Hepatocellular carcinoma organoid-T cell co-culture system as well as construction method and application thereof

Abstract

The invention relates to a hepatocellular carcinoma organoid-T cell co-culture system as well as a construction method and application thereof. The system comprises a liver cancer organoid culture medium and a T cell amplification culture medium which are mixed according to a specific ratio, and can be used for rapidly evaluating the efficacy of the immunosuppressor in vitro or screening candidate drugs. The construction method of the system comprises the steps of construction of hepatocellular carcinoma (HCC) organs, amplification of T cells, co-culture of hepatocellular carcinoma organs-T cells, and detection of efficacy killing evaluation of a PD-1 inhibitor. According to the HCC organ-T cell co-culture system constructed by the invention, the HCC organs maintain genomes, forms and pathophysiology characteristics of parental tumors of the HCC organs, the PD-1 inhibitor drug sensitivity result evaluated by the co-culture system is consistent with the clinical result, and a new model is provided for PD1 drug screening.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and more particularly to an organoid-immune cell co-culture system for evaluating the efficacy of PD-1 inhibitors and a construction and application thereof. Technical Background

[0002] Studies have shown that cancer has caused a serious social burden worldwide. In 2020, there were 19.29 million new cases of cancer worldwide, of which my country ranked first in the world in both new cases and deaths. The threat of liver cancer faced by my country is particularly severe, with nearly 410,000 cases each year, accounting for nearly half of the cases worldwide. At the same time, China faces an extremely high death toll of 390,000, ranking second in the number of cancer deaths in the country. Liver cancer is difficult to detect in the early stages, and most of them are already in the late stages once discovered. Even if it is discovered early, after complete resection surgery, liver cancer patients still have a recurrence rate of 60% to 70% within 5 years. Developing more and more effective drugs will have direct and far-reaching social value.

[0003] Immunotherapy has flourished in recent years, showing exciting clinical effects, with programmed death receptor 1 (PD1) inhibitors developing the fastest. PD1 inhibitors have been included in the guidelines of the Chinese Society of Clinical Oncology (CSCO) as a second-line treatment for advanced HCC. Due to the good prospects of PD1 inhibitors, more pharmaceutical research and development companies are making plans for PD1 inhibitors. In the future, a large number of models are urgently needed for the drug efficacy evaluation of PD1 inhibitors, including preclinical and clinical treatment.

[0004] Organoid technology is believed to be able to effectively reflect a variety of biological processes in the body and was selected by Science as one of the top ten technological breakthroughs in 2013. Human organoids can highly simulate the growth and development of human organs in vitro, and their responsiveness to drugs is highly consistent with the patient's tumor. With the rapid development of immunology and immunotherapy, it is becoming increasingly possible and important to deeply understand the interaction and mechanism between immune cells and tumor cells in order to improve the clinical effect of immunotherapy and develop new treatment methods.

[0005] Therefore, there is an urgent need for a liver cancer organoid and T cell co-culture system to quickly evaluate the efficacy of candidate PD1 drugs in vitro, screen potential clinical development drugs, improve the success rate of drug development, reduce drug development costs, shorten drug development time, and ultimately benefit more clinical patients. Summary of the invention

[0006] The present invention is based on the above-mentioned problems and provides a hepatocellular carcinoma organoid-T cell co-culture system for evaluating the efficacy of drugs including PD-1 inhibitors, as well as its construction and application.

[0007] In a first aspect, the present invention provides a liver cancer organoid-T cell co-culture system, comprising a liver cancer organoid culture medium and a T cell expansion culture medium mixed in a specific ratio.

[0008] Among them, the liver cancer organoid culture medium includes 500-800ng / ml R-spondin1, 1% N2, 1% B27, 1% GlutaMAX Supplement, 1% HEPES, 1-10mM nicotinamide, 1% penicillin-streptomycin, 1.5mM N-acetylcysteine, A83015μM, 15nM gastrin I, 30-50ng / ml EGF, 30-50ng / ml Noggin, 15μM Y-27632, 5μM forskolin, and 30-50ng / ml FGF10.

[0009] The T cell expansion medium includes liquid basal medium, IL-2 200-500 units / ml, and CD3 / CD28 magnetic beads 3-5ug / ml.

[0010] Preferably, the preferred composition of the liver cancer organoid culture medium is as follows: R-spondin 1500 ng / ml, N2 1%, B27 1%, GlutaMAX Supplement 1%, HEPES 1%, nicotinamide 1-10 mM, penicillin-streptomycin 1%, N-acetylcysteine ​​1.5 mM, A830 15 μM, gastrin I 15 nM, EGF 30 ng / ml, Noggin 30 ng / ml, Y-27632 15 μM, forskolin 5 μM, FGF 10 30 ng / ml.

[0011] In the T cell expansion medium, the liquid basal medium is selected from a combination of MEM cell culture medium, DMEM cell culture medium, DMEM / F12 cell culture medium, IMDM cell culture medium, 199 cell culture medium, F10 cell culture medium, F12 cell culture medium, and 1640 cell culture medium.

[0012] Preferably, the liver cancer organoid culture medium and the T cell expansion medium are mixed in a volume ratio of 10:90.

[0013] In a second aspect of the present invention, the application of the above-mentioned culture system in rapid in vitro evaluation of immunosuppressant efficacy or drug screening is provided. The culture medium of liver cancer organoids can rapidly culture liver cancer organoids, and liver cancer organoids of a size of 100-200 μm that meet the requirements of co-culture can be cultured in 14-20 days, and then cultured with T cells under co-culture conditions, and candidate drugs are added to achieve rapid in vitro evaluation of the efficacy of candidate drugs (such as PD-1 inhibitors), that is, to evaluate the killing power of T cells on liver cancer organoids.

[0014] In a third aspect, the present invention provides a method for rapidly evaluating the efficacy of immunosuppressants or performing drug screening in vitro using any of the above-mentioned culture systems, which mainly comprises the following steps:

[0015] A. Take liver cancer tissue to establish liver cancer organoids;

[0016] B. Isolation and expansion of T cells from peripheral blood;

[0017] C. Mix the liver cancer organoids cultured in step (A) and the T cells isolated in step (B) at a ratio of 1:10-20, then add them to the above co-culture system for co-culture, then add the candidate reagent, and co-culture for more than 48 hours to evaluate the apoptosis of the liver cancer organoids.

[0018] The preferred conditions for the above steps are as follows:

[0019] In step A, the method for establishing liver cancer organoids is as follows: hepatocellular carcinoma tissue is prepared into a 70-100 μm pellet suspension; after centrifugation at 300g for 5 minutes, it is resuspended with matrix gel and inoculated into a multi-porous cell culture plate. After the matrix gel solidifies, liver cancer organoid culture medium is added, and cultured in an incubator. The culture medium is replaced every three days for 10-14 days.

[0020] The method for preparing hepatocellular carcinoma tissue into a suspension of 70-100 μm pellets is as follows: the liver cancer tissue sample is washed three times with PBS buffer and then cut into pieces with an area of ​​1 mm 2 The fragments were enzymatically digested with tissue organoid digestion solution at 37°C for 20-25 min; the enzymatic digestion was terminated with PBS buffer, and the suspension was prepared by centrifugation at 4°C and 300g, and rinsing with PBS once more.

[0021] In step B, the method for isolating and expanding T cells from peripheral blood is as follows: dilute peripheral blood with PBS buffer at a ratio of 1:1 and gently mix; add Ficoll cell separation solution, centrifuge at 2000rpm for 20min, separate the second layer of white suspension to obtain PBMC; centrifuge at 1500rpm for 10min, discard the supernatant, add T cell expansion medium for expansion, and then add T cell complete basal medium containing IL-2200-500 units / ml and CD3 / CD28 magnetic beads 3-5μg / ml for culture.

[0022] The specific steps of step C are as follows:

[0023] 1) Collect liver cancer organoids, remove the culture medium with a pipette, add 4°C organoid subculture buffer to each well and leave for 2 minutes;

[0024] 2) Gently pipette the matrix gel, collect it in a centrifuge tube, and let it stand at 4°C for 10 minutes;

[0025] 3) Centrifuge for 5 min and discard the supernatant, add an appropriate amount of organoid digestion solution for digestion, add organoid subculture buffer to terminate digestion, centrifuge for 5 min and discard the mixed solution, add an appropriate amount of organoid culture buffer, resuspend and transfer to a centrifuge tube, centrifuge at 300g for 5 min, and discard the supernatant;

[0026] 4) After the liver cancer organoids are collected, the liver cancer organoid culture medium is resuspended and the liver cancer organoid culture medium is added to each well of the 96-well cell culture plate;

[0027] 5) Incubate the organoids with T cell culture medium containing 200 ng / mL IFNγ for 24 h and discard the supernatant.

[0028] The beneficial protection and effects of the present invention are as follows:

[0029] The present invention co-cultures liver cancer organoids with T cells, wherein the culture medium of the liver cancer organoids can quickly culture liver cancer organoids, and liver cancer organoids with a size of 100-200 μm that meets the co-culture requirements can be cultured in 14-20 days, and then cultured with T cells under co-culture conditions, thereby providing a method for rapidly evaluating the efficacy of immunosuppressants including PD-1 inhibitors in vitro, i.e., a method for the lethality of T cells against liver cancer organoids.

[0030] In addition, the HCC organoid-T cell co-culture system constructed by the present invention, the HCC organoids retain the genomic, morphological and pathophysiological characteristics of their parental tumors, and the PD-1 inhibitor sensitivity results evaluated by the co-culture system are consistent with the clinic, providing a new model for PD1 drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1This is a schematic diagram of the construction method of the liver cancer organoid and T co-culture model for evaluating the efficacy of PD1 in an embodiment of the present invention;

[0032] Figure 2 This is the growth morphology of liver cancer organoid culture for 9 days (representing the bright field morphology of 0 day, 3 day, 6 day, and 9 day respectively);

[0033] Figure 3 HE image of liver cancer organoid;

[0034] Figure 4 This is a graph showing the growth status of T cells 7 days after activation and expansion;

[0035] Figure 5 This is a growth status diagram of liver cancer organoids co-cultured with T cells (co-culture 0h, 48h);

[0036] Figure 6 LDH results for Anti-PD1 screening of co-cultures. DETAILED DESCRIPTION

[0037] The present invention is now described in detail with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0038] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0039] The liver cancer organoid-T cell co-culture system involved in this embodiment includes liver cancer organoid culture medium and T cell expansion culture medium mixed in a volume ratio of 10:90.

[0040] Among them, the concentrations of each component of the liver cancer organoid culture medium are as follows: R-spondin 1500ng / ml, N21%, B271%, GlutaMAX Supplement 1%, HEPES1%, nicotinamide 1-10mM, penicillin-streptomycin 1%, N-acetylcysteine ​​1.5mM, A83015μM, gastrin I15nM, EGF 30ng / ml, Noggin 30ng / ml, Y-2763215μM, forskolin 5μM, FGF1030ng / ml.

[0041] The components of T cell activation and expansion medium are as follows: 1640 medium, IL-2 200-500 units / ml, and CD3 / CD28 magnetic beads 3-5 μg / ml.

[0042] The method flow for rapid in vitro evaluation of the efficacy of immunosuppressants using the above culture system is shown in Figure 1, as follows:

[0043] Example 1 Method for establishing liver cancer organoids

[0044] The specific operations are as follows:

[0045] 1) Obtain liver cancer organoid tissue samples, wash the liver cancer tissue samples with PBS buffer for 3 times, cut the liver cancer tissue samples into pieces with an area of ​​1 mm 2 The fragments were enzymatically digested with tissue organoid digestion solution at 37°C for 20-25 min;

[0046] 2) Terminate the enzymatic digestion with PBS buffer, centrifuge at 4°C, 300 g, and rinse once with PBS;

[0047] 3) Resuspend the cell pellet in Matrigel and inoculate it into a 24-well TC cell culture plate. After the Matrigel solidifies, add the organoid culture medium. Observe and replace the culture medium every 3 days.

[0048] The results are as follows Figure 2 , Figure 3 shown. Figure 2 The figure shows the bright field morphology of liver cancer organoid culture at 0 days, 3 days, 6 days, and 9 days. As the culture time increases, the volume of the organoid gradually increases and the bright field intensity gradually increases; Figure 3 The figure shows the HE identification of cultured liver cancer organoids, with obvious characteristics of liver cancer cells.

[0049] Example 2 Activation and expansion of T cells

[0050] The specific operations are as follows:

[0051] 1) Take fresh venous blood and dilute it with PBS buffer in a ratio of 1:1, mix gently and set aside.

[0052] 2) Take a 15 ml centrifuge tube, add 4 ml of Ficoll cell separation solution, slowly add 8 ml of venous blood mixture, and let it stand for use.

[0053] 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.

[0054] 4) Centrifuge at 1500 rpm for 10 minutes, discard the supernatant, add an appropriate amount of T cell expansion medium, and culture and observe.

[0055] 5) T cell complete basal medium, add IL-2 200-500 units / ml (to stimulate T cell growth and proliferation), CD3 / CD28 magnetic beads 3ug / ml (to activate T cells).

[0056] The results are as follows Figure 4 Shown is the growth status diagram of T cells 7 days after activation and expansion.

[0057] Example 3 Establishment of a co-culture system for evaluating PD1 liver cancer organoids and T cell immunity

[0058] The specific operations are as follows:

[0059] 1) Collect liver cancer organoids, remove the culture medium with a pipette, add 1-2 ml of 4°C organoid subculture buffer to each well and let stand for 2 minutes;

[0060] 2) Use a pipette to gently blow up the matrix gel, collect it in a 15 ml centrifuge tube, and let it stand at 4°C for 10 min;

[0061] 3) Centrifuge for 5 minutes and discard the supernatant, add an appropriate amount of organoid 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 culture buffer to resuspend and transfer into a 1.5 ml centrifuge tube, centrifuge at 300g for 5 minutes, and discard the supernatant;

[0062] 4) After the liver cancer organoids were collected, the liver cancer organoid culture medium was resuspended, and 100 ul of liver cancer organoid culture medium was added to each well of the 96-well cell culture plate;

[0063] 5) Incubate the organoids with T cell culture medium containing 200 ng / mL IFNγ for 24 h and discard the supernatant;

[0064] 6) The collected T cells were co-cultured with liver cancer organoids, with the ratio of T cells to organoids being 10-20:1; 100ul of co-culture medium and PD1 antibody were added, and the volume ratio of co-culture system medium HCC organoid medium and T cell expansion medium was 10:90;

[0065] (7) High-content photography was performed continuously for 48 h. After the experiment, the supernatant was collected and the LDH method was used to detect apoptosis in liver cancer organoids to evaluate the efficacy of the candidate PD1 antibody.

[0066] The results are as follows Figure 5 , Figure 6 As shown, Figure 5 The images of liver cancer organoids and T cells at 0h and 48h of co-culture are shown. The liver cancer organoids gradually begin to show apoptosis. The cell killing effect of PD-1 antibodies on different "liver cancer organoids + T cells" co-culture systems is shown in Table 1:

[0067] Table 1 The cell killing effect of PD-1 antibody on the "liver cancer organoid + T cell" co-culture system

[0068] Active toxicity% System 1 System 2 System 3 Cell killing 30.79％ 45.51％ 36.02％ Cell viability 69.21％ 54.49％ 63.98％

[0069] Figure 6 LDH results for screening of Anti-PD-1 for co-cultures.

[0070] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A liver cancer organoid-T cell co-culture system, characterized in that: It includes liver cancer organoid culture medium and T cell expansion culture medium mixed in a specific ratio. The hepatoma organoid culture medium includes 500-800 ng / ml R-spondin1, 1% N2, 1% B27, 1% GlutaMAXSupplement, 1% HEPES, 1-10 mM nicotinamide, 1% penicillin-streptomycin, 1.5 mM N-acetylcysteine, A83015 μM, 15 nM gastrin I, 30-50 ng / ml EGF, 30-50 ng / ml Noggin, 15 μM Y-27632, 5 μM forskolin, 30-50 ng / ml FGF10, The T cell expansion culture medium comprises a liquid basal culture medium, IL-2 200-500 units / ml, and CD3 / CD28 magnetic beads 3-5 μg / ml.

2. The liver cancer organoid-T cell co-culture system according to claim 1, characterized in that: in, The liver cancer organoid culture medium includes R-spondin 1500ng / ml, N2 1%, B27 1%, GlutaMAX Supplement 1%, HEPES 1%, nicotinamide 1-10mM, penicillin-streptomycin 1%, N-acetylcysteine ​​1.5mM, A830 15μM, gastrin I 15nM, EGF 30ng / ml, Noggin 30ng / ml, Y-27632 15μM, forskolin 5μM, FGF 10 30ng / ml, The liquid basal culture medium is selected from one or more combinations of MEM cell culture medium, DMEM cell culture medium, DMEM / F12 cell culture medium, IMDM cell culture medium, 199 cell culture medium, F10 cell culture medium, F12 cell culture medium, and 1640 cell culture medium.

3. The liver cancer organoid-T cell co-culture system according to claim 1, characterized in that: in, The liver cancer organoid culture medium and T cell expansion culture medium were mixed at a volume ratio of 10:

90.

4. Use of the culture system according to any one of claims 1 to 3 in rapid in vitro evaluation of the efficacy of immunosuppressants or screening of candidate drugs.

5. A method for rapidly evaluating the efficacy of immunosuppressants or performing drug screening in vitro using the culture system according to any one of claims 1 to 3, characterized in that: The steps include: A. Take liver cancer tissue to establish liver cancer organoids; B. Isolation and expansion of T cells from peripheral blood; C. The liver cancer organoids cultured in step (A) are mixed with the T cells separated in step (B) at a ratio of 1:10-20, and then added to the co-culture system for co-culture. The candidate reagent is then added, and the apoptosis of the liver cancer organoids is evaluated after co-culture for more than 48 hours.

6. The method according to claim 5, characterized in that: in, In step A, the method for establishing liver cancer organoids is as follows: hepatocellular carcinoma tissue is prepared into a 70-100 μm pellet suspension; after centrifugation, it is resuspended with matrix gel and inoculated into a porous cell culture plate. After the matrix gel solidifies, liver cancer organoid culture medium is added, and cultured in an incubator. The culture medium is replaced every three days for 10-14 days.

7. The method according to claim 6, Features: in, The method for preparing hepatocellular carcinoma tissue into a suspension of 70-100 μm pellets is as follows: the liver cancer tissue sample was washed three times with PBS buffer and cut into pieces with an area of ​​1 mm 2 The fragments were enzymatically digested with tissue organoid digestion solution at 37°C for 20-25 min; Enzymatic digestion was terminated with PBS buffer, and the suspension was prepared by centrifugation at 4°C and 300 g and washing with PBS once more; The suspension was centrifuged at 300 g for 5 min and then resuspended in Matrigel.

8. The method according to claim 5, characterized in that: in, In step B, the method for isolating and expanding T cells from peripheral blood is as follows: dilute peripheral blood with PBS buffer at a ratio of 1:1 and gently mix; add Ficoll cell separation solution, centrifuge at 2000rpm for 20min, separate the second layer of white suspension to obtain PBMC; centrifuge at 1500rpm for 10min, discard the supernatant, add T cell expansion medium for expansion, and then add T cell complete basal medium containing IL-2200-500 units / ml and CD3 / CD28 magnetic beads 3ug / ml for culture.

9. The method according to claim 5, Features: Wherein, the specific steps of step C are as follows: 1) Collect liver cancer organoids, remove the culture medium with a pipette, add 4°C organoid subculture buffer to each well and leave for 2 minutes; 2) Gently pipette the matrix gel, collect it in a centrifuge tube, and let it stand at 4°C for 10 minutes; 3) Centrifuge for 5 min and discard the supernatant, add an appropriate amount of organoid digestion solution for digestion, add organoid subculture buffer to terminate digestion, centrifuge for 5 min and discard the mixed solution, add an appropriate amount of organoid culture buffer, resuspend and transfer to a centrifuge tube, centrifuge at 300g for 5 min, and discard the supernatant; 4) After the liver cancer organoids are collected, the liver cancer organoid culture medium is resuspended and the liver cancer organoid culture medium is added to each well of the 96-well cell culture plate; 5) Incubate the organoids with T cell culture medium containing 200 ng / mL IFNγ for 24 h and discard the supernatant.

10. The method according to claim 5, characterized in that: in, The immunosuppressant is a PD-1 inhibitor.

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