CN110643574A - Preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes - Google Patents

Abstract

The invention discloses a preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes. The efficiency of the feeder cells manufactured by the method for amplifying the tumor infiltrating lymphocytes is better than the efficiency of the feeder cells manufactured by gamma ray irradiation for amplifying the tumor infiltrating lymphocytes, and the cost is obviously reduced by the method. The preparation method is simple and feasible, and can be widely popularized and applied.

Description

Preparation method of feeder cells for rapid culture of tumor-infiltrating lymphocytes

technical field

The invention belongs to the technical field of cell culture, and in particular relates to a preparation method of feeder cells for rapid culture of tumor-infiltrating lymphocytes.

Background technique

Tumor infiltrated lymphocytes (TIL) are lymphocytes that leave the blood circulation and migrate to the vicinity of the tumor, including T cells, B cells, NK cells, etc. They can act to kill cancer cells. The amount of TIL in the tumor is an important indicator for predicting the prognosis of cancer patients and the response to immunotherapy. TIL therapy is mainly to rapidly expand the TIL isolated from the fresh tumor tissue of the patient in an in vitro cell culture environment, expand the number of TIL to the amount that can be reinfused into the patient, and then reinfuse it into the patient.

The currently known technique for in vitro expansion of TIL is to pre-culture the isolated TIL in vitro for 1-2 weeks in a certain ratio (usually 1:200) with peripheral blood lymphocytes irradiated with 30-50 Gy γ rays ( PeripheralBlood Mononuclear Cells, PBMC) for co-cultivation, the main principle is to use γ-ray irradiation PBMC to inhibit growth and become feeder cells, in the process of co-culture with TIL, the growth factors secreted by feeder cells will promote the rapid proliferation of TIL and to reach the number of cells used for treatment. However, this method requires special irradiation equipment, and the irradiation cost is relatively high, and general research institutions do not have such equipment, so it is not economical for general research institutions to use this method to expand a large number of TILs in vitro.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a low-cost preparation of feeder cells for the rapid culture of tumor-infiltrating lymphocytes in view of the problems in the prior art that the feeder cells are prepared by irradiating PBMC with γ-rays, and the irradiation cost is high and is not conducive to widespread use. method.

The technical scheme adopted in the present invention is as follows:

A method for preparing feeder cells for rapid culture of tumor-infiltrating lymphocytes, comprising the following steps:

S1. Preparation of Feeder Cells

1) Isolation and culture of peripheral blood lymphocytes: extract autologous peripheral blood from patients with non-small cell lung cancer, dilute with phosphate buffer, add lymphocyte separation medium to separate lymphocytes, and then use serum-free X-VIVO medium in Culture in 5% CO ₂ , 37°C incubator;

2) Feeder cell preparation: After overnight culture, the isolated lymphocytes were counted, the cell density was adjusted to 1.5×10 ⁶ cells/mL, mitomycin C was added to treat for 1.5-2 hours, centrifuged, and the supernatant was discarded. , then resuspend and wash the cells with X-VIVO medium, centrifuge again, discard the supernatant, add medium to resuspend the cells, and obtain feeder cells;

S2. Isolation and pre-culture of tumor-infiltrating lymphocytes

The clinically collected non-small cell lung cancer tissues were washed with sterile phosphate buffered saline and cut into 1-2mm ³ tissue pieces, then added with tumor-infiltrating lymphocyte culture medium, and cultured in a 5% CO ₂ , 37°C incubator Discard the tissue block after 2-3 days, continue culturing the tumor-infiltrating lymphocytes, and add fresh medium every 2-3 days. After culturing for 10-14 days, collect the cells, after centrifugation, discard the supernatant, and then resuspend with the medium and washing the cells to obtain pre-cultured tumor-infiltrating lymphocytes;

S3. Co-culture of feeder cells with tumor-infiltrating lymphocytes

The feeder cells prepared in S1 and the pre-cultured tumor-infiltrating lymphocytes prepared in S2 were centrifuged separately, resuspended and washed twice with medium, and then added to AIM-V/TIL medium with IL-2 at the same time. And CD3/CD28 antibody, put into 5% CO ₂ , 37 ℃ incubator for culture, add fresh medium every 2-3 days.

In the present invention, mitomycin C is used to treat PBMC to obtain feeder cells, and then the feeder cells are mixed with pre-cultured TIL in proportion to carry out co-cultivation. The experimental results show that the feeder cells produced by the method of the present invention are more efficient in amplifying TIL than feeder cells produced by γ-ray irradiation, and the cost of this method is significantly reduced. The preparation method of the method of the invention is simple and feasible, and can be widely applied.

The traditional method is to irradiate PBMCs with γ-rays to inhibit their growth, and then co-culture with TILs. During the culture, the irradiated PBMCs secrete a large number of growth factors to promote the rapid growth of TILs. However, general research units do not have the conditions for γ-ray irradiation, and the cost of γ-ray irradiation is high.

The present invention uses mitomycin C to prepare feeder cells, and treatment of PBMC with mitomycin C makes the growth of PBMC cells impaired, but the cells still survive and secrete growth factors that promote the growth of TILs. This method has low cost, and the research results show that the effect of promoting the proliferation of TILs after co-culture of PBMCs treated with mitomycin C is better than that of PBMCs treated with γ-ray irradiation.

Further, the phosphate buffer in S1 and the patient's autologous peripheral blood are diluted at a volume ratio of 1:1-1.2.

Further, 40ng/mL of IL-2 and 200ng/mL of CD3/CD28 antibody were also added to the X-VIVO medium in S1.

Further, the final concentration of mitomycin C in S1 was 50 μg/mL.

Further, the tumor-infiltrating lymphocyte medium in S2 included 1640 medium, 10 wt% human A/B serum, 1 wt% penicillin/streptomycin solution, 10 mM L-glutamine and 10 mM 2-mercaptoethanol.

Further, in S3, the feeder cells prepared in S1 and the pre-cultured tumor-infiltrating lymphocytes prepared in S2 were added to the AIM-V/TIL medium in a ratio of 200-230:1.

Further, the AIM-V/TIL medium was prepared from the AIM-V medium and the tumor-infiltrating lymphocyte medium in a volume ratio of 1:1.

Further, AIM-V/TIL medium was also supplemented with 5000 U/mL IL-2 and 200 ng/mL CD3/CD28 antibody.

Further, after the cell density reaches 2×10 ⁶ cells/mL in S3, 1:1 cell passage is performed; meanwhile, cell counts are performed 1-2 times a week.

To sum up, due to the adoption of the above-mentioned technical solutions, the beneficial effects of the present invention are:

1. The present invention utilizes mitomycin C to treat peripheral blood lymphocytes to prepare feeder cells for rapid expansion culture of TIL in vitro, compared with the feeder cells produced by traditional methods using γ-ray irradiation to expand tumor-infiltrating lymphocytes. Compared with the efficiency, the effect is better, and the cost is significantly reduced, and the application scope is effectively expanded;

2. In the present invention, after PBMC is treated with mitomycin C, the growth of PBMC cells is damaged, but the cells still survive and secrete growth factors that promote the growth of TIL. The effect of proliferation was better than that of PBMC treated with γ-irradiation.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Figure 1 is a graph of cell proliferation;

Figure 2 is a graph showing the content of killer T cells (CD3 ⁺ CD8 ⁺ ) after co-culture of feeder cells prepared by γ-ray irradiation with TIL for 14 days;

Figure 3 is a graph showing the content of killer T cells (CD3 ⁺ CD8 ⁺ ) after co-culture of feeder cells and TIL prepared by the method of the present invention for 14 days;

Figure 4 is a graph of T cell viability.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention, that is, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The components of the embodiments of the invention generally described and illustrated in the drawings herein may be arranged and designed in a variety of different configurations.

Thus, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present invention.

It should be noted that relational terms such as the terms "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any relationship between these entities or operations. any such actual relationship or sequence exists. Moreover, the terms "comprising", "comprising" or any other variation thereof are intended to encompass a non-exclusive inclusion such that a process, method, article or device that includes a list of elements includes not only those elements, but also includes not explicitly listed or other elements inherent to such a process, method, article or apparatus. Without further limitation, an element qualified by the phrase "comprising a..." does not preclude the presence of additional identical elements in a process, method, article or apparatus that includes the element.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example

A preferred embodiment of the present invention provides a method for preparing feeder cells for rapid culture of tumor-infiltrating lymphocytes, the specific steps are as follows:

1. Preparation of feeder cells:

1) Isolation and culture of the patient's autologous peripheral blood lymphocytes: After extracting 50 mL of the patient's autologous peripheral blood, dilute it with phosphate buffer at 1:1, then add lymphocyte separation medium to separate lymphocytes, and then use serum-free X-VIVO medium (added with 40 ng/mL IL-2 and 200 ng/mL CD3/CD28 antibody) was cultured in a 5%, CO ₂ , 37°C incubator.

2) Feeder cell preparation: After overnight culture of the isolated lymphocytes, count the cells, adjust the cell density to 1.5 × 10 ⁶ cells/mL, add mitomycin C (final concentration of 50 μg/mL) for 1.5 hours, 1000 rpm After centrifugation at 1000 rpm/min for 5 min, the supernatant was discarded, and the cells were then resuspended and washed with X-VIVO medium. After centrifugation at 1000 rpm/min for 5 min, the supernatant was discarded, and medium was added to resuspend the cells for the co-cultivation step with TIL.

2. Isolation and pre-culture of TIL

In a biological safety cabinet, the clinically collected non-small cell lung cancer tissue was washed twice with sterile phosphate buffered saline and then cut into 1-2 mm tissue pieces, and the tissue pieces ^were placed in a 48-well cell culture plate (1 piece/well). ), then add TIL medium (1640 medium+10wt% human A/B serum+1wt% penicillin/streptomycin solution+10mM L-glutamine+2-mercaptoethanol), put in 5%, CO ₂ , 37℃ Culture in the incubator, discard the tissue block after 2-3 days, continue the culture of TIL in the well, add fresh medium every 2-3 days, collect the cells after culturing for 10-14 days, and discard the supernatant after centrifugation at 1000rpm/min for 5min , cells were then resuspended and washed with medium for co-culture.

3. Co-culture of feeder cells with TIL

The prepared feeder cells and pre-cultured TIL were centrifuged, resuspended and washed twice with the medium, and then added to the AIM-V/TIL medium mixed at a ratio of 200:1 according to the number of cells. , add 5000U/mL IL-2 and 200ng/mL CD3/CD28 antibody at the same time, put it into a 5%, CO ₂ , 37 ℃ incubator for cultivation, add fresh medium every 2-3 days, until the cell density reaches 2× After 10 ⁶ cells/mL, cells were passaged 1:1; cell counts were performed 1-2 times per week.

Experimental example 1

The feeder cells produced by γ-ray irradiation and the method of the embodiment of the present invention were used to prepare feeder cells, respectively, and the feeder cells prepared by the two methods were respectively co-cultured with non-small cell lung cancer TIL.

It can be seen from the figure that the difference in the proliferation efficiency of the cells is small, and the cell growth fold is higher after the feeder cells prepared by the method of the embodiment of the present invention are co-cultured with non-small cell lung cancer TIL for 21 days.

Experimental example 2

Feeder cells produced by γ-ray irradiation and the method of the embodiment of the present invention were used to prepare feeder cells, respectively, and flow cytometry was used to detect the content of killer T cells (CD3+CD8+) after co-culture of feeder cells prepared by the two methods with TIL for 14 days , the results are shown in Figures 2 and 3; the results show similar effects.

Experimental example 3

The feeder cells were prepared by the method of the embodiment of the present invention, and then co-cultured with TIL, and the cell viability was detected. The results are shown in FIG. 4 . It can be seen from the figure that the cell survival rate of TIL is above 85% after the feeder cells prepared by the present invention are co-cultured with TIL.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (9)

1. A method for preparing feeder cells for rapid culture of tumor infiltrating lymphocytes is characterized by comprising the following steps:

s1. preparation of feeder cells

1) Separation and culture of peripheral blood lymphocytes: collecting autologous peripheral blood of patient with non-small cell lung cancer, diluting with phosphate buffer solution, adding lymphocyte separation solution to separate lymphocytes, and culturing with serum-free X-VIVO medium in 5% CO₂Culturing in an incubator at 37 ℃;

2) preparing feeder cells: after overnight culture, the lymphocytes obtained after the above separation were subjected to cell counting to adjust the cell density to 1.5X 10⁶Adding mitomycin C into cells/mL, treating for 1.5-2 hours, centrifuging, removing supernate, then using X-VIVO to culture basic suspension and washing cells, centrifuging again, removing supernate, adding the basic suspension cells to obtain feeder cells;

s2, separation and pre-culture of tumor infiltrating lymphocytes

Washing clinically collected non-small cell lung cancer tissue with sterile phosphate buffer solution, and cutting into 1-2mm³Then adding a tumor infiltrating lymphocyte culture medium, and adding 5% CO₂Culturing in 37 deg.C incubator for 2-3 days, discarding tissue block, culturing tumor infiltrating lymphocytes, and adding fresh culture every 2-3 daysCulturing for 10-14 days, collecting cells, centrifuging, removing supernatant, suspending and washing the cells by using a culture medium to obtain pre-cultured tumor infiltrating lymphocytes;

s3, co-culture of feeder cells and tumor infiltrating lymphocytes

The feeder cells obtained in S1 and the pre-cultured tumor-infiltrating lymphocytes obtained in S2 were centrifuged, suspended and washed twice with medium, added to AIM-V/TIL medium, and IL-2 and CD3/CD28 antibodies were added to the medium, followed by addition of 5% CO₂Culturing in 37 deg.C incubator, and adding fresh culture medium every 2-3 days.

2. The method of claim 1 for the preparation of feeder cells for the rapid culture of tumor-infiltrating lymphocytes, wherein: the phosphate buffer solution in the S1 is mixed with the peripheral blood of the patient in a volume ratio of 1: 1-1.2 dilution.

3. The method of claim 1 for the preparation of feeder cells for the rapid culture of tumor-infiltrating lymphocytes, wherein: the S1 medium X-VIVO was further supplemented with IL-2 at 40ng/mL and CD3/CD28 at 200 ng/mL.

4. The method of claim 1 for the preparation of feeder cells for the rapid culture of tumor-infiltrating lymphocytes, wherein: the final concentration of mitomycin C in S1 was 50. mu.g/mL.

5. The method of claim 1 for the preparation of feeder cells for the rapid culture of tumor-infiltrating lymphocytes, wherein: the culture medium of the tumor infiltrating lymphocytes in the S2 comprises 1640 culture medium, 10 wt% of human A/B serum, 1 wt% of penicillin/streptomycin solution, 10mM of L-glutamine and 10mM of 2-mercaptoethanol.

6. The method of claim 1 for the preparation of feeder cells for the rapid culture of tumor-infiltrating lymphocytes, wherein: the cell number of the feeder cells prepared in S1 and the pre-cultured tumor infiltrating lymphocytes prepared in S2 in the S3 was 200-230:1, and the feeder cells were added to the AIM-V/TIL medium.

7. The method of claim 6, wherein the feeder cells for the rapid culture of tumor infiltrating lymphocytes are selected from the group consisting of: the AIM-V/TIL culture medium is prepared from an AIM-V culture medium and a tumor infiltrating lymphocyte culture medium according to the volume ratio of 1: 1.

8. The method of claim 7, wherein the feeder cells are selected from the group consisting of: the AIM-V/TIL medium was also supplemented with 5000U/mL IL-2 and 200ng/mL of CD3/CD28 antibody.

9. The method of claim 1 for the preparation of feeder cells for the rapid culture of tumor-infiltrating lymphocytes, wherein: the density of cells to be treated in S3 reaches 2X 10⁶After cells/mL, carrying out 1:1 cell passage; cell counts were also performed 1-2 times per week.

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