CN115247149B

CN115247149B - Culture medium composition suitable for NK cells and culture method

Info

Publication number: CN115247149B
Application number: CN202211004571.3A
Authority: CN
Inventors: 牟春琳; 王秀娥; 秦臻; 李政楠; 张雅楠; 武嘉钊
Original assignee: Huayu Life Technology Tianjin Co ltd; Tianjin Huayu Pharmaceutical Co ltd; Huayu Biotechnology Tianjin Co ltd
Current assignee: Huayu Life Technology Tianjin Co ltd; Tianjin Huayu Pharmaceutical Co ltd; Huayu Biotechnology Tianjin Co ltd
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2023-06-16
Anticipated expiration: 2042-08-22
Also published as: CN115247149A

Abstract

The invention relates to the technical field of biology, in particular to a culture medium composition suitable for NK cells and a culture method. The culture medium composition comprises basal culture medium, 3-10 v/v% serum, IL2, IL18 and non-cytokine type pro-inflammatory factors.

Description

Culture medium composition suitable for NK cells and culture method

Technical Field

The invention relates to the technical field of biology, in particular to a culture medium composition suitable for NK cells and a culture method.

Background

Natural killer cells (natural killer cell, NK cells) are an important lymphocyte subpopulation that plays an important role in the human innate immune system, the primary source of human mature NK paper cells being peripheral blood. As early as 70 s, researchers have described them, but until the last 15 years, NK cells have not come to know their complexity and therapeutic potential in helping combat cancer and other diseases. Since NK cells account for only 5% -10% of peripheral blood mononuclear cells, purification and in vitro amplification of NK cells are important methodology bases for NK cell basis and clinical research.

However, since NK cells derived from peripheral blood are difficult to be amplified in large quantities in vitro, and the separation and purification of NK cells from peripheral blood have a certain technical difficulty, the pollution of lymphocytes is difficult to be avoided, and a large number of uniform NK cells cannot be obtained and reach the clinically required standard, the NK cells cannot be widely applied to clinic; in addition, in some cases, NK cell mediated antitumor response is weak and may be related to factors such as poor NK cell killing, poor survival in vivo, or limited migration to tumor sites.

Therefore, the purification culture effect of NK cells is still not ideal and cannot meet the practical application.

Disclosure of Invention

The invention provides a culture medium composition suitable for NK cells, which comprises a basal medium, 3-10 v/v% of serum, IL2, IL18 and non-cytokine pro-inflammatory factors.

According to still another aspect of the present invention, there is also provided a medium combination kit for NK cells comprising the medium composition for NK cells as described above and a CD3 antibody.

According to still another aspect of the present invention, there is also provided a NK cell culturing method comprising the steps of:

a) Inoculating mononuclear cells into a container containing a CD3 antibody, and culturing for 3 to 5 days using the culture medium composition of NK cells as described above;

b) The culture medium composition of NK cells as described above was used for 9 to 11 days in a container not containing CD3 antibody.

The culture medium composition provided by the invention has simple and clear components, the purity of NK cells obtained by culture is higher, the proliferation efficiency is higher, and the cell killing capacity is very strong, and particularly, the invention has unexpectedly found that the group added with serum amyloid A can obviously improve the killing capacity of NK cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic representation of NK cell culture; a is example 1; b is example 2; c is a comparative example;

FIG. 2 shows the results of flow cytometric analysis of example 1 of the present invention;

FIG. 3 shows the results of flow cytometric analysis of example 2 of the present invention;

FIG. 4 is a flow cytometric analysis of the comparative example;

FIG. 5 shows a cell proliferation curve corresponding to the different embodiments;

FIG. 6 shows the detection of NK cell killing ability according to various embodiments.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).

The symbol "%" as used herein, unless otherwise indicated, generally refers to weight/weight percent when the total material is solid; where the total material is liquid, it is generally referred to as weight/volume percent. Of course, where the total material is liquid and the solute is liquid, the percentage characterizing the liquid solute is generally referred to as the volume/volume percentage.

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.

Concentration values are referred to in this invention, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated.

In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention. In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

The first aspect of the present invention relates to a culture medium composition suitable for NK cells comprising basal medium, 3v/v% to 10v/v% serum, IL2, IL18 and non-cytokine type pro-inflammatory factors.

According to the invention, in the NK cell culture process, non-cytokine pro-inflammatory factors (particularly LPS and SSA) are added to be matched with IL2, IL18 and CD3 antibodies for the first time, so that the purity and proliferation capacity of NK cells can be obviously improved, and the killing capacity of the NK cells can be improved.

In the present invention, the culture medium composition may be packaged in the form of a solution, or may be packaged in the form of a dry powder composition (e.g., a lyophilized powder or a spray-dried powder) and then dissolved at the time of use. When the composition is a solution, the ratio of each component in the composition or the solution satisfies a predetermined concentration when the composition or the solution is formulated as a working concentration. Usually in the form of working concentrations or mother liquor. In the present invention, the "working concentration" is used to define the proportion of the main active ingredient of the medium composition at the time of culturing NK cells, and the concentration of the main active ingredient in the medium composition may be the working concentration or may be a mother liquor (

e.g. mother liquor

2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100-fold concentrated) which can be diluted to that concentration.

It will be appreciated that for IL2, IL18, or CD3 antibodies, SSA, etc., components, they may include full length components or variants that retain their activity. Variants may comprise substitutions, deletions, additions or modifications of one or more amino acids relative to the wild-type protein, etc. Their origin may be recombinant expression, the original source preferably being the same as the NK cells being cultured, e.g. of human origin.

The use of amino acid supplements, including any amino acid, including glycine, alanine, valine, leucine, isoleucine, arginine, lysine, aspartic acid, cysteine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, asparagine, glutamine, histidine and serine, is not excluded from the present application, or is an addition of some conventional nutritional supplements, such as salts, antibiotics, vitamins and amino acids, to the medium of the present application. Antibiotics such as gentamicin, penicillin, streptomycin, ampicillin, kanamycin, and the like.

The medium of the present application may also contain a buffer, the type of which is not particularly limited and may be of a type well known to those skilled in the art. A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. Various buffers that may be employed depending on, for example, the desired pH of the buffer (as well as the microbial growth and metabolic characteristics, microbial culture system, pH control and the medium used) are described in buffers, aguide for the Preparation and Use of Buffers in Biological Systems, gueffroy, d., edit Calbiochem Corporation (1975). In one embodiment, the buffer has a pH in the range of about 2 to about 9, or about 3 to about 8, or about 4 to about 7, or about 5 to about 7. Non-limiting examples of buffers that will control the pH in this range include MES, MOPS, MOPSO, tris, HEPES, phosphate, acetate, citrate, succinate and ammonium buffers, as well as combinations of these. In some embodiments, the buffer is selected from the group consisting of 3- (N-morpholino) propanesulfonic acid (MOPS) free acid, 3- (N-morpholino) propanesulfonic acid (MOPS) Na, hydroxyethylpiperazine ethanesulfonic acid (HEPES), and sodium bicarbonate.

In the present invention, unless otherwise specified, the culture environment of the cells is about 5% CO ₂ And at about 37 ℃.

In some embodiments, formulation aids, such as defoamers, may also be added to the medium. In some embodiments, the defoamer of the medium formulation comprises an ionic or nonionic surfactant.

In some embodiments, the non-cytokine based proinflammatory factor comprises lipopolysaccharide and/or serum amyloid a (SSA).

In some embodiments, the non-cytokine based proinflammatory factor is added in an amount of 0.1 to 1 μg/mL, e.g., 0.3 μg/mL, 0.5 μg/mL, 0.7 μg/mL.

In some embodiments, the IL2 is added in an amount of 500 to 1000U/mL, such as 600U/mL, 700U/mL, 800U/mL, 900U/mL.

In some embodiments, the IL18 is added in an amount of 200 to 600U/mL, such as 300U/mL, 400U/mL, 500U/mL.

It is readily understood that the basal medium is a medium that facilitates NK cell culture, and in some embodiments, is selected from the group consisting of X VIVO-15, RPMI1640, AIM-V, alpha-MEM medium.

The concentration of serum may be 4v/v%, 5v/v%, 6v/v%, 7v/v%, 8v/v%, 9v/v%. In some embodiments, the serum is autologous serum.

The second aspect of the present invention relates to a medium combination kit suitable for NK cells comprising the medium composition suitable for NK cells as described above and CD3 antibodies.

A third aspect of the present invention relates to a NK cell culturing method comprising the steps of:

In some embodiments, the CD3 antibodies are coated in the container.

In some embodiments, the CD3 antibody is added to the container in free form.

The CD3 antibody is used in conventional amounts at NK cell culture, and in some embodiments, the CD3 antibody is added in an amount of 50 to 500ng/mL, for example 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL.

In some embodiments, the peripheral blood mononuclear cells have an seeding density of (1-5) x 10 ⁶ Per mL, e.g. 2X 10 ⁶ 3X 10 per mL ⁶ Per mL, 4X 10 ⁶ And each mL.

In some embodiments, the cells of step b) are passaged once after culturing day 2-4.

In some embodiments, the cells of step b) are supplemented with IL2, IL18 and non-cytokine based pro-inflammatory factors to about the initial concentration of step b) after 2-4 days of culture.

Mononuclear cells may be derived from peripheral blood or cord blood.

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

In the following examples:

human recombinant serum amyloid a (SSA) was purchased from Peprotech (Rocky Hill, NJ, USA).

Lipopolysaccharide (LPS) was purchased from Solarbio, a technology Co., ltd.

NK cell serum-free medium XVO-15 is a product of Lonza company of America.

Recombinant human IL-2 and group human IL-18 were purchased from Tokyo Hospital Seisakusho Biotech Co.

Anti-human CD3 antibodies are self-purified by the applicant.

Example 1

1. Isolation of umbilical cord blood mononuclear cells (PBMC)

Collecting PBMC of a patient through a blood cell separator, transferring the collected blood sample to a centrifuge tube, centrifuging for 10min at 700g, and sucking the upper plasma for later use; reducing the blood sample to the original volume by using normal saline, and uniformly mixing; slowly adding diluted blood on Ficol1 separating liquid, centrifuging for 20min at 900 g; sucking the milky mononuclear cell layer at the interface of the liquid separation, washing with 0.9% physiological saline, and centrifuging for 2 times; PBMCs were resuspended in serum-free medium.

NK cell culture

The concentration of PBMC cells was adjusted to 1X 10 ⁶ Adding 5% autologous plasma, recombinant human IL-2 700U/mL, recombinant human IL18 400U/mL, SSA 0.5 μg/mL, and anti-human CD3 mAb 200ng/mL X VIVO-15 medium into antibody plated culture flask, and placing in 5% CO ₂ The culture was continued at 37℃for 4 days. On day 5, the medium was changed to a medium containing no CD3 antibody, and the rest was the same as before. The culture period was continued for 10 days (i.e., co-culture for 14 days) depending on the medium color and NK colony size half-dose (cytokine and SSA supplementation) or the change of the fluid (3 days of fluid change).

Example 2

The difference is only that SSA is replaced by LPS as in example 1

Comparative example

The only difference from example 1 is that no SSA was added.

Experimental example

NK cell culture results

The cell pictures obtained at 14 days of culture for example 1, example 2 and comparative example are shown in FIG. 1. Each group is generally typical of NK cells, with some cells deforming to form raised or thick, long pseudopodia.

Flow cytometric analysis was performed on the cells obtained by culturing each group for 14 days to detect the molecular expression on the cell surface, and the results are shown in table 1 and fig. 2 to 4.

TABLE 1

From the results, the purity of NK cells is obviously increased to more than 95% in the example group added with inflammatory factors.

2. The cell numbers of each group were examined on

days

6, 8, 10, 12, and 14 of culture, and the results are shown in FIG. 5. It can be seen that the comparative example of example 1 and example 2 significantly improved in amplification rate. Wherein the final amplification factor of the example 1 group is 674 times, the final amplification factor of the example 2 group is 512 times, and the final amplification factor of the comparative example group is 310 times.

NK cell killing Activity comparison

The killing experiment method comprises the following steps:

the test subjects were cells on day 14 of culture. Different groups of NK cells and tumor cells (K562-Luc) are respectively added into a white impermeable 96-well plate according to the effective target ratio (E/T ratio) of 1:1, 5:1, 10:1 and 20:1, the number of the tumor cells is fixed to 5000 per well, namely, the number of the NK cells is 5000, 25000, 50000 and 100000 respectively, a corresponding number of tumor cell blank groups and NK cell blank groups are simultaneously arranged, three repeated holes are arranged per group of cells, the cell volume of each hole is adjusted to be 100uL, and the cells are subjected to static culture for 24 hours after uniform mixing.

Bright-Lumi is as follows: 1 volume ratio is added into a culture medium of a white 96-well plate, and a luminescence value of the luminecent is directly read by an enzyme-labeled instrument.

Cell killing efficiency was calculated according to chemiluminescent values, and the counting formula was as follows:

1- (Mixed group emission value-blank NK group emission value)/blank tumor group emission value×100%

The killing efficiency of the three groups of cells is shown in figure 6. From the results, the killing activity of the three groups of cells is higher, and surprisingly, the killing activity of NK cells is remarkably improved by the SSA group compared with the embodiment group added with LPS.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.

Claims

1. The culture medium composition suitable for NK cells is characterized by comprising a basal medium, 3-10 v/v% of serum v/v%, 500-1000U/mL of IL2, 200-600U/mL of IL18 and 0.1-1 mug/mL of serum amyloid A.

2. The medium composition suitable for NK cells according to claim 1, wherein the basal medium is selected from X VIVO-15, RPMI1640, AIM-V, alpha-MEM medium.

3. The medium composition suitable for NK cells according to claim 1 or 2, wherein the serum is autologous serum.

4. A culture medium combination kit suitable for NK cells, comprising the culture medium composition suitable for NK cells according to any one of claims 1 to 3 and a CD3 antibody.

An nk cell culture method characterized by comprising the steps of:

a) Inoculating peripheral blood mononuclear cells into a container containing a CD3 antibody, and culturing for 3-5 days by using the culture medium composition of NK cells according to any one of claims 1-3;

b) Culturing for 9-11 days in a container without CD3 antibody using the NK cell culture medium composition of any one of claims 1-3.

6. The NK cell culture method according to claim 5, wherein the inoculation density of the peripheral blood mononuclear cells is (1 to 5). Times.10 ⁶ And each mL.

7. The NK cell culture method of claim 5 wherein the CD3 antibodies are coated in the vessel or the CD3 antibodies are added in free form to the vessel.

8. The NK cell culture method of claim 7, wherein the amount of CD3 antibody added is 50ng/mL to 500ng/mL.