CN107502592A - A kind of immunocyte serum free medium and preparation method thereof - Google Patents

Abstract

The invention discloses an immune cell serum-free culture medium and a preparation method thereof. The main components of the serum-free medium of the present invention are: polyamine, insulin, cholesterol, ferric citrate, inositol, antioxidant and interleukin-15. The serum-free culture medium of the invention is safe for human body and can increase the proliferation speed and activation rate of CIK cells. And it has the advantages of simple ingredients, stable properties, small batch-to-batch difference, convenient quality control, high efficiency of inducing CIK proliferation, high positive ratio of CD3 ⁺ CD56 ⁺ , and stable quality of CIK cells obtained by culture, which is of great importance for the promotion and application of immunotherapy significance.

Description

A kind of immune cell serum-free culture medium and preparation method thereof

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a serum-free culture medium for immune cells and its preparation method and application.

Background technique

Cytokine-induced tumor killer cells (CIKs) are a group of heterogeneous cell populations obtained by co-culturing human peripheral blood mononuclear cells with various cytokines in vitro, because they have both strong anti-tumor activity of T lymphocytes and The non-major histocompatibility antigen (MHC)-restricted tumor killing characteristics of NK cells have become one of the important means of tumor adoptive immunotherapy. CIK cells have received extensive attention due to their strong proliferative ability, cytotoxicity and broad tumor killing spectrum. They have shown good application prospects in the immunotherapy of some solid tumors, hematological tumors and hematological diseases after bone marrow transplantation. Whether a sufficient number of effector cells can be obtained and the killing activity of effector cells are closely related to the clinical efficacy of CIK cell therapy. Studies have shown that the main effector cells of CIK are CD3+/CD56+ double positive cells. The ratio of CD3+/CD56+ cells to the total number of cells after CIK cell culture is an important observation index.

The general process of traditional CIK cell therapy is to infuse the mononuclear cells collected from the patient's peripheral blood into the patient after being modified, stimulated, cultured and proliferated by IFN-γ, CD3 monoclonal antibody, IL-1α and IL-2 in vitro , thereby killing the tumor. The current culture method has slow cell proliferation, long doubling time, and unstable ratio of CD3+/CD56+ double positive cells. Most laboratories culture human immune cells in vitro mainly by adding human AB serum or FBS to maintain the growth of immune cells. However, animal or human serum contains pathogens or uncertain substances, and the source of human AB serum is difficult, which will bring dangerous or uncertain hazards to cultured cells. Moreover, the components of the traditional culture medium are unstable and cannot be used for quality control in the later stage, and cannot be mass-produced in industry. Therefore, there is an urgent need to develop a serum-free medium that can overcome the above-mentioned defects.

Contents of the invention

The object of the present invention is to provide an immune cell serum-free culture medium and its preparation method and application. The culture medium is safe for human body and can increase the double-positive rate of CIK cell phenotype (CD3+CD56+).

An immune cell serum-free medium, the medium components include basal medium, polyamine, insulin, cholesterol, ferric citrate, inositol and antioxidant; the basal medium is RPMI-1640.

Each liter of medium includes: cholesterol 13.2mg/L; insulin 20mg/L; iron citrate 2mg/L; NaHCO ₃ 2g/L; ethanolamine 1.22mg/L; linoleic acid 1mg/L; inositol 19mg/L; L-glutamic acid 584mg/L; sodium pyruvate 110mg/L; 2-mercaptoethanol 0.78mg/L; 1-thioglycerol 5.41mg/L; non-essential amino acid 20ml/L; vitamin 10ml/L; polyamine additive 3.31mg /L; antioxidant 0.52mg/L; oleic acid 1mg/L.

The non-essential amino acid is one or one of glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid, glutamic acid above.

The vitamins are vitamin A, vitamin B1, vitamin B2, vitamin PP, vitamin B4, vitamin B5, vitamin B6, biotin, vitamin B9, vitamin B12, inositol, vitamin C, vitamin D, vitamin E, vitamin K One or more than one.

The medium components also include one or more of IL-15 (interleukin-15), IFN-γ, CD3, IL-1α, and IL-2 factors.

Beneficial effects of the present invention: the serum-free medium of the present invention is safe for human body, can stabilize CIK cell proliferation rate and increase cell CD3 ⁺ CD56 ⁺ , and has simple ingredients, stable properties, small batch-to-batch difference, convenient quality control, and induction of CIK The advantages of high proliferation efficiency, high CD3 ⁺ CD56 ⁺ positive ratio, and stable quality of CIK cells obtained from culture are of great significance for the promotion and application of immunotherapy.

Description of drawings

Fig. 1 is the comparative figure of the CIK cell phenotype flow cytometry detection result that culture medium No. 1-3 and the serum-free medium of embodiment 1 cultivate;

Fig. 2 is a comparison chart of flow cytometry detection results of CIK cell phenotypes cultured in the serum-free medium of Example 1 and the H3 medium of Takara Company.

Fig. 3 is a comparison chart of CIK cell phenotype flow cytometry detection results after IL-15 is added to the serum-free medium in Example 1.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Example 1

Stir and dissolve a bag of 1 liter RPMI 1640 (Gibco) with ultrapure water, add the substances added in the following table (Table 1) in sequence, and finally set the volume to 1L, stir and dissolve, adjust the pH value to about 7.0, and use a 0.22μm filter membrane Sterilize by filtration and store at 4°C. Wherein the pH value is kept between 6.8-7.2, and the optimum pH value is kept at 7.0.

Table 1. Preparation of serum-free medium for immune cells

Take 60ml of peripheral blood from the patient, transfer it to two 50ml centrifuge tubes, centrifuge at 2200rpm for 10min, absorb the upper yellow plasma, transfer to a new 50ml centrifuge tube, inactivate at 56°C for 25min, refrigerate at 4°C for 10min, 3000rpm, 4°C, After centrifugation for 15 min, the supernatant was collected and stored at 4°C for later use. Slowly add the red blood cells in the lower layer to two 50ml centrifuge tubes filled with lymphocyte separation medium, and centrifuge at 400g for 30min. Take the white cells in the middle layer, wash them twice with NaCl, plant the cells in No. 1-4 medium, add IFN-γ, CD3, IL-1α, and IL-2 factors, and continue to culture for 14 days.

Medium No. 1: RPMI 1640.

Medium 2: RPMI 1640+albumin.

Medium 3: RPMI 1640+UltraKURE.

Medium No. 4: RPMI 1640+Formulation in Table 1 (C922).

Example 2

The preceding medium preparation steps and cell culture steps are the same as in Example 1. The cells were planted in H3 and medium, and IFN-γ, CD3, IL-1α, IL-2 factors were added, and the culture was continued for 14 days. Compare the effects of Takara's commercialized H3 medium and our medium in this example on cell proliferation rate, survival rate and cell phenotype.

Example 3

The preceding medium preparation steps and cell culture steps are the same as in Example 1. The cells were planted in the medium and the medium plus interleukin 15 (0.5ng/mL), and IFN-γ, CD3, IL-1α, IL-2 factors were added, and the culture was continued for 14 days. Compare the effects of IL-15 on our culture medium of the present invention on cell proliferation rate, survival rate and cell phenotype.

The experimental results showed that after 14 days of culture, the percentage of CD3 ⁺ CD56 ⁺ double-positive cells: groups 1-3 were all less than 3%, and culture medium 4 was 27.2% (Fig. 1). Compared with the commercialized H3, the cell expansion rate of the H3 group is almost the same, and the cell survival rate is about 98%. But in terms of cell phenotype, the percentage of CD3 ⁺ CD56 ⁺ double-positive cells in H3 group was 17.3%, while that in No. 4 medium was 23.7% (Fig. 2). 6 percentage points higher than H3. Adding IL-15 (0.5ng/mL) to No. 4 medium can effectively increase the percentage of CD3 ⁺ CD56 ⁺ double-positive cells, and the activation rate of CIK cells has more than doubled (Figure 3).

Example 4

The preceding medium preparation steps and cell culture steps are the same as in Example 1. The cells were planted in the medium and the medium plus the extract of Chenopodium quinoa (3mg/L), and IFN-γ, CD3, IL-1α, IL-2 factors were added, and the culture was continued for 14 days. To compare the effect of the extract of quinoa quinoa on our culture medium of the present invention on cell proliferation rate, survival rate and cell phenotype.

The extraction method of the quinoa quinoa extract is as follows: dry the leaves of the quinoa quinoa extract, add 3-5 times the weight of the aqueous solution to reflux for extraction three times, combine the filtrates, and evaporate to dryness.

Example 5

The preceding medium preparation steps and cell culture steps are the same as in Example 1. The cells were seeded in the medium and the medium plus cyclophosphamide, and IFN-γ, CD3, IL-1α, IL-2 factors were added, and the culture was continued for 14 days. Compare the effects of cyclophosphamide IL-15 on our culture medium of the present invention on cell proliferation rate, survival rate and cell phenotype.

The experimental results showed that after 14 days of culture, the percentage of CD3 ⁺ CD56 ⁺ double-positive cells: No. 4 culture medium was 27.2% (Fig. 1), Example 4 was 36.2%, and Example 5 was 37.1%. Adding quinoa quinoa extract and cyclophosphamide to No. 4 medium can effectively increase the percentage of CD3 ⁺ CD56 ⁺ double-positive cells, and the activation rate of CIK cells is more than doubled.

Claims (5)

1. a kind of immunocyte serum free medium, it is characterised in that medium component includes basal medium, polyamine, pancreas islet Element, cholesterol, ferrum citricum, inositol and antioxidant；The basal medium is RPMI-1640.

2. immunocyte serum free medium according to claim 1, it is characterised in that every liter of culture medium includes：Cholesterol 13.2mg/L；Insulin 20mg/L；Ferrum citricum 2mg/L；NaHCO₃2g/L；Monoethanolamine 1.22mg/L；Linoleic acid 1mg/L；Flesh Alcohol 19mg/L；Glutaminol 584mg/L；Sodium Pyruvate 110mg/L；2 mercaptoethanol 0.78mg/L；1 thioglycerol 5.41mg/ L；Nonessential amino acid 20ml/L；Vitamin 10ml/L；Polyamine additive 3.31mg/L；Antioxidant 0.52mg/L；Oleic acid 1mg/L。

3. immunocyte serum free medium according to claim 2, it is characterised in that the nonessential amino acid is sweet ammonia In acid, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, aspartic acid, glutamic acid It is one or more kinds of.

4. immunocyte serum free medium according to claim 2, it is characterised in that the vitamin is vitamin A, dimension Raw plain B1, vitamin B2, nicotinic acid, adenine phosphate, vitamin B5, vitamin B6, biotin, FA, vitamin B12, inositol, vitamin C, vitamin D, vitamin E, the one or more in vitamin K.

5. immunocyte serum free medium according to claim 1, it is characterised in that the medium component also includes IL-15, IFN-γ, CD3, the one or more in IL-1 α, the IL-2 factors.