CN105219710B

CN105219710B - Method for culturing immune cell population with high killing activity

Info

Publication number: CN105219710B
Application number: CN201410247638.5A
Authority: CN
Inventors: 谌兵来; 王玲
Original assignee: Shanghai Knockgene Biotechnology Co Ltd
Current assignee: Shanghai houchao Biotechnology Co., Ltd
Priority date: 2014-06-05
Filing date: 2014-06-05
Publication date: 2020-01-10
Anticipated expiration: 2034-06-05
Also published as: CN105219710A

Abstract

The invention discloses a method for culturing a high killing activity immune cell population, which comprises the step of culturing human peripheral blood lymphocytes in a culture medium containing a basal culture medium, IL-2, human CD137L, human SCF and optional ConA, wherein the concentration of the IL-2 is 10-100ng/ml, the concentration of the human CD137L is 10-100ng/ml, the concentration of the human SCF is 10-100ng/ml, and the concentration of the ConA is 10-100 ng/ml. The method of the invention is simple and easy to operate, has long culture time, and can obtain immune cell populations with high killing activity.

Description

Method for culturing immune cell population with high killing activity

Technical Field

The invention relates to the fields of cytobiology and clinical medicine, in particular to a method for efficiently culturing human peripheral blood lymphocytes in vitro in the aspect of tumor immune cell therapy.

Background

The occurrence and development of tumors are closely related to the immune state of a host, and the traditional tumor treatment means comprises surgery, chemotherapy and radiotherapy, so that the risk of tumor migration and recurrence exists, and the side effects of seriously damaging normal tissues and immune functions are also realized. The tumor cell immunotherapy can remove residual tumor cells in the body of a patient after operation and chemoradiotherapy by recovering and enhancing the immune monitoring capability and the immune killing function of the patient with tumor, achieves the purposes of preventing tumor recurrence, metastasis and radically treating tumor, and has the characteristics of strong specificity, small toxic and side effects and the like. The tumor cell immunotherapy mainly takes immune active cells as a carrier, and achieves the purpose of resisting tumors by activating immune cells in vivo or in vitro to secrete cytokines to regulate the functions of other immune cells or directly killing tumor cells.

According to the characteristics of immune cell therapy and the mechanism of anti-tumor, cellular immunotherapy can be divided into active immunotherapy and adoptive cellular immunotherapy, or specific immunotherapy and non-specific immunotherapy. At present, research on tumor cell immunotherapy is mainly focused on adoptive immunotherapy, which uses immune cells expanded in vitro, such as lymphokine-activated killer cells (LAK cells), tumor-infiltrating lymphocytes (TIL cells), cytokine-induced killer cells (CIK cells), dendritic cells (DC cells), natural killer cells (NK cells), genetically modified T cells, and the like, to perform tumor therapy.

In recent years, there are several major methods for in vitro culture of peripheral blood lymphocytes, which are developed at home and abroad and applied to clinical applications:

(1) the classical DC-CIK culture method combines the existing commercial lymphocyte culture medium on the market, adopts the conventional DC-CIK lymphocyte culture method, applies the anti-human CD3 antibody, IL2, GM-CSF, INF-gamma and other main stimulating factors to respectively stimulate and culture DC cells and CIK cells, and co-cultures the DC cells and the CIK cells after the DC cells are activated by tumor antigens.

(2) The TIL cells are heterogeneous lymphocytes existing in tumor stroma, have stronger capability of gathering to tumor sites in vivo than CIK and NK, only kill the tumor cells with antigen specificity, and have no killing effect on tumor cells of other tissue sources. The main operation is to separate TIL cells from fresh tumor tissue and stimulate them with interleukin-2 in vitro, the TIL cells become cytotoxic T lymphocytes after activation and amplification, and the cytotoxic T lymphocytes can kill tumor cells expressing specific antigens after in vitro screening and large-scale amplification.

(3) Autologous NK cells (natural killer cells) have been cultured, NK cells have been used for cell immunotherapy of tumors for more than twenty years, and the main antitumor component of LAK cells is NK cells. Mainly adopts the steps of establishing an NK cell line and adopting co-culture with the constructed engineering cells to directionally stimulate the NK cells in autologous lymphocytes to multiply in large quantity. Much research has been carried out on engineered cells for co-culture, and combinatorial expression schemes for various cytokines have also been developed.

However, the conventional culture methods still have many problems, which are mainly shown in the following aspects: the culture method is complex and the steps are complicated. DC-CIK needs to separate DC cells from CIK cells for culture, and needs to specially prepare tumor antigens for deactivation; the TIL cell culture needs to be matched with clinical operation, and the separation and further culture of TIL are needed to the obtained tumor tissue, so the operation is complicated; after the NK cell is successfully established, the operation is simple, but the risk is high, most of the established cell lines cannot be used clinically, and in addition, the engineering cell co-culture is adopted, so that the engineering cell establishment is difficult, and even if the establishment is successful, the engineering cell is required to be subjected to special fire extinguishing treatment when being used for the co-culture. Therefore, the conventional lymphocyte culture method generally has the problems of complicated operation and short culture time. In addition, in recent years in China, immune cell therapy is performed clinically, but poor or no effect is generally reflected. Therefore, in response to these problems, there is a need in the art to develop novel lymphocyte culture methods.

Disclosure of Invention

The present invention aims to provide a method for in vitro culturing human peripheral blood lymphocytes, which can culture human peripheral blood lymphocytes in vitro for a long period of time (at least 4 weeks) to obtain a specific cell population capable of efficiently killing tumor cells.

In a first aspect of the invention, there is provided a lymphocyte culture medium comprising a basal medium, IL-2 (human interleukin-2), human CD137L (4-1BBL), human SCF and optionally ConA (concanavalin A), wherein,

the concentration of the IL-2 (human interleukin 2) is 10-100 ng/ml;

the concentration of the human CD137L (4-1BBL) is 10-100 ng/ml;

the concentration of the human SCF is 10-100 ng/ml;

the concentration of the ConA (canavalin A) is 10-100 ng/ml.

In another preferred embodiment, the basal medium is selected from the group consisting of: RPMI-1640, DMEM, IMDM.

In another preferred embodiment, the concentration of the IL-2 (human interleukin 2) is 20-80 ng/ml;

the concentration of the human CD137L (4-1BBL) is 20-80 ng/ml;

the concentration of the human SCF is 20-80 ng/ml;

the concentration of the ConA (canavalin A) is 20-80 ng/ml.

In another preferred embodiment, the concentration of IL-2 (human interleukin 2) is 30-50 ng/ml;

the concentration of the human CD137L (4-1BBL) is 30-50 ng/ml;

the concentration of the human SCF is 30-50 ng/ml;

the concentration of the ConA (canavalin A) is 40-60 ng/ml.

In another preferred embodiment, the concentration of IL-2 (human interleukin 2) is 40 ng/ml; the concentration of the human CD137L (4-1BBL) is 40 ng/ml; the concentration of the human SCF is 40 ng/ml; the concentration of the ConA (canavalin A) is 50 ng/ml.

In another preferred embodiment, the cells are human peripheral blood mononuclear cells.

In another preferred embodiment, the cells are human peripheral blood lymphocytes.

In a second aspect of the present invention, there is provided a method for culturing cells in vitro, comprising the step of culturing the cells using the lymphocyte culture medium according to the first aspect.

In another preferred example, the method comprises the steps of:

(a) providing a cell;

(b) culturing is initiated by adding a first lymphocyte culture medium to the cells, the first lymphocyte culture medium comprising basal medium, IL-2, human CD137L, human SCF, and ConA, wherein,

the concentration of the IL-2 is 10-100 ng/ml;

the concentration of the human CD137L is 10-100 ng/ml;

the concentration of the human SCF is 10-100 ng/ml;

the concentration of the ConA is 10-100 ng/ml;

(c) adding a second lymphocyte culture medium to the culture process for fluid replacement, wherein the second lymphocyte culture medium comprises a basal medium, IL-2, human CD137L and human SCF,

the concentration of the IL-2 is 10-100 ng/ml;

the concentration of the human CD137L is 10-100 ng/ml;

the concentration of the human SCF is 10-100 ng/ml.

In another preferred embodiment, the peripheral blood mononuclear cells are provided in step (a).

In another preferred embodiment, the peripheral blood lymphocytes are provided in step (a).

In another preferred example, the following method is adopted for liquid supplementing in the step (c): in the first week of culture, supplementing the culture medium once every 2-4 days in an equal volume (according to the existing culture medium volume of the cultured cells); during the second week of culture, supplementing the culture medium once every 2-4 days, wherein the volume of the supplemented culture medium is 1-2 times of the volume of the culture medium for culturing the cells; and during the third-fourth week of culture, supplementing the culture medium once a week, and supplementing the culture medium by 2-3 times according to the existing culture medium volume of the cultured cells.

In another preferred embodiment, the method obtains a cell population comprising 5% to 40% CIK cells, 5% to 20% NK cells, 30% to 70% DC cells, based on the total number of cells in the cell population.

In a third aspect of the present invention, there is provided a cell population obtained by culturing the cells in the lymphocyte culture medium according to the first aspect or the method according to the second aspect.

In another preferred embodiment, the cell population comprises 5% to 40% of CIK cells, 5% to 20% of NK cells and 30% to 70% of DC cells based on the total number of cells in the cell population.

In another preferred embodiment, the cell population comprises 5% to 10%, 10% to 15%, 30% to 40%, 5% to 15%, or 20% to 25% CIK cells based on the total number of cells in the cell population.

In another preferred embodiment, the population of cells comprises 5% to 10% or 10% to 15% NK cells based on the total number of cells in the population.

In another preferred embodiment, the population of cells comprises 50% to 60%, 30% to 45%, or 40% to 55% DC cells, based on the total number of cells in the population.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising the cell population of the third aspect and a pharmaceutically acceptable carrier.

The lymphocyte culture method is simple and easy to operate, a cytokine combination adding scheme is adopted, after lymphocytes are separated, culture media with different factor adding schemes are directly adopted at different periods, the existing culture media are not required to be removed, the cell culture time can be at least one month, and the method has more excellent killing activity in an in-vitro killing experiment by comparing the existing commercial culture media and methods.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a comparison of experimental and control groups during in vitro culture of human peripheral blood lymphocytes, wherein the experimental group employs a 3+1 factor addition protocol; the control group was added with only the factor IL-2, and the results of photographing were taken on

days

1, 7, 14, 21 and 28, respectively.

FIG. 2 is a graph showing the number of proliferated cells cultured in vitro with human peripheral blood lymphocytes.

FIG. 3 is a photograph taken under a microscope of a lymphocyte killing test.

FIG. 4 shows comparative results of killing experiments in vitro with cultured human peripheral blood lymphocytes.

FIG. 5 is an agarose gel electrophoresis image.

Detailed Description

The present inventors have extensively and intensively studied and, for the first time, have unexpectedly developed a novel method for in vitro culture of human peripheral blood lymphocytes, which can be used for in vitro culture of human peripheral blood lymphocytes for a long period of time (at least 4 weeks) by adding human IL-2 (human interleukin 2), human CD137L (human 4-1BBL), human SCF (human stem cell factor) and ConA (concanavalin a), to obtain a specific cell population capable of efficiently killing tumor cells, wherein the cell population is characterized in that: 15% -40% of CIK cells; 5% -20% of NK cells; DC: 30 to 70 percent. On the basis of this, the present invention has been completed.

Lymphocytes

Lymphocytes (also known as lymphocytes) are a type of white blood cell. The cell component is generated by lymphoid organs and is important for the immune response function of the body. Including T cells, B cells, NK cells and other subclasses, which mediate the immunological functions of cellular immunity, humoral immunity, killing tumor cells and virus infected cells, and the like.

Peripheral blood is blood other than bone marrow, i.e., blood that has been released into the circulatory system by the hematopoietic organ to participate in circulation, as distinguished from immature blood cells within the hematopoietic organ or blood cells that have not been released into the circulation. Peripheral blood lymphocytes are mainly composed of T cells and B cells.

CIK cell

The CIK cell, namely Cytokine-Induced Killer (CIK), is a novel immunocompetent cell, and has strong CIK proliferation capacity, strong cytotoxic effect and certain immunological characteristics. The cell expresses two membrane protein molecules of CD3 and CD56 simultaneously, so the cell is also called NK cell (natural killer cell) like T lymphocyte, and has the advantages of strong antitumor activity of the T lymphocyte and non-MMC limitation tumor killing of the NK cell.

NK cells

NK cells, natural killer cells (NK), are important immune cells of the body. NK cells belong to the group of non-specific immune cells which can directly kill certain tumor and virus-infected target cells without antigen pre-sensitization, and thus play an important role in the immune process against tumors and early viral or intracellular parasitic infection. NK cells do not express specific antigen recognition receptors, are a type of lymphoid cells different from T, B lymphocytes, and can express a variety of surface markers, many of which can also be expressed on the surface of other immune cells. Clinically, TCR-, mIg-, CD56+, CD16+ lymphoid cells were identified as NK cells.

Interleukin (interleukin )

Interleukins or interleukins (interleukins) are a group of cytokines (secreted signaling molecules). Expression in leukocytes was first discovered as a means of intercellular signaling. In fact, interleukins can be produced by a variety of cells. The function of the immune system is largely dependent on interleukins. Some rare interleukin deficiencies are common in autoimmune diseases or immunodeficiency.

Interleukin-2 (IL-2) is a glycoprotein with a molecular weight of 14500, a kind of interleukin among cytokines, and plays an important role in the immune system, and is responsible for regulating the immune activity of leukocytes (leukocytes, usually lymphocytes). IL-2 production is a part of the immune response of the body when infected with a microorganism to distinguish "self" from "non-self". IL-2 acts by binding to IL-2 receptors on the surface of lymphocytes. IL-2 can stimulate the growth and differentiated T cell response and can be used in immunotherapy for the treatment of cancer.

Human CD137L (human 4-1BBL)

4-1BBL is a type II transmembrane glycoprotein with a molecular weight of 34KD, which is mainly expressed on professional APC cells (such as mononuclear macrophages, DC cells and B cells), activated T cells and some tumor cells, and has 36% homology between human and mouse. In recent years, the 4-1BB/4-1BBL signaling pathway has become a new target for tumor immunotherapy design.

Human SCF (human stem cell factor)

Stem cell factor is an acidic glycoprotein produced by stromal cells in the bone marrow microenvironment. The glycosyl is connected on N and O groups of a peptide bond, has the relative molecular mass of 31000-36000 and consists of two same subunits which are combined in a non-covalent manner. Isoelectric point PI is 3.8. SCF has a total of 273 amino acids. From-25 to-1 is a signal peptide, +1 to +189 is a membrane-outside functional region, +190 to +216 is a transmembrane region, and +217 to +248 is a cytoplasmic functional region. Murine and human SCF have 83% homology.

ConA (Canavalia gladiata protein A)

Concanavalin A (Con A), also called concanavalin and concanavalin, is a phytohemagglutinin, has strong mitogenic action and good lymphocyte transformation reaction promoting action, has the optimum lymphocyte transformation concentration of 40-100 mug/ml, can precipitate hepatic glycogen and agglutinate red blood cells of animals such as sheep, horses, dogs, rabbits, pigs, rats, mice, guinea pigs and the like and human red blood cells. Can also selectively activate suppressive T cell (Ts) cell, and has important effect on regulating organism immune response.

Lymphocyte culture medium

The lymphocyte culture medium comprises a basal medium, IL-2 (human interleukin 2), human CD137L (4-1BBL) and human SCF, wherein,

the concentration of the IL-2 (human interleukin 2) is 10-100 ng/ml;

the concentration of the human CD137L (4-1BBL) is 10-100 ng/ml;

the concentration of the human SCF is 10-100 ng/ml.

The lymphocyte culture medium further comprises ConA (Canavarin A), and the concentration of the ConA (Canavarin A) is 10-100 ng/ml.

In another preferred embodiment, the concentration of IL-2 (human interleukin 2) is 20-80ng/ml, preferably 30-50 ng/ml; more preferably 40 ng/ml.

In another preferred embodiment, the concentration of the human CD137L (4-1BBL) is 20-80ng/ml, preferably 30-50 ng/ml; more preferably 40 ng/ml.

In another preferred embodiment, the concentration of said human SCF is 20-80ng/ml, preferably 30-50 ng/ml; more preferably 40 ng/ml.

In another preferred embodiment, the concentration of the ConA (canavalin A) is 20-80ng/ml, preferably 40-60 ng/ml; more preferably 50 ng/ml.

In the present invention, the basic medium is a medium which is a main component of lymphocyte culture, provides basic nutrients for cell growth, and contains no components such as serum and antibiotics for maintaining basic cell survival. The basic medium is not particularly limited, and various basic media known in the art for culturing lymphocytes can be selected for use in the present invention.

In another preferred embodiment, the basal medium (also referred to as minimal medium) is a commercially available serum-free medium specifically for lymphocyte culture.

In another preferred embodiment, the basal medium is RPMI-1640, DMEM or IMDM.

In another preferred embodiment, the basic medium is serum-free RPMI-1640, serum-free DMEM or serum-free IMDM.

Method for in vitro cell culture

The culture method of the present invention comprises the step of culturing the cells using the culture medium of the present invention.

Preferably, the method comprises the steps of:

(1) providing peripheral blood mononuclear cells;

(2) culturing is started by adding a first medium to peripheral blood mononuclear cells, the first medium comprising a basal medium, IL-2 (human interleukin 2), human CD137L (4-1BBL), human SCF and ConA (concanavalin A), wherein,

the concentration of the IL-2 (human interleukin 2) is 10-100 ng/ml;

the concentration of the human CD137L (4-1BBL) is 10-100 ng/ml;

the concentration of the human SCF is 10-100 ng/ml;

the concentration of the ConA (canavalin A) is 10-100 ng/ml;

(3) adding a second culture medium comprising a basal medium, IL-2 (human interleukin 2), human CD137L (4-1BBL), and human SCF during the culturing process for supplementing the culture solution,

the concentration of the IL-2 (human interleukin 2) is 10-100 ng/ml;

the concentration of the human CD137L (4-1BBL) is 10-100 ng/ml;

the concentration of the human SCF is 10-100 ng/ml.

In a preferred embodiment, the cultivation method of the present invention comprises the steps of:

1. separating 2ml of freshly extracted anticoagulant (heparin or EDTA anticoagulant, non-citric acid anticoagulant) by using Ficoll lymphocyte separation liquid, and separating to obtain Peripheral Blood Mononuclear Cells (PBMC);

2. counting the separated lymphocytes, taking out a part of the lymphocytes for flow-type flow, and detecting the proportion of three cell populations including NK cells, CIK cells and DC cells;

3. the remaining lymphocytes adjusted the cell concentration at 1X 10⁶One group was cultured in a basal medium supplemented with IL2(40ng/ml) and the other group was cultured in a basal medium supplemented with IL2(40ng/ml) + CD137L (40ng/ml) + SCF (40ng/ml) + ConA (50ng/ml) (i.e., 3+1 factor protocol) under 37 ℃ and 5% CO after counting in a blood count plate under a microscope₂An incubator;

4. when the cells are cultured for the first week, slowly proliferating the cells, supplementing the cells once every three days, and supplementing the cells with the same volume of the supplemented cells according to the volume of the existing culture solution for culturing the cells;

5. when the cells are cultured for the second week, the cells enter a proliferation period, fluid supplementation is carried out once every three days, and the volume of the fluid supplementation is 1-2 times that of the existing culture fluid for culturing the cells;

6. when the cells are cultured for the third and the fourth weeks, the cells enter a rapid proliferation stage, large-volume fluid supplement can be performed once a week, and the fluid supplement volume is 2-3 times the volume of the existing culture fluid for culturing the cells; meanwhile, when the cells are cultured to the end of the third week or the fourth week, cell detection experiments (proliferation, flow and in-vitro tumor cell killing detection) can be carried out for clinical injection. For example, by the third week of culture, cell counting, flow assay and killing assay can be performed, optionally with clinical injection or continued culture; and for the cells which are continuously cultured, the cells are cultured to the fourth week and then subjected to cell counting, flow detection and killing detection selection for clinical injection or continuous culture.

The medium added in the above step for replacement contained basal medium, IL2(40ng/ml), CD137L (40ng/ml) and SCF (40 ng/ml).

By adopting the method, the obtained lymphocyte mixed population can keep better killing activity aiming at the tumor cells from the second week and has the continuity of proliferation and the long-acting property of killing power.

Cell population

The cell population of the present invention is obtained by culturing cells using the medium of the present invention.

In a preferred embodiment, the cell population of the present invention comprises 5% to 40% of CIK cells, 5% to 20% of NK cells, and 30% to 70% of DC cells, based on the total number of cells in the cell population.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising the cell population of the invention and a pharmaceutically acceptable carrier.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with the active ingredient (cell population) of the invention and with each other without significantly diminishing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (e.g. sodium carboxymethylcellulose)Sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (e.g., (ii)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

Detection method

Flow cytometry

In the present invention, flow cytometry is used for lymphocyte typing. Flow Cytometry (FCM) is a technique that can rapidly, accurately, objectively, simultaneously detect and quantify multiple characteristics of individual particles (typically cells) and can sort specific populations.

Lymphocytes are the most important large cell population of the immune system of the organism, develop and differentiate into subgroups with different functions, different cell subgroups have their specific surface antigen markers, through choosing different monoclonal antibodies and fluorescent dyes, FCM can be utilized to simultaneously determine a plurality of different characteristics on one cell, thereby determining the surface antigen expression condition and judging the lymphocyte subgroups.

NK, CIK and DC cell specific surface markers:

NK cell surface markers: CD3-CD56+

The CIK cell surface markers are: CD3+ CD56+

DC cell surface markers: CD86+ HLA-DR + (roughly)

In cell staining, CD3 and CD56 double staining, and CD86 and HLA-DR double staining were used.

Mycoplasma detection

In the invention, the lymphocyte is detected by adopting a PCR method, primers are designed for specific sequences of mycoplasma, when mycoplasma pollution exists, the target DNA specificity is copied through PCR specific amplification, and then the positive result appears in the running strip through agarose electrophoresis observation detection. On the contrary, when there is no mycoplasma contamination, PCR cannot be amplified due to the absence of the template, and a negative result occurs because no band is left by agarose electrophoresis.

Limulus reagent

The limulus reagent is lyophilized product of hemocyte lysate of Tachypleus tridentatus of Tachypleus of Tachypleidae, and contains factor C, factor B, prothrombin, coagulogen, etc. Under appropriate conditions (temperature, pH value and non-interfering substances), bacterial endotoxin activates factor C to cause a series of enzymatic reactions, activates proclotting enzyme to form coagulase, and the coagulase decomposes artificially synthesized chromogenic substrates into polypeptide and yellow paranitroaniline (pNA, lambda max ═ 405 nm). Within a certain period of time, the generation amount of pNA is positively correlated with the endotoxin concentration of bacteria, so that the endotoxin concentration of the test article can be quantified. Meanwhile, paranitroaniline (pNA) can also be dyed into rose red (λ max is 545nm) by using a diazotized reagent, so that the interference of the color of a test sample on an absorption peak at 405nm is avoided.

Gram staining method

The Gram staining method (Gramstain) was created by Danish physician Gram in 1884, and it was discovered that Gram-positive bacteria, because of their thicker cell walls, more layers of peptidoglycan and dense cross-linking, can firmly retain the compound of crystal violet and iodine in the wall after Gram staining, and are not easy to be removed by the destaining solution and still become purple, on the basis of which the Gram staining solution reagent is formed. The gram staining method is a differential staining method (belonging to a counterstaining method) widely used in bacteriology, can be used for specimen smears or colony smears, and can be used for clinical classification and identification by clearly observing the form, arrangement and certain structural characteristics of bacteria after staining the bacteria in sharp contrast with the environment. The staining results classified the bacteria into two broad categories, gram-positive (purple) and gram-negative (red).

MTT colorimetric method

In the invention, an improved cytotoxicity MTT experiment is adopted to evaluate the killing performance of the lymphocyte.

MTT colorimetry is a method of detecting cell survival and growth. MTT is known as 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide salt, and the trade name is as follows: thiazole blue. Is a yellow dye. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystal Formazan (Formazan) and deposit the Formazan in the cells, but dead cells do not have the function. Dimethyl sulfoxide can dissolve formazan in cells, and the light absorption value is measured by an enzyme labeling instrument at 490nm wavelength, which can indirectly reflect the number of living cells. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number. The method is widely used for activity detection of some bioactive factors, large-scale screening of anti-tumor drugs, cytotoxicity test, tumor radiosensitivity determination and the like.

The reagent used in the present invention is CellTiter

An AQueous single solution cell proliferation assay kit (MTS) for the colorimetric detection of viable cell numbers in cell proliferation, cytotoxicity or chemosensitivity assays. CellTiter

The AQueous single solution reagent contains a tetrazolium compound, an inner salt (MTS) and an electron coupling reagent (ethiophenazine, PES). MTS is an MTT analogue, and the action principle is similar to that of MTT. The chemical stability of PES has been enhanced, which allows it to be mixed with MTS to form a stable solution. Compared with the MTT method, the MTS reagent is simpler to operate, and the result is more stable. When in detection, only a small amount of CellTiter is needed

The AQueous single solution was added directly to the medium in the wells of the plate, incubated for 3 hours, and then the absorbance value was read at 490nm with a microplate reader.

In addition, the principle of detecting the killing effect of the lymphocytes is as follows: the lymphocyte and the tumor cell are co-cultured, the lymphocyte can kill the tumor cell, the killing capacity of the lymphocyte under different culture conditions is different, the number of the survival tumor cells and the activity of single cells are different, and the cell activity of the single tumor cell is calculated by subtracting the total cell activity of the lymphocyte and the tumor cell in the co-culture group and the cell activity of the lymphocyte in the single group through detecting, so that the corresponding killing capacity of the lymphocyte is strong and weak.

The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

The invention has the advantages that:

(1) providing a novel cell culture medium and a culture method;

(2) by adopting the culture medium and/or the culture method, a cell group with a specific composition can be obtained, wherein the content of the CIK cells is 15-40%; 5% -20% of NK cells; DC cell: 30% -70%;

(3) compared with the cell population obtained by the prior art, the cell population has very obvious capacity of efficiently killing tumor cells;

(4) the invention well solves the problem of the number proliferation of the lymphocytes cultured in vitro, and can also well maintain the proliferation continuity and the long-acting property of the lymphocytes in the in vitro culture process;

(5) the invention greatly improves the killing property of the in vitro cultured lymphocytes to the tumor cells.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Example 1

Human peripheral blood lymphocyte isolation assay

(lymphocyte separation Using Ficoll solution)

1.1 serum isolation: collecting anticoagulated blood in a 50ml centrifuge tube, centrifuging at room temperature of 300g for 5min, collecting upper layer serum, subpackaging, and storing at-20 deg.C;

1.2 anticoagulation dilution: adding equal-volume salt balance solution into the residual blood for dilution, then adding the diluted anticoagulated blood and the Ficoll solution, wherein the volume ratio of the diluted anticoagulated blood to the Ficoll solution is 4:3, the temperature is 20 ℃, 400g, and centrifuging for 30 min;

1.3 lymphocyte isolation: removing the uppermost cell sap layer as much as possible by using a disposable pasteur pipette tip, and carefully sucking the lymphocyte layer to a 50ml centrifuge tube by using a pipette;

1.4 washing lymphocytes: adding salt balance solution to 20ml, centrifuging at 20 deg.C and 400g for 10 min;

1.5 removing the supernatant, and repeating the previous step;

1.6 removing supernatant, using IMDM base culture medium to re-suspend cells to obtain human peripheral blood lymphocytes, and counting;

1.7 cells were cultured in groups.

Example 2

Lymphocyte culture

2.1 the lymphocytes isolated in example 1 were counted and the cell concentration was adjusted to 1X 10⁶One group was divided into control groups and cultured in a basal medium supplemented with IL2(40ng/ml), and the other group was cultured in a medium supplemented with IL2(40ng/ml) after counting 2ml (using a blood count plate under a microscope) and dividing into groupsAssay group, cultured in basal medium supplemented with IL2(40ng/ml) + CD137L (40ng/ml) + SCF (40ng/ml) + ConA (50ng/ml) (3+1 factor addition protocol). Culturing lymphocytes with cell culture plate at 37 deg.C and 5% CO₂The cell culture box of (1);

2.2 during the first week of culture, fluid replacement is carried out every three days without removing the original culture fluid during fluid replacement, a control group is directly supplemented with a fresh culture fluid containing IL2(40ng/ml), an experimental group is directly supplemented with a fresh culture fluid containing IL2(40ng/ml) + CD137L (40ng/ml) + SCF (40ng/ml), and the volume of the fluid replacement is equal to the volume of the existing culture fluid for culturing cells;

2.3 when the cells are cultured for the second week, the cells enter a proliferation period, fluid supplement is carried out every three days, the fluid supplement condition is the same as that of the first week, and the fluid supplement volume is 1-2 times of the volume of the fluid supplement according to the existing culture fluid volume of the cultured cells;

2.4 when the cells are cultured for the third and the fourth weeks, the cells enter a rapid proliferation stage, and large-volume fluid supplement can be performed once a week, wherein the fluid supplement condition is the same as that of the first week, and the fluid supplement volume is 2-3 times the volume of the existing culture fluid for culturing the cells.

FIG. 1 is a comparison graph of experimental group and control group during in vitro culture of human peripheral blood lymphocytes, the photographed results of

days

1, 7, 14, 21 and 28 are taken, respectively, and FIG. 2 is the total number of lymphocytes of the experimental group and the control group.

From the comparison results in FIG. 1, it is clear that when lymphocytes are cultured using the medium of the present invention, the lymphocytes grow to form cell masses, and the cell masses of the lymphocyte group having a good growth state are visible to the naked eye and are large in number, as compared with the medium containing only IL-2, indicating that the proliferation state of lymphocytes is good. As can be seen from FIG. 2, the number of lymphocytes obtained by culturing the cells in the medium of the present invention was much greater than that of the control group after the culture for a long period of time.

Example 3

Lymphocyte typing using flow cytometry

3.1 culturing the lymphocytes to 7 th, 14 th, 21 th and 28 th days, carrying out flow detection on the cultured lymphocytes, centrifuging the cultured lymphocytes for 5min by adopting 300g, and collecting the cells; using basis weight of culture mediumAfter suspension, the cells were counted and adjusted to 2X 10³Mu/l;

3.2 staining 50ul cell suspension, adding 1 ul CD3 (anti-human CD3FITC) and 1 ul CD56 (anti-human CD56 APC) antibody into each tube, mixing uniformly, and incubating at room temperature for 40 min;

3.3 adding 1ml PBS for washing, 500g, centrifuging for 5min, and removing the supernatant;

3.4 repeat step 3.3;

3.5 Add 300 u l PBS heavy suspension cell, the heavy suspension cell transfer to the flow loading tube, the machine for cell sorting, wherein CD3-CD56+ is NK cells, CD3+ CD56+ is CIK cells.

The results are shown in Table 1.

TABLE 1 results of in vitro cell flow typing of human peripheral blood lymphocytes

[ CIK cells ]

	Day 7	Day 14	Day 21	Day 28
					Experimental group	8.31％	14.05％	34.39％	32.70％
Control group	8.74％	6.07％	9.83％	8.10％

[ NK cells ]

	Day 7	Day 14	Day 21	Day 28
					Experimental group	12.15％	11.26％	6.90％	5.61℃
Control group	14.47％	12.75％	4.10％	1.95％

The results show that the proportion of CIK cell subsets in the lymphocytes of the control group is basically unchanged and the proportion of NK cell subsets is reduced along with the proliferation of cell numbers and the differentiation of the lymphocytes in the whole culture process; the proportion of CIK cell subset in the lymphocyte of the experimental group is obviously increased, almost accounts for 1/3 of the whole cell population, and the proportion of NK cell subset is also reduced, but the reduction amplitude is smaller than that of the control group. The experimental method disclosed by the invention is proved to have a promoting effect on the proliferation and differentiation of CIK and NK cells, particularly on the CIK cells.

Example 4

Killing experiment

Using the method of example 1, freshly drawn heparin anticoagulated blood was isolated and the resulting lymphocytes were divided into 4 aliquots for cell culture: two of them (labeled as company A-1 and company A-2) used the known culture medium and culture method of company A, and the other two (labeled as experimental group-1 and experimental group-2) used the culture medium and culture method of example 2 of the present invention, each method using two replicate groups.

The culture protocol provided by company a was used as follows:

① diluting HER2 monoclonal antibody (1:2000 dilution ratio) with 3ml of physiological saline, adding the diluted antibody into a T25 culture flask, and slightly shaking the culture flask to allow the solution to spread on the culture surface of the culture flask;

② incubation at room temperature for 1h, removal of antibody solution, washing the flask twice with physiological saline;

③ when separating lymphocytes with Ficoll liquid, collecting the upper layer diluted plasma;

④ the isolated lymphocytes were added to the treated flasks and cultured with 7ml of Medium A (IL 2 with 1000 UI/ml);

⑤ to the third day, supplemented with 13ml of Medium A (containing 1000UI/ml of IL 2);

⑥ to day 5, transferring the cells to a T75 flask, and adding 47ml of medium A (containing 1000UI/ml of IL2 and 5ml of diluted plasma);

⑦ to day 7, 133ml of Medium B (containing 1000UI/ml of IL2) was added;

⑧ to day 9, transferring the cells into a culture bag, and adding 200ml of culture solution B (containing 1000UI/ml of IL 2);

⑨ to day 11, 200ml of Medium B (containing 1000UI/ml of IL2) was added;

⑩ to day 15, the cells were tested, and the lymphocytes were tested on day 15, 22 and 29, respectively, throughout the experiment.

Killing experiment of lymphocytes on human lung cancer cell line H1299:

the reagent adopted in the experiment is CellTiter

AQueous single solution cell proliferation detection kit (MTS), only a small amount of CellTiter is needed for detection

The specific experimental steps are as follows: the experiment was performed using 96-well cell culture plates, one day ahead, with H1299 cells plated in 96-well plates at 4000/well in 50 μ l of medium, and on the day of the experiment, lymphocyte counts were collected according to lymphocyte: tumor cells were plated at a ratio of 20:1 with 80000 lymphocytes per well in 50 μ l of medium in wells plated with H1299 cells, 6 replicate wells for each group of lymphocytes, and a single tumor cell, a single lymphocyte, and a blank medium control were set. After the 96-well cell culture plate with the well-paved cells is placed in a cell culture box for culturing for 24 hours, the cells are observed and photographed under a microscope, and the photographing result is shown in figure 3; then 20ul of CellTiter was added to each well

And (3) continuously culturing the Aqueous solution of aquous in a cell culture box for 3H, measuring the absorbance value of the solution at 490 wavelengths by using an enzyme-labeling instrument, and calculating the survival rate of H1299 cells in different lymphocyte groups according to the obtained absorbance value, wherein the experimental result is shown in figure 4.

As can be seen from the results in fig. 3, the lymphocytes of the company a-1 and the company a-2 are small compared to the H1299 of the control group, and a large amount of H1299 cells still exist in the co-culture group after the killing of the lymphocytes, while the lymphocytes of the experimental group-1 and the experimental group-2 are large, and the co-culture group basically only leaves the lymphocytes after the killing of the lymphocytes, it can be seen that the lymphocytes obtained by using the culture medium and the culture method of the company a have substantially no killing property on the human lung cancer cell line H1299 used for the detection, and the lymphocytes obtained by using the culture medium and the culture method of the present invention have a very good killing effect on the H1299 compared to that obtained by using the culture medium and the culture method of the present invention.

As can be seen from the results of FIG. 4, the killing efficiency of tumor cells by company A-1 and company A-2 groups was substantially below 20% by comparing the survival rate of tumor cells of the control group; the killing efficiency of the experimental group-1 and the experimental group-2 on the tumor cells is basically over 80 percent and even reaches 100 percent.

The experimental results are analyzed by SPSS-one-way analysis of variance (ANOVA), and each experimental group is compared with a control group for analysis; "+" indicates p < 0.05, resulting in statistical significance of the difference; ". indicates p < 0.01, and the difference was statistically significant.

Example 5

Detection of cultured lymphocytes

5.1 lymphocyte typing by flow cytometry

TABLE 2 in vitro culture of peripheral blood lymphocytes and cell flow typing results

[ day 15 ]

	Company A-1	Company A-2	Experimental group-1	Experimental group-2
					NK cell (%)	38.3	47.3	7.7	8.3
CIK cell (%)	8.3	5.0	7.4	13.0
					DC cell (%)	74.5	66.7	54.9	53.1

[ day 22 ]

	Company A-1	Company A-2	Experimental group-1	Experimental group-2
					NK cell (%)	29.4	28.6	10.6	12.6
CIK cell (%)	8.9	10.2	22.8	23.4
					DC cell (%)	40.7	45.7	33.5	41.3

[ 29 th day ]

	Company A-1	Company A-2	Experimental group-1	Experimental group-2
					NK cell (%)	19.8	18.1	11.7	13.2
CM cells (%)	6.5	5.4	36.4	36.7
					DC cell (%)	30.3	26.6	46.8	50. To master

The lymphocyte typing was examined by the same method as in example 3, and as a result, as shown in table 2, the NK cell ratio reached a maximum value of 47.3% at 2 weeks in the culture medium and culture protocol of company a, after which the culture was continued, the lymphocytes began to die largely, the NK cell ratio was also decreased, and the CIK ratio was 10.2% at the maximum during the whole culture. By using the culture medium and the culture scheme of the invention, the proportion of NK cells is increased slightly, but the proportion of CIK cells is increased to 36.7%, and lymphocytes are proliferated all the time in the whole culture process.

5.2 the PCR method is adopted to detect mycoplasma of the lymphocyte:

(1) sampling:

directly taking the culture medium supernatant of the cultured cells.

(2) PCR (10ul reaction):

preparing a reaction system, calculating the number of PCR samples according to the number of the detection samples, 1 negative control (water) and 1 positive control (a sample polluted by mycoplasma in cell culture), increasing the number of several tubes on the basis, adding the other components except the detection culture medium together according to the calculated amount, mixing uniformly, subpackaging, and finally adding the detection culture medium. The reaction system and reaction conditions are shown in tables 3 and 4 below.

TABLE 3 PCR reaction System and reaction conditions when primer # 5 is used

TABLE 4 PCR reaction System and reaction conditions when primer # 6 was used

(3) And (4) detecting a result:

the PCR results were examined by 1% agarose gel electrophoresis, and the results are shown in FIG. 5, in which 1/2 is the results of examination of the 5# primers of both company A-1 and company A-2; 3/4 is the result of 5# primer detection for both experimental group-1 and experimental group-2; 5/6 are two positive controls for

primer #

5, and 7 is a negative control for primer # 5. 8/9 is the result of the detection of the 6# primer from two groups, company A-1 and company A-2; 10/11 is the result of 5# primer detection in the two groups of experiment group-1 and experiment group-2, 12 is the negative control of 6# primer, 13/14 is the two positive controls of 6# primer.

As can be seen from the electrophoresis result, a positive control group has a band, and the size of the band accords with the primer prediction result, which indicates that the PCR detection system is working; no band appears in the negative control group, which indicates that the reaction system of the PCR is normal and pollution-free; the results of the positive and negative controls indicate that the PCR detection method for Mycoplasma is reliable. No bands were present in the cultured lymphocyte samples, indicating that no mycoplasma contamination of the cultured lymphocytes occurred.

5.3 the Limulus reagent gel method is adopted to detect the endotoxin of the lymphocyte:

(1) one piece of endotoxin work (120 EU) was taken and 1ml of test water was added to give a concentration of 120 EU/ml.

(2) Endotoxin standard solutions of 0.12EU/ml, 0.06EU/ml, 0.03EU/ml and 0.015EU/ml were prepared by using a detection liquid (kit with endotoxin-free water) as a solvent (dilution) instead of the water for bacterial endotoxin detection.

(3) Experimental grouping (16 tubes): sterile water was added to the negative control, and endotoxin standards at 0.12EU/ml were added to the positive control. Dividing the test solvent into endotoxin standard substances (4 concentrations) prepared by Text2 stock solution (culture medium supernatant of experimental group cells in lymphocyte in-vitro culture experiment, serving as a detected sample), endotoxin standard substances (4 concentrations) prepared by Text2 stock solution diluting 2 times, endotoxin standard substances (4 concentrations) prepared by Text2 stock solution diluting 5 times, and directly detecting Text 2.

(4) Taking a pyrogen-free test tube to sequentially dilute the concentration of the endotoxin works to four concentration gradients of 0.12EU/ml, 0.06EU/ml, 0.03EU/ml and 0.015 EU/ml.

(5) The limulus reagent was taken out, and 1.7ml of test water was added thereto and gently shaken to completely dissolve the reagent.

(6) 100ul limulus reagent was added to each tube and mixed well, and then incubated at 37 ℃ for 1 h.

(7) The tube was removed from the water bath and slowly inverted 180 °, if the contents of the tube were firm gel, not deformed, not sliding down the tube wall as positive, not forming gel or forming gel but not remaining intact as negative.

Experimental observation results show that the positive control group generates solid gel, and the generated gel does not deform or slide from the tube wall in the process of slowly inverting the test tube, which indicates that the experimental system is working; the negative control group produced no gel, and positive and negative results indicated that the test was reliable. None of the experimental groups, including the Text2 stock solution, produced gels indicating that no endotoxin contamination occurred in the cultured lymphocytes.

5.4 bacteriological detection of lymphocytes by gram staining:

(1) sample preparation:

positive control:

a. directly taking escherichia coli for experiments, adding a small amount of serum to increase the liquid viscosity, and smearing;

b. uniformly mixing a certain amount of samples for detection with escherichia coli, adding a small amount of serum to increase the liquid viscosity, and smearing;

detecting a sample: directly taking cultured lymphocytes, adding a small amount of serum to increase the liquid viscosity, and smearing;

(2) performing smear, and waiting for the natural drying of the smear after the smear is performed;

(3) dyeing process

① adding radix Gentianae purple liquid (CrystalViolet), dyeing for 10 s, washing with water, and drying;

② adding iodine solution (IodineSolution) for dyeing for 10 seconds, washing with water, and spin-drying;

③ adding a decolorizing solution (Decolorizer) for decolorizing for 10-20 seconds, then washing with water, and spin-drying;

④ adding Safranil solution (Safranin solution) for 10 s, washing with water;

⑤ the positive control contained small red colored E.coli cells observed under a microscope after drying on filter paper or air.

The observation result under the mirror shows that the positive control group a adopted in the experiment shows red under the mirror, which is consistent with the theory and indicates that the detection volume is working; and the detection sample is human lymphocyte which cannot be stained, and the lymphocyte is observed under the microscope without staining, so that the result is negative, which indicates that the detection method is feasible. In the positive control group b, a part was observed to be red and a part was not colored, which was expected. The results showed that bacterial contamination of the cultured lymphocytes did not occur.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A lymphocyte culture medium, comprising basal medium, IL-2, human CD137L, human SCF and ConA, wherein,

the concentration of the IL-2 is 10-100 ng/ml;

the concentration of the human CD137L is 10-100 ng/ml;

the concentration of the human SCF is 10-100 ng/ml;

the concentration of the ConA is 10-100 ng/ml.

2. The lymphocyte culture medium of claim 1, wherein the basal medium is selected from the group consisting of: RPMI-1640, DMEM, IMDM.

3. The lymphocyte culture medium of claim 1, wherein the IL-2 is at a concentration of 20-80 ng/ml;

the concentration of the human CD137L is 20-80 ng/ml;

the concentration of the human SCF is 20-80 ng/ml;

the concentration of the ConA is 20-80 ng/ml.

4. The lymphocyte culture medium of claim 1 or 3, wherein the concentration of IL-2 is from 30 to 50 ng/ml;

the concentration of the human CD137L is 30-50 ng/ml;

the concentration of the human SCF is 30-50 ng/ml;

the concentration of the ConA is 40-60 ng/ml.

5. The lymphocyte culture medium of claim 1 or 3, wherein the concentration of IL-2 is 40 ng/ml; the concentration of the human CD137L is 40 ng/ml; the concentration of the human SCF is 40 ng/ml; the concentration of the ConA was 50 ng/ml.

6. An in vitro method for culturing cells comprising the step of culturing the cells, which are human peripheral blood mononuclear cells or human peripheral blood lymphocytes, with the lymphocyte culture medium according to any one of claims 1 to 5, comprising the steps of:

(a) providing a cell;

(b) culturing is initiated by adding to the cells a first lymphocyte culture medium consisting of basal medium, IL-2, human CD137L, human SCF and ConA,

the concentration of the IL-2 is 10-100 ng/ml;

the concentration of the human CD137L is 10-100 ng/ml;

the concentration of the human SCF is 10-100 ng/ml;

the concentration of the ConA is 10-100 ng/ml;

(c) adding a second lymphocyte culture medium to perform fluid replacement during the culture process, wherein the second lymphocyte culture medium consists of a basal medium, IL-2, human CD137L and human SCF,

the concentration of the IL-2 is 10-100 ng/ml;

the concentration of the human CD137L is 10-100 ng/ml;

the concentration of the human SCF is 10-100 ng/ml.

7. The method of claim 6, wherein the replenishing is performed in step (c) by: during the first week of culture, performing equal-volume fluid replacement once every 2-4 days according to the existing culture solution volume of the cultured cells; during the second week of culture, supplementing the culture medium once every 2-4 days, wherein the volume of the supplemented culture medium is 1-2 times of the volume of the culture medium for culturing the cells; and during the third-fourth week of culture, supplementing the culture medium once a week, and supplementing the culture medium by 2-3 times according to the existing culture medium volume of the cultured cells.