freezing;
and after the freezing storage is finished, transferring the freezing storage tube into liquid nitrogen for freezing storage.
Preferably, the aforementioned use, wherein in use, the vial is removed from liquid nitrogen, thawed in a water bath at 37-40 ℃ and introduced directly into the human body.
By the technical scheme, the NK cell frozen stock solution and the preparation method and the application thereof provided by the invention at least have the following advantages:
1. the NK cell frozen stock solution provided by the invention is composed of 0.9% sodium chloride injection, human serum albumin, glycerol, vitamin C and trehalose, does not contain dimethyl sulfoxide and animal-derived components except for human bodies, and has the advantages of high recovery rate, simple components, good stability and the like.
2. The NK cell frozen stock solution provided by the invention can promote the proliferation of cells, improve the activity of the cells and greatly improve the survival rate of the cells after the cells are recovered through the combined action of several components, namely human serum albumin, glycerol, vitamin C and trehalose; after the cell freezing solution is frozen, the survival rate of the cell freezing solution does not change obviously. The experimental results of the embodiment show that the total cell concentration, the viable cell concentration, the cell survival rate, the average diameter, the average roundness and the agglomeration rate are not significantly changed before freezing, after 1-month thawing and 3-month thawing, and the NK cell freezing solution can effectively protect NK cells in the freezing process.
3. The NK cell frozen stock solution provided by the invention only contains 0.9% sodium chloride injection, human serum albumin, glycerol, vitamin C and trehalose, expensive raw materials do not need to be purchased, and the cost is economic; meanwhile, the components can enter human tissues, and the content of the components is controlled within a safe range of human dosage, so that the NK cells frozen and stored by the NK cell frozen and stored solution can be directly input into a human body after being melted without being input into the human body after being processed by complex centrifugation, cleaning and the like the cells frozen and stored in the prior art, the cells can be prevented from being damaged due to centrifugation, and the method is economical in cost, convenient, rapid and efficient and free of safety risk.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a flow chart of NK cell surface marker antigens of the present invention;
FIG. 2 is a diagram of NK cells before cryopreservation of NK cell cryopreservation solution No. 1 in an example of the present invention;
FIG. 3 is a diagram showing NK cells after thawing of NK cell cryopreservation solution No. 1 after cryopreservation for 1 month in the examples of the present invention;
FIG. 4 is a diagram showing NK cells obtained after thawing of NK cell cryopreservation solution No. 1 after cryopreservation for 3 months in the examples of the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention for achieving the predetermined objects, the following detailed description will be given to the NK cell cryopreserved solution and the preparation method thereof and the application thereof, and the specific implementation, structure, characteristics and effects thereof according to the present invention with reference to the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
One embodiment of the present invention provides an NK cell cryopreservation solution, comprising: 0.9% sodium chloride injection, human serum albumin, glycerol, vitamin C and trehalose.
The NK cell frozen stock solution is generally prepared for use or refrigerated storage.
In the embodiment, the human serum albumin is used as an impermeable cryoprotectant, so that the content of free water in the solution is reduced, and the formation of ice crystals is reduced; meanwhile, because of the large molecular weight, the concentration of electrolyte in the solution is reduced, thereby reducing solute damage and greatly improving the survival rate of cells;
the glycerol is used as the osmotic freezing protective agent, can permeate into cells before the cell frozen suspension is completely solidified, generates a certain molar concentration inside and outside the cells, and reduces the concentration of electrolyte in unfrozen solution inside and outside the cells, thereby protecting the cells from being damaged by high-concentration electrolyte. The water in the cells can not be excessively exosmosed, and the cells are prevented from being excessively dehydrated and shrunk.
The vitamin C can effectively inhibit cell oxidation, maintain the activity of cells and has no harm to human bodies.
Trehalose is a non-reducing sugar formed by two glucose molecules through a, a,1, 1-glycosidic bonds, has very stable self-property, can form a protective film on the surface of a cell, effectively protects protein molecules from deformation, and maintains the life process and biological characteristics of a living body. Because the main body of the NK cell frozen stock solution contains a large amount of human serum albumin, the survival rate of the cells after frozen stock can be greatly improved only by assisting human autologous serum with a very small amount (less than 1%) of trehalose, and the cost is more economic.
The components are combined integrally, and the lack of any component can not achieve good effect of freezing and storing NK cells.
The components in the embodiment are all selected from medical injection grades, are clear, can be directly input into a human body, are convenient and quick, have no safety risk, and have good cryopreservation effect, and the state of the cells after cryopreservation recovery is basically close to the state before cryopreservation.
In some embodiments, the NK cell cryopreservation comprises, per 100 parts by volume: 10-50 parts of 0.9% sodium chloride injection, 40-88 parts of human serum albumin, 1-20 parts of glycerol and 1-20 parts of vitamin C;
the NK cell cryopreservation solution further comprises: 10-60mg/mL of trehalose.
Further, every 100 parts of the NK cell freezing medium comprises the following components in parts by volume: 30-50 parts of 0.9% sodium chloride injection, 40-60 parts of human serum albumin, 1-10 parts of glycerol and 1-10 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 30-50 mg/mL.
Preferably, the NK cell frozen stock solution comprises the following components in parts by volume per 100 parts of the NK cell frozen stock solution: 40-50 parts of 0.9% sodium chloride injection, 40-50 parts of human serum albumin, 6-15 parts of glycerol and 1-10 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 30-50 mg/mL.
Preferably, the NK cell frozen stock solution comprises the following components in parts by volume per 100 parts of the NK cell frozen stock solution: 20-40 parts of 0.9% sodium chloride injection, 50-70 parts of human serum albumin, 5-15 parts of glycerol and 5-15 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 40-60 mg/mL.
Preferably, the NK cell frozen stock solution comprises the following components in parts by volume per 100 parts of the NK cell frozen stock solution: 20-30 parts of 0.9% sodium chloride injection, 40-60 parts of human serum albumin, 6-10 parts of glycerol and 10-20 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 20-40 mg/mL.
In some preferred embodiments, the NK cell cryopreservation comprises, per 100 parts by volume: 50 parts of 0.9% sodium chloride injection, 40 parts of human serum albumin, 6 parts of glycerol and 4 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 50 mg/mL.
In other preferred embodiments, the NK cell cryopreservation comprises, per 100 parts by volume: 40 parts of 0.9% sodium chloride injection, 50 parts of human serum albumin, 4 parts of glycerol and 6 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 20 mg/mL.
The invention also provides a preparation method of the NK cell frozen stock solution, which comprises the following steps:
dissolving trehalose in 0.9% sodium chloride injection, adding human serum albumin, glycerol and vitamin C into the solution obtained by dissolving, and uniformly stirring to obtain a mixed solution;
sterilizing the mixed solution to obtain an NK cell frozen stock solution; every 100 parts of the NK cell frozen stock solution comprises the following components in parts by volume: 10-50 parts of 0.9% sodium chloride injection, 40-88 parts of human serum albumin, 1-20 parts of glycerol and 1-20 parts of vitamin C;
the NK cell cryopreservation solution further comprises: 10-60mg/mL of trehalose.
In the step, except that the trehalose is granules, the other components are all liquid, so the trehalose is dissolved in 0.9% sodium chloride injection and then is mixed with human serum albumin, glycerol and vitamin C.
In some embodiments, the sterilization employs a bacterial filter having a filter size of 0.22 μm.
The invention also provides another embodiment of the NK cell frozen stock solution for the cryopreservation of NK cells.
In some embodiments, the method of cryopreserving NK cells with the NK cell cryopreserving solution comprises the steps of:
adding 0.9% sodium chloride injection into NK cells to prepare cell suspension;
centrifuging the cell suspension, removing supernatant, adding the NK cell frozen stock solution, and adjusting cell concentration to make the concentration of NK cells in the frozen stock solution be 2 × 107-4×107Precooling each cell/mL, and transferring the cells into a freezing tube;
cooling the freezing tube to-90 ℃ for freezing;
and after the freezing storage is finished, transferring the freezing storage tube into liquid nitrogen for freezing storage.
Further, when in use, the freezing tube is taken out from liquid nitrogen, melted in water bath at 37-40 ℃ and directly infused into human body.
The NK cell frozen stock solution provided by the invention only contains 0.9% sodium chloride injection, human serum albumin, glycerol, vitamin C and trehalose, expensive raw materials do not need to be purchased, and the cost is economic; meanwhile, the components can enter human tissues, and the content of the components is controlled within a safe range of human dosage, so that NK cells after the NK cell frozen stock solution is frozen can be directly input into a human body after being melted, and the cells after being frozen in the prior art can be input into the human body after being processed through complex centrifugation, cleaning and the like, so that the cells are prevented from being damaged due to centrifugation, the cost is low, the use is convenient, fast and efficient, the components are all medical injection levels, the components are clear, and the input dosage of the human body is met, therefore, the NK cell frozen stock solution can be directly input into the human body, and the injection is convenient, fast and free of safety risks.
The present invention will be further described with reference to the following specific examples, which should not be construed as limiting the scope of the invention, but rather as providing those skilled in the art with certain insubstantial modifications and adaptations of the invention based on the teachings of the invention set forth herein.
The invention will be described herein below by means of specific examples. Unless otherwise specified, the methods can be performed according to the methods listed in the experimental manuals such as "guide to cell experiments" (scientific Press, Beijing, China, 2001) and "immunoassay technology" (scientific Press, Beijing, China, 1991) which are familiar to those skilled in the art, and the references cited therein. Wherein, the used reagent raw materials are all commercial products and can be purchased and obtained through public channels.
The culture process of NK cells is as follows: 50ml of peripheral blood (donated by armed police department) was drawn and diluted with sterile PBS (dilution ratio 1:1), 20ml of human lymphocyte isolate (commercially available) was added to each 50ml centrifuge tube, and 50ml of diluted peripheral blood was slowly added along the tube wall so as not to disrupt the aqueous phase of the isolate, followed by centrifugation at 3000 rpm for 30 minutes. Carefully sucking the upper layer of plasma out of another 50ml centrifuge tube, inactivating at 56 deg.C for 20min (mainly in 56 deg.C water bath, and extinguishing fire to remove heat sensitive substances such as complement in serum and inactivate mycoplasma in serum). The light yellow mononuclear cell layer was aspirated into a 15ml centrifuge tube, washed 3 times with sterile PBS (commercially available), and the supernatant was discarded, and the pellet was peripheral blood mononuclear cells. Resuspending mononuclear cells with 40ml NK culture medium (purchased from market), adding inactivated autologous serum and recombinant human IL-2 (purchased from market), mixing, and adding 75cm2The mixture was placed in a culture flask at 37 ℃ and 5% (v/v) CO2And (5) standing and culturing in a cell culture box. Day 3, NK broth (to supplement nutrients required for NK cell culture) and recombinant human IL-2 (mainly, IL-2) were supplementedActivating NK cells and enhancing the therapeutic effect of NK cells), and distributing the culture solution in the culture flask into two large culture bags on average to continue culturing, and supplementing 100ml of NK culture solution and recombinant human IL-2 into each large culture bag every 3-4 days. The culture is carried out for about 14 days in total, all operations are carried out in a standard GMP laboratory, and quality monitoring including bacterial and fungal contamination detection, cell endotoxin detection, cell viability, cell flow detection, cell killing detection and the like is carried out.
NK cells were detected at 14 days of culture:
1. detection of NK cells for bacteria and fungi: and (4) negativity.
2. And (3) detecting endotoxin in NK cells: less than 0.05 EU.
3. And (3) detecting the NK cell survival rate: 96.1 percent.
4. NK cell flow detection, main detection items are CD3-/CD16+ CD56+ NK: 92.88% (see fig. 1).
5. NK cell killing detection: taking NK cells (density 1X 10)6One/ml) and K562 cells (density 1X 10)5Pieces/ml) are mixed according to the effective target ratio of 40:1, cultured in a 96-well plate, the total volume of each well is 2ml, meanwhile, an effect cell hole, a target cell hole and a blank control hole are arranged, an experimental hole is an NK cell hole, and the mixture is placed at 37 ℃ and 5% (v/v) CO2After culturing for 24h in a cell culture box, adding 20 mu L of CCK-8 reagent, placing the mixture in the cell culture box, incubating for 4h, and measuring the absorbance (A) value at 450 nm. Calculating the killing property of the NK cells according to the formula: killing rate ═ 1-9 (experimental well a value-effector cell well a value)/target cell well a value]X 100%. Calculated killing rate: 91.5 percent.
Examples
The embodiment provides a preparation method of an NK cell frozen stock solution, which comprises the following steps:
(1) respectively measuring 0.9% sodium chloride injection, human serum albumin, glycerol, vitamin C and trehalose according to the formula amount;
(2) dissolving trehalose in 0.9% sodium chloride injection;
(3) adding human serum albumin, glycerol and vitamin C into the solution obtained in the step (2), and uniformly stirring to obtain a mixed solution;
(4) sterilizing the mixed solution obtained in the step (3) by using a bacterial filter with the diameter of 0.20-0.25 mu m to obtain NK cell frozen stock solution;
preparing NK cell frozen stock solutions No. 1-12 listed in Table 1 according to the above preparation method;
the cryopreservation test was performed on NK cell cryopreservation solution Nos. 1 to 12 prepared in this example:
the NK cells are cryopreserved by using the NK cell cryopreserving liquid prepared in the embodiment, and the method comprises the following steps of:
1) observing the cultured NK cells by a microscope to prepare a cell suspension;
2) adding the cell suspension into a centrifuge tube, and centrifuging for 5min at the rotating speed of 2000 rpm;
3) centrifuging, removing supernatant, adding 45mL physiological saline, sucking one drop of cell, and counting 3.5 × 107The cell viability of each cell is 95 percent, and the cells are centrifuged for 5min under the rotating speed condition of 2000 rpm;
4) the normal saline was discarded, and NK cell frozen stock solution No. 1-12 prepared in this example was added thereto, and precooled at 4 ℃ to adjust the cell concentration to 2X 107-4×107Each cell/mL of cryopreservation solution, and about 1.5mL of cell solution is added into each cryopreservation tube;
5) putting the freezing tube into a program cooling instrument, starting a prefabrication program, and completing freezing in about 1.5 hours;
6) and transferring the cryopreservation tube into liquid nitrogen for cryopreservation, wherein the cryopreservation time is 1 month and 3 months respectively.
7) Cell recovery: and (3) after freezing for a specified time, taking out the freezing tube from the liquid nitrogen, quickly putting the tube into a water bath kettle at 37-40 ℃, taking out the tube from the water bath kettle when the liquid level of the cells is required to be completely immersed below the water level and the tube opening of the freezing tube is higher than the water level, quickly shaking to melt the cells within 1min, and taking out the tube from the water bath kettle when ice crystals with the size of rice grains exist in the liquid. And adding the recovered cells into a corresponding culture medium for culture observation. The cells were observed with a microscope the next day after recovery, and with a 4X objective, the cells were homogeneous in morphology and did not show abnormal morphology in each field.
8) Cell count after recovery: 20 microliter of the recovered cell suspension is sucked, 20 microliter of prepared 0.08 percent trypan blue working solution is added, the mixture is gently mixed to prevent bubbles from generating, 20 microliter of the prepared working solution is sucked, the mixture is added into a special counting plate, a Countstar cell counter is used for detecting the cell number and the survival rate, the cell number and the survival rate are repeated twice, and the experimental results are shown in table 2. The cells were observed with a microscope, and as shown in FIG. 2, FIG. 3 and FIG. 4, they are a photograph of NK cells before cryopreservation in NK cell cryopreservation solution No. 1, a photograph of NK cells after 1 month recovery from cryopreservation and a photograph of NK cells after 3 months recovery from cryopreservation in examples of the present invention, respectively.
TABLE 1 formulation No. 1-12 of NK cell freezing medium in this example
TABLE 2 Experimental results of NK cell cryopreservation of NK cell numbers 1 to 12 in this example
The experimental results in table 2 show that, before cryopreservation, after 1-month resuscitation and 3-month resuscitation, the total cell concentration, the viable cell concentration, the cell survival rate, the average diameter, the average roundness and the agglomeration rate are not significantly changed, which indicates that the NK cell cryopreservation solution of the embodiment can effectively protect NK cells in the cryopreservation process, and the NK cells after cryopreservation can be directly infused into a human body after being thawed.
The NK cell cryopreservation solution provided by the embodiment of the invention has the advantages of low toxic and side effects, simple components and low cost, maintains high cell recovery and survival rate, does not influence the characteristics of cells and the proliferation capacity of the cells, and is beneficial to rapid application and popularization of a cell cryopreservation technology.
Comparative example
NK cell frozen stocks No. 13 to No. 17 listed in Table 3 were prepared according to the preparation methods of the above examples, and No. 13 to No. 17 of the comparative examples were each reduced in one component as compared with the examples;
the cryopreservation test was carried out on NK cell cryopreserved solution No. 13-17 prepared in comparative example according to the test methods of the above examples, and the experimental results are shown in Table 4.
TABLE 3 formulation No. 13-17 of NK cell frozen stock solution for comparative example
| Numbering | Sodium chloride | Human serum albumin | Glycerol | Vitamin C | Trehalose |
| Number 13 | / | 90mL | 6mL | 4mL | 5g |
| Number 14 | 90mL | / | 6mL | 4mL | 5g |
| Number 15 | 50mL | 40mL | / | 10mL | 5g |
| Number 16 | 50mL | 40mL | 10mL | / | 5g |
| Number 17 | 50mL | 40mL | 6mL | 4mL | / |
TABLE 4 experimental results of NK cell cryopreservation of NK cell cryopreserved solution No. 13-17 of comparative example
As can be seen from the experimental results in Table 4, the total cell concentration, viable cell concentration, cell survival rate, average diameter, average roundness and agglomeration rate were all significantly reduced as compared with the examples before and after 1 month recovery from cryopreservation, indicating that the components of the NK cell cryopreservation solution of this example are an integral whole, and the components supplement each other and are all indispensable.
In the foregoing embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. An NK cell cryopreservation liquid, which is characterized by consisting of 0.9% sodium chloride injection, human serum albumin, glycerol, vitamin C and trehalose.
2. The NK cell cryopreservation solution of claim 1, comprising per 100 parts by volume of the NK cell cryopreservation solution: 10-50 parts of 0.9% sodium chloride injection, 40-88 parts of human serum albumin, 1-20 parts of glycerol and 1-20 parts of vitamin C;
the NK cell cryopreservation solution further comprises: 10-60mg/mL of trehalose.
3. The NK cell cryopreservation solution of claim 1,
every 100 parts of the NK cell frozen stock solution comprises the following components in parts by volume: 30-50 parts of 0.9% sodium chloride injection, 40-60 parts of human serum albumin, 1-10 parts of glycerol and 1-10 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 30-50 mg/mL.
4. The NK cell cryopreservation solution of claim 1,
every 100 parts of the NK cell frozen stock solution comprises the following components in parts by volume: 50 parts of 0.9% sodium chloride injection, 40 parts of human serum albumin, 6 parts of glycerol and 4 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 50 mg/mL.
5. The NK cell cryopreservation solution of claim 1,
every 100 parts of the NK cell frozen stock solution comprises the following components in parts by volume: 40 parts of 0.9% sodium chloride injection, 50 parts of human serum albumin, 4 parts of glycerol and 6 parts of vitamin C;
the NK cell cryopreservation solution further comprises: trehalose 20 mg/mL.
6. A preparation method of NK cell frozen stock solution is characterized by comprising the following steps:
dissolving trehalose in 0.9% sodium chloride injection, adding human serum albumin, glycerol and vitamin C into the solution obtained by dissolving, and uniformly stirring to obtain a mixed solution;
sterilizing the mixed solution to obtain an NK cell frozen stock solution;
every 100 parts of the NK cell frozen stock solution comprises the following components in parts by volume: 10-50 parts of 0.9% sodium chloride injection, 40-88 parts of human serum albumin, 1-20 parts of glycerol and 1-20 parts of vitamin C;
the NK cell cryopreservation solution further comprises: 10-60mg/mL of trehalose.
7. The method for producing an NK cell cryopreserving solution according to claim 6,
the sterilization adopts a bacterial filter, and the filtering size of the bacterial filter is 0.22 mu m.
8. Use of the NK cell cryopreservation solution of any one of claims 1 to 6 for cryopreservation of NK cells.
9. The use according to claim 8, wherein the method of cryopreserving NK cells using said NK cell cryopreserving solution comprises the steps of:
adding 0.9% sodium chloride injection into NK cells to prepare cell suspension;
centrifuging the cell suspension, removing supernatant, adding the NK cell frozen stock solution, and adjusting cell concentration to make the concentration of NK cells in the frozen stock solution be 2 × 107-4×107Precooling each cell/mL, and transferring the cells into a freezing tube;
cooling the freezing tube to-90 ℃ for freezing;
and after the freezing storage is finished, transferring the freezing storage tube into liquid nitrogen for freezing storage.
10. Use according to claim 9,
when in use, the freezing tube is taken out from liquid nitrogen, melted in water bath at 37-40 ℃ and directly transfused into a body.
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