CN113684179A - Cell amplification medium for gamma delta T, combination product and culture method thereof - Google Patents

Abstract

The invention relates to a cell expansion medium for gamma delta T, a combination product and a culture method thereof. The gamma delta T cell expansion medium comprises a basal medium and additional components, wherein the additional components comprise: 5-15% (v/v) FBS, IL 2200 IU-800 IU/mL, IL 211 ng-20 ng/mL, and coenzyme Q105-20. mu. mol/L. Compared with the traditional method, the method can obviously improve the amplification efficiency and the cell purity of the gamma delta T cells, a large amount of high-purity gamma delta T cells can be obtained by culture, and the killing activity of the gamma delta T cells to tumor cells can be effectively maintained. The gamma delta T cell can be used for foreign body feedback after being modified by a genetic engineering means to express the chimeric antigen receptor, and does not generate graft-versus-host disease, thereby having wider application prospect.

Description

Cell amplification medium for gamma delta T, combination product and culture method thereof

Technical Field

The invention relates to the technical field of cell culture, in particular to a cell amplification culture medium for gamma delta T, a combined product and a culture method thereof.

Background

T cells can be classified into α β T cells and γ δ T cells according to the TCR receptor. Wherein, the alpha beta T cells account for more than 95 percent of the total number of the T cells, and the gamma delta T cells account for 1 to 5 percent of the total number of the T cells and are mainly distributed in mucosa-associated lymphoid tissues. The gamma delta T cell is an immune cell which can kill cancer cells and tumor stem cells and can recognize cancer antigens. Although the proportion of γ δ T cells is low, meta-analysis of the expression profile of about 18000 human tumors from 39 malignancies indicates that the expression of γ δ T cells within tumors is one of the most favorable prognostic indicators for survival. In addition, various studies have shown that γ δ T cells recognize and kill tumor cells.

A variety of common antigens are used to stimulate the expansion of γ δ T cells, such as isopentenyl pyrophosphate (IPP), (E)4 hydroxy 3 methyl but 2 alkenyl pyrophosphate (HMBPP), zoledronate (Zometa), and bromohydroproteinate pyrophosphate (BrHPP), while zoledronate is widely used to stimulate γ δ T cell expansion. The traditional method is to add zoledronic acid and IL2 into the culture medium of PBMC, which can obtain gamma delta T with higher purity, but has the defect that a large amount of gamma delta T cells cannot be obtained, so that the application in clinical treatment is difficult.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a gamma delta T cell amplification culture medium, which comprises a basic culture medium and additional components, wherein the additional components comprise:

5-15% (v/v) FBS, IL 2200 IU-800 IU/mL, IL 211 ng-20 ng/mL, and coenzyme Q105-20. mu. mol/L.

Optionally, the γ δ T cell expansion medium as described above, the additional components comprising:

7-13% (v/v) FBS, IL 2400 IU/mL-600 IU/mL, IL 217 ng/mL-13 ng/mL, and coenzyme Q107-13 μmol/L.

Optionally, the γ δ T cell expansion medium as described above, the additional component further comprising vitamin C.

Optionally, the concentration of the vitamin C in the γ δ T cell amplification medium is 5 μmol/L to 20 μmol/L.

Optionally, the γ δ T cell expansion medium as described above, and the basal medium is Optivitro medium.

A second object of the present invention is to provide a combination product comprising the γ δ T cell expansion medium and the γ δ T cell activation medium as described above;

the gamma delta T cell activation culture medium comprises 5-15% (v/v) FBS, IL 2200 IU/mL-800 IU/mL, IL 211 ng/mL-20 ng/mL and bisphosphonate 1-10 mu M.

Optionally, the combination product as described above, wherein the bisphosphonate comprises one or more of zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, and milo phosphoric acid.

A third object of the present invention is to provide a γ δ T cell culture method, comprising:

b) the γ δ T cells were cultured with γ δ T cell expansion medium as described above.

Optionally, the cultivation method as described above, further comprising step a) before b): γ δ T cells were pre-cultured with γ δ T cell activation medium as described above.

Optionally, in the above-mentioned culture method, the pre-cultured cells are peripheral blood mononuclear cells, and the seeding density is (2-3) × 10⁶/mL。

Optionally, in the culture method, the pre-culture time is 2-4 days.

Optionally, the culture method as described above, the seeding density of the cells initially cultured in step b) is (1-2). times.10⁶/mL。

Optionally, in the above culture method, in step b), the cells are changed every 2 to 3 days.

The fourth purpose of the invention is to provide the application of the method in preparing the chimeric antigen receptor gamma delta T cells.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a culture medium capable of efficiently amplifying gamma delta T cells and a corresponding culture method. The method adds the coenzyme Q10 and other components in the process of amplifying and culturing the gamma delta T cells, and compared with the traditional method, the method can obviously improve the amplification efficiency and the cell purity of the gamma delta T cells and culture a large amount of high-purity gamma delta T cells. The inventors have also surprisingly found that the culture medium can effectively maintain the killing activity of the gamma delta T cells on the tumor cells. The gamma delta T cell has wide application, can be used for foreign body feedback after being modified and expressed by a genetic engineering means, and does not generate Graft Versus Host Disease (GVHD).

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a flow chart of a culture method using high efficiency amplification of γ δ T cells according to an embodiment of the present invention;

FIG. 2 is a gamma delta T cell expansion fold curve according to an embodiment of the present invention;

FIG. 3 is a graph of D14 γ δ T cell expansion fold according to an embodiment of the present invention;

FIG. 4 shows the results of an experiment for killing target cells by γ δ T in one embodiment of the present invention;

FIG. 5 shows the results of an experiment for killing target cells by γ δ T in one embodiment of the present invention.

Detailed Description

Reference will now 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. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing the invention are to be interpreted as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions serve to better understand the teachings of the present invention by way of further guidance. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

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

The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range, as well as the recited endpoints. In the description of the invention, "a plurality" means at least two, e.g., two, three, etc., unless explicitly specified otherwise. In the description of the present invention, "a plurality" means at least one, e.g., one, two, etc., unless specifically limited otherwise.

The invention relates to a gamma delta T cell amplification culture medium, which comprises a basic culture medium and additional components, wherein the additional components comprise:

5-15% (v/v) FBS, IL 2200 IU-800 IU/mL, IL 211 ng-20 ng/mL, and coenzyme Q105-20. mu. mol/L.

Coenzyme Q10(CO Q10) is an important antioxidant, and can be used as an inhibitor of mitochondrial permeability transition pore, mitochondrial membrane potential and erythropoiesis, and can protect various cells from apoptosis caused by oxidative stress, such as T lymphocytes and B lymphocytes.

CO Q10 derivatives comprising CO Q10 complexed or bound to other molecules may also be used in the γ δ T cell expansion media of the present invention. For example, esterified ubiquinones such as acetyl CO Q10, succinyl CO Q10, or propionyl CO Q10 may be used. Alternatively, CO Q10 complexes, such as the polyoxyethylene- α -tocopherol sebacate (PTS) -CO Q10 complex or the CO Q10-cyclodextrin complex, may be used. In the following description of the preferred embodiments, it will be appreciated that in the described embodiments, a CO Q10 derivative may be used in place of CO Q10.

It is understood that the components of IL2, IL21 and the like can be isolated natural proteins, can also be recombinant expression proteins, and also comprise mutants with similar functions with wild-type IL2 and IL 21.

Through the reasonable proportion of the components, the invention can obviously improve the amplification efficiency and the cell purity of the gamma delta T cells, and a large amount of high-purity gamma delta T cells are obtained by culturing. The inventors have also surprisingly found that the culture medium can effectively maintain the killing activity of the gamma delta T cells on the tumor cells.

In some embodiments, the concentration of IL2 is 300IU/mL, 400IU/mL, 500IU/mL, 600IU/mL, 700 IU/mL.

In some embodiments, the concentration of IL21 is 2ng/mL, 4ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL, 10ng/mL, 11ng/mL, 12ng/mL, 13ng/mL, 15ng/mL, 17ng/mL, 19 ng/mL.

In some embodiments, the concentration of coenzyme Q10 is 6. mu. mol/L, 7. mu. mol/L, 8. mu. mol/L, 9. mu. mol/L, 10. mu. mol/L, 11. mu. mol/L, 12. mu. mol/L, 13. mu. mol/L, 14. mu. mol/L, 15. mu. mol/L, 16. mu. mol/L, 17. mu. mol/L, 18. mu. mol/L, 19. mu. mol/L.

In some embodiments, the additional components include:

7-13% (v/v) FBS, IL 2400 IU/mL-600 IU/mL, IL 217 ng/mL-13 ng/mL, and coenzyme Q107-13 μmol/L.

In some embodiments, the additional component further comprises vitamin C.

Vitamin C is a potent antioxidant and free radical scavenger, and is involved in a variety of physiological processes. Research shows that vitamin C can promote the proliferation of T cells, regulate the functions of T cells and play an important role in immunotherapy.

Vitamin C derivatives comprising vitamin C complexed or bound to other molecules, or vitamin C can be obtained by solubilization or the like, can also be used in the γ δ T cell expansion medium of the present invention. For example, esterified ubiquinones such as Sodium Ascorbyl Phosphate (SAP) or Magnesium Ascorbyl Phosphate (MAP), Ascorbyl palmitate, Ascorbyl glucoside, Tetra-isopalmitoyl Ascorbyl, Ascorbyl2-phosphate 6-palmitate may be used. In the following description of the preferred embodiments, it will be understood that in the described embodiments, vitamin C derivatives may be used instead of vitamin C.

In some embodiments, the concentration of vitamin C is 5 μmol/L to 20 μmol/L, and may also be 6 μmol/L, 7 μmol/L, 8 μmol/L, 9 μmol/L, 10 μmol/L, 11 μmol/L, 12 μmol/L, 13 μmol/L, 14 μmol/L, 15 μmol/L, 16 μmol/L, 17 μmol/L, 18 μmol/L, or 19 μmol/L.

It is readily understood that the basal medium is a medium that favors activation, growth, or proliferation of γ δ T cells, and in some embodiments, is Optivitro medium.

According to a further aspect of the invention, it also relates to a combination product comprising a γ δ T cell expansion medium as described above and a γ δ T cell activation medium;

the gamma delta T cell activation culture medium comprises 5-15% (v/v) FBS, IL 2200 IU/mL-800 IU/mL, IL 211 ng/mL-20 ng/mL and bisphosphonate 1-10 mu M.

In some embodiments, the γ δ T cell activation medium comprises 7% to 13% (v/v) FBS, IL 2400 IU/mL to 600IU/mL, IL 217 ng/mL to 13ng/mL, and bisphosphonate 3 μ M to 8 μ M.

It will be appreciated that the bisphosphonate may be a first, second or third generation bisphosphonate, particularly a soluble salt, further the bisphosphonate includes one or more of zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, and miltiorrhynchosic acid.

The present invention does not exclude the use of other media, or the addition of conventional nutritional supplements such as salts, antibiotics, vitamins, and amino acids to the media of the present application, and the amino acid supplement may include 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. Antibiotics such as gentamicin, penicillin, streptomycin, ampicillin, kanamycin, and the like.

The medium of the present invention may also contain a buffer, and the type thereof is not particularly limited and may be one well known to those skilled in the art. A "buffer" is a solution that resists pH changes by the action of its acid-base conjugate components. Various Buffers that can be employed depending on, for example, the desired pH of the buffer (as well as the microbial growth and metabolic characteristics, the microbial culture system, pH control and the medium used) are described in Buffers. In one embodiment, the buffer has a pH in the range of about 2 to about 9, alternatively about 3 to about 8, alternatively about 4 to about 7, alternatively about 5 to about 7. Non-limiting examples of buffers that will control pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, and 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.

According to a further aspect of the present invention, it also relates to a γ δ T cell culture method comprising:

b) the γ δ T cells were cultured with γ δ T cell expansion medium as described above.

In some embodiments, step a) is further included before b): γ δ T cells were pre-cultured with γ δ T cell activation medium as described above.

In some embodiments, the pre-cultured cells are peripheral blood mononuclear cells seeded at a density of (2-3). times.10⁶/mL。

In the present invention, Peripheral Blood Mononuclear Cells (PBMC), which are cells isolated from animals, or γ δ T cells, may be used, which are cells from mammals (e.g., human cells) or cells from non-human mammals (e.g., monkeys, mice, rats, rabbits, pigs, sheep, cows, horses, cats, or dogs). PBMCs can be isolated by well known methods, such as density gradient centrifugation. Preferably, the PBMCs are derived from healthy donors.

In the invention, the gamma delta T cell can be one or a mixture of V delta 1T cells, V delta 2T cells and V delta 3T cells.

In some embodiments, the pre-incubation time is 2-4 days.

In some embodiments, the density of the cells seeded at the initial culture in step b) is (1-2). times.10⁶/mL。

In some embodiments, in step b), the cells are plated every 2 to 3 days.

In some embodiments, in step b), the culturing is for a period of greater than or equal to 18 days, e.g., 20 days, 22 days, 24 days.

In the present invention, the culture environment of the cells is, but not particularly limited to, about 5% CO₂And about 37 degrees celsius.

According to a further aspect of the invention, the use of the method as described above for the preparation of chimeric antigen receptor γ δ T cells is also contemplated.

As used herein, "Chimeric Antigen Receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a polypeptide other than the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric Antigen Receptors (CARs)" are sometimes referred to as "chimeric receptors", "T-bodies", or "Chimeric Immunoreceptors (CIRs)". "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide that can bind to a particular antigen. By "intracellular domain" is meant any oligopeptide or polypeptide known to function in a cell as a domain that transmits signals to cause activation or inhibition of a biological process.

In some embodiments, the extracellular domain of the chimeric antigen receptor (typically sc-Fv) is capable of specifically recognizing a tumor antigen. "tumor antigen" refers to an antigenic biomolecule, the expression of which is newly recognized to be associated with cellular carcinogenesis. Detection, e.g., immunological detection of tumor antigens, can be used to distinguish cancerous cells from their parent cells. Tumor antigens in the present invention include tumor-specific antigens (antigens that are present only in tumor cells and not in other normal cells) and tumor-associated antigens (antigens that are also present in other organs and tissues or in heterogeneous and allogeneic normal cells, or antigens that are expressed during development and differentiation). In the present invention, the tumor (or cancer) is a hematological tumor or a solid tumor, and the tumor antigen can be selected from common tumor targets, such as: alpha-fetoprotein, alpha-actinin-4, A, an antigen specific for an A antibody, ART-4, B, Ba 733, BAFF-R, BAGE, BCMA, BrE antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m, CCL, CD1, CD11, CD32, CD40, CD 66/B/c/e, CD70, CD79, CD117(c-kit), CD123, CD126, CD132, CD138, CD147, CD154, CD319(CS, CD 319), CLAMF, CLL-1, CDK 2, CDK 2/c-kit, CD-1, CDK, CD-1, CDK, CD-1, CDK, CD-1, CDK, CD-c-1, CD-c/e, CD-1, CD-c/e, CD-kit, CD-K, CD-1, CD-K, CD-K, CD-K, CD-K, CD-K, CD-K, CD-K, CD-1, CD-K, CD-K, HIF-1 α, Colon Specific Antigen P (CSAP), CEA (CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Fibroblast Growth Factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO- β, GPRC5D, HLA-DR, HM1.24, Human Chorionic Gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN- γ, IFN- α, IFN- β, IFN- λ, IL-4R, IL-6R, IL-13R, IL-15-32-17R, IL-18R 15R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor 1(IGF-1), Ig kappa, IL1RAP, LewisY, LMP1, KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR/FUT, macrophage Migration Inhibitory Factor (MIF), MAGE-3, MART1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MMG49, MIP-1-8-1B, MIF, MIC 1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MIPM-3, MUA 66, NCA 635, NCA90, NKG2, NKG D, MUC5, MUC13, MUC 39 2, prostate growth factor 599, and prostate growth factor 599, PSA, PRAME, PSMA, PlGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, ROR1, T101, SAGE, S100, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF- α, Tn antigen, Thomson-Fredenbis antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factor C3, C3a, C3B, C5a, C5, angiogenic markers, bc1-2, bc1-6, Kras, oncogenic markers, and oncogenic gene products.

According to a further aspect of the present invention, there is also provided a method of treating cancer in a patient in need thereof, the method comprising culturing γ δ T cells obtained by the cell culture method as described above, and allowing the cells to express a chimeric antigen receptor to obtain host cells;

administering to the patient a therapeutically effective amount of a host cell as described above, thereby treating the cancer.

In the above method, the donor and the recipient of the γ δ T cell may be the same or different individuals.

A "patient" is a mammal, including, but not limited to, humans, monkeys, pigs and other farm animals, sport animals, pets, primates, horses, dogs, cats, rodents (including mice, rats, guinea pigs), and the like.

The phrase "effective amount," as used herein, means an amount of a compound or composition within the scope of reasonable medical adjustment that is large enough to significantly effectively alleviate the symptom or condition being treated, but small enough to avoid serious side effects (at a reasonable benefit/risk ratio). The safe and effective amount of active ingredient in the host cell used in the methods of the invention will vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the disease, the time of treatment, the concurrent condition, the particular active ingredient used, the particular pharmaceutically acceptable excipient used and such factors including the knowledge and skill of the attending physician.

In some embodiments, the cultured cells are allogeneic to the patient, i.e., two different individuals of the same species.

Embodiments of the present invention will be described in detail with reference to examples.

Examples

The flow chart of the γ δ T cell culture method used in this example is shown in fig. 1. The detailed steps are as follows:

1. PBMC separation: collecting 50ml of peripheral blood, and under the normal working state of a clean bench, respectively adding 15ml of lymphocyte separation solution into 250 ml sterilized centrifuge tubes. The peripheral whole blood is slowly injected into the upper layer of the liquid surface of the lymphocyte separating medium in 2 centrifuge tubes, and 25-30ml of peripheral blood is added into each centrifuge tube. Centrifugation, 700g × 20 min, ramp-up 1, ramp-down 2, room temperature. After centrifugation, the blood was separated into 4 layers consisting of plasma (upper layer), mononuclear cells between the plasma and the separation liquid (layer 2), the separation liquid (layer 3), and a red blood cell layer (bottom layer). Layer 2 mononuclear cells were collected with a pipette into a new centrifuge tube. 20ml of PBS was added to dilute the cell suspension and centrifuged at 500g for 10 min.

2.γ δ T cell activation: resuspending cells with γ δ T cell activation medium (Optivitro medium containing 10% (v/v) FBS, 5 μ M zoledronic acid, 500IU/mL IL2, 10ng/mL IL21), cell counting, adjusting cell density to 2-3 × 10⁶/mL, seeded in cell culture flasks at 37 ℃ with 5% CO₂Culturing in an incubator.

3.γ δ T cell expansion culture: the cells were equally divided into 4 groups, changed to γ δ T cell expansion medium (Optivitro medium containing 10% (v/v) FBS, 500IU/mL IL2, 10ng/mL IL21, 10 μmol/L coenzyme Q10, 10 μmol/L vitamin C), and cell density was adjusted to 1-2 × 10⁶/mL, seeded in cell culture flasks at 37 ℃ with 5% CO₂Culturing in an incubator.

4. Appropriate amounts of samples were taken at D0, D5, D7, D10, D12, D14, D16, D18, D20, D22, D24 for counting.

The results of the experiment are shown in fig. 2 and 3.

In the present invention, IL2+ IL21 denotes a medium in which a conventional method is employed in the scale-up culture, that is, a medium in which IL2 and IL21 are added to a basal medium;

IL2+ IL21+ VC indicates that a medium supplemented with IL2, IL21, and vitamin C in a basal medium was used for the expansion culture;

IL2+ IL21+ CO Q10 indicates that a medium prepared by adding IL2, IL21 and coenzyme Q10 to a basal medium was used for the expansion culture;

IL2+ IL21+ VC + CO Q10 indicates that a medium supplemented with IL2, IL21, vitamin C and coenzyme Q10 in a basal medium was used in the expansion culture.

The results show that: the addition of coenzyme Q10 significantly enhanced the expansion of γ δ T cells and maintained better activity after D18 days. The proliferation efficiency of the gamma delta T cells is low by using a culture medium of a traditional method, namely a culture medium without adding vitamin C and coenzyme Q10, and the proliferation efficiency of the gamma delta T cells can be effectively improved by adding the culture medium with the vitamin C and the coenzyme Q10. At D14, the fold expansion of γ δ T cells cultured using medium supplemented with vitamin C and coenzyme Q10 was significantly higher than γ δ T cells cultured using conventional medium.

5. Gamma delta T cells and Daudi-luc cells are inoculated on a 96-well plate according to the effective target ratio of 10:1 in D10, D12, D14, D16 and D18, the volume is 100 mu L/well, the cells are co-cultured for 4 to 6h, and luciferase detection reagent with the same volume is added for detection.

As shown in fig. 4 and 5, γ δ T cultured using these four media was effective in killing Daudi-luc cells, indicating that the addition of vitamin C and coenzyme Q10 maintained the killing activity function of γ δ T cells. Especially on days D18 and D22, the cell killing activity without added antioxidant ingredient was significantly reduced, which may be related to the antioxidant effect of vitamin C and coenzyme Q10. Furthermore, the addition of coenzyme Q10(IL2+ IL21+ CO Q10) is more apt to maintain this killing activity than the addition of vitamin C (IL2+ IL21+ VC).

In conclusion, the addition of vitamin C and coenzyme Q10 can efficiently amplify the gamma delta T cells, and can better maintain the tumor cell killing activity of the gamma delta T cells in the later culture period. Therefore, the culture medium can be used for harvesting a large amount of high-quality gamma delta T cells, and a large amount of high-quality cell materials are provided for foreign body feedback after the chimeric antigen receptor is modified by genetic engineering.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.

Claims (13)

1. A γ δ T cell expansion medium, comprising a basal medium and additional components, the additional components comprising:

5-15% (v/v) FBS, IL 2200 IU-800 IU/mL, IL 211 ng-20 ng/mL, and coenzyme Q105-20. mu. mol/L.

2. The γ δ T-cell expansion medium according to claim 1, wherein the additional components comprise:

7-13% (v/v) FBS, IL 2400 IU/mL-600 IU/mL, IL 217 ng/mL-13 ng/mL, and coenzyme Q107-13 μmol/L.

3. The γ δ T-cell expansion medium according to claim 1, wherein the additional component further comprises vitamin C, preferably at a concentration of 5 μmol/L to 20 μmol/L.

4. The γ δ T-cell expansion medium according to any one of claims 1 to 3, wherein the basal medium is an Optivitro medium.

5. A combination comprising the γ δ T cell expansion medium according to any one of claims 1 to 4 and a γ δ T cell activation medium;

the gamma delta T cell activation culture medium comprises 5-15% (v/v) FBS, IL 2200 IU/mL-800 IU/mL, IL 211 ng/mL-20 ng/mL and bisphosphonate 1-10 mu M.

6. A combination product according to claim 5, wherein the bisphosphonate comprises one or more of zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, and milo phosphoric acid.

7. A γ δ T cell culture method, comprising:

b) culturing γ δ T cells in the γ δ T cell expansion medium according to any one of claims 1 to 4.

8. The method of claim 7, further comprising step a) before b): preculture of γ δ T cells with the γ δ T cell activation medium of claim 5 or 6.

9. The culture method according to claim 8, wherein the pre-cultured cells are peripheral blood mononuclear cells, and the seeding density is (2-3). times.10⁶/mL。

10. The culture method according to claim 9, wherein the pre-culture time is 2 to 4 days.

11. The method according to any one of claims 7 to 10, wherein the seeding density of the cells initially cultured in step b) is (1 to 2). times.10⁶/mL。

12. The method according to claim 11, wherein the cells are changed every 2 to 3 days in step b).

13. Use of a method according to any one of claims 7 to 12 for the preparation of chimeric antigen receptor γ δ T cells.

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