Method for amplifying tumor infiltrating lymphocytes in vitro

Abstract

The invention relates to the technical field of cell culture. The invention provides a method for in-vitro amplification of tumor infiltrating lymphocytes, which comprises the following steps: (1) inoculating tumor cells into a first culture medium, and culturing to obtain a first cell population; (2) transferring the first cell population into a second culture medium, culturing, and separating to obtain tumor infiltrating lymphocytes; and (3) transferring the tumor infiltrating lymphocyte obtained in the step (2) into a third culture medium for culturing, and harvesting the amplified tumor infiltrating lymphocyte. According to the amplification method disclosed by the invention, a large number of tumor infiltrating lymphocytes can be obtained in an in-vitro environment, and the activity of the tumor infiltrating lymphocytes and the killing ability of the tumor infiltrating lymphocytes to tumor cells are remarkably improved. The use amount of tumor infiltrating lymphocytes is reduced, and the final use effect is improved.

Description

Technical Field

[0001] The invention relates to the technical field of cell cultivation, and in particular to a method for amplifying tumor-infiltrating lymphocytes in vitro. Background Art

[0002] The phenotype of tumor infiltrating lymphocytes (TIL) is heterogeneous. Generally speaking, the vast majority of cells in TIL are CD3 positive. There are differences in the ratio of CD4+T cells and CD8+T cells in TIL cells from different tumor sources. In most cases, CD8+T cells are dominant. The percentage of CD25+ cells in freshly isolated TIL is low, and the percentage of CD25+ cells gradually increases with the extension of in vitro IL-2 culture time. The markers of NK cells (CD16, CD56) tend to increase first and then decrease during the in vitro IL-2 culture of TIL.

[0003] There are two problems with the current application of TIL. One is the problem of the number of TIL cells. The number of starting TILs cells that can be collected from the tumor tissue of many patients with advanced tumors is limited. The total number of TIL cells obtained using general amplification methods is limited. In this way, the number of cells that can be re-infused to patients is also limited. The second is that the killing ability of conventional TIL cells is limited. If the problem of insufficient number of TIL cells is superimposed, it is difficult to produce obvious clinical efficacy. Therefore, a TIL cell with high killing ability is needed that can obtain a large number of TIL cells. Summary of the invention

[0004] The purpose of the present invention is to provide a method for in vitro amplification of tumor infiltrating lymphocytes, which adopts a new combination of factors to promote the in vitro amplification of tumor infiltrating lymphocytes, enhance biological functions, and improve toxicity to tumor cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for amplifying tumor-infiltrating lymphocytes in vitro, comprising the following steps:

[0007] (1) inoculating tumor cells into a first culture medium to obtain a first cell population;

[0008] (2) transferring the first cell population into a second culture medium, and isolating tumor infiltrating lymphocytes after culturing;

[0009] (3) The tumor infiltrating lymphocytes obtained in step (2) are transferred to a third culture medium for culture, and the amplified tumor infiltrating lymphocytes are harvested.

[0010] Preferably, the types of tumor cells in step (1) include renal cancer cells, liver cancer cells, ovarian cancer cells or lung cancer cells.

[0011] Preferably, in step (1), the first culture medium is based on AIM-V culture medium or RPMI-1640 culture medium, and further contains IL-2 4000-4500 U / mL, penicillin 110-130 μg / mL, streptomycin 110-130 μg / mL, and L-glutamine 1-1.2 mM.

[0012] Preferably, the inoculation density of tumor cells in the first culture medium in step (1) is 3×10 6 ~4×10 6 Pieces / mL.

[0013] Preferably, the culturing time in step (1) is 72 to 96 hours.

[0014] Preferably, the second culture medium in step (2) is based on AIM-V culture medium containing 6-10% serum or RPMI-1640 culture medium containing 6-10% serum, and further contains IL-152 600-3000 U / mL, penicillin 110-130 μg / mL, streptomycin 110-130 μg / mL, and CD28 antibody 1000-1200 U / mL.

[0015] Preferably, the separation in step (2) is performed when the tumor cell death rate in the first cell population reaches 10-30%.

[0016] Preferably, the third culture medium in step (3) is based on AIM-V culture medium containing 6-10% serum or RPMI-1640 culture medium containing 6-10% serum, and further contains IL-212200-2400U / mL, penicillin 110-130μg / mL, streptomycin 110-130μg / mL, TNFα 140-160pg / mL, and sodium pyruvate 2.4-2.6mM.

[0017] Preferably, the culturing time in step (3) is 48 to 60 hours.

[0018] The present invention provides a method for amplifying tumor-infiltrating lymphocytes in vitro, comprising the following steps:

[0019] (1) Inoculate tumor cells into a first culture medium and culture to obtain a first cell population; (2) Transfer the first cell population to a second culture medium and culture to obtain tumor infiltrating lymphocytes; (3) Transfer the tumor infiltrating lymphocytes obtained in step (2) to a third culture medium for culture and harvest the amplified tumor infiltrating lymphocytes. The amplification method of the present invention can obtain a large number of tumor infiltrating lymphocytes in an in vitro environment, significantly improve the activity of tumor infiltrating lymphocytes and their ability to kill tumor cells. Reduce the amount of tumor infiltrating lymphocytes used and improve the final use effect. DETAILED DESCRIPTION

[0020] The present invention provides a method for amplifying tumor-infiltrating lymphocytes in vitro, comprising the following steps:

[0021] (1) inoculating tumor cells into a first culture medium to obtain a first cell population;

[0022] (2) transferring the first cell population into a second culture medium, and isolating tumor infiltrating lymphocytes after culturing;

[0023] (3) The tumor infiltrating lymphocytes obtained in step (2) are transferred to a third culture medium for culture, and the amplified tumor infiltrating lymphocytes are harvested.

[0024] In the present invention, the types of tumor cells in step (1) preferably include renal cancer cells, liver cancer cells, ovarian cancer cells or lung cancer cells.

[0025] In the present invention, the first culture medium in step (1) is preferably based on AIM-V culture medium or RPMI-1640 culture medium, and further contains IL-24000-4500U / mL, penicillin 110-130μg / mL, streptomycin 110-130μg / mL, and L-glutamine 1-1.2mM, and is further preferably based on AIM-V culture medium or RPMI-1640 culture medium, and further contains IL-24200-4300U / mL, penicillin 120μg / mL, streptomycin 120μg / mL, and L-glutamine 1.1mM.

[0026] In the present invention, the inoculation density of tumor cells in the first culture medium in step (1) is preferably 3×10 6 ~4×10 6 / mL, more preferably 3.5×10 6 Pieces / mL.

[0027] In the present invention, the culturing time in step (1) is preferably 72 to 96 hours, more preferably 84 hours.

[0028] In the present invention, the second culture medium in step (2) is preferably based on AIM-V culture medium containing 6-10% serum or RPMI-1640 culture medium containing 6-10% serum, and further contains IL-152600-3000U / mL, penicillin 110-130μg / mL, streptomycin 110-130μg / mL, and CD28 antibody 1000-1200U / mL, and is further preferably based on AIM-V culture medium containing 8% serum or RPMI-1640 culture medium containing 8% serum, and further contains IL-152800U / mL, penicillin 120μg / mL, streptomycin 120μg / mL, and CD28 antibody 1100U / mL.

[0029] In the present invention, the timing of separation in step (2) is preferably when the tumor cell death rate in the first cell population reaches 10-30%, and more preferably when the death rate reaches 20%.

[0030] In the present invention, the third culture medium in step (3) is preferably based on AIM-V culture medium containing 6-10% serum or RPMI-1640 culture medium containing 6-10% serum, and further contains IL-212200-2400U / mL, penicillin 110-130μg / mL, streptomycin 110-130μg / mL, TNFα140-160pg / mL, and sodium pyruvate 2.4-2.6mM, and further preferably based on AIM-V culture medium containing 8% serum or RPMI-1640 culture medium containing 8% serum, and further contains IL-212300U / mL, penicillin 120μg / mL, streptomycin 120μg / mL, TNFα150pg / mL, and sodium pyruvate 2.5mM.

[0031] In the present invention, the culturing time in step (3) is preferably 48 to 60 hours, more preferably 54 hours.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] A method for amplifying tumor-infiltrating lymphocytes in vitro comprises the following steps:

[0035] (1) Lung cancer cells were cultured at a rate of 3×10 6 Inoculate the cells at a density of 100 cells / mL into the first culture medium and culture for 96 hours to obtain the first cell population;

[0036] (2) transferring the first cell population to a second culture medium, culturing until the tumor cell death rate in the first cell population reaches 10%, and isolating tumor infiltrating lymphocytes;

[0037] (3) The tumor infiltrating lymphocytes obtained in step (2) are transferred to a third culture medium and cultured for 60 hours, and the expanded tumor infiltrating lymphocytes are harvested.

[0038] Note: In this example, the first culture medium is based on AIM-V culture medium and also contains IL-2 4000U / mL, penicillin 130μg / mL, streptomycin 110μg / mL, and L-glutamine 1.2mM;

[0039] The second culture medium is based on AIM-V culture medium containing 10% serum, and further contains IL-152600U / mL, penicillin 110μg / mL, streptomycin 110μg / mL, and CD28 antibody 1200U / mL;

[0040] The third culture medium is based on RPMI-1640 culture medium containing 6% serum, and further contains IL-212 200 U / mL, penicillin 130 μg / mL, streptomycin 110 μg / mL, TNFα 140 pg / mL, and sodium pyruvate 2.6 mM.

[0041] Example 2

[0042] A method for amplifying tumor-infiltrating lymphocytes in vitro comprises the following steps:

[0043] (1) Ovarian cancer cells were cultured at a rate of 4 × 10 6 Inoculate the cells at a density of 100 cells / mL into the first culture medium and culture for 72 hours to obtain the first cell population;

[0044] (2) transferring the first cell population to a second culture medium, culturing until the tumor cell death rate in the first cell population reaches 30%, and isolating tumor infiltrating lymphocytes;

[0045] (3) The tumor infiltrating lymphocytes obtained in step (2) are transferred to a third culture medium and cultured for 48 hours, and the expanded tumor infiltrating lymphocytes are harvested.

[0046] Note: In this example, the first culture medium is based on RPMI-1640 culture medium, and also contains IL-24500U / mL, penicillin 110μg / mL, streptomycin 130μg / mL, and L-glutamine 1mM;

[0047] The second culture medium is based on AIM-V culture medium containing 6% serum, and further contains IL-15 3000U / mL, penicillin 130μg / mL, streptomycin 130μg / mL, and CD28 antibody 1000U / mL;

[0048] The third culture medium is based on RPMI-1640 culture medium containing 10% serum, and further contains IL-212400 U / mL, penicillin 110 μg / mL, streptomycin 130 μg / mL, TNFα 160 pg / mL, and sodium pyruvate 2.6 mM.

[0049] Example 3

[0050] A method for amplifying tumor-infiltrating lymphocytes in vitro comprises the following steps:

[0051] (1) Ovarian cancer cells were cultured at a rate of 3.5×10 6 The cells were inoculated into the first culture medium at a density of 100 cells / mL and cultured for 84 hours to obtain the first cell population;

[0052] (2) transferring the first cell population to a second culture medium, culturing until the tumor cell death rate in the first cell population reaches 20%, and isolating tumor infiltrating lymphocytes;

[0053] (3) The tumor infiltrating lymphocytes obtained in step (2) are transferred to a third culture medium and cultured for 54 hours, and the expanded tumor infiltrating lymphocytes are harvested.

[0054] Note: In this example, the first culture medium is based on RPMI-1640 culture medium, and also contains IL-24 300U / mL, penicillin 120μg / mL, streptomycin 120μg / mL, and L-glutamine 1.1mM;

[0055] The second culture medium is based on AIM-V culture medium containing 8% serum, and further contains IL-152800U / mL, penicillin 120μg / mL, streptomycin 120μg / mL, and CD28 antibody 1100U / mL;

[0056] The third culture medium is based on RPMI-1640 culture medium containing 8% serum, and further contains IL-212300 U / mL, penicillin 120 μg / mL, streptomycin 120 μg / mL, TNFα 150 pg / mL, and sodium pyruvate 2.5 mM.

[0057] Comparative Example 1

[0058] The CD28 antibody was replaced with the PD-1 antibody, and the other conditions were the same as those in Example 3.

[0059] Comparative Example 2

[0060] After obtaining tumor infiltrating lymphocytes in step (2), the obtained tumor infiltrating lymphocytes are mixed with dendritic cells in a ratio of 1:2, and the tumor infiltrating lymphocytes are separated after co-culturing for 24 hours, and then the culturing in step (3) is performed. The other conditions are the same as those in Example 3.

[0061] Test example

[0062] Tumor cells were cultured using the amplification methods of Examples 1 to 3 and Comparative Examples 1 to 2, respectively, and the number and cell viability of tumor infiltrating lymphocytes harvested from each group after culture were detected. The results are shown in Table 1.

[0063] Table 1

[0064] Group Total number of cells Cell viability (%) Example 1 <![CDATA[1.35×10 11 ]]> 95.11 Example 2 <![CDATA[9.83×10 10 ]]> 96.37 Example 3 <![CDATA[1.75×10 11 ]]> 97.09 Comparative Example 1 <![CDATA[6.77×10 10 ]]> 86.41 Comparative Example 2 <![CDATA[7.51×10 10 ]]> 88.09

[0065] As can be seen from the above embodiments, the present invention provides a method for amplifying tumor-infiltrating lymphocytes in vitro, comprising the following steps: (1) inoculating tumor cells into a first culture medium and culturing to obtain a first cell population; (2) transferring the first cell population to a second culture medium and isolating and obtaining tumor-infiltrating lymphocytes after culturing; (3) transferring the tumor-infiltrating lymphocytes obtained in step (2) to a third culture medium for culturing, and harvesting the amplified tumor-infiltrating lymphocytes. The amplification method of the present invention can obtain a large number of tumor-infiltrating lymphocytes in an in vitro environment, significantly improving the activity of tumor-infiltrating lymphocytes and their ability to kill tumor cells. The amount of tumor-infiltrating lymphocytes used can be reduced, and the final use effect can be improved.

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for amplifying tumor-infiltrating lymphocytes in vitro, characterized in that: The steps include: (1) inoculating tumor cells into a first culture medium to obtain a first cell population; (2) transferring the first cell population into a second culture medium, and isolating tumor infiltrating lymphocytes after culturing; (3) The tumor infiltrating lymphocytes obtained in step (2) are transferred to a third culture medium for culture, and the amplified tumor infiltrating lymphocytes are harvested.

2. The method according to claim 1, characterized in that The types of tumor cells in step (1) include renal cancer cells, liver cancer cells, ovarian cancer cells or lung cancer cells.

3. The method according to claim 2, characterized in that In step (1), the first culture medium is based on AIM-V culture medium or RPMI-1640 culture medium, and further contains IL-2 4000-4500 U / mL, penicillin 110-130 μg / mL, streptomycin 110-130 μg / mL, and L-glutamine 1-1.2 mM.

4. The method according to claim 3, characterized in that The inoculation density of tumor cells in the first culture medium in step (1) is 3×10 6 ~4×10 6 Pieces / mL.

5. The method according to claim 4, characterized in that The culturing time in step (1) is 72 to 96 hours.

6. The method according to claim 5, characterized in that The second culture medium in step (2) is based on AIM-V culture medium containing 6-10% serum or RPMI-1640 culture medium containing 6-10% serum, and also contains IL-152 600-3000 U / mL, penicillin 110-130 μg / mL, streptomycin 110-130 μg / mL, and CD28 antibody 1000-1200 U / mL.

7. The method according to claim 6, characterized in that The timing of separation in step (2) is when the tumor cell death rate in the first cell population reaches 10-30%.

8. The method according to claim 7, characterized in that The third culture medium described in step (3) is based on AIM-V culture medium containing 6-10% serum or RPMI-1640 culture medium containing 6-10% serum, and also contains IL-212200-2400U / mL, penicillin 110-130μg / mL, streptomycin 110-130μg / mL, TNFα 140-160pg / mL, and sodium pyruvate 2.4-2.6mM.

9. The method according to any one of claims 1 to 8, characterized in that: The culturing time in step (3) is 48 to 60 hours.