Co-culture-based tils cell amplification method

Abstract

The invention discloses a co-culture-based tils cell amplification method, which comprises: S1, tumor tissue treatment: obtaining a fresh tumor tissue, and avoiding cell activity reduction caused by long-time storage; s2, tumor cell culture: carrying out enzyme digestion on one part of tumor tissue, separating out tumor cells, carrying out primary culture, and establishing a tumor cell line; s3, TILs separation: carrying out mechanical separation and density gradient centrifugation on the other part of tumor tissue to separate out TILs; s4, TILs pre-stimulation, wherein the separated TILs are subjected to pre-stimulation in a culture medium containing IL-2, and activation and proliferation of the TILs are promoted; s5, co-culture: mixing the pre-stimulated TILs and tumor cells according to a ratio of 1: 1 or 2: 1, and carrying out co-culture; and S6, TILs amplification: after co-culture is finished, separating out TILs, and further amplifying in a culture medium containing IL-2 to obtain high-activity TILs. According to the invention, the activity of TILs, the tumor antigen specificity and the amplification efficiency can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a TILs cell expansion method based on co-culture. Background Art

[0002] Tumor-infiltrating lymphocytes (TILs) are lymphocytes isolated from tumor tissue that have the potential to recognize and kill tumor cells. In recent years, TILs therapy has shown promising application prospects in the treatment of various solid tumors. However, the in vitro expansion efficiency and anti-tumor activity of TILs remain key factors limiting their clinical application.

[0003] Traditional TILs culture methods typically involve direct enzymatic digestion of tumor tissue, isolation of TILs, and subsequent in vitro expansion. This method has the following disadvantages: (1) Low TILs activity: Immunosuppressive factors in the tumor microenvironment inhibit the activity and proliferation of TILs.

[0004] (2) Insufficient tumor antigen stimulation: Traditional methods are difficult to provide continuous and effective tumor antigen stimulation, resulting in limited TILs' ability to recognize and kill tumor cells.

[0005] (3) Low expansion efficiency: The in vitro expansion efficiency of TILs is low, and it is difficult to obtain a sufficient number of cells for treatment. Summary of the Invention

[0006] The object of the present invention is to provide a TILs cell expansion method based on co-culture to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions: A co-culture-based TILs cell expansion method comprises the following steps: S1: Tumor tissue processing: Obtain fresh tumor tissue to avoid prolonged storage that may lead to a decrease in cell activity; S2: Tumor cell culture: A tumor tissue is enzymatically digested to isolate tumor cells, which are then cultured to establish a tumor cell line. S3: TILs isolation: Another sample of tumor tissue was subjected to mechanical dissociation and density gradient centrifugation to isolate TILs; S4: TILs pre-stimulation: The isolated TILs are pre-stimulated in a culture medium containing IL-2 to promote the activation and proliferation of TILs; S5: Co-culture: Pre-stimulated TILs and tumor cells were mixed at a ratio of 1:1 or 2:1 for co-culture; S6: TILs expansion: After the co-culture is completed, TILs are isolated and further expanded in a culture medium containing IL-2 to obtain highly active TILs.

[0008] As a further solution of the present invention: in S1, the fresh tumor tissue obtained is divided into two parts, one for tumor cell culture and the other for TILs isolation.

[0009] As a further solution of the present invention: In S2, the culture conditions of tumor cells need to be optimized according to the tumor type to ensure high activity and proliferation ability of the cells, and the antigen expression of tumor cells should be regularly tested to ensure that they can provide effective antigen stimulation.

[0010] As a further solution of the present invention: in S3, excessive mechanical damage is avoided during the separation process to maintain the integrity of the cells, and penicillin and streptomycin antibiotics are added during the separation and culture process to avoid microbial contamination and ensure the purity of TILs.

[0011] As a further solution of the present invention: in S4, the concentration of IL-2 is 500-1000 IU / mL, the pre-stimulation time is 3-5 days, and the concentration and pre-stimulation time are adjusted according to the initial state of TILs to avoid excessive stimulation leading to cell apoptosis.

[0012] As a further embodiment of the present invention: in S5, during the co-culture process, tumor cells provide continuous tumor antigen stimulation to promote the activation and proliferation of TILs, and the co-culture time is 7-10 days.

[0013] As a further solution of the present invention: in S6, the culture medium needs to be replaced regularly during the expansion process to maintain high cell activity.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Improve TILs activity: By co-culturing tumor cells, simulating the tumor microenvironment, providing continuous tumor antigen stimulation, and effectively improving the activity and anti-tumor ability of TILs; Enhanced tumor antigen specificity: During co-culture, TILs can recognize and kill tumor cells, thereby screening TILs with tumor antigen specificity; Improve expansion efficiency: Co-culture of tumor cells can promote the proliferation of TILs, significantly improve the expansion efficiency of TILs, and obtain a sufficient number of cells for treatment; Easy to operate: The method of the present invention is easy to operate, easy to standardize, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Flowchart of the co-culture-based TILs cell expansion method.

[0016] Figure 2 This is a graph showing the activity detection results after TILs expansion in the co-culture-based TILs cell expansion method.

[0017] Figure 3 Schematic diagram of the killing effect of TILs on tumor cells in the co-culture-based TILs cell expansion method. DETAILED DESCRIPTION

[0018] Various exemplary embodiments, features, and aspects of the present application will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise indicated.

[0019] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0020] In addition, numerous specific details are provided in the following specific examples to better illustrate the present application. Those skilled in the art will appreciate that the present application can be practiced without certain specific details. In some instances, methods, means, and components well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0021] Example 1: Expansion of melanoma TILs S1: Tumor tissue processing: Fresh melanoma tissue was obtained and divided into two parts.

[0022] S2: Tumor cell culture: A tumor tissue was enzymatically digested to isolate the tumor cells, which were then cultured to establish a melanoma cell line.

[0023] S3: TILs isolation: Another sample of tumor tissue was subjected to mechanical separation and density gradient centrifugation to isolate TILs.

[0024] S4: TILs pre-stimulation: The isolated TILs were pre-stimulated in culture medium containing 1000 IU / mL IL-2 for 3 days.

[0025] S5: Co-culture: Pre-stimulated TILs were mixed with melanoma cells at a 1:1 ratio and co-cultured for 7 days.

[0026] S6: TILs expansion: After 7 days of co-culture, TILs were isolated and further expanded in culture medium containing 1000 IU / mL IL-2 for 14 days to obtain highly active TILs.

[0027] Example 2: Amplification of lung cancer TILs S1: Tumor tissue processing: Fresh lung cancer tissue was obtained and divided into two parts.

[0028] S2: Tumor cell culture: A tumor tissue is enzymatically digested to separate the tumor cells, which are then cultured in primary culture to establish a lung cancer cell line.

[0029] S3: TILs isolation: Another sample of tumor tissue was subjected to mechanical separation and density gradient centrifugation to isolate TILs.

[0030] S4: TILs pre-stimulation: The isolated TILs were pre-stimulated in culture medium containing 500 IU / mL IL-2 for 5 days.

[0031] S5: Co-culture: Pre-stimulated TILs were mixed with lung cancer cells at a ratio of 2:1 and co-cultured for 10 days.

[0032] S6: TILs expansion: After 10 days of co-culture, TILs were isolated and further expanded for 21 days in culture medium containing 500 IU / mL IL-2 to obtain highly active TILs.

[0033] See Figure 2 , amplification efficiency comparison: See Figure 3 , Cytotoxicity assay: When the effector-target ratio was 10:1, the killing rate of autologous tumor cells by TILs of the present invention reached 85%±4%, while that of the traditional method was 52%±6%.

[0034] The killing rate of allogeneic tumor cells remained ≤15%, indicating high antigen specificity.

[0035] The present invention can effectively improve the activity, tumor antigen specificity and amplification efficiency of TILs, and provides a new technical means for the clinical application of TILs therapy.

[0036] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be regarded as illustrative and non-limiting in all respects, and the scope of the present invention is determined by the appended claims. The present invention is not limited to the above description and therefore it is intended that all Variations are encompassed by the invention.

[0037] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A TILs cell expansion method based on co-culture, characterized in that: The following steps are involved: S1: Tumor tissue processing: Obtain fresh tumor tissue to avoid prolonged storage that may lead to a decrease in cell activity; S2: Tumor cell culture: A tumor tissue is enzymatically digested to isolate tumor cells, which are then cultured to establish a tumor cell line. S3: TILs isolation: Another sample of tumor tissue was subjected to mechanical dissociation and density gradient centrifugation to isolate TILs; S4: TILs pre-stimulation: The isolated TILs are pre-stimulated in a culture medium containing IL-2 to promote the activation and proliferation of TILs; S5: Co-culture: Pre-stimulated TILs and tumor cells were mixed at a ratio of 1:1 or 2:1 for co-culture; S6: TILs expansion: After the co-culture is completed, TILs are isolated and further expanded in a culture medium containing IL-2 to obtain highly active TILs.

2. The TILs cell expansion method based on co-culture according to claim 1, characterized in that In S1, fresh tumor tissue was obtained and divided into two parts, one for tumor cell culture and the other for TILs isolation.

3. The TILs cell expansion method based on co-culture according to claim 1, characterized in that In S2, the culture conditions of tumor cells need to be optimized according to the tumor type to ensure high activity and proliferation ability of the cells, and the antigen expression of tumor cells needs to be tested regularly to ensure that they can provide effective antigen stimulation.

4. The TILs cell expansion method based on co-culture according to claim 1, characterized in that In S3, excessive mechanical damage is avoided during the separation process to maintain cell integrity. Penicillin and streptomycin antibiotics are added during the separation and culture process to avoid microbial contamination and ensure the purity of TILs.

5. The TILs cell expansion method based on co-culture according to claim 1, characterized in that In S4, the concentration of IL-2 is 500-1000 IU / mL, and the pre-stimulation time is 3-5 days. The concentration and pre-stimulation time are adjusted according to the initial state of TILs to avoid overstimulation leading to cell apoptosis.

6. The method for expanding TILs cells based on co-culture according to claim 1, characterized in that In S5, during the co-culture process, tumor cells provide continuous tumor antigen stimulation, promoting the activation and proliferation of TILs, and the co-culture time is 7-10 days.

7. The method for expanding TILs cells based on co-culture according to claim 1, characterized in that: In S6, the culture medium needs to be replaced regularly during the expansion process to maintain high cell activity.

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