CN118406649B

CN118406649B - CIK cell serum-free culture medium and application thereof

Info

Publication number: CN118406649B
Application number: CN202410876318.XA
Authority: CN
Inventors: 王叶晨; 林家会; 马士棋; 陈旭; 陈刚
Original assignee: Shanghai Geotech Biotech Co ltd; Suzhou Ecosai Biotechnology Co ltd
Current assignee: Shanghai Geotech Biotech Co ltd; Suzhou Ecosai Biotechnology Co ltd
Priority date: 2024-07-02
Filing date: 2024-07-02
Publication date: 2024-09-17
Anticipated expiration: 2044-07-02
Also published as: CN118406649A

Abstract

The invention belongs to the technical field of biology, and relates to a CIK cell serum-free culture medium and application thereof. Based on RPMI 1640, the invention improves the purity and the expansion times of CIK cells obtained by culture by adding glucose, L-alanyl-L-glutamine, N-acetylcysteine, PMA, ionomycin and 2-mercaptoethanol. The CIK cells cultured by the optimized CIK cell serum-free culture medium can be amplified by more than 600 times on the 13 th day, the CD3 ⁺CD56⁺ proportion can reach 56%, the cell activity rate in the culture process can be stably improved, the D13 cell activity rate can reach more than 95%, and compared with RPMI 1640, the method can better meet the demands of scientific research and clinical application.

Description

CIK cell serum-free culture medium and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a serum-free culture medium for CIK cells and application thereof.

Background

Cytokine-induced killer cells, which are known as CIK (cytokine-induced killers) cells, are immune cells obtained by culturing human Peripheral Blood Mononuclear Cells (PBMC) with various cytokines (e.g., IFN-gamma, anti-human CD3 mab, IL-1α, IL-2, etc.) in vitro for a period of time, and are also known as NK cell-like T lymphocytes because their cell phenotype is CD3 ⁺CD56⁺ as identified by flow cytometry.

CIK cells have strong anti-tumor activity of T lymphocytes and non-MHC restriction tumor killing characteristics of NK cells, can accurately identify and kill tumor cells without injuring normal cells by mistake, have the advantages of strong proliferation capacity, strong killing property, broad tumor killing spectrum, small toxic and side effects and the like, and are widely applied to adoptive immunotherapy of cancers such as pancreatic cancer, gastric cancer, non-Hodgkin lymphoma and the like. Clinical researches show that the CIK cell adoptive immunotherapy can overcome the defects of incomplete focus clearance, cancer cell metastasis, large toxic and side effects and the like in the traditional treatment modes of operation, chemotherapy and the like, thereby effectively prolonging the life cycle of patients and improving the quality of life. However, CIK cells are extremely rare in normal human peripheral blood, and account for only 1% -5% of PBMCs, so CIK cells required for immunotherapy often need to be obtained by extracting PBMCs of patients and performing in vitro expansion and differentiation. The research shows that the CIK cells cultured in vitro are rapidly amplified, and the multiplication number of the CIK cells can reach hundreds of times as compared with the multiplication number of the CIK cells before culture.

The main problem of the common immune cell general serum-free culture medium (such as RPMI 1640) in vitro culture of CIK cells in the current market is that even if proper cytokines are selected for stimulation, the obtained PBMC is a heterogeneous cell population containing various cell types after expansion, wherein CD3 ⁺CD56⁺ cells only account for 20-40%, the total cell expansion multiple is more difficult to reach more than 500 times, and the high purity and the quantity requirement of the CIK cells in clinical treatment are not facilitated. Therefore, there is a need to develop a serum-free medium that can harvest a large number of high purity CIK cells to improve the clinical efficacy of CIK cell immunotherapy and reduce the potential impact of contaminating cells.

Disclosure of Invention

The invention aims to provide a serum-free culture medium for CIK cells and application thereof.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

A CIK cell serum-free culture medium comprises a basal culture medium and an additive composition, wherein the basal culture medium is RPMI 1640 culture medium, and the additive composition consists of 1-4 mg/mL glucose, 100-400 mug/mL L-alanyl-L-glutamine, 0.5-3 mg/mL N-acetylcysteine, 10-80 ng/mL PMA, 200-600 ng/mL ionomycin and 1-8 mug/mL 2-mercaptoethanol.

Preferably, the additive composition consists of 2.5mg/mL glucose, 200. Mu.g/mL L-alanyl-L-glutamine, 1.5mg/mL N-acetylcysteine, 50ng/mL PMA, 500ng/mL ionomycin, 4. Mu.g/mL 2-mercaptoethanol.

The use of a serum-free medium for CIK cells, comprising any one of the foregoing, for obtaining CIK cells by in vitro culture of PBMC extracted from human peripheral blood.

The beneficial effects of the invention are mainly as follows:

the invention mainly aims to improve the efficiency of the existing immune cell universal serum-free culture medium for culturing CIK cells, and provides the serum-free culture medium for optimizing the CIK cells, which has definite chemical components, is convenient for harvesting and amplifying times after culturing, has higher CD3 ⁺CD56⁺ proportion and can be safely applied to CIK cells of clinical adoptive immunotherapy. The optimized CIK cell serum-free culture medium containing the L-alanyl-L-glutamine, the N-acetylcysteine and the PMA has definite components, all the sources of the added components are non-animal sources, the infection risk of diseases such as hepatitis B and the like is avoided, the pharmaceutical grade is met, and the human safety applied to clinical research is ensured.

The invention supplements nutrient substances required by cells in the culture process by adding glucose and L-alanyl-L-glutamine into the basic culture medium, so that the metabolism of the cells generates more energy for biosynthesis of lipid, protein, nucleic acid and other carbohydrates, thereby improving the proliferation multiple of the cells and meeting the number of harvested cells required by scientific research and clinical treatment.

The invention also adds N-acetylcysteine as a powerful antioxidant, which can promote the differentiation of T cells to CIK cells, improve the CIK proportion and enhance the cytotoxicity. The invention adds PMA (phorbol ester) and ionomycin, wherein PMA is a DAG (diglyceride glycerol) simulator, ionomycin is Ca ²⁺ transporter, and the two factors act together to induce up-regulation of expression of protective protein in CIK cells, inhibit cell death, maintain high cell activity rate, and stimulate cell activation and proliferation to ensure stable cell expansion.

In summary, the optimized CIK cell serum-free culture medium containing L-alanyl-L-glutamine, N-acetylcysteine and PMA has the advantages that the cell activity rate, the expansion multiple and the CIK proportion are improved on the basis of the original culture medium, the culture effect is excellent, the total cell expansion multiple of the CIK culture day 13 can be 600 times, the cell expansion multiple of the CIK culture day 13 is improved by 27% compared with the primary RPMI 1640, the CD3 ⁺CD56⁺ proportion of the CIK culture day 13 can be 56%, the cell activity rate of the CIK culture day 13 is improved by 59% compared with the primary RPMI 1640, the cell activity rate of the CIK culture medium is stably improved, and the cell quality standard required by scientific research and clinical application can be better met.

Drawings

FIG. 1 is a schematic representation of the activity of human peripheral blood mononuclear cells under conditions of serum-free medium culture of chemically defined CIK cells of example 3;

FIG. 2 is a schematic representation of proliferation results of human peripheral blood mononuclear cells under conditions of chemically defined CIK cell serum-free medium culture in example 3;

FIG. 3 is a graphical representation of the results of cell marker flow assay of human peripheral blood mononuclear cells under conditions of clear chemical composition CIK cell serum-free medium culture in example 3.

Detailed Description

The technical scheme of the invention is further described below with reference to specific embodiments. The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The basal medium used in the examples of the present invention was RPMI 1640 medium purchased from the Siemens Fei (Thermo Fisher) official network under the brand Ji Buke (Gibco) and under the product number 11875085; heat-inactivated FBS (escitalopram, cat No. FSP 500); glucose (aletin, cat No. G640146); L-alanyl-L-glutamine (Allatin, cat. A432688); n-acetylcysteine (aladine, cat No. a 105422); PMA (aledine, cat No. P167764); ionomycin (aletin, cat No. I139530); 2-mercaptoethanol (Alatine, cat# M301574); CD3 antibodies (hundred organism Biolegend, cat No. 300439); CD56 antibody (Bai jin Bio-led, cat. 362508).

1. The PBMC extracted from human peripheral blood is subjected to an in vitro culture experiment, and the experimental method is as follows:

(1) On the day of cell inoculation, extracting fresh human blood, separating out human peripheral blood mononuclear cells by using a Ficoll density gradient separation method, re-suspending the human peripheral blood mononuclear cells by using a CIK cell serum-free culture medium, inoculating the human peripheral blood mononuclear cells into a 12-hole plate at a density of 1.7-2.5X10 ⁶ cells/mL, simultaneously adding 50-200 ng/mL of IFN-gamma and 10% heat-inactivated FBS into each hole, uniformly mixing, and culturing under the condition of 5% CO ₂ at 37 ℃;

(2) On day 1 after inoculation, 100-500 ng/mL of anti-human CD3 monoclonal antibody, 50-200 ng/mL of IL-1 alpha and 500-1000IU/mL of IL-2 are added into each hole, and after uniform mixing, the mixture is placed at 37 ℃ and cultured under the condition of 5% CO ₂;

(3) On day 3 after inoculation, 2 times of the volume of fresh CIK serum-free medium and 10% heat-inactivated FBS are added to each hole, and after uniform mixing, the mixture is placed at 37 ℃ and cultured under the condition of 5% CO ₂;

(4) Performing AOPI-13 days after inoculation, counting the cultured cells at intervals of 2 days, calculating cell expansion times, supplementing fresh CIK serum-free medium and 5% heat-inactivated FBS according to the counting result, and adjusting the cell density to 0.7X10- ⁶ cell number/mL;

(5) Cells were harvested from culture to day 13 and analyzed for CD3 ⁺CD56⁺ cell fraction by flow cytometry on days 11, 13.

Example 1: a CIK cell serum-free medium, wherein the basal medium is RPMI 1640 medium;

The content of each component of the additive component is as follows, based on the volume of the basal medium: glucose 1mg/mL, L-alanyl-L-glutamine 100 μg/mL, N-acetylcysteine 0.5mg/mL, PMA 10ng/mL, ionomycin 200ng/mL, 2-mercaptoethanol 1 μg/mL.

Example 2: a CIK cell serum-free medium, wherein the basal medium is RPMI 1640 medium;

The content of each component of the additive component is as follows, based on the volume of the basal medium: glucose 2mg/mL, L-alanyl-L-glutamine 150 μg/mL, N-acetylcysteine 1mg/mL, PMA 30ng/mL, ionomycin 400ng/mL, 2-mercaptoethanol 3 μg/mL.

Example 3: a CIK cell serum-free medium, wherein the basal medium is RPMI 1640 medium;

The content of each component of the additive component is as follows, based on the volume of the basal medium: glucose 2.5mg/mL, L-alanyl-L-glutamine 200. Mu.g/mL, N-acetylcysteine 1.5mg/mL, PMA 50ng/mL, ionomycin 500ng/mL, 2-mercaptoethanol 4. Mu.g/mL.

Example 4: a CIK cell serum-free medium, wherein the basal medium is RPMI 1640 medium;

The content of each component of the additive component is as follows, based on the volume of the basal medium: glucose 3mg/mL, L-alanyl-L-glutamine 300 μg/mL, N-acetylcysteine 2mg/mL, PMA 70ng/mL, ionomycin 500ng/mL, 2-mercaptoethanol 5 μg/mL.

Example 5: a CIK cell serum-free medium, wherein the basal medium is RPMI 1640 medium;

The content of each component of the additive component is as follows, based on the volume of the basal medium: glucose 4mg/mL, L-alanyl-L-glutamine 400 μg/mL, N-acetylcysteine 3mg/mL, PMA 80ng/mL, ionomycin 600ng/mL, 2-mercaptoethanol 8 μg/mL.

Example 6: a CIK cell serum-free medium, wherein the basal medium is RPMI 1640 medium;

The content of each component of the additive component is as follows, based on the volume of the basal medium: glucose 2.5mg/mL, L-alanyl-L-glutamine 200 μg/mL, N-acetylcysteine 1.5mg/mL, PMA 50ng/mL, 2-mercaptoethanol 4 μg/mL.

Example 7: a CIK cell serum-free medium, wherein the basal medium is RPMI 1640 medium;

The content of each component of the additive component is as follows, based on the volume of the basal medium: glucose 2.5mg/mL, L-alanyl-L-glutamine 200. Mu.g/mL, N-acetylcysteine 1.5mg/mL, ionomycin 500ng/mL, 2-mercaptoethanol 4. Mu.g/mL.

2. Comparative test of Medium Performance of examples

Serum-free media for CIK cells were prepared according to the formulations described in examples 1-7, numbered M1, M2, M3, M4, M5, M6, M7, respectively. Cells were cultured using serum-free media M1, M2, M3, M4, M5, M6, M7 and commercial serum-free medium RPMI 1640 according to the experimental method of the in vitro culture experiments on PBMCs extracted from human peripheral blood to compare the culture efficacy of the culture media of the respective examples.

The experimental results are as follows:

Detection of Activity of CIK cells

Cells were cultured by the experimental method of in vitro culture experiments with PBMC extracted from human peripheral blood, and cells were sampled and stained for AOPI days 5,7,9, 11, and 13 after inoculation, and cell viability data obtained are shown in Table 1.

The experimental results show that: the cell viability of the culture medium cultured with M1, M2, M3, M4, M5, M6, M7 and commercial serum-free medium was steadily increased at days 5-13 after inoculation, and reached 90% or more at day 7, with the highest cell viability at day 13 of M3 reaching 96.5%. The decrease in cell viability at day 13 compared to M3, M6 and M7 cultured cells indicates that PMA and ionomycin alone were impaired in their ability to increase cell viability.

TABLE 1

Proliferation efficiency assay of CIK cells

Cells were cultured by experimental methods of in vitro culture experiments with PBMCs extracted from human peripheral blood, and AOPI staining counts were performed by sampling on days 5,7,9, 11, and 13 after inoculation, respectively, and total cell expansion fold data obtained after calculation are shown in table 2.

The experimental results show that: cells cultured with the CIK cell serum free medium (M3) described in example 3 had a total cell expansion up to 600 fold on day 13 post inoculation, superior to other formulations. The decrease in total cell expansion on day 13 compared to M3, M6 and M7 cultured cells indicates that PMA and ionomycin alone are impaired in their ability to activate and promote cell expansion.

TABLE 2

Detection of surface markers of CIK cells

Cells were cultured by experimental methods of in vitro culture experiments with PBMC extracted from human peripheral blood, and the cell fraction of CD3 ⁺CD56⁺ was sampled and examined on days 11 and 13 after inoculation, respectively. About 0.5X10 ⁶ cells were taken, added with an appropriate amount of PBS, centrifuged for 5min at 500g, the supernatant was discarded, 100. Mu.L of PBS was added for resuspension, the fluorescent-labeled CD3 antibody and CD56 antibody were added to the cell suspension, incubation was performed at 4℃for 15min, PBS was added for washing once after the incubation was completed, the supernatant was discarded, and the supernatant was resuspended with 400. Mu.L of PBS and subjected to detection analysis by flow cytometry. The CD3 ⁺CD56⁺ cell fraction data are shown in table 3.

The experimental results show that: cells cultured with the CIK cell serum free medium (M3) described in example 3 had a CD3 ⁺CD56⁺ ratio of up to 38% on day 11 post-inoculation and a CD3 ⁺CD56⁺ ratio of up to 57% on day 13 post-inoculation, which is superior to other formulations. The decrease in the proportion of CD3 ⁺CD56⁺ on day 13 compared to M3, M6 and M7 cultured cells indicates that PMA and ionomycin alone have impaired ability to repeatedly activate and induce T cell differentiation to CIK cells.

TABLE 3 Table 3

3. Repeated and stability experiments with example 3 as experimental group and commercial serum-free medium as control group were verified

In vitro culture of PBMC extracted from human peripheral blood was performed on cultured cells in vitro using the commercial serum-free medium RPMI 1640 as a control group and example 3 as an experimental group.

The experimental results are as follows:

Detection of Activity of CIK cells

Cells were cultured as described above, and AOPI staining counts were taken on days 5,7,9, 11, and 13 after inoculation, respectively, and cell viability curves were plotted according to the results, as shown in FIG. 1.

The experimental results show that: the cell viability in culture with the CIK cell serum-free medium described in example 3 was steadily increased from day 5 to day 13 after inoculation, and reached 95% or more at day 13.

Proliferation efficiency assay of CIK cells

Cells were cultured as described above, and AOPI staining counts were taken on days 5,7,9, 11, and 13 after inoculation, respectively, and cell proliferation fold change curves were plotted according to the results, as shown in fig. 2.

The experimental results show that: the cell expansion factor of the culture with the CIK cell serum-free medium described in example 3 was always higher than that of the commercial serum-free medium at days 5-13 after inoculation, and could reach 600 times at day 13, significantly better than that of the commercial serum-free medium.

Detection of surface markers of CIK cells

Cells were cultured as described above and samples were taken at 11 and 13 days post inoculation to detect the CD3 ⁺CD56⁺ cell fraction. About 0.5X10 ⁶ cells were taken, added with an appropriate amount of PBS, centrifuged for 5min at 500g, the supernatant was discarded, 100. Mu.L of PBS was added for resuspension, the fluorescent-labeled CD3 antibody and CD56 antibody were added to the cell suspension, incubation was performed at 4℃for 15min, PBS was added for washing once after the incubation was completed, the supernatant was discarded, and the supernatant was resuspended with 400. Mu.L of PBS and subjected to detection analysis by flow cytometry. CD3 ⁺CD56⁺ cells were as shown in figure 3.

The experimental results show that: cells cultured with the CIK cell serum-free medium described in example 3 had a CD3 ⁺CD56⁺ ratio of up to 35% on day 11 after inoculation and a CD3 ⁺CD56⁺ ratio of up to 56% on day 13 after inoculation, which was significantly superior to commercial serum-free medium.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The CIK cell culture medium is characterized by comprising a basal culture medium and an additive composition, wherein the basal culture medium consists of RPMI 1640 culture medium, 50-200 ng/mL IFN-gamma, 10% heat-inactivated FBS, 100-500 ng/mL anti-human CD3 monoclonal antibody, 50-200 ng/mL IL-1 alpha and 500-1000IU/mL IL-2, and the additive composition consists of 2.5-3 mg/mL glucose, 200-300 mug/mL L-alanyl-L-glutamine, 1.5-2 mg/mL N-acetylcysteine, 50-70 ng/mL PMA, 500ng/mL ionomycin and 4-5 mug/mL 2-mercaptoethanol;

the 100-500 ng/mL anti-human CD3 monoclonal antibody, 50-200 ng/mL IL-1 alpha and 500-1000IU/mL IL-2 are added on day 1 after cell inoculation;

the 50-200 ng/mL IFN-. Gamma.and 10% heat-inactivated FBS were added on the day of cell inoculation.

2. The CIK cell culture medium of claim 1, wherein said additive composition consists of 2.5mg/mL glucose, 200 μg/mL L-alanyl-L-glutamine, 1.5mg/mL N-acetylcysteine, 50ng/mL PMA, 500ng/mL ionomycin, 4 μg/mL 2-mercaptoethanol.

3. Use of a CIK cell culture medium according to any one of claims 1 or 2 for in vitro culture of PBMCs extracted from human peripheral blood to obtain CIK cells.