Culture medium for immune cells as well as preparation method and application of culture medium

Abstract

The invention provides a culture medium for immune cells as well as a preparation method and application thereof. On the basis of a serum-free culture medium, a specific additive is added into the culture medium and is used as a nutrient substance for growth of immune cells, so that the concentrations of various components in the final culture medium are fixed, the culture efficiency of the immune cells is improved, the cell activity is increased, and the repeatability of subsequent experiments is ensured. Meanwhile, due to the use of a specific additive, animal serum protein added into a conventional culture medium is avoided, so that the risk of infection of exogenous viruses and pathogenic factors is avoided, the immune cells obtained by culture have no sensitization, and the condition that a receptor is allergic to exogenous serum is avoided. In addition, the cost of the immune cell culture medium is reduced, and the preparation process of the culture medium is simplified. Compared with the prior art, the additive provided by the invention can be preserved for a long time in a low-temperature environment, the activity is kept unchanged, and the additive can be taken and used when a culture medium is prepared, so that the preservation cost of immune cells is effectively reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of culture medium, and specifically relates to a culture medium for immune cells, a preparation method thereof and an application thereof. Background Art

[0002] B cells are a type of immune cell and a member of the lymphocyte subfamily of white blood cells. Their main function is to secrete antibodies in humoral immunity. In mammals, B cells originate from the hematopoietic stem cell series in the bone marrow and mature in the bone marrow.

[0003] At present, serum is still the most basic additive in immune cell culture, especially when the cell growth is poor, it is often cultured with serum-containing culture medium first, and then replaced with serum-free culture medium after the cells grow vigorously. Traditional cell culture medium contains heterologous serum components, such as bovine serum. Due to the inconsistency of activity and factors of different batches of bovine serum, there is a risk of contamination of exogenous viruses and pathogenic factors when using bovine serum in cell culture. The residual bovine serum in the product can easily cause an allergic reaction to the serum in the vaccine recipient, resulting in poor reproducibility of the product and experimental results. If the patient's own serum is used, a large amount of the patient's blood must be used, which increases the burden on the patient.

[0004] Since animal serum contains animal components and is complex, it contains many components that are beneficial to cells, but also contains components that are harmful to cells, such as polyamine oxidase, etc. If it is used in clinical cell therapy systems, it may lead to some potential risks. In addition, in large-scale production, the source of serum is becoming increasingly difficult and expensive, which is one of the main parts of the production cost of animal cell culture. Therefore, various studies hope to proliferate immune cells in culture media that do not use or minimize the use of serum. Serum-free culture media and reagents are widely used to culture mammalian and invertebrate cells to prepare monoclonal antibodies, viral antigens, and recombinant proteins.

[0005] In addition, the current common method for preparing immune cells is to first separate mononuclear cells from peripheral blood, use a culture medium suitable for lymphocytes, add an appropriate amount of cytokines for stimulation and induction, and finally obtain a certain number of immune cells. However, the proliferation multiples and cytotoxicity of the immune cells finally obtained are not ideal. Summary of the invention

[0006] In order to overcome the above technical problems, the present invention provides a culture medium for immune cells, a preparation method thereof and an application thereof.

[0007] The first invention of the present invention is to provide a culture medium for immune cells.

[0008] Furthermore, the culture medium is a serum-free culture medium containing specific additives.

[0009] Furthermore, the serum-free culture medium is MEM culture medium, DMEM culture medium, IMDM culture medium, RPMI1640 culture medium, Ham's F-12 culture medium, DMEM / F12 culture medium or M199 culture medium, preferably DMEM culture medium; the specific additive is at least one of cell growth factor, interleukin, antioxidant and non-essential amino acid.

[0010] Furthermore, the cell growth factor is transforming growth factor (TGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin growth factor and / or hepatocyte growth factor (HGF), preferably insulin growth factor (IGF).

[0011] Furthermore, the interleukin is interleukin-4 (IL-4) and / or interleukin-21 (IL-21).

[0012] Furthermore, the antioxidant is vitamin C, N-acetylcysteine, L-cysteine, catalase, superoxide dismutase and / or 2-mercaptoethanol.

[0013] Furthermore, the non-essential amino acids are alanine, asparagine, aspartic acid, glycine, glutamic acid, proline and / or serine.

[0014] In some embodiments, the cell growth factor is insulin-like growth factor I and hepatocyte growth factor, the interleukin is interleukin-4 and interleukin-21, the antioxidant is 2-mercaptoethanol, and the non-essential amino acid is L-glutamic acid.

[0015] In some embodiments, the concentration of insulin-like growth factor I is 10-30 ng / mL, the concentration of hepatocyte growth factor is 10-20 ng / mL, the concentration of interleukin-4 is 40-60 ng / mL, the concentration of interleukin-21 is 20-40 ng / mL, the concentration of 2-mercaptoethanol is 50-60 μM, and the concentration of L-glutamate is 1-3 mM.

[0016] In some embodiments, the concentration of insulin-like growth factor I is 20 ng / mL, the concentration of hepatocyte growth factor is 15 ng / mL, the concentration of interleukin-4 is 50 ng / mL, the concentration of interleukin-21 is 30 ng / mL, the concentration of 2-mercaptoethanol is 55 μM, and the concentration of L-glutamate is 2 mM.

[0017] The second invention of the present invention is to provide a method for preparing the culture medium.

[0018] Furthermore, the method comprises:

[0019] S1, adding antioxidants and non-essential amino acids to the DMEM medium, and letting them stand for 3-10 minutes after they are dissolved;

[0020] In some embodiments, after the antioxidant and non-essential amino acids are completely dissolved, the mixture is allowed to stand for 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

[0021] S2, adding cell growth factors and interleukins to the culture medium obtained in S1, and letting them stand for 3-10 minutes after they are dissolved;

[0022] In some embodiments, after the cell growth factors and interleukins are completely dissolved, the mixture is allowed to stand for 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

[0023] S3, make up the volume of serum-free medium obtained in S2 to 500 mL, and then shake and mix at 0-10°C.

[0024] In some embodiments, the volume of the serum-free medium is supplemented to 500 mL, and then shaken and mixed at 0-10° C. for 0.5-1.5 hours, preferably at 4° C. for 1 hour, filtered through a 0.22 μm filter membrane, sterilized, and set aside.

[0025] Preferably, in the final culture medium, the final concentration of the antioxidant is 50-60 μM, the final concentration of the non-essential amino acid is 20-40 ng / mL, the final concentration of the cell growth factor is 10-30 ng / mL, and the final concentration of the interleukin is 20-60 ng / mL.

[0026] Preferably, the above additives can be placed in a frozen environment, such as stored at -40°C or lower, so that they can be taken at any time without losing their vitality when the culture medium is needed.

[0027] Preferably, the additive is stored in a -80°C refrigerator for a long period of time, such as more than one year or longer.

[0028] The third invention point of the present invention is to provide the use of the culture medium in culturing immune cells.

[0029] Preferably, the immune cells are selected from B cells or T cells.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention adds specific additives on the basis of serum-free culture medium, which serve as nutrients for the growth of immune cells, so that the concentrations of various components in the final culture medium are fixed, the efficiency of immune cell culture is improved, cell activity is increased, and the repeatability of subsequent experiments is ensured. At the same time, the use of specific additives avoids the animal serum proteins added in conventional culture medium, so there is no risk of infection by exogenous viruses and pathogenic factors, and the immune cells obtained by culture are non-sensitizing, avoiding the situation where the receptor is allergic to exogenous serum. In addition, the cost of immune cell culture medium is reduced, and the configuration process of the culture medium is simplified.

[0032] 2. The in vitro expansion, activation and maturation of immune cells, especially B cells, provide technical support and experimental basis for studying transplantation mechanisms and potential therapies. However, T cells cannot proliferate in large quantities after transfection using ordinary culture medium. The prior art mostly adds serum on the basis of ordinary culture medium, but the batch-to-batch differences in serum components are large, and there is a risk of contamination by exogenous factors. The additives in the culture medium provided by the present invention include cell growth factors, interleukins, antioxidants and non-essential amino acids, among which cytokines can promote further expansion and differentiation of B cells, and interleukins can promote the massive proliferation of B cells. The above-mentioned additive ingredients are immunoglobulins with verified activity, so the culture medium is safe and effective, the formula is clearer, and the consistency and stability of the cell culture effect are also guaranteed.

[0033] 3. Compared with the prior art, the additive provided by the present invention can be stored for a long time in a low temperature environment, such as -80°C, while maintaining its activity. The culture medium can be used as needed when preparing the culture medium, which effectively reduces the storage cost of immune cells. At the same time, the immune cell culture medium and culture method provided by the present invention have no effect on the proportion of B cell expansion culture using different serum-free culture media, and can be adapted to more different basic serum-free culture media. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the cell growth situation of Example 3. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below. However, it should be understood that the description herein is only used to explain the present invention and is not intended to limit the scope of the present invention.

[0036] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0037] Example 1

[0038] This embodiment provides a culture medium for immune cells.

[0039] The culture medium is obtained by adding specific additives to a known basic cell culture medium.

[0040] Preferably, the basal culture medium is a serum-free culture medium.

[0041] Preferably, the serum-free culture medium includes MEM culture medium, DMEM culture medium, IMDM culture medium, RPMI1640 culture medium, Ham's F-12 culture medium, DMEM / F12 culture medium and M199 culture medium.

[0042] More preferably, the culture medium is a serum-free immune cell culture medium, which can improve the proliferation efficiency of immune cells. However, if ordinary culture medium is used, the transfected T cells cannot proliferate in large quantities. The existing technology mostly adds serum to ordinary culture medium, but the serum components vary greatly between batches, and there is a risk of contamination by exogenous factors.

[0043] In some embodiments, the additives selected by the present invention can stimulate B cells, leading to further maturation and final differentiation of B cells into terminally differentiated plasma cells, thereby achieving massive expansion and differentiation of B cells.

[0044] Preferably, the serum-free immune cell culture medium further comprises specific additives, which are at least one of cell growth factors, interleukins, antioxidants and non-essential amino acids.

[0045] Preferably, the cell growth factor is transforming growth factor (TGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin growth factor (IGF) and / or hepatocyte growth factor (HGF). Cell growth factors can promote the growth of cells in serum-free cell culture medium, regulate the cell's uptake, utilization and storage of glucose, amino acids and lipids, and inhibit the decomposition of glycogen, protein and fat. Hepatocyte growth factor (HGF) was discovered as a substance that can stimulate the proliferation of hepatocytes. HGF can also act on epithelial cells, hematopoietic cells, vascular endothelial cells and other cells. It is a multifunctional factor that can regulate the growth, movement and morphogenesis of various cells.

[0046] More preferably, the cell growth factors are insulin-like growth factor I (IGF1) and hepatocyte growth factor (HGF).

[0047] Preferably, the interleukin is interleukin-4 (IL4) and / or interleukin-21 (IL21). Interleukin is a type of cytokine produced by a variety of cells and acts on a variety of cells. It plays an important role in a series of processes such as the maturation, activation and immunoregulation of immune cells. Interleukin also participates in a variety of physiological and pathological reactions of the body. Interleukin exerts its biological effects by specifically binding to cytokine receptors on the surface of target cells. These effects include promoting the proliferation and differentiation of target cells, enhancing the effects of anti-infection and killing tumor cells, promoting and inhibiting the synthesis of other cytokines, promoting the inflammatory process, affecting cell metabolism, etc. The effect of interleukin has a networking feature, that is, each interleukin can act on a variety of cells, and each cell can be regulated by a variety of interleukins. Different interleukins have a mutually synergistic or mutually restrictive effect, thereby forming a complex interleukin immunoregulatory network.

[0048] Preferably, the antioxidant is at least one of vitamin C, N-acetylcysteine, L-cysteine, catalase, superoxide dismutase and 2-mercaptoethanol; more preferably, the antioxidant is 2-mercaptoethanol. Adding antioxidants to the culture medium is an effective measure to prevent browning.

[0049] Preferably, the non-essential amino acid is alanine, asparagine, aspartic acid, glycine, glutamic acid, proline, serine and / or. Adding non-essential amino acids to the culture medium reduces the metabolic burden of cells and is beneficial to cell proliferation.

[0050] More preferably, the non-essential amino acid is L-glutamic acid.

[0051] Furthermore, the present invention also provides the content of the above-mentioned specific additives in the culture medium.

[0052] Taking serum-free basal medium DMEM as an example, based on the DMEM medium, the concentration range of the above additives is as follows in terms of final concentration:

[0053] Table 1 Additive ingredients and final concentration range

[0054] Ingredient name Final concentration range Insulin-like growth factor 1 (IGF1) 10-30 ng / mL Interleukin-4 (IL4) 40-60 ng / mL Interleukin-21 (IL21) 20-40 ng / mL Hepatocyte Growth Factor (HGF) 10-20 ng / mL 2-Mercaptoethanol 50-60μM L-Glutamic Acid 1-3mM

[0055] Preferably, in the final culture medium, the final concentration of IGF1 is 20 ng / mL, the final concentration of IL4 is 50 ng / mL, the final concentration of IL21 is 30 ng / mL, the final concentration of HGF is 15 ng / mL, the final concentration of 2-mercaptoethanol is 55 μM, and the final concentration of L-glutamate is 2 mM.

[0056] The proliferation of B cells is achieved through the induction of immune cells by cell growth factors; the programmed cell death of B cells is inhibited by antioxidants; interleukins can transmit biological information between white blood cells, activate and regulate the function of immune cells, and activate and proliferate B cells, thereby playing an important immune role in the body's inflammatory response; the addition of antioxidants prevents the browning of the culture medium; and non-essential amino acids reduce the metabolic burden of cells, which is beneficial to cell proliferation.

[0057] In summary, the culture medium can efficiently expand immune cells and improve the growth rate and cytotoxicity of immune cells. In addition, the culture medium does not contain serum, which greatly avoids the defects of serum-containing culture medium such as instability between serum batches, cytotoxicity, and a large amount of heterologous proteins, providing a good tool for the clinical treatment of immune cells.

[0058] Example 2

[0059] This embodiment provides a method for preparing the culture medium for immune cells as described in Example 1.

[0060] The method includes:

[0061] S1, add 2-mercaptoethanol and L-glutamic acid to the DMEM medium, wait for them to completely dissolve, and let stand for 6 minutes;

[0062] S2, add interleukin-21 and interleukin-4, wait for them to completely dissolve, and let stand for 6 minutes;

[0063] S3, add insulin-like growth factor I and hepatocyte growth factor (HGF), and make up the volume of DMEM medium to 500 mL, then shake and mix at 4°C for 1 hour, filter through a 0.22 μm filter membrane, sterilize, and set aside.

[0064] Preferably, in the final culture medium, the final concentration of insulin-like growth factor I is 20 ng / mL, the final concentration of interleukin-4 is 50 ng / mL, the final concentration of interleukin-21 is 30 ng / mL, the final concentration of hepatocyte growth factor is 15 ng / mL, the final concentration of 2-mercaptoethanol is 55 μM, and the final concentration of L-glutamate is 2 mM.

[0065] Preferably, the additives can be placed in a frozen environment, such as stored at -40°C or lower, and can be used at any time when the culture medium is needed without losing activity.

[0066] Preferably, the additive is stored in a -80°C refrigerator for a long period of time, such as more than one year or longer.

[0067] Example 3

[0068] This example provides a method for culturing immune cells using the immune cell culture medium described in Example 1.

[0069] Taking the cultivation of B cells as an example, the method includes:

[0070] S1. Obtaining B cells: Using the EasySep cell isolation kit, human B cells were isolated from the spleen and stored in a liquid nitrogen tank.

[0071] S2, B cell resuscitation: Take out the above B cells from the liquid nitrogen tank, quickly put them into a 37°C water bath, shake them from time to time, completely melt them within 1 minute, and then take them out under a sterile environment.

[0072] S3, preliminary treatment of B cells: centrifuge B cells at 1000 rpm for 5 min, discard the supernatant, add appropriate amount of the culture medium prepared above, and inoculate B cells into a 125 mL cell culture shake flask at a concentration of 1*10 6 The culture shake flask was placed in a shaking incubator at 37°C, at a speed of 120 rpm and a carbon dioxide concentration of 8%.

[0073] S4, further processing of B cells: on the second day after recovery, centrifuge the culture medium and discard the supernatant, replace with ordinary culture medium, continue culturing, and observe the growth. The cell density is controlled at about 2*10 6 The B cells were then divided into 9 portions and placed in 9 T25 cell culture flasks (cell growth area of ​​25 cm 2 in culture flasks).

[0074] S5, group culture:

[0075] Cytokine group: three samples, using the immune cell culture medium described in Example 2;

[0076] Serum group: three samples, the culture medium composition was DMEM medium containing 10% fetal bovine serum;

[0077] Control group: cultured in DMEM.

[0078] S6, Experimental records: Observe and count the cells in each group at a fixed time every day, and record the cell growth. If the cell density is high, dilute it in time, and finally get the following Figure 1 Growth statistics shown.

[0079] The results show that if Figure 1 As shown, B cells in the experimental group can proliferate in large quantities, similar to the serum group, with a growth advantage far higher than that of the control group, and the growth state is good.

[0080] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0081] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A culture medium for immune cells, It is characterized in that The culture medium is a serum-free culture medium which contains specific additives.

2. The immune cell culture medium according to claim 1, It is characterized in that The serum-free culture medium is MEM culture medium, DMEM culture medium, IMDM culture medium, RPMI1640 culture medium, Ham's F-12 culture medium, DMEM / F12 culture medium or M199 culture medium, preferably DMEM culture medium; the specific additive is at least one of cell growth factor, interleukin, antioxidant and non-essential amino acid.

3. The immune cell culture medium according to claim 2, It is characterized in that The cell growth factor is at least one of transforming growth factor (TGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin growth factor and hepatocyte growth factor (HGF); the interleukin is at least one of interleukin-4 (IL-4) and interleukin-21 (IL-21).

4. The immune cell culture medium according to claim 2, It is characterized in that The antioxidant is at least one of vitamin C, N-acetylcysteine, L-cysteine, catalase, superoxide dismutase and 2-mercaptoethanol; the non-essential amino acid is at least one of alanine, asparagine, aspartic acid, glycine, glutamic acid, proline and serine.

5. The immune cell culture medium according to claim 2, It is characterized in that The cell growth factors are insulin-like growth factor I and hepatocyte growth factor, the interleukins are interleukin-4 and interleukin-21, the antioxidant is 2-mercaptoethanol, and the non-essential amino acid is L-glutamic acid.

6. The immune cell culture medium according to claim 5, It is characterized in that In the immune cell culture medium, the concentration of insulin-like growth factor I is 10-30 ng / mL, the concentration of hepatocyte growth factor is 10-20 ng / mL, the concentration of interleukin-4 is 40-60 ng / mL, the concentration of interleukin-21 is 20-40 ng / mL, the concentration of 2-mercaptoethanol is 50-60 μM, and the concentration of L-glutamate is 1-3 mM.

7. A method for preparing a culture medium for immune cells according to any one of claims 1 to 6, It is characterized in that The method comprises: S1, adding antioxidants and non-essential amino acids to serum-free medium, allowing them to dissolve and then stand for 3-10 minutes; S2, adding interleukin and cell growth factor to the culture medium obtained in S1, and letting them stand for 3-10 minutes after they are dissolved; S3, make up the volume of serum-free medium obtained in S2 to 500 mL, and then shake and mix at 0-10°C.

8. The preparation method according to claim 7, It is characterized in that The final concentration of the antioxidant in the culture medium is 50-60 μM, the final concentration of the non-essential amino acid in the culture medium is 20-40 ng / mL, the final concentration of the cell growth factor in the culture medium is 10-30 ng / mL, and the final concentration of the interleukin in the culture medium is 20-60 ng / mL.

9. Use of the immune cell culture medium according to any one of claims 1 to 6, or the immune cell culture medium prepared by the preparation method according to claim 7 or 8, in culturing immune cells.

10. The use according to claim 9, It is characterized in that The immune cells are selected from B cells or T cells.

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