WO2015103792A1

WO2015103792A1 - Method for preserving a synergist of lymphocyte in vitro culture

Info

Publication number: WO2015103792A1
Application number: PCT/CN2014/070543
Authority: WO
Inventors: 张明杰; 伍震懿
Original assignee: 深圳市汉科生物工程有限公司
Priority date: 2014-01-13
Filing date: 2014-01-13
Publication date: 2015-07-16
Also published as: CN105051183B; CN105051183A

Abstract

Provided is a method for preserving a synergist of a lymphocyte in vitro culture. The method comprises: suspending the synergist of a lymphocyte in vitro culture in a preservation solution to obtain a suspension of the synergist of the lymphocyte in vitro culture; and cryopreserving the suspension of the synergist of the lymphocyte in vitro culture at a predetermined temperature, wherein the predetermined temperature is -20°C to 4°C.

Description

Method for preserving lymphocyte in vitro culture synergist

Technical field

The present invention relates to the field of cell biology, and in particular to a method for preserving a synergistic agent for in vitro culture of lymphocytes. Background technique

The proliferation and differentiation of lymphocytes in vitro is regulated by a variety of factors. The most common regulators are soluble cytokines, various natural cells and genetically engineered cells. In the in vitro culture of cells, since solid-phase cytokines such as feeder cells or antigen-presenting cells (APCs) regulate lymphocytes more strongly than free cytokines, servated cells are currently used or APC promotes the proliferation and activation of lymphocytes.

In practical applications, in order to not affect the proliferation and activation of lymphocytes in vitro, it is necessary to inactivate the cells and APC. The current conventional methods include gamma ray irradiation, UV irradiation, electroshock, and mitomycin chemical treatment. However, the above methods are disadvantageous in that they cannot be prepared in a large amount, the equipment is expensive, the operation is unsafe, the degree of inactivation is poor, the residual cells are damaged, or the cells are completely damaged.

Moreover, the spheroid cells or APC prepared by the above method are all cells, and in order to ensure their biological activities, they must be stored in a preservation solution containing dimethyl sulfoxide (DMSO) or glycerin, and a preservation solution containing sputum cells or APC is The -80 °C refrigerator can only be stored for a short period of time. The long-term storage can only be liquid nitrogen. This dosage form and storage conditions are not a problem for laboratories with good conditions, but they cannot be satisfied by ordinary clinical laboratories or cell clinical treatment. This preservation method has the disadvantages of transportation, high cost, and, in terms of cell activity, from the ultra-low temperature during storage to the thawing during use, the protein and the cytoskeleton are damaged, which seriously affects the activity of the product.

Therefore, the effective and safe preservation of lymphocyte in vitro synergists is a difficult problem in today's biology. Summary of the invention

The present invention aims to solve at least one of the above technical problems to some extent. To this end, an object of the present invention is to provide a method and preparation for preserving a lymphocyte in vitro culture synergist.

In the present invention, a spheroid cell or an antigen presenting cell is prepared by washing to prepare a cell empty shell. It was found that this cell empty shell was used as a synergistic agent for lymphocyte in vitro culture, and the activity of the original spheroid cells or APC was maintained. At the same time, it was found that the composition of the cell culture in vitro synergist is protein, and the protein is stored in a preservation solution containing glycerin, and can maintain activity for a long period of time at minus 20 degrees Celsius to minus 4 degrees Celsius; or by adding human serum albumin. The lyophilized powder is prepared by a freeze-drying process, and the lyophilized powder is stored at 4 degrees Celsius for long-term storage. At the same time, the lyophilized powder type and the liquid dosage type lymphocyte in vitro synergist of the present invention can be transported at 4 ° C, which is convenient and inexpensive, and therefore provides protection for large-scale applications.

In one aspect of the invention, the invention provides a method of preserving a lymphocyte in vitro culture synergist. According to an embodiment of the present invention, the method comprises: suspending a lymphocyte in vitro culture synergist in a preservation solution to obtain a drench The bain cells are cultured in vitro with a synergist suspension; the lymphocyte in vitro culture synergist suspension is cryopreserved at a predetermined temperature, wherein the predetermined temperature is from minus 20 degrees Celsius to minus 4 degrees Celsius. The inventors have surprisingly found that the method of the present invention can effectively preserve lymphocyte in vitro culture synergist, and the method has low storage conditions, and the lymphocyte in vitro culture synergist activity remains high and the activity retention time is long. °C transportation, convenient and cheap.

The method for preserving a lymphocyte in vitro culture synergist according to an embodiment of the present invention may further have the following additional technical features:

According to an embodiment of the present invention, the lymphocyte in vitro culture synergist is a cell prepared by using a feeder cell or APC.

According to an embodiment of the present invention, the preservation solution is a RPMI 1640 culture solution containing 5-15% by volume of glycerol.

According to an embodiment of the present invention, the method further comprises: pre-drying the lymphocyte in vitro culture synergist suspension before the cryopreservation of the lymphocyte in vitro culture synergist suspension at a predetermined temperature, Wherein, the preservation solution is a RPMI 1640 culture solution containing 1-10% by volume of human serum albumin.

According to an embodiment of the present invention, the cell vacant shell is prepared by subjecting the cells to a washing treatment to obtain washed cells; and lysing the washed cells to obtain a cell empty shell, wherein The surface of the cell has a cytokine capable of promoting lymphocyte proliferation and differentiation. The inventors have found that the cell shell can be obtained quickly and efficiently by the method of the present invention, and the method is simple in operation, easy to control, and easy to mass-produce.

It should be noted that the surface of the cell has a cytokine capable of promoting lymphocyte proliferation, and the cell itself can express the cytokine on its surface, or can be genetically engineered (for example, transient transfection or stabilization). Expression) The cytokine is expressed on the surface of the cell, and the cytokine may also be adsorbed or cross-linked on the cell surface. Further, the cells may be primary cells (e.g., peripheral blood mononuclear cells) or passaged cells (e.g., K562 cells).

According to an embodiment of the present invention, the washing treatment further comprises: suspending the cells in an isotonic solution to obtain a cell suspension; and centrifuging the cell suspension to obtain the washed cells.

According to an embodiment of the invention, the isotonic solution is pre-cooled to 4 degrees Celsius before the cells are suspended in an isotonic solution.

According to an embodiment of the invention, the isotonic solution is an isotonic phosphate buffer (PBS) having a pH of 7.4.

According to an embodiment of the present invention, the lysing treatment further comprises: suspending the washed cells in a hypotonic solution according to a predetermined volume ratio, and allowing the obtained cell suspension to stand for 2 hours to obtain a cell lysate; The cell lysate is subjected to centrifugation to obtain the cell empty shell.

According to an embodiment of the invention, the predetermined volume ratio is 1:40. Thereby, it is advantageous to increase the efficiency of cell lysis. According to an embodiment of the invention, the hypotonic solution is pre-cooled to 4 degrees Celsius before suspending the washed cells in a hypotonic solution.

According to an embodiment of the invention, the hypotonic solution is a hypotonic Tris hydrochloric acid buffer. Thereby, it is beneficial to increase the cells The efficiency of the lysis.

According to an embodiment of the present invention, the cytokine capable of promoting lymphocyte proliferation is at least one selected from the group consisting of IL-4, IL-7, IL-15, IL-2K CD19, CD64, CD86 and 4-1BBL.

In another aspect of the invention, the invention provides a method of preserving a lymphocyte in vitro culture synergist. According to an embodiment of the present invention, the method comprises: suspending a lymphocyte in vitro culture synergist in a preservation solution to obtain a lymphocyte in vitro culture synergist suspension; and performing the lymphocyte in vitro culture synergist suspension Freeze-drying to obtain a lyophilized powder type lymphocyte in vitro culture synergist; storing the lyophilized powder type lymphocyte in vitro culture synergist at minus 20 degrees Celsius to minus 4 degrees Celsius, wherein the preservation solution is RPMI 1640 medium containing 1% by volume to 10% by volume of human serum albumin.

In one embodiment of the invention, the cell empty shell is prepared by the following steps: a) cell empty shell preparation reagent: 1) isotonic PBS: 0.15 mol/L sodium chloride, pH 7.4; 2) hypotonic Tris hydrochloric acid Buffer: 10 mmol/L, pH 7.4. b) Operation steps: 1) Collection and washing of cells: The cells are suspended in about 3 times the amount of pre-diluted pH 7.4 isotonic PBS, centrifuged at 600 ° C (1500 rpm) for 10 minutes, the supernatant is removed, and the washing is repeated 1- 3 times. 2) Lysis and washing of cell membrane: To the washed cells, add about 10 mmol/L hypotonic Tris-HCl buffer to the ratio of 1:40 by volume, while stirring slowly, set about 4 ° C in the refrigerator. After 2 hours, it was completely lysed; then, it was centrifuged at 12,000 g (9000 rpm) for 10 minutes at 4 ° C to precipitate a cell membrane. The washing was repeated, centrifuged 3-5 times, and finally the cell empty shell was obtained. 3) Dilute and dispense with 2 × 10 ⁷ /ml cell empty shells in a refrigerated storage solution, -20 degrees Celsius to 4 degrees Celsius in refrigerator, or use 2 x 10 ⁷ /ml cell empty shell with cryoprotectant The concentration is diluted and dispensed, and after lyophilization, the refrigerator is stored in a refrigerator at 4 ° C.

In order to verify the product form of the lymphocyte in vitro culture synergist of the present invention and the product stability thereof, the present invention also employs a method of activating and activating NK cells from peripheral monocytes (PBMC) as an example. Those skilled in the art will appreciate that activated CTLs and Tregs can be amplified in a similar manner using the corresponding cell vesicles (servo cells or APCs). Specific method steps: 1) Preparation of autologous plasma: 50 ml of anticoagulated peripheral blood, 700 G, 20 min at room temperature; aspirate plasma, placed in a water bath at 56 ° C, 30 min; then stand at 4 ° C for 15 min; finally 4 ° C, 900G, centrifuged for 30min, taken from autologous plasma at 4 ° C for storage. 2) The cell layer after taking the plasma was added to D-PBS and mixed, and the PBMC was separated by lymphocyte separation solution 800G for 20 minutes; 3) One T175 culture flask was selected, and 40 ml of lymphocyte complete culture solution was added to the separated PBMC (containing about 200 IU/ml IL-2, autologous plasma 1-10%, gentamicin 80 U/ml RPMI 1640) was made into a cell suspension (about 5 X 10 ⁶ lymphocytes) and added to the T175 flask; Add 2x10 ⁷ cell empty shells, and store them in saturated humidity, 37 ° C, 5.0% [0 ₂ incubator; 4) On the fourth day of the culture, add about 40 ml of lymphocyte complete medium. 5) Transfer the cells in the T175 flask to the culture bag about the seventh day of the culture, add the complete lymphocyte culture solution to about 400 ml, and add about 8 X 10 ⁷ fronts. The cell is empty; 6) the culture is passaged into two culture bags on the tenth day of the culture, wherein each culture bag contains about 640 ml of the complete lymphocyte culture solution; The culture product was collected around the twelfth day of the culture to obtain NK cells. Or if you need a larger amount of NK cells, add the culture solution to 1280 mM. l / bag; 8) On the twelfth day of the culture, transfer to two culture bags, add lymphocyte complete medium to each about 850 ml / bag; 9) around the 16th day of the culture Approximately 500 ml of a total of approximately 1000 ml of cell suspension was taken from each of the two culture bags, and the cells were collected and returned to the patient. At the same time, add about 150 ml/bag of fresh lymphocyte complete medium; 10) About 1000 ml of cell suspension is left around the 18th day of the culture, and the cells are collected and returned to the patient. It will be understood by those skilled in the art that NK cells can be cultured for different lengths of time depending on the number of NK cells required.

In order to verify the product application effect of the dosage form of the spheroid cell or APC of the present invention and the preservation method thereof, the cytotoxicity test method is also provided in the embodiment of the present invention, and the specific method steps are as follows: 1) one tube effector cell is recovered one day before the assay, for example NK cells were cultured in RPMI 1640 complete medium (RPMI 1640 medium containing 10% fetal bovine serum, 80 U/ml gentamicin); 2) One cell line was used as a target cell for each assay. Need 6x l0 ⁵ effector cells and target cells 3x l0 ^5; 3) with complete RPMI 1640 diluted Calcein-AM, was prepared CAM; 4) the lx lO ⁶ target cells were resuspended in 1 mL CAM. Incubate at 37 °C for 1 hour and shake at the right time. It was then washed twice with RPMI 1640 complete medium and centrifuged for 5 min at 1200 rpm. Count, adjust the concentration to lx lO ⁵ /mL; 5) Dilute the effector cells to lx lO ⁶ / mL, set 3 wells on the U-bottom 96-well cell culture plate, add 200 uL of target cells per well, corresponding effect target Proportion (E:T) 10:1; 6) Add 100 μL of 2% Triton X-100 to the largest release well, and add 100 μL of complete culture to the remaining wells. 7) The effector cells were diluted 5 times. The final dilution ratio (E:T) was 0.3125:1; 8) 100 μΐ target cells per well and 100 g centrifugation for 1 min to guide cell contact. Incubate for 4 hours at 37 °C 5% C0 ₂ float tank; 9) Gently pipe the cells with a 100 μ sampler to suspend the released calcein; centrifuge at 100 g for 5 min to pellet the cells. Gently pipette the supernatant 100 and transfer to a new plate to prevent foaming. If foam is formed, use a needle to pierce; 10) read the plate with a fluorescence plate reader (excitation light 485 nm, emission 530 nm); 11) Calculate the specific cytotoxic percentage: [(test group - natural release group) / (Maximum release group - natural release group)] χΐοο.

In order to better explain the effect of different preservation preparations, in the embodiment of the present invention, the preparation method is described by taking K15-41BBL spheroid cells as an example; K15-41BBL is human IL- 15 and 4-1BBL expressed cell lines on the surface of K562 cells. We prepared spheroid cells by gamma irradiation treatment, and prepared them into cell empty shells according to our invention, and formulated them into different dosage forms; respectively, at 4 ° C, -20 ° C, and -80 ° The C refrigerator was chilled for a different time and then amplified to activate the NK cells in the activated PBMC by detecting the amplification activation effect. DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the effect of a synergistic agent of different dosage forms of lymphocytes in vitro on the expansion and activation of NK cells in PBMC according to Examples 1 and 2 of the present invention;

Fig. 2 is a graph showing the killing effect of NK cells obtained against K562 cancer cells according to Example 3 of the present invention. detailed description

The invention provides a method for preserving a lymphocyte in vitro culture synergist, including a preparation method and application thereof. this invention The lymphocyte in vitro culture synergist is a cell empty shell with an active factor; the preservation method includes two dosage forms of a water agent and a dry powder, specifically RPMI 1640 water hydrate containing 5-15 vol% glycerol [J, and A lyophilized powder of RPMI 1640 containing 1-10% human serum albumin is exemplified. The embodiments of the present invention are described in detail below, and are not to be construed as limiting the invention. For example, in the following examples, a cryopreservation solution is prepared using glycerin, but those skilled in the art will appreciate that other aqueous in vitro culture synergists can be prepared using other materials that promote cryopreservation and are suitable for cell culture. In the present invention, 5-15% by volume of glycerol is an optimum concentration, but it will be understood by those skilled in the art that concentrations outside this range can also be used. As another example, human serum albumin is used as a lyoprotectant in the following examples, but it will be understood by those skilled in the art that any other material having a lyophilization protective effect and suitable for cell culture can also be used to prepare a powder type lymphocyte. The cell culture in vitro synergist. In the present invention, 1-10% of human serum albumin concentration is an optimum concentration, but those skilled in the art will understand that concentrations outside this range may also be used; further, in the following examples, in the preparation of refrigerated Both the preservation solution and the cryoprotectant are added with RPMI 1640, but those skilled in the art will appreciate that no other materials may be added, or any other suitable material may be added. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in the art or in accordance with the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained commercially.

Example 1

Preparation of water-based NK cell in vitro culture synergist and its test conditions

Cell empty shells were prepared using K15-41BBL according to the following procedure, wherein K15-41BBL is a cell line prepared by expressing human IL-15 and 4-1BBL on the surface of K562 cells. details as follows:

K15-41BBL cells were suspended in 3-fold amount of isotonic PBS pre-cooled to 4 ° C, pH 7.4, centrifuged at 4 ° C, 1500 rpm for 10 minutes, the supernatant was removed, and washing was repeated 1-3 times. The washed K15-41BBL cells were obtained; then the washed K15-41BBL cells were added to a hypotonic Tris-HCl buffer pre-cooled to 4 ° C and a concentration of 10 mmol/L at a ratio of 1:40 by volume. Stir slowly, then, the resulting mixture was allowed to stand in a refrigerator at 4 ° C for 2 hours to completely lyse the cells; then, centrifuge at 10 ° C, 9000 rpm for 10 minutes to precipitate the cell shell, further Repeat washing and centrifugation 3-5 times to obtain K15-41BBL cell empty shell, namely K15-41BBL-empty shell.

Then, at a concentration of lxlO ⁷ /ml, the K14-41BBL cell empty shell suspension was suspended in RPMI 1640 cryopreservation solution containing no glycerol, containing 5% by volume, 10% by volume, and 15% by volume of glycerol, which was lymph In vitro culture synergist; stored at 4 degrees Celsius, minus 20 degrees Celsius, and minus 80 degrees Celsius for one day, one month, six months, one year, two years, etc., and then tested for the function of activating NK cells in activated PBMC At the same time, the killing activity of the obtained NK cells against K562 cancer cells was tested.

Example 2

Preparation and test conditions of lyophilized powder type NK cell in vitro culture synergist

A K15-41BBL-shell was prepared as in Example 1. Press K15-41BBL at a concentration of lxlO ⁷ /ml The cell empty shell pellet was suspended in RPMI 1640 lyophilized solution containing human serum albumin, containing 1% by volume, 5% by volume, and 10% by volume of Human serum Albumin (HAS), and then lyophilized. It is a lyophilized powder type lymphocyte in vitro culture synergist; it is stored at 4 degrees Celsius, minus 20 degrees Celsius, and minus 80 degrees Celsius for one day, one month, half year, one year, two years, etc., and then tested for amplification. The function of NK cells in PBMC was activated, and the killing activity of the obtained NK cells against K562 cancer cells was also tested.

Example 3

Amplification and activation of NK cells in PBMC by different dosage forms of lymphocyte in vitro culture synergists preserved under different conditions and their results

The NK cell expansion in PBMC was stimulated by K15-41BBL-empty shells prepared in Examples 1 and 2 with different storage time and different dosage forms, and each experiment was repeated 3 times. The specific steps are as follows:

1) Preparation of autologous plasma: 50ml of anticoagulated peripheral blood, 700G, 20min at room temperature; aspirate the plasma, placed in a water bath at 56 ° C, 30 min; then stand at 4 ° C for 15 min; finally 4 ° C, 900 G, centrifuge for 30 min , taking autologous plasma

Store at 4 ° C for later use.

2) The cell layer after taking the plasma is added to D-PBS and mixed, and the PBMC is separated by the lymphocyte separation solution 800G for 20 minutes; 3) One T175 culture flask is selected, and 40 ml of lymphocyte complete culture solution is added to the separated PBMC (including approximately

200 IU/ml IL-2, autologous plasma 1-10% by volume, gentamicin 80U/ml) made into cell suspension (about 5 X 10 ⁶ lymphocytes), added to T175 flask; add 2x at the same time L0 ⁷ cell empty shells with different storage time and different dosage forms, cultured in saturated humidity, 37 ° C, 5.0% C0 ₂ incubator;

4) on the fourth day of the culture, add about 40 ml of lymphocyte complete medium;

5) Transfer the cells in the T175 flask to the culture bag about the seventh day of the culture, add the complete lymphocyte culture solution to about 400 ml, and add about 8×10 ⁷ different preservations. Cell empty shell of time and different dosage forms;

6) The culture is subcultured into two culture bags on the tenth day of the culture, wherein each culture bag contains about 640 ml of the complete lymphocyte culture solution;

7) On the twelfth day of the culture, the culture products were collected, and the effect of cell storage of different retention time and different dosage forms on the activation and activation of NK cells in PBMC was tested. The experimental results are shown in Figure 1.

From the results of Fig. 1, it can be seen that, in addition to the glycerin-free aqueous solution and the lyophilized powder without HAS, 5 vol%, 10 vol%, and 15 vol% glycerol were added as a cryopreservative, and 1 was added. 5% by volume, 5% by volume, and 10% by volume of BSA as a lyophilized powder of lyoprotectant, the K15-41BBL empty shell stimulated PBMC was well preserved at 4 ° C, -20 ° C, and -80 ° C. Amplify the function of activated NK cells. The glycerol-free water and the HAS-free lyophilized powder gradually decreased in function with the storage time.

Example 4

Cytotoxicity test and results of NK cells obtained from Example 3 against K562 cancer cells

K562 cancer cells were used as target cells, and in vitro, the lymphocytes preserved under different conditions were cultured in vitro in vitro. Stimulator stimulation, amplification of activated NK cells from PBMC as effector cells, cytotoxicity experiments, each experiment was repeated 3 times, the specific steps are as follows:

Dilute the fluorescent dye Calcein-AM with RPMI1640 complete medium (RPMI 1640 containing 10% fetal bovine serum, 80U/ml gentamicin) to prepare CAM solution, and suspend 1×10 ⁶ target cells in 1 mL. The CAM solution was incubated for 1 hour under saturated humidity, 37 ° C, and 5 vol% carbon dioxide, and shaken at the appropriate time during the culture, and then washed twice with RPMI 1640 complete medium, and centrifuged at 1200 rpm for 5 min. Count, adjust the concentration of target cells to 1 × 10 ⁵ /mL. The effector cells were diluted to a cell concentration of 1 X 10 ⁶ /mL.

Set 3 wells on a U-bottom 96-well cell culture plate, add 200 uL of target cells per well, corresponding to the target ratio (E:T) 10: 1, add 100 uL of 2% Triton X-100 to 1 well to complete The target cells were lysed as the maximum release positive control, and 100 μL of RPMI 1640 complete medium was added to the remaining 2 wells as a natural release negative control.

The effector cells were diluted 5 times, and the target ratio (E:T) of the last dilution was 0.3125:1.

According to the total number of effector cells in different concentrations, set the required number of wells to be tested in a 96-well plate, 100 target cells per well, centrifuge at 100g for 1 min, guide the cells to contact, and then place in saturated humidity, 37 ° C, 5 vol% [0 _{2 in a} float tank for 4 hours, then, gently pipette the cells with a 100-sampler to suspend the released calcein fluorescent dye, then centrifuge at 100 g for 5 min to pellet the cells and gently pipette them. After clearing the solution for 100 μΐ, transfer to a new plate and read the plate with a fluorescence plate reader (excitation light 485 nm, emission 530 nm). According to the formula kill rate = [(test group - natural release group) / (maximum release group - natural release group)] X 100, calculate the NK cells in PBMC after different storage time, different dosage forms of cell empty shell stimulation amplification Killing rate of K562 cells. The experimental results are shown in Figure 2.

It can be seen from the results in Fig. 2 that the NK cells in PBMC are highly cytotoxic to K562 cells after being stimulated by different storage time and different dosage forms of K15-41BBL-vacant, and there is no significant difference between them. .

Although there is no significant difference from the results of the cytotoxicity test, the total amount of NK cells obtained from different groups is different. Therefore, the water supplement with cryopreservative and the lyophilized powder with lyoprotectant are significantly better than the lyophilized powder. Added control group. In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms is not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. Further, those skilled in the art can combine and combine the various embodiments or examples described in the specification and the features of the different embodiments or examples without departing from the scope of the invention.

Although the embodiments of the present invention have been shown and described, it is understood that the above described embodiments are illustrative and are not to be construed as limiting the scope of the invention. The embodiments are subject to variations, modifications, substitutions and variations.

Claims

claims

1. A method for preserving lymphocyte culture enhancer in vitro, which is characterized by including:

Suspend the lymphocyte in vitro culture enhancer in the preservation solution to obtain the lymphocyte in vitro culture enhancer suspension;

The lymphocyte in vitro culture enhancer suspension is cryopreserved at a predetermined temperature,

Wherein, the predetermined temperature is minus 20 degrees Celsius to above zero 4 degrees Celsius.

2. The method according to claim 1, characterized in that the lymphocyte in vitro culture enhancer is a cell empty shell.

3. The method according to claim 1, characterized in that the preservation solution is RPMI 1640 containing 5-15% by volume glycerol.

4. The method according to claim 1, further comprising: prior to cryopreservation of the lymphocyte in vitro culture enhancer suspension at a predetermined temperature, pre-conditioning the lymphocyte in vitro culture enhancer suspension. The agent suspension is freeze-dried.

Wherein, the preservation solution is RPMI 1640 culture solution containing 1-10% human serum albumin by volume.

5. The method according to claim 2, characterized in that the empty cell shell is prepared by the following steps: washing the cells to obtain washed cells; lysing the washed cells , in order to obtain empty cell shells, wherein the surface of the cells has cytokines that can promote lymphocyte proliferation and differentiation.

6. The method according to claim 5, characterized in that the washing treatment further includes:

The cells are suspended in an isotonic solution to obtain a cell suspension; the cell suspension is centrifuged to obtain the washed cells.

7. The method according to claim 6, characterized in that, before suspending the cells in the isotonic solution, the isotonic solution is pre-cooled to 4 degrees Celsius.

8. The method according to claim 7, wherein the isotonic solution is an isotonic phosphate buffer with a pH of 7.4.

9. The method according to claim 5, characterized in that the cracking treatment further includes:

The washed cells were suspended in a hypotonic solution according to a predetermined volume ratio, and the resulting cell suspension was allowed to stand for 2 hours to obtain the cell lysate; the cell lysate was centrifuged to obtain the Empty cell shell.

10. The method according to claim 9, characterized in that the predetermined volume ratio is 1:40.

11. The method according to claim 9, characterized in that, before suspending the washed cells in the hypotonic solution, the hypotonic solution is pre-cooled to 4 degrees Celsius.

12. The method according to claim 11, characterized in that the hypotonic solution is hypotonic Tris hydrochloride buffer.

13. The method according to claim 5, further comprising: washing the empty cell shells. polyester treatment.

14. The method according to claim 5, wherein the cytokine capable of promoting lymphocyte proliferation and differentiation is selected from the group consisting of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4 -1 At least one of BBL.

15. A method for preserving lymphocyte culture enhancer in vitro, which is characterized by including:

The lymphocyte in vitro culture enhancer suspension is freeze-dried to obtain a lymphocyte in vitro culture enhancer in the form of a freeze-dried powder;

The freeze-dried powder lymphocyte in vitro culture enhancer is stored at minus 20 degrees Celsius to above zero degrees Celsius, wherein the preservation solution is RPMI 1640 culture medium containing 1-10 volume % human serum albumin.