Serum-free culture medium for CIK cell culture, culture method, and use

Abstract

A serum-free culture medium for CIK cell culture, a culture method, and a use. The serum-free culture medium comprises a basic culture medium, and recombinant human transferrin, recombinant human insulin, recombinant human albumin, ethanolamine, a lipid mixture, a trace element, α-cyclodextrin, a non-essential amino acid, sodium bicarbonate, nicotinamide mononucleotide, and an astaxanthin extract are added into the basic culture medium. The culture medium does not contain any human-derived or animal-derived component, all protein components are recombinant human protein components, and the use of human AB serum or animal-derived serum is not required, so that the biological safety is high. Moreover, the proliferation multiple of CIK cells in the culture medium can be increased, the cell survival rate and the positive rate are remarkably improved, a CIK cell population having a high positive rate (CD3+ CD56+) can be stably cultured, CIK cells can be safely used for subsequent clinical treatment and the like, and the application prospect is good.

Description

Serum-free culture medium, culture method and application for CIK cell culture Technical Field

[0001] The present application relates to the technical field of lymphocyte culture, and in particular to a serum-free culture medium, culture method and application for CIK cell culture. Background Art

[0002] Generally, the growth of animal cells cultured in vitro depends on the presence of serum. Due to the difficulty of obtaining it and the limited quantity, human AB serum and animal serum such as bovine and equine serum are mostly used. Both human and animal serum have the potential risk of contamination by exogenous viruses (such as HIV-1, HCV, HBV, HCMV, EBV, HPV, HHV-6 / 7, human parvovirus B19, HTLV1 and bovine viruses, etc.) and infection by pathogenic factors. In addition, due to the inconsistency of biological activity and factor content between different batches of serum, there are large differences between batches.

[0003] Serum-free culture medium is a basic culture medium with the addition of serum substitutes with clear ingredients, such as proteins, lipids, hormones, and trace elements. These replace serum to provide the nutrients necessary for cell proliferation, reduce the adverse factors caused by unknown components in the serum, stabilize cell culture conditions, reduce batch-to-batch differences, and simplify the procedures for identifying various cell products, thus avoiding the hazards caused by viral contamination from the source.

[0004] CIK cells (cytokine-induced killer) are cytokine-induced killer cells. Because they express both CD3+ and CD56+ membrane proteins, they are also known as NK cell-like T lymphocytes. They combine the potent anti-tumor activity of T lymphocytes with the non-MHC-restricted tumor-killing properties of NK cells. Therefore, the use of CIK cells is considered the preferred option for the next generation of adoptive cell immunotherapy for anti-tumor diseases. However, the proportion of CD3+CD56+ CIK cells in normal human peripheral blood and human umbilical cord blood is relatively small, only approximately 1%-5%. Therefore, it is necessary to culture CIK cells in vitro.

[0005] Commercially available serum-free lymphocyte culture media have low CIK cell positivity rates, are often unstable, and exhibit slow proliferation. They still require supplementation with substances such as human AB serum or fetal bovine serum to promote cell proliferation, making them incapable of industrial, large-scale, stable culture and unable to eliminate the risk of potential pathogen contamination. Therefore, there is an urgent need to develop a serum-free culture medium that is safe, requires no additional serum, and offers excellent proliferation rates and positivity to meet market demand.

[0006] Summary of the Invention

[0007] In order to address the deficiencies of the prior art, the present application provides a serum-free culture medium for CIK cell culture. The culture medium does not contain human and animal-derived components, and all protein components are of human recombinant origin. It does not need to be used with human AB serum or animal-derived serum, and can stably culture CIK cell populations with a high positive rate (CD3+CD56+), and can be safely used for subsequent clinical treatment and other purposes.

[0008] To this end, the first aspect of the present application provides a serum-free culture medium for CIK cell culture, wherein the serum-free culture medium includes a basal culture medium, and the basal culture medium is supplemented with recombinant human transferrin, recombinant human insulin, recombinant human albumin, ethanolamine, a lipid mixture, trace elements, α-cyclodextrin, non-essential amino acids, sodium bicarbonate, nicotinamide mononucleotide and astaxanthin extract.

[0009] In this application, the proteins contained in the serum-free culture medium are all human recombinant sources, with high biosafety and stable culture performance. The addition of recombinant human transferrin, recombinant human insulin and recombinant human albumin in the culture medium can provide the nutrients necessary for cell growth and metabolism; wherein, recombinant human transferrin can reversibly bind to trivalent iron ions, and it plays a vital role in the transport and metabolism of iron ions, and is also an important extracellular antioxidant; recombinant human insulin can promote sugar and amino acid transport, increase anabolism and reduce catabolism, and stimulate cell growth; recombinant human albumin can maintain the osmotic pressure and pH value of the culture fluid system, promote the binding and transportation of important ligands such as lipids, metal ions, amino acids and other factors, and has antioxidant function. Ethanolamine is an important compound that stimulates cell growth and is a precursor for the synthesis of brain phosphate; lipids in lipid mixtures are important components of cell membranes and are involved in signal transduction; trace elements are major components of cellular metabolism, regulating various enzymatic reactions and promoting cell growth metabolism and product synthesis; non-essential amino acids are the main nitrogen source for cells and can serve as energy sources for cell growth and precursors of purine and pyrimidine nucleotides; α-cyclodextrin can solubilize lipids and has excellent inclusion properties, which is beneficial for protecting the stability of nutrients; sodium bicarbonate (NaHCO3) is a non-toxic natural buffer that can stabilize changes in the pH value of the culture medium system.

[0010] More importantly, the serum-free culture medium described in the present application also includes nicotinamide mononucleotide and astaxanthin extract. Nicotinamide mononucleotide (β-NMN) is a precursor of NAD+. β-NMN adenylyltransferase converts β-NMN into nicotinamide adenine dinucleotide (NAD+). NAD+ participates in various redox reactions such as mitochondrial electron transport. As a substrate for various enzymes, it participates in biological processes such as cell signal transduction, DNA damage repair, and epigenetic modification. It can activate lymphocytes and promote lymphocyte proliferation. Astaxanthin extract is a new type of antioxidant with extremely strong antioxidant capacity. It can scavenge free radicals and pro-inflammatory factors from the cell surface and inside the phospholipid membrane, reduce oxidative stress and maintain mitochondrial integrity. In addition to its antioxidant function, astaxanthin also has an immunomodulatory effect, acting on mitogens to induce lymphocyte proliferation, increase cytotoxicity, and increase the secretion of cytokines IFN-γ and IL-2. The inventors of the present application have discovered through research that the addition of nicotinamide mononucleotide and astaxanthin extract to serum-free culture medium can significantly improve the proliferation multiples, cell survival rate, and positive rate (CD3+CD56+) of CIK cells in the culture medium, thereby achieving better CIK cell culture effects.

[0011] The present application does not clearly limit the specific type of the basal culture medium in the serum-free culture medium. In some embodiments, the basal culture medium is selected from any one of RPMI1640, DMEM, DMEM / F12 and IMDM.

[0012] In the present application, the recombinant human transferrin, recombinant human insulin and recombinant human albumin in the culture medium are all commercially available products, and those skilled in the art can obtain them through conventional purchasing channels.

[0013] In some embodiments, the basal culture medium includes 1-20 mg / L recombinant human transferrin, 1-20 mg / L recombinant human insulin, 1-10 g / L recombinant human albumin, 0.5-5 mg / L ethanolamine, 1-5 mL / L lipid mixture, 1-10 mL / L trace elements, 0.5-1.5 g / L α-cyclodextrin, 1-5 mL / L non-essential amino acids, 2-4 g / L sodium bicarbonate, 1-10 mg / L nicotinamide mononucleotide, and 5-20 mg / L astaxanthin extract.

[0014] The present application controls the addition amount of each component in the basal culture medium within the above range, so that the prepared serum-free culture medium can have a better culture effect on CIK cells.

[0015] In some specific embodiments, the content of nicotinamide mononucleotide in the basal medium can be 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, or 10 mg / L. In some preferred embodiments, the content of nicotinamide mononucleotide in the basal medium is 2 to 6 mg / L. In some most preferred embodiments, the content of nicotinamide mononucleotide in the basal medium is 4 mg / L.

[0016] In some specific embodiments, the content of astaxanthin extract in the basal medium is 5 mg / L, 8 mg / L, 10 mg / L, 12 mg / L, 15 mg / L, 18 mg / L or 20 mg / L. In some preferred embodiments, the content of astaxanthin extract in the basal medium is 8-15 mg / L. In some most preferred embodiments, the content of astaxanthin extract in the basal medium is 10 mg / L.

[0017] This application optimizes the content of nicotinamide mononucleotide and astaxanthin extract in the culture medium, so that the prepared serum-free culture medium has the best CIK cell culture effect, and the proliferation multiples, cell survival rate and positive rate (CD3+CD56+) of CIK cells in the culture medium are significantly improved.

[0018] In some embodiments, the lipid mixture is selected from one or more of arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, and tocopheryl acetate.

[0019] In some preferred embodiments, the lipid mixture is a mixture of arachidonic acid, linoleic acid, linolenic acid and oleic acid, and the mass ratio of arachidonic acid, linoleic acid, linolenic acid and oleic acid in the mixture is (1-3):1:1:(1-3).

[0020] In some more preferred embodiments, the mass ratio of arachidonic acid, linoleic acid, linolenic acid and oleic acid in the mixture is 2:1:1:2.

[0021] The present application can further enhance the culture effect of serum-free culture medium on CIK cells by selecting the lipid mixture with the above composition and ratio.

[0022] In some embodiments, the trace element is selected from one or more of sodium selenite, ferric citrate, ammonium metavanadate, manganese chloride, copper sulfate and zinc sulfate.

[0023] In this application, selenium, iron, vanadium, manganese, copper, and zinc in the above-mentioned trace elements all promote cell growth, metabolism, and product synthesis. For example, selenium in sodium selenite, as a cofactor for glutathione peroxidase, has anti-peroxide ability, participates in the elimination of fatty acid peroxides in cells, and increases cell growth rate and activity; iron in ferric citrate is an important component of iron-sulfur proteins, peroxidases, and catalases, and plays an important role in gas transport, biological oxidation, and enzymatic reactions; zinc in zinc sulfate is a component of zinc-containing metalloenzymes, which is related to protein folding and can effectively bind to the disulfide bonds of proteins, affecting protein stability and activity. It plays an indispensable role in RNA / DNA synthesis, mRNA stability, and cell anti-apoptosis. Zinc ions also participate in the activation of glutathione and antioxidant enzymes such as superoxide dismutase and catalase, which can protect cells from ROS attacks.

[0024] In some specific embodiments, the trace elements include sodium selenite, ammonium metavanadate and manganese chloride, and the content of sodium selenite in the basal culture medium is 20-50 ng / L, the content of ammonium metavanadate is 10-20 mg / L, and the content of manganese chloride is 10-20 mg / L.

[0025] In some embodiments, the non-essential amino acids are selected from one or more of L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine, L-proline and L-glutamic acid.

[0026] In some preferred embodiments, the non-essential amino acids are a mixture of L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine and L-proline, and the mass ratio of L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine and L-proline in the mixture is (0.2-1):(1-2):1:(1-2):1:(1-2).

[0027] The present application can further enhance the culture effect of serum-free culture medium on CIK cells by selecting the non-essential amino acids with the above composition and ratio.

[0028] In some embodiments, the pH value of the serum-free culture medium is 7.0-7.4.

[0029] This application controls the pH of the serum-free culture medium to between 7.0 and 7.4, preferably 7.1, by adding a pH regulator to the basal culture medium. CIK cells are more suited to growing in slightly alkaline conditions, and controlling the culture medium pH within the above range is more conducive to the cultivation of CIK cells. The pH regulator described in this application can be sodium hydroxide or hydrochloric acid, etc.

[0030] In this application, the preparation method of the serum-free medium is a conventional method in the art. In some embodiments, the method of preparing 1 L of the serum-free medium may include the following steps:

[0031] S1, dissolving recombinant human insulin in purified water containing 1 mM hydrochloric acid to prepare a recombinant human insulin solution;

[0032] S2, dissolving the culture medium powder required to prepare 1 L of basal culture medium solution in 900 mL of water for injection, stirring until dissolved, and sequentially adding sodium bicarbonate, recombinant human transferrin, recombinant human insulin solution, recombinant human albumin, ethanolamine, lipid mixture, trace elements, α-cyclodextrin, non-essential amino acids, nicotinamide mononucleotide, and astaxanthin extract, and stirring evenly;

[0033] S3, dilute the solution prepared in step S2 to 1 L with water for injection and stir evenly;

[0034] S4, adjusting the pH value of the solution prepared in step S3 to 7.0-7.4 with sodium hydroxide or hydrochloric acid, and then performing 0.22 uM filtration sterilization on the solution to prepare the serum-free culture medium.

[0035] In the present application, the prepared serum-free culture medium can be stored in a light-proof environment at 2-8°C.

[0036] The second aspect of the present application provides a method for culturing CIK cells, which comprises: inoculating peripheral blood mononuclear cells derived from human peripheral blood or umbilical cord blood mononuclear cells into the serum-free culture medium as described in the first aspect of the present application, and culturing CIK cells after adding stimulating factors; the stimulating factors are selected from one or more of IL-2, IL-15, IL-1α and CD3.

[0037] In the present application, the CIK cell culture conditions are conventional conditions in the art, for example, the culture conditions are: temperature of 37.0±0.5° C., and carbon dioxide volume fraction of 5.0±0.2%.

[0038] In some embodiments, the stimulatory factors include IL-2, IL-15, IL-1α and CD3 at the same time, and the amounts of IL-2, IL-15, IL-1α and CD3 added to the culture medium are 800-1200 IU / mL, 15-25 ng / mL, 5-15 ng / mL and 40-60 ng / mL, respectively.

[0039] In the present application, the seeding density of the peripheral blood mononuclear cells and the cord blood mononuclear cells in the serum-free culture medium can be independently (0.5-2)×10 6 pieces / mL.

[0040] The present application controls the cell seeding density within the above range, which is more conducive to the culture of CIK cells.

[0041] The culture medium used in the culture method described in this application is the serum-free culture medium described in this application. This culture medium does not contain human and animal-derived components, and all protein components are of human recombinant origin. It does not need to be used with human AB serum or animal-derived serum. Therefore, the CIK cell population cultured by this method has high biosafety; at the same time, the serum-free culture medium contains a specific composition and is supplemented with specific concentrations of nicotinamide mononucleotide and astaxanthin extract, so that when culturing CIK cells by this method, the proliferation multiples, cell survival rate and positive rate (CD3+CD56+) are higher.

[0042] The third aspect of the present application provides a use of CIK cells cultured by the culture method described in the second aspect of the present application in the preparation of anti-tumor drugs.

[0043] Since the CIK cells cultured by the culture method described in the present application have high biological safety and cell positive rate (CD3+CD56+), the cultured CIK cells can be well used in the preparation of anti-tumor drugs and then safely used in subsequent clinical treatments.

[0044] The beneficial technical effects of this application are as follows: the culture medium provided by this application does not contain human or animal-derived components, all protein components are of human recombinant origin, and does not require use with human AB serum or animal-derived serum, with high biosafety and stable culture performance. At the same time, the serum-free culture medium contains a specific composition, and is supplemented with specific concentrations of nicotinamide mononucleotide and astaxanthin extract, which can significantly improve the proliferation rate, cell survival rate, and positive rate (CD3+CD56+) of CIK cells in the culture medium. It can stably culture a CIK cell population with a high positive rate (CD3+CD56+), and can be safely used for subsequent clinical treatment and other purposes, with good application prospects. DETAILED DESCRIPTION

[0045] To make this application easier to understand, the following examples will be used to further illustrate this application. These examples are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained through commercial channels or conventional methods.

[0046] The basal culture medium, recombinant human transferrin, recombinant human insulin, recombinant human albumin, nicotinamide mononucleotide and astaxanthin extract used in the following examples were all purchased from SIGMA.

[0047] Example 1: Preparation of serum-free culture medium

[0048] Composition: In this embodiment, the basal medium used in preparing the serum-free medium is DMEM, and the basal medium is supplemented with 5 mg / L recombinant human transferrin, 7 mg / L recombinant human insulin, 7 g / L recombinant human albumin, 2 mg / L ethanolamine, 3 mL / L lipid mixture, 5 mL / L trace elements, 1.5 g / L α-cyclodextrin, 3 mL / L non-essential amino acids, 3 g / L sodium bicarbonate, 4 mg / L nicotinamide mononucleotide, and 10 mg / L astaxanthin extract; wherein the lipid mixture contains arachidonic acid, linoleic acid, linolenic acid, and oleic acid, and the mass ratio of arachidonic acid, linoleic acid, linolenic acid, and oleic acid is 2:1: 1:2; the trace elements contain sodium selenite, ammonium metavanadate and manganese chloride, and the contents of the three in the culture medium are 20ng / L, 20mg / L and 10mg / L respectively; the non-essential amino acids contain L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine and L-proline, and the contents of L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine and L-proline in the culture medium are 0.1g / L, 0.5g / L, 0.5g / L, 1.0g / L, 0.5g / L and 0.5g / L respectively, and the mass ratio of the six is ​​0.2:1:1:2:1:1.

[0049] Preparation:

[0050] (1) Weigh recombinant human insulin powder and dissolve it in 50 mL of purified water containing 1 mM HCl. Mix thoroughly by inversion until the recombinant human insulin is completely dissolved and the solution becomes clear, thereby preparing a recombinant human insulin solution.

[0051] (2) Weigh 13.4 g of the powder required to prepare 1 L of DMEM basal medium and dissolve it in 900 mL of water for injection. Stir until dissolved. Then add recombinant human transferrin, recombinant human insulin solution, recombinant human albumin, ethanolamine, lipid mixture, trace elements, α-cyclodextrin, non-essential amino acids, sodium bicarbonate, nicotinamide mononucleotide, and astaxanthin extract in sequence and stir evenly.

[0052] (3) The solution prepared in step (2) was diluted to 1 L with water for injection and stirred evenly;

[0053] (4) preparing sodium hydroxide or hydrochloric acid to a concentration of 1 M, and adjusting the pH of the solution prepared in step (3) to 7.1 with 1 M sodium hydroxide or hydrochloric acid;

[0054] (5) The solution prepared in step (4) was filtered using a disposable filter device with a 0.22 μM pore size and PVDF membrane in a biosafety cabinet in a clean room to prepare a serum-free medium. The prepared serum-free medium was placed in a sterile PETG container and stored in a sealed container at 4°C in a dark environment.

[0055] Example 2: Preparation of serum-free culture medium

[0056] Composition: basically the same as Example 1, except that the amount of nicotinamide mononucleotide added to the basal culture medium is 1 mg / L.

[0057] Preparation: Same as Example 1.

[0058] Example 3: Preparation of serum-free culture medium

[0059] Composition: basically the same as Example 1, except that the amount of nicotinamide mononucleotide added to the basal culture medium is 6 mg / L.

[0060] Preparation: Same as Example 1.

[0061] Example 4: Preparation of serum-free culture medium

[0062] Composition: basically the same as Example 1, except that the amount of nicotinamide mononucleotide added to the basal culture medium is 10 mg / L.

[0063] Preparation: Same as Example 1.

[0064] Example 5: Preparation of serum-free culture medium

[0065] Composition: basically the same as Example 1, except that the content of astaxanthin extract in the basal culture medium is 5 mg / L.

[0066] Preparation: Same as Example 1.

[0067] Example 6: Preparation of serum-free culture medium

[0068] Composition: basically the same as Example 1, except that the content of astaxanthin extract in the basal culture medium is 8 mg / L.

[0069] Preparation: Same as Example 1.

[0070] Example 7: Preparation of serum-free culture medium

[0071] Composition: basically the same as Example 1, except that the content of astaxanthin extract in the basal culture medium is 20 mg / L.

[0072] Preparation: Same as Example 1.

[0073] Example 8: Preparation of serum-free culture medium

[0074] Composition: basically the same as Example 1, except that the lipid mixture contains arachidonic acid, linoleic acid, linolenic acid and oleic acid, and the mass ratio of arachidonic acid, linoleic acid, linolenic acid and oleic acid is 1:1:1:3.

[0075] Preparation: Same as Example 1.

[0076] Example 9: Preparation of serum-free culture medium

[0077] Composition: basically the same as Example 1, except that the lipid mixture contains arachidonic acid, linoleic acid, linolenic acid and myristic acid, and the mass ratio of arachidonic acid, linoleic acid, linolenic acid and myristic acid is 2:1:1:2.

[0078] Preparation: Same as Example 1.

[0079] Example 10: Preparation of serum-free culture medium

[0080] Composition: Basically the same as Example 1, except that the non-essential amino acids contain L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine and L-proline, and the contents of L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine and L-proline in the culture medium are 0.5 g / L, 1.0 g / L, 0.5 g / L, 0.5 g / L, 0.5 g / L and 1.0 g / L, respectively, and the mass ratio of the six is ​​1:2:1:1:1:2.

[0081] Comparative Example 1: Preparation of serum-free culture medium

[0082] Composition: basically the same as Example 1, except that nicotinamide mononucleotide is not added to the basal culture medium.

[0083] Preparation: Same as Example 1.

[0084] Comparative Example 2: Preparation of serum-free culture medium

[0085] Composition: basically the same as Example 1, except that no astaxanthin extract is added to the basal culture medium.

[0086] Preparation: Same as Example 1.

[0087] Comparative Example 3: Preparation of serum-free culture medium

[0088] Composition: basically the same as Example 1, except that nicotinamide mononucleotide and astaxanthin extract are not added to the basal culture medium.

[0089] Preparation: Same as Example 1.

[0090] Application Example 1: CIK Cell Culture

[0091] The collected human peripheral blood was spread on lymphocyte separation medium and centrifuged at 800g for 20 minutes to obtain the cells of the intermediate buffy coat layer. The peripheral blood mononuclear cells (PBMCs) in the intermediate buffy coat layer were washed twice with physiological saline, centrifuged at 500g for 10 minutes, the supernatant was removed, the cell pellet was resuspended, and the cells were counted. 6 CIK cells were cultured at a density of 1 × 10 cells / mL in a culture flask containing the serum-free medium prepared in Example 1, supplemented with 1000 IU / mL IL-2, 20 ng / mL IL-15, 10 ng / mL IL-1α, and 50 ng / mL CD3 stimulatory factor. The culture conditions were: temperature 37.0°C, carbon dioxide volume fraction 5.0%. Counting and rehydration were performed every other day to adjust the cell density to 1 × 10 cells / mL. 6 The cells were cultured for 14 days, and the cell proliferation rate, cell survival rate and cell positive rate were detected. The results are shown in Table 1.

[0092] Application Example 2-10: CIK Cell Culture

[0093] The culture process was basically the same as that of Application Example 1, except that the serum-free culture medium prepared in Example 1 was replaced by the serum-free culture medium prepared in Examples 2-10, respectively.

[0094] Comparative Application Examples 1-3: CIK Cell Culture

[0095] The culture process was basically the same as that of Application Example 1, except that the serum-free culture medium prepared in Example 1 was replaced by the serum-free culture medium prepared in Comparative Examples 1-3, respectively.

[0096] Comparative Application Example 4: CIK Cell Culture

[0097] The culture process is basically the same as that of Application Example 1, except that the serum-free culture medium prepared in Example 1 is replaced by the commercial V-VIVO15 culture medium (Cat. No. 04-418Q) produced by Lonza.

[0098] Table 1

[0099] From the test results in Table 1, it can be seen that when the serum-free culture medium prepared in Examples 1-10 of the present application is used for CIK cell culture, the cell proliferation multiples, cell survival rate and cell positive rate (CD3+CD56+) are significantly better than those prepared in the serum-free culture medium of Comparative Examples 1-3, and the cell proliferation multiples and cell positive rate (CD3+CD56+) are better than those of the commercial V-VIVO15 culture medium, indicating that the serum-free culture medium prepared in the examples of the present application has a better CIK cell culture effect.

[0100] The test results of Application Example 1 and Comparative Application Examples 1-3 show that when serum-free culture medium contains nicotinamide mononucleotide and astaxanthin extract, CIK cells cultured in this culture medium have higher cell proliferation multiples, cell survival rate, and cell positive rate (CD3+CD56+). The test results of Application Example 1-7 show that when the addition of nicotinamide mononucleotide to the culture medium is 4 mg / L and the addition of astaxanthin extract is 10 mg / L, the culture effect of CIK cells is further improved.

[0101] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the invention may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same function.

Claims

1. A serum-free medium for CIK cell culture, characterized in that: The serum-free culture medium comprises a basal culture medium, and the basal culture medium is supplemented with recombinant human transferrin, recombinant human insulin, recombinant human albumin, ethanolamine, a lipid mixture, trace elements, α-cyclodextrin, non-essential amino acids, sodium bicarbonate, nicotinamide mononucleotide and astaxanthin extract.

2. The serum-free medium according to claim 1, characterized in that The basic culture medium comprises 1-20 mg / L of recombinant human transferrin, 1-20 mg / L of recombinant human insulin, 1-10 g / L of recombinant human albumin, 0.5-5 mg / L of ethanolamine, 1-5 mL / L of lipid mixture, 1-10 mL / L of trace elements, 0.5-1.5 g / L of α-cyclodextrin, 1-5 mL / L of non-essential amino acids, 2-4 g / L of sodium bicarbonate, 1-10 mg / L of nicotinamide mononucleotide and 5-20 mg / L of astaxanthin extract.

3. The serum-free medium according to claim 1 or 2, characterized in that The lipid mixture is selected from one or more of arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid and tocopherol acetate.

4. The serum-free medium according to claim 3, characterized in that The lipid mixture is a mixture of arachidonic acid, linoleic acid, linolenic acid and oleic acid, and the mass ratio of arachidonic acid, linoleic acid, linolenic acid and oleic acid in the mixture is (1-3):1:1:(1-3).

5. The serum-free medium according to claim 1 or 2, characterized in that The trace elements are selected from one or more of sodium selenite, ferric citrate, ammonium metavanadate, manganese chloride, copper sulfate and zinc sulfate.

6. The serum-free medium according to claim 1 or 2, characterized in that The non-essential amino acids are selected from one or more of L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine, L-proline and L-glutamic acid.

7. The serum-free medium according to claim 6, characterized in that The non-essential amino acids are a mixture of L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine and L-proline, and the mass ratio of L-alanine, L-asparagine, L-aspartic acid, L-glycine, L-serine and L-proline in the mixture is (0.2-1):(1-2):1:(1-2):1:(1-2).

8. The serum-free medium according to claim 1 or 2, characterized in that The pH value of the serum-free culture medium is 7.0-7.

4.

9. A method for culturing CIK cells, characterized in that: The culture method comprises: inoculating peripheral blood mononuclear cells derived from human peripheral blood or umbilical cord blood mononuclear cells derived from umbilical cord blood into the serum-free culture medium as described in any one of claims 1 to 8, and culturing CIK cells after adding stimulating factors; the stimulating factors are selected from one or more of IL-2, IL-15, IL-1α and CD3.

10. Use of CIK cells cultured by the culture method according to claim 9 in preparing anti-tumor drugs.

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