Method for in-vitro expansion culture of memory immune cells

Abstract

Disclosed in the present invention is a method for in-vitro expansion culture of memory immune cells. The method comprises: separating immune cells from peripheral blood or an apheresis sample by using a lymphocyte separation solution; in a complete culture medium, activating the immune cells obtained in the step 1) by using a first solution; after the immune cells are activated, supplementing a second solution during culture, and adjusting the cell culture density at regular intervals; and comparing the expansion fold and phenotypes of cells by using different culture containers, so as to determine the optimum culture container and obtain memory immune cells. In the present invention, on the basis of traditional immune cell culture media, a culture system (the type and concentration of cytokines, cell culture density and culture containers) is optimized. The method can be used in production as a preparation method for memory immune cells, thereby increasing the proportion and number of specific T cells.

Description

A method for in vitro expansion and cultivation of memory immune cells Technical Field

[0001] The present invention belongs to the field of biotechnology, relates to immune cells, and particularly to a method for amplifying and culturing memory immune cells in vitro. Background Art

[0002] Adoptive immune cell therapy, involving the ex vivo expansion and reinfusion of tumor-targeted T cells, such as tumor-infiltrating lymphocytes (TILs) and genetically reprogrammed T cells with conventional T cell receptors (TCRs) or chimeric antigen receptors (CARs), is emerging as a second-line treatment for patients with advanced cancer. However, dysregulated metabolic activity of tumor cells leads to an immunosuppressive tumor microenvironment (TME), which includes soluble factors secreted by tumor or stromal cells, as well as suppressive immune cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), leading to metabolic stress and exhaustion of immune cell therapy.

[0003] Current adoptive immune cell therapies mostly focus on targeting tumor cells, using gene editing to introduce an antibody sequence, ignoring the functionality of the T cells themselves. The vast majority of T cells obtained are terminally differentiated T cells (Teff), which are difficult to survive in the body for a long time. Summary of the Invention

[0004] To address the problem of immune cells easily differentiating into terminal effector T cells during in vitro culture, the present invention has developed a method for in vitro expansion and cultivation of memory immune cells. This method uses peripheral blood or apheresis blood as a cell source, isolates lymphocytes from these cells, and then further induces, expands, and cultivates memory T cells in complete immune cell culture medium. The total culture time is 12 days, during which fresh complete culture medium is continuously added to adjust the culture density based on cell density.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for in vitro expansion and cultivation of memory immune cells, the method comprising the following steps:

[0007] 1) Isolate immune cells from peripheral blood or single blood using lymphocyte separation medium;

[0008] 2) activating the immune cells obtained in step 1) with the first solution in complete culture medium;

[0009] 3) After immune cell activation, add the second solution during the culture period and adjust the cell culture density at regular intervals;

[0010] 4) Use different culture containers to compare cell expansion times and phenotypes to determine the optimal culture container and obtain memory immune cells.

[0011] As a preferred embodiment of the present invention, in step 2), the first solution includes the following components: immune cell culture medium, IL-2: 20-100 IU / mL, IL-21: 10-50 ng / mL, IL-7: 30-120 IU / mL and activated magnetic beads.

[0012] As a preferred embodiment of the present invention, the immune cell culture medium is one of ImmunoCult-XF, TexMACS or X-VIVO.

[0013] As a preferred embodiment of the present invention, in step 3), the second solution comprises the following components: immune cell culture medium, IL-2: 50-300 IU / mL, IL-21: 20-100 ng / mL, IL-7: 50-200 IU / mL and serum replacement.

[0014] As a preferred embodiment of the present invention, the immune cell culture medium is one of ImmunoCult-XF, TexMACS or X-VIVO.

[0015] As a preferred embodiment of the present invention, the first solution in step 2) activates the immune cells on day D0, and the cell density is adjusted to 1 to 4E 6 / mL.

[0016] As a preferred embodiment of the present invention, the culture time in step 3) is D12 days, 2-3 times the volume of the second solution is added on D2, and the cell density is adjusted to 5E on D4. 5 / mL; adjust the cell density to 6E on D6 5 / mL; adjust the cell density to 7E on D8 5 / mL; adjust the cell density to 8E at D10 5 / mL.

[0017] As a preferred embodiment of the present invention, in step 4), the culture container includes a G-Rex culture container, an Origen culture bag, a Takara culture bag and a traditional culture bottle.

[0018] As a preferred embodiment of the present invention, step 1) specifically comprises: taking a 50 mL centrifuge tube, adding a separation solution of equal volume to the blood sample; slowly adding the blood sample to the separation solution with a pipette, and centrifuging at 500-1000 g for 20-30 minutes; after centrifugation, aspirating the second layer of circular milky white lymphocytes into a new centrifuge tube, adding 10 mL of PBS, and mixing the cells; centrifuging at 250 g for 10 minutes, and discarding the supernatant to obtain immune cells.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) Based on traditional immune cell culture medium, this invention optimizes the culture system (cytokine types and concentrations, cell culture density and culture container) and can be used as a preparation method for memory immune cells in production, thereby increasing the proportion and number of specific T cells.

[0021] 2) The present invention uses peripheral blood or single-collected blood as a cell source, isolates lymphocytes therefrom, and then further induces, expands, and cultures memory T cells in a complete immune cell culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 is a schematic diagram of cell expansion times in different groups.

[0023] FIG2 is a schematic diagram of cell viability in different groups.

[0024] FIG3 is a schematic diagram of cell phenotypes in different groups. Implementation Method

[0025] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further described below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the embodiments, other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0026] The present invention provides a method for in vitro expansion and culture of memory immune cells. Lymphocytes are isolated from peripheral blood or apheresis blood as a cell source, and then further induced, expanded, and cultured in complete immune cell culture medium to produce memory T cells. The total culture period is 12 days, during which fresh complete culture medium is continuously added to adjust the culture density based on cell density. Example

[0027] The method for in vitro expansion and cultivation of memory immune cells disclosed in this embodiment comprises the following steps:

[0028] 1. Isolate immune cells from peripheral blood or single blood using lymphocyte separation medium:

[0029] (1) The lymphocyte separation medium used was GE Ficoll-Paque (Cat. No.: 17-1440-02) from GE.

[0030] (2) Take a 50mL centrifuge tube and add a volume of separation solution equal to the volume of the blood sample.

[0031] (3) Use a pipette to slowly add the blood sample to the separation solution and centrifuge at 500-1000g for 20-30 minutes.

[0032] (4) After centrifugation, aspirate the second layer of circular milky white lymphocytes into a new centrifuge tube, add 10 mL of PBS, and mix the cells.

[0033] (5) Centrifuge at 250g for 10 minutes and discard the supernatant.

[0034] 2. Activate immune cells with activation magnetic beads:

[0035] 1) Resuspend the cells with the first solution and adjust the cell density to 1-4E 6 / mL, temperature is 37℃, 5% CO2.

[0036] 2) First Solution Components: ImmunoCult-XF, TexMACS, or X-VIVO immune cell culture medium (all three are acceptable; ImmunoCult-X was used in this example), IL-2: 20-100 IU / mL, IL-21: 10-50 ng / mL, IL-7: 30-120 IU / mL, and activated Transact magnetic beads (Miltenyi, 130-111-160).

[0037] 3. After immune cell activation, continuously adjust cell culture density:

[0038] The activation of immune cells by the first solution was recorded as D0 day, and then culture was continued to D12 day.

[0039] 1) Add 2 to 3 times the volume of solution 2 to D2;

[0040] 2) Adjust the cell density to 5E on D4 5 / mL;

[0041] 3) Adjust the cell density to 6E on D6 5 / mL;

[0042] 4) Adjust the cell density to 7E on D8 5 / mL;

[0043] 5) Adjust the cell density to 8E at D10 5 / mL.

[0044] Second solution composition: immune cell culture medium such as ImmunoCult-XF, TexMACS, or X-VIVO (all three are acceptable; ImmunoCult-X is used in this example), IL-2: 50-300 IU / mL, IL-21: 20-100 ng / mL, IL-7: 50-200 IU / mL, and serum replacement (SR): 2%.

[0045] 4. Compare cell expansion times and phenotypes using different culture vessels:

[0046] 1) Different container groups are as follows:

[0047] Group 1 is the G-Rex culture container, Group 2 is the Origen culture bag, Group 3 is the Takara culture bag, and Group 4 is the traditional culture flask.

[0048] result:

[0049] See Figures 1 and 2 for amplification fold and viability.

[0050] As shown in Figure 1, judging from the final D12 cell expansion multiples, Group 4 had the highest expansion multiple, reaching about 300 times; followed by the two culture bag groups (Groups 2 and 3), with cell expansion multiples close to each other, at about 120 times; the worst was Group 1, which ultimately expanded less than 50 times.

[0051] As shown in Figure 2, cell viability: Group 4 was relatively stable, and finally maintained a viability of about 90%; the other three groups showed a downward trend after D6, and the final cell viability was about 80%.

[0052] As shown in Figure 3, the cell phenotypes were detected on D8 / 10 / 12 days, respectively. Group 1 was relatively stable, with more than 95% being memory T cells (Tm); Group 4 showed a downward trend, with the final memory T cell ratio being around 70%; the phenotypes of groups 2 and 3 both declined on D10 and rebounded on D12, with group 3 (90%) being better than group 2 (80%).

[0053] In summary, as a preferred embodiment, when the culture container is an Origen culture bag or a Takara culture bag, the cells are first activated with the first solution and then amplified and cultured with the second solution until day 12. The final amplification multiple, viability, and phenotype all show good performance.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for in vitro amplification and cultivation of memory immune cells, characterized in that: The method comprises the following steps: 1) Use lymphocyte separation fluid to separate immune cells from peripheral blood or single blood; 2) Activating the immune cells obtained in step 1) with the first solution in complete culture medium; 3) After the immune cells are activated, the second solution is added during the culture period, and the cell culture density is adjusted at regular intervals; 4) Use different culture containers, compare cell proliferation multiples and phenotypes to determine the optimal culture container, and obtain memory immune cells.

2. The method for in vitro expansion and cultivation of memory immune cells according to claim 1, characterized in that: In step 2), the first solution includes the following components: immune cell culture medium, IL-2: 20-100 IU / mL, IL-21: 10-50 ng / mL, IL-7: 30-120 IU / mL and activated magnetic beads.

3. The method for in vitro expansion and cultivation of memory immune cells according to claim 2, characterized in that: The immune cell culture medium is one of ImmunoCult-XF, TexMACS or X-VIVO.

4. The method for in vitro expansion and cultivation of memory immune cells according to claim 1, characterized in that: In step 3), the second solution includes the following components: immune cell culture medium, IL-2: 50-300 IU / mL, IL-21: 20-100 ng / mL, IL-7: 50-200 IU / mL and serum substitute.

5. The method for in vitro expansion and cultivation of memory immune cells according to claim 4, characterized in that: The immune cell culture medium is one of ImmunoCult-XF, TexMACS or X-VIVO.

6. The method for in vitro expansion and cultivation of memory immune cells according to claim 1, characterized in that: Note that the first solution in step 2) activates immune cells on day D0, and adjusts the cell density to 1-4E 6 / mL.

7. The method for in vitro expansion and cultivation of memory immune cells according to claim 6, characterized in that: Step 3) The total culture time is D12 days. Add 2-3 times the volume of the second solution on D2, and adjust the cell density to 5E on D4. 5 / mL; adjust the cell density to 6E at D6 5 / mL; adjust the cell density to 7E at D8 5 / mL; adjust the cell density to 8E at D10 5 / mL.

8. The method for in vitro expansion and cultivation of memory immune cells according to claim 1, characterized in that: In step 4), the culture containers include G-Rex culture containers, Origen culture bags, Takara culture bags and traditional culture bottles.

9. The method for in vitro expansion and cultivation of memory immune cells according to claim 1, characterized in that: Step 1) is specifically as follows: take a 50mL centrifuge tube and add a separation solution of the same volume as the blood sample; use a pipette to slowly add the blood sample to the separation solution, and centrifuge at 500-1000g for 20-30min; after centrifugation, aspirate the second layer of circular milky white lymphocytes into a new centrifuge tube, add 10mL PBS, and mix the cells; centrifuge at 250g for 10min, discard the supernatant, and obtain immune cells.

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