Pancreatic cancer organoid and T cell co-culture method

Abstract

The invention discloses a method for co-culturing pancreatic cancer organoid and T cells. The method comprises the following steps: step 1, mixing the pancreatic cancer organoid with the T cells; 2, co-culturing by adopting a mixed culture medium of a pancreatic cancer organoid culture medium and a T cell amplification culture medium; wherein the volume ratio of the pancreatic cancer organoid culture medium to the T cell amplification culture medium in the mixed culture medium is (20: 80)-(40: 60). According to the method for co-culturing the pancreatic cancer organoid and the T cells, the pancreatic cancer organoid and the T cells can grow, the activity of the pancreatic cancer organoid and the T cells can be maintained, particularly, the cell state of the T cells can be rapidly amplified into clusters, and the drug effect of an immune drug can be efficiently evaluated by adding the specific immune drug.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a method for co-culturing pancreatic cancer organoids and T cells. Background Art

[0002] Pancreatic cancer seriously endangers human health. Early pancreatic cancer patients have no obvious symptoms, and most pancreatic cancer patients have developed to the middle and late stages when diagnosed. Pancreatic cancer cells are less sensitive to chemotherapy drugs, so their chemotherapy effects are poor, resulting in a low five-year survival rate. Current clinical research data show that immunotherapy in pancreatic cancer treatment has good therapeutic effects and application prospects. For example, CD19-CAR-T, PD1 / PD-L1 immune checkpoint inhibitors, etc. have shown exciting effects in clinical medicine, among which immune checkpoint inhibitors and T cell agonist antibodies are becoming hot areas. However, there is currently no suitable in vitro model for evaluating immune drugs. Organoid culture is a variety of cell groups contained in specific tissues and organs cultured in a three-dimensional environment. The composition is similar to that of organs in the body, and its culture system is more closely connected to the in vivo microenvironment. By constructing a pancreatic cancer organoid and T cell co-culture method model in vitro, the efficacy of immune drugs such as PD1 / PDL1 can be better predicted, which will promote the widespread use of organoid technology in precision medicine, drug screening and development, gene therapy and other fields. Summary of the invention

[0003] The purpose of the present invention is to provide a method for co-culturing pancreatic cancer organoids and T cells in response to the deficiencies in the prior art.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] A method for co-culturing pancreatic cancer organoids and T cells is provided, comprising the following steps:

[0006] Step 1: Mix pancreatic cancer organoids with T cells;

[0007] Step 2, co-culturing using a mixed culture medium of pancreatic cancer organoid culture medium and T cell expansion culture medium;

[0008] Wherein, the volume ratio of pancreatic cancer organoid culture medium to T cell expansion culture medium in the mixed culture medium is 20:80 to 40:60.

[0009] Furthermore, the volume ratio of pancreatic cancer organoid culture medium to T cell expansion culture medium in the mixed culture medium is 20:80.

[0010] Furthermore, the pancreatic cancer organoid is obtained by the following method:

[0011] S1, collect pancreatic cancer organoids after P2 culture, remove the culture medium with a pipette, add 1-2 ml of organoid subculture buffer to each well and let it stand for 2-10 min;

[0012] S2, gently blow the base gel with a pipette, collect it in a centrifuge tube, and let it stand for 8-10 minutes;

[0013] S3, centrifuge for 5-10 minutes and discard the supernatant, add pancreatic cancer organoid tissue digestion solution and digest for 2-3 minutes, add organoid subculture buffer to terminate digestion, centrifuge for 5-10 minutes and discard the mixed solution, add organoid subculture buffer to resuspend and transfer to a centrifuge tube, centrifuge at 300g for 5-10 minutes, and discard the supernatant;

[0014] S4, after pancreatic cancer organoids were collected, they were resuspended in pancreatic cancer organoid culture medium, and 80-100 μl of pancreatic cancer organoid culture medium was added to each well of the cell culture plate;

[0015] S5, add T cell culture medium containing IFNγ, incubate for 24 h, and discard the supernatant.

[0016] Furthermore, in S1, the temperature of the organoid subculture buffer is 4°C.

[0017] Furthermore, in S4, 100 μl of pancreatic cancer organoid culture medium is added to each well of a 96-well cell culture plate.

[0018] Furthermore, in S4, the concentration of IFNγ in the T cell culture medium is 200 ng / mL.

[0019] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0020] The method of co-culturing pancreatic cancer organoids and T cells provided by the present invention will enable both pancreatic cancer organoids and T cells to grow and maintain the activity of pancreatic cancer organoids and T cells, especially the cell state of T cells can be rapidly expanded into clusters, and the addition of specific immune drugs can efficiently evaluate the efficacy of immune drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are bright-field images of pancreatic cancer organoids co-cultured with T cells for 48 hours.

[0022] Figure 2 These are the growth morphology images of pancreatic cancer organoids cultured for 9 days (bright field morphology images at 0day, 3day, 6day, and 9day).

[0023] Figure 3 HE and histochemistry images of pancreatic cancer organoids.

[0024] Figure 4 Bright field images of T cells 10 days after activation and expansion are shown. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0026] The raw materials and reagents involved in the embodiments of the present invention are all commercially available products unless otherwise specified, and the operations involved are all routine operations in the art unless otherwise specified.

[0027] The names, specifications and sources of the raw materials used in the embodiments of the present invention are as follows:

[0028] 1) Advanced DMEM (Dulbecco's modified Eagle medium) / F12 culture medium, purchased from Gibco, catalog number 12634010.

[0029] 2) Matrigel, purchased from Corning, was packaged and stored at -20 degrees Celsius before use, and was thawed in a refrigerator at 4 degrees Celsius before use.

[0030] 3) Pancreatic cancer organoid culture medium was purchased from Shanghai Jiefang Kairui Biotechnology Co., Ltd. with the catalog number JFKR-PADC-100.

[0031] 4) Pancreatic cancer organoid tissue digestion fluid was purchased from Shanghai Jiefang Kairui Biotechnology Co., Ltd. with the catalog number JFKR-ZXHY-100.

[0032] 5) Organoid subculture buffer, purchased from Shanghai Jiefang Kairui Biotechnology Co., Ltd., with the catalog number JFKR-HCY-500.

[0033] 6) T cell expansion medium, T cell complete basal medium (15-040-CV) supplemented with IL-2 (corning, 354043) 200 U / mL.

[0034] 7) T cell culture medium: T cell complete basal medium (15-040-CV) supplemented with CD3 / CD28 magnetic beads (ThermoFisher, 11132D) 3ug / mL.

[0035] 8) T cell complete basal medium, purchased from Corning, catalog number 15-040-CV.

[0036] Example 1

[0037] This embodiment provides a method for co-culturing pancreatic cancer organoids and T cells, and the specific steps are as follows:

[0038] 1) Collect pancreatic cancer organoids after P2 culture, remove the culture medium with a pipette, add 1-2 ml of 4°C organoid subculture buffer to each well and leave for 2 minutes;

[0039] 2) Gently pipette the base gel, collect it in a 15 ml centrifuge tube, and let it stand at 4°C for 10 minutes;

[0040] 3) Centrifuge for 5 minutes and discard the supernatant, add an appropriate amount of pancreatic cancer organoid tissue digestion solution and digest for 2-3 minutes, add organoid subculture buffer to terminate digestion, centrifuge for 5 minutes and discard the mixture, add an appropriate amount of organoid subculture buffer to resuspend and transfer to a 1.5 ml centrifuge tube, centrifuge at 300g for 5 minutes, and discard the supernatant;

[0041] 4) After pancreatic cancer organoids were collected, they were resuspended in pancreatic cancer organoid culture medium and 100 μl of pancreatic cancer organoid culture medium was added to each well of a 96-well cell culture plate;

[0042] 5) Add T cell culture medium containing 200 ng / mL IFNγ, incubate for 24 h, and discard the supernatant;

[0043] 6) Mixing the pancreatic cancer organoids with T cells and co-culturing them in a mixed culture medium of pancreatic cancer organoid culture medium and T cell expansion culture medium; wherein the volume ratio of the pancreatic cancer organoid culture medium to the T cell expansion culture medium in the mixed culture medium is 20:80.

[0044] Figure 1 Shown are bright-field images of pancreatic cancer organoids co-cultured with T cells for 48 hours.

[0045] The specific operations of the P1 and P2 culture methods of the above pancreatic cancer organoids are as follows:

[0046] 1) Obtain pancreatic cancer organoid tissue samples, wash the pancreatic cancer tissue organoid samples repeatedly with pre-cooled PBS buffer, cut them into pieces and cut them into 1mm 2 Add 3-5 ml of pancreatic cancer tissue organoid digestion solution to the fragments; the enzymatic digestion conditions are: 37°C, shaking digestion for 10-20 minutes;

[0047] 2) Add 3 times the volume of pre-cooled PBS buffer to terminate the primary digestion, repeatedly blow and vortex with a pipette until the tissue fragments disappear, take out the partially digested solution, and when it is observed as a uniform cell cluster under a microscope, resuspend the cell cluster by low-temperature low-speed centrifugation, count the number of cell clusters under a microscope, and transfer the corresponding cell clusters to a 1.5 ml or 2 ml centrifuge tube according to the number of wells to be inoculated, and centrifuge at 300 g for 5 min;

[0048] 3) The cell mass after enzymatic hydrolysis was added to the basement gel to be resuspended and inoculated into the TC cell culture plate. When the basement gel solidified, the pancreatic cancer organoid culture medium was added and cultured. During the period, the medium was regularly observed and replaced.

[0049] 4) After culturing the pancreatic cancer organoids for 5-7 days, the organoids were subcultured and expanded: 0.8-1 ml of pre-cooled PBS buffer was added to the TC cell culture plate, the base gel was transferred to a 15 ml or 50 ml centrifuge tube, and the tube was repeatedly pipetted and transferred to a refrigerator for 10 min, centrifuged at 300 g for 5 min, and the supernatant was removed to obtain pancreatic cancer organoids;

[0050] 5) The obtained pancreatic cancer organoids were enzymatically digested (add 1-2 ml of TrypLE Express, digest for 2-3 minutes, digest while blowing, add 6-8 ml of pre-cooled PBS buffer, centrifuge at 300g for 5 minutes, and terminate the enzymatic digestion). After digestion, the pancreatic cancer organoids were resuspended in Matrigel, and the cell concentration was adjusted to 200-300 cells / 25 μl, and inoculated into a 24-well plate. After the Matrigel solidified, the culture medium of the pancreatic cancer tissue organoids was added.

[0051] The results are as follows Figure 2 , Figure 3 As shown: Figure 2 This is the growth morphology of pancreatic cancer organoid culture for 9 days (0day, 3day, 6day, 9day bright field morphology), Figure 3 HE and histochemistry images of pancreatic cancer organoids.

[0052] The above T cells are activated and expanded by the following method, and the specific operation is as follows:

[0053] 1) Take fresh venous blood, dilute it with PBS buffer at a ratio of 1:1, and mix gently for later use;

[0054] 2) Take a 15 ml centrifuge tube, add 4 ml of Ficoll cell separation solution, slowly add 8 ml of the above venous blood mixture, and let it stand for use;

[0055] 3) Centrifuge at 2000 rpm for 20 min. At this time, the 15 ml centrifuge tube is observed to have 4 layers. Gently pipette the second layer of white suspension into a 15 ml centrifuge tube and mix well for later use.

[0056] 4) Centrifuge at 1500 rpm for 10 min, discard the supernatant, add an appropriate amount of T cell expansion medium, and culture for observation;

[0057] 5) T cell complete basal medium (IL-2 200-500 units / ml was added to stimulate T cell growth and proliferation; CD3 / CD28 magnetic beads 3ug / ml was added to activate T cells) was then added for culture.

[0058] The results are as follows Figure 4 Shown is an electron micrograph of T cells activated and expanded for 10 days.

[0059] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for co-culturing pancreatic cancer organoids and T cells, characterized in that: The steps include: Step 1: Mix pancreatic cancer organoids with T cells; Step 2, co-culturing using a mixed culture medium of pancreatic cancer organoid culture medium and T cell expansion culture medium; Wherein, the volume ratio of pancreatic cancer organoid culture medium to T cell expansion culture medium in the mixed culture medium is 20:80 to 40:

60.

2. The method for co-culturing pancreatic cancer organoids and T cells according to claim 1, characterized in that: The volume ratio of pancreatic cancer organoid culture medium to T cell expansion culture medium in the mixed culture medium is 20:

80.

3. The method for co-culturing pancreatic cancer organoids and T cells according to claim 1, characterized in that: The pancreatic cancer organoids are obtained by the following method: S1, collect pancreatic cancer organoids after P2 culture, remove the culture medium with a pipette, add 1-2 ml of organoid subculture buffer to each well and let it stand for 2-10 min; S2, gently blow the base gel with a pipette, collect it in a centrifuge tube, and let it stand for 8-10 minutes; S3, centrifuge for 5-10 minutes and discard the supernatant, add pancreatic cancer organoid tissue digestion solution and digest for 2-3 minutes, add organoid subculture buffer to terminate digestion, centrifuge for 5-10 minutes and discard the mixed solution, add organoid subculture buffer to resuspend and transfer to a centrifuge tube, centrifuge at 300g for 5-10 minutes, and discard the supernatant; S4, after pancreatic cancer organoids were collected, they were resuspended in pancreatic cancer organoid culture medium, and 80-100 μl of pancreatic cancer organoid culture medium was added to each well of the cell culture plate; S5, add T cell culture medium containing IFNγ, incubate for 24 h, and discard the supernatant.

4. The method for co-culturing pancreatic cancer organoids and T cells according to claim 3, characterized in that: In S1, the temperature of the organoid subculture buffer is 4°C.

5. The method for co-culturing pancreatic cancer organoids and T cells according to claim 3, characterized in that: In S4, 100 μl of pancreatic cancer organoid culture medium was added to each well of a 96-well cell culture plate.

6. The method for co-culturing pancreatic cancer organoids and T cells according to claim 3, characterized in that: In the S4, the concentration of IFNγ in the T cell culture medium is 200 ng / mL.

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