Medium composition for culturing t cells

Abstract

The present invention relates to a medium composition for culturing T cells and, more specifically, to a medium composition for culturing T cells, the medium composition containing L-alanine. The medium composition of the present invention can effectively proliferate T cells within a short period of time, thus making it possible to more quickly obtain a desired number of cells when developing a therapeutic agent, and enhances activity, such that markers indicating T cell activity are maintained at a high level. Therefore, the medium composition of the present invention can be effectively used in the fields of T cell research and therapeutic agent development.

Description

Medium composition for T cell culture

[0001] The present invention relates to a medium composition for culturing T cells, and more specifically, to a medium composition for T cell proliferation containing L-alanine as an active ingredient.

[0002] T cells (T cells) are a central cell population in the immune system, serving as a defense system against various pathogens. T cells are broadly classified into CD4-positive helper T cells and CD8-positive cytotoxic T cells. The former promote immune responses, while the latter eliminate virus-infected cells and tumor cells. Helper T cells are further classified into type 1 helper T cells, which promote cell-mediated immunity, and type 2 helper T cells, which promote humoral immunity.

[0003] T cell cultures have primarily utilized human-derived materials. However, the use of human-derived materials presents a challenge: the difficulty of completely removing foreign substances after culture. Furthermore, the inherent nature of human-derived materials posed quality control and safety issues. However, when human-derived materials were not used, low proliferation potential was a problem. Therefore, the need for the development of a medium composition capable of culturing T cells without using human-derived materials emerged.

[0004] Therefore, there is a need to develop a medium composition that can obtain T cells with a high differentiation efficiency and yield compared to existing medium compositions.

[0005] L-Alanine (C3H7NO2) is an amino acid derived from muscle and serves as an important energy source for the human body. L-Alanine is a key amino acid with wide-ranging applications in fields such as chemistry, food, and medicine. As a sweet-tasting amino acid, it is particularly used in the food industry as a flavor enhancer and nutritional supplement.

[0006]

[0007] The purpose of the present invention is to provide a medium composition for proliferating T cells with high efficiency and yield while maintaining the characteristics of T cells without using human-derived materials.

[0008] In particular, the present invention aims to provide a medium composition for culturing T cells, which comprises L-alanine as an active ingredient.

[0009] In addition, the present invention aims to provide a medium containing the above medium composition and a T cell culturing method using the medium.

[0010]

[0011] To solve the above problem, the present invention provides a medium composition for T cell culture, which includes L-alanine as an effective ingredient.

[0012] The T cell culture medium according to the present invention can produce T cells with high cell growth rates and cell viability without the addition of additional active ingredients. Furthermore, the medium composition of the present invention can be used to culture T cells with high levels of markers indicating T cell activity, making it useful for T cell research and development.

[0013] Figure 1 shows the cell growth rate (Fold Increase) and cell viability (Viability) when the medium composition of the present invention is added to the basic medium.

[0014] The purpose of the present invention is to provide a medium composition for culturing T cells, which comprises L-alanine as an active ingredient.

[0015] In the present invention, L-alanine may be included at a concentration of 0.1 to 1.5 mM, preferably at a concentration of 0.5 to 1.0 mM, and more preferably at a concentration of 0.6 to 1.0 mM, but is not limited thereto.

[0016] The medium composition of the present invention can be added to a basic medium.

[0017] In the present invention, the "basic medium" is a mixture containing essential sugars, amino acids, water, etc. necessary for the survival and proliferation of cells. The basic medium of the present invention may be manufactured and used by artificial synthesis, or a commercially manufactured medium may be used.

[0018] The above-mentioned basic medium may be a general cell culture medium and may contain all components essential for cell culture. The above-mentioned basic medium may be any one selected from the group consisting of, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), a-MEM (a-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), KnockOut DMEM (GIBCO), and MCDB, and is preferably RPMI 1640, but is not limited thereto.

[0019] In addition, the medium of the present invention may additionally include a nutrient mixture. The nutrient mixture is a mixture containing various amino acids, vitamins, mineral salts, etc. that are generally used in cell culture. The nutrient mixture may be prepared by mixing the amino acids, vitamins, mineral salts, etc., or a commercially manufactured nutrient mixture may be used. Commercially manufactured nutrient mixtures include, for example, F-12, M199, MCDB110, MCDB202, MCDB302, etc., and preferably, F-12 nutrient mixture, M199, MCDB media, etc. can be used.

[0020] As used herein, T cells refer to helper T cells such as CD4+, Th1, Th2 or Th17 helper T cells, cytotoxic T cells such as CD4+ or CD8+ cytotoxic T cells, regulatory T cells such as FOXP3+ T regulatory cells or gamma delta T cells, and CAR-T cells.

[0021] Hereinafter, preferred embodiments are presented to aid in understanding the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. This means that the description of the present invention is for illustrative purposes only, and those skilled in the art will readily understand that modifications to other specific forms are possible without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0022] Manufacturing Example 1. Method for obtaining L-alanine

[0023] L-alanine was purchased from ThermoFisher and stored at room temperature. Using a microbalance, we weighed 0.5 mM and 1 mM L-alanine in basal medium, respectively, and dissolved them in 1 mL of manufacturing water to prepare 0.5 mM and 1 mM stocks. The prepared stocks were added to the basal medium to form L-alanine-supplemented medium (0.5 mM and 1 mM, respectively).

[0024] Example 1. Thawing and culture of T cells

[0025] The T cells (Human Pan T cells) used in this invention were purchased from Stemcell technologies and transferred to a cell preservation nitrogen tank in the laboratory. After thawing the cryopreserved cells, centrifugation was performed to remove the supernatant. The cell pellet was suspended in basal medium (RPMI 1640) without L-alanine and used in the following experiments. 100 μl of a solution containing 1 μg / ml anti-CD3 was added to each 48-well plate and refrigerated for at least 18 hours to prepare.

[0026] Example 2. Confirmation of the effect of promoting T cell proliferation when using the medium composition of the present invention.

[0027] 2-1. Method

[0028] A proliferation medium was prepared by adding 25 μg / ml of antibiotic (gentamicin) to the medium without L-alanine and the medium with L-alanine. The prepared T cells were seeded at 1 X 10 6 / ml was suspended in each medium, and the solution in the prepared plate was removed and left at room temperature for more than 30 minutes. 1 μg / ml Anti-CD28 and 300 IU IL-2 were added to the cells suspended in each medium. 3 X 10 5 After dividing the cells into 5 wells, they were cultured in a 5% carbon dioxide incubator at 37°C for 3 days. After 3 days, the number of proliferated cells was measured and recorded. 5 X 10 5Each medium was prepared with 300 IU IL-2 added to make the concentration / ml, and then dispensed into plates or flasks according to the cell number. The cells were cultured for 2 days in a 5% carbon dioxide incubator at 37℃. After 2 days, the number of proliferated cells was measured and recorded, and the process was repeated until day 10 to measure cell viability. The cell growth rate (Fold Increase) was calculated by measuring the number of cells proliferating on each day of culture compared to the number of cells seeded at the beginning of culture.

[0029]

[0030] 2-2. Results

[0031] Table 1 below<L-알라닌 첨가 유무에 따른 T 세포의 세포성장률(Fold Increase) 변화> And as can be confirmed in Figure 1, there was no significant difference in the cell growth rate (Fold Increase) until the 6th day of culture, but on day 8, the media with 0.5 mM and 1 mM L-alanine added were approximately 1.7 times and approximately 1.8 times higher than the control group without L-alanine, respectively, and on day 10, the media with 0.5 mM and 1 mM L-alanine added were approximately 1.3 times and approximately 1.4 times higher than the control group without L-alanine.

[0032] Also, Table 2 below<L-알라닌 첨가 유무에 따른 T 세포의 세포생존률(Viability) 변화> As can be seen in Figure 1, the media added with 0.5 mM and 1 mM of L-alanine had cell viability of 94.4% and 94.6% on the 10th day of culture, respectively, while the control group without L-alanine had cell viability of 90.70%, which was 4% higher than the control group.

[0033] That is, it was confirmed that L-alanine has the effect of increasing the proliferation of T cells.

[0034]

[0035]

[0036]

[0037] Example 3. Confirmation of the effect of promoting T cell proliferation according to the concentration of L-alanine.

[0038] 3-1. Method

[0039] Prepare a proliferation medium by adding 25 μg / ml of antibiotic (gentamicin) to a medium without L-alanine and a medium with L-alanine at different concentrations (0.5 and 1 mM). Prepared T cells were cultured at 1 x 10 6 / ml was suspended in each medium, and the solution in the prepared plate was removed and left at room temperature for more than 30 minutes. 1 μg / ml Anti-CD28 and 300 IU IL-2 were added to the cells suspended in each medium. 3 X 10 5 After dividing the cells into 5 wells, they were cultured in a 5% carbon dioxide incubator at 37°C for 3 days. After 3 days, the number of proliferated cells was measured and recorded. 5 X 10 5 Each medium was prepared with 300 IU IL-2 to reach a concentration of 100 IU / ml, and then dispensed into plates or flasks according to the cell count. The cells were cultured in a 5% carbon dioxide incubator at 37°C for 2 days. After 2 days, the number of proliferated cells was measured and recorded, and the process was repeated until day 10.

[0040]

[0041] 3-2. Results

[0042] As can be seen in Fig. 1, L-alanine can improve the cell growth rate (Fold Increase) and cell viability (Viability) of T cells in the concentration range of 0.5 to 1 mM, respectively.

[0043]

[0044] The T cell culture medium according to the present invention can produce T cells with high cell growth rates and cell viability without the addition of additional active ingredients. Furthermore, the medium composition of the present invention can be used to culture T cells with high levels of markers indicating T cell activity, making it useful for T cell research and development.

Claims

1. A medium composition for T cell culture, comprising L-alanine as an active ingredient.

2. In paragraph 1, A medium composition for culturing T cells, characterized in that the T cells are T cells selected from the group consisting of helper T cells, cytotoxic T cells, regulatory T cells, and CAR-T cells.

3. In paragraph 1, A medium composition for T cell culture, characterized in that the L-alanine is contained in an amount of 0.1 to 1.5 mM.

4. In paragraph 3, A medium composition for T cell culture, characterized in that the above L-alanine is contained in an amount of 0.5 to 1.0 mM.

5. In paragraph 1, A medium composition for T cell culture, characterized in that it additionally includes a basic medium.

6. In paragraph 5, A medium composition for T cell culture, characterized in that the basic medium comprises at least one selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), a-MEM (a-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), KnockOut DMEM (GIBCO), and MCDB.

7. In paragraph 1, A medium composition for T cell culture, characterized in that the cell viability of T cells cultured with the above medium composition increases by 4% or more on the 10th day of culture compared to the cell viability in a basic medium without additives.

8. In paragraph 1, A medium composition for T cell culture, characterized in that the cell growth rate (Fold Increase) of T cells cultured with the above medium composition is 1.3 times or more on the 10th day of culture compared to the cell growth rate (Fold Increase) in the basic medium without additives.

9. A method for culturing T cells, comprising a step of culturing T cells using the medium composition of paragraph 1.

10. In paragraph 9 A method for culturing T cells, characterized in that the T cells are T cells selected from the group consisting of helper T cells, cytotoxic T cells, regulatory T cells, and CAR-T cells.

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