Construction method of large-scale liver cancer model co-cultured with immune cells

Abstract

The invention discloses a construction method of a large-scale liver cancer model co-cultured with immune cells, and relates to the technical field of liver cancer model construction. The method comprises the following steps: preparing primary hepatoma cells from in-vitro hepatoma tumor tissues, mixing the primary hepatoma cells with supporting hydrogel, refrigerating, and printing in a 12-pore plate to obtain a hepatoma printing body; placing the liver cancer printing body in a 100mM calcium chloride solution to quickly crosslink the liver cancer printing body, then culturing the liver cancer printing body by using a DMEM + 10% FBS culture medium, replacing a fresh culture medium every 2 days, and culturing for 5 days to obtain a large-scale liver cancer model; the CD8 + T cells and the large-scale liver cancer model are co-cultured for 48 h according to different proportions of 1: 1, 2: 1 and 5: 1, and the large-scale liver cancer model co-cultured with immune cells is obtained. The liver cancer model has the advantages of being uniform in size, stable in character, high in throughput and capable of easily reflecting cell activity. The model can reflect the effects of immunotherapy drugs such as PD1 and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of liver cancer model construction, and more specifically to a method for constructing a large-scale liver cancer model co-cultured with immune cells. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, accounting for approximately 90% of all primary liver cancers. HCC is often diagnosed at an advanced stage, with a five-year survival rate of only 14% to 18%, and a very poor prognosis. Despite significant advances in cancer treatment, effective treatment options for HCC remain insufficient, making it an urgent challenge in global health.

[0003] In recent years, immunotherapy has made breakthrough progress in the field of cancer treatment and has completely changed the pattern of traditional treatment. Among them, immune checkpoint blockade (ICB), as a core treatment strategy, has shown significant clinical efficacy in the treatment of metastatic cancers of various hematological malignancies and solid tumors. Programmed cell death protein 1 (PD-1) and its ligand PD-L1 (Programmed Death-Ligand 1) are key targets of ICB therapy. By blocking the PD-1 / PD-L1 signaling pathway, they can effectively restore the anti-tumor immune activity of T cells. Based on this mechanism, PD-1 / PD-L1 inhibitors have achieved outstanding clinical success in the treatment of various malignant tumors such as non-small cell lung cancer, advanced melanoma, liver cancer, bladder cancer and metastatic renal cell carcinoma.

[0004] However, although immunotherapy has shown significant efficacy in some patients, the overall response rate is still low, and only a few patients can benefit from it. This individual difference may be closely related to the heterogeneity of the tumor microenvironment, the infiltration of immunosuppressive cells, and the immune escape mechanism of tumor cells themselves. Therefore, before clinical treatment, how to accurately screen patients who may respond to immunotherapy has become a key issue that needs to be urgently addressed in the current field of immunotherapy. Developing a preclinical model that can simulate the patient's tumor immune microenvironment and predict the response to immunotherapy is of great significance for optimizing treatment plans and improving the success rate of treatment. .

[0005] Over the past decade, patient-derived three-dimensional (3D) organoid cultures have been developed, typically grown in an extracellular matrix such as Matrigel. +The most widely studied method for co-culture of T cells, whether the organoids and immune cells are autologous or allogeneic, is direct co-culture. Typically, organoids and immune cells are first cultured in their own unique culture media before co-culture begins. For immune cells, the typical culture media used are: RPMI1640, DMEM or MEM. Organoids are cultured in a culture medium containing specific growth factors and stem cell factors that are required for the organoid subtype used. Both culture media contain specific substances that allow each specific cell type to grow efficiently. Organoids from different tissue source types have different requirements for the type of growth factors. Therefore, there is currently no universal expansion culture medium for immune cells and organoids. Therefore, for each co-culture / disease environment, precise experiments should be performed before attempting co-culture to find the best culture medium in which immune cells are not harmed and organoids can still proliferate, which is very technically difficult. In addition, Matrigel, as an extract of mouse tumor transplants, has certain immunogenicity and will affect the chemotaxis of immune cells when co-cultured with immune cells. Therefore, it is critical to develop a new co-culture model of tumor cells and immune cells.

[0006] Therefore, providing a large-scale liver cancer model co-cultured with immune cells is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a large-scale liver cancer model co-cultured with immune cells.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A method for constructing a large-scale liver cancer model co-cultured with immune cells, the process comprising:

[0010] Step 1: preparing primary liver cancer cells from in vitro liver cancer tumor tissue;

[0011] Step 2: Mix the supporting hydrogel with primary liver cancer cells until the final concentration of primary liver cancer cells is 5×10 6 / mL, refrigerate to obtain a hydrogel containing liver cancer cells; then print it in a 12-well plate to obtain a liver cancer print body;

[0012] Step 3: Place the liver cancer print in a 100 mM calcium chloride solution to allow rapid cross-linking, and then culture it in a DMEM+10% FBS culture medium, replace the culture medium with fresh one every 2 days, and culture for 5 days to obtain a large-scale liver cancer model;

[0013] Step 4: Isolation of CD8 + T cells;

[0014] Step 5: Mix the activated CD8 + T cells were co-cultured with the large-scale liver cancer model for 48 hours to obtain a large-scale liver cancer model co-cultured with immune cells.

[0015] Preferably, the process of preparing primary liver cancer cells in step 1 is:

[0016] 1) Digest the ex vivo liver cancer tumor tissue in 1 mg / mL collagenase IV solution for 40 min to obtain a cell suspension; filter the cell suspension with a 100 μm filter membrane, and centrifuge at 4°C and 1000 rpm for 5 min to collect the primary cells digested from the tissue;

[0017] 2) Wash the primary cells twice with 10× Wash Buffer. Aspirate the supernatant after centrifugation each time, re-add 10× Wash Buffer to resuspend the cells, centrifuge at 4°C, 1000 rpm for 5 min, and then wash the primary cells once with preheated serum-containing culture medium.

[0018] Preferably, in step 2, the supporting hydrogel is prepared by mixing 3-6% gelatin and 0.05-0.1% sodium alginate in a volume ratio of 2:1.

[0019] Preferably, in step 2, the temperature of the refrigeration step is 4° C. and the refrigeration step is performed for 20 min.

[0020] Preferably, in step 2, the printing is performed using an extrusion nozzle with a specification of 23G-27G, the printing nozzle temperature is adjusted to 10-15°C, the printing platform temperature is adjusted to 5-10°C, and the printing temperature is adjusted to 1-2 mm. 3 The hydrogel containing liver cancer cells was printed in a 12-well plate at an extrusion speed of / s.

[0021] Preferred: In step 4, CD8 + T cells are human CD8 + T cells or mouse CD8 + T cells.

[0022] Preferred: Human CD8 + T cells were cultured from human peripheral blood mononuclear cells.

[0023] Preferred: Mouse CD8 + T cells were isolated from mouse spleen.

[0024] Preferably: In step 4, the activated CD8 + T cells were co-cultured with the large-scale liver cancer model for 48 hours to obtain a large-scale liver cancer model co-cultured with immune cells.

[0025] The present invention also provides a large-scale liver cancer model prepared by any of the above methods.

[0026] It can be seen from the above technical solutions that compared with the prior art, the liver cancer model constructed by the present invention has the advantages of uniform size, stable properties, high throughput, and easy to reflect cell activity. The model can reflect the effects of immunotherapy drugs such as PD1. By establishing this large-scale liver cancer model that can be co-cultured with immune cells, immunotherapy drugs such as PD1, PDL1, CTLA-4, etc. can be preclinically tested to reflect the patient's responsiveness to these drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The attached figure is a proliferation diagram of the printed body composed of mouse liver cancer cells.

[0029] Figure 2 The attached figure shows the proliferation diagram of the print body composed of human liver cancer cell line.

[0030] Figure 3 The picture is CD8 + T cell activation diagram.

[0031] Figure 4 The attached pictures are different scales CD8 + A diagram showing changes in printed structure after co-culture of T cells with liver cancer model.

[0032] Figure 5 The attached pictures are different scales CD8 + The extent of T cells' killing of liver cancer cells in the printed body.

[0033] Figure 6 The attached figure shows that PD1 antibody can enhance CD8 + The killing ability of T cells on liver cancer cells in the printed body. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Materials used in the embodiments of the present invention:

[0036] 3-6% gelatin and 0.05-0.1% sodium alginate were mixed in a volume ratio of 2:1 as the supporting hydrogel.

[0037] Liver cancer cell materials include: mouse liver cancer cell line, human liver cancer cell line, and primary liver cancer cells from liver cancer patients.

[0038] The immune cells include: immune cells isolated from mouse spleen and immune cells derived from the peripheral blood of liver cancer patients.

[0039] There is no limitation on the source of the above cells; any commercially available cells that meet the experimental requirements may be used.

[0040] Hepatoma cell line culture system: 90% DMEM + 10% FBS + 1% penicillin / streptomycin.

[0041] Furthermore, the method for culturing primary liver cancer cells from liver cancer patients in the embodiment of the present invention is:

[0042] 1) The ex vivo liver cancer tumor tissue was placed in 10×Wash Buffer and stored and transported at 4°C (10×Wash Buffer contains 84% ​​DMEM, 5% FBS, 10% penicillin / streptomycin, and 1% Hepes).

[0043] Complete the isolation of primary cells in a clean bench within 48 hours: Place the tissue in a culture dish, cut off a portion with a scalpel and place in a 4% formaldehyde solution for fixation, then cut the tissue into 1 mm 3 The cells were divided into pieces of different sizes, two parts were kept for DNA and RNA extraction, and the remaining tissue was placed in 1 mg / mL collagenase IV solution in a 37°C water bath for cell digestion.

[0044] After digestion for 40 min, the cell suspension was filtered through a 100 μm filter membrane and centrifuged at 4°C and 1000 rpm for 5 min to collect the cells digested from the tissue.

[0045] 2) Wash the primary cells twice with 10× Wash Buffer. After each wash, aspirate the supernatant after centrifugation and resuspend the cells in 10× Wash Buffer. Centrifuge at 4°C, 1000 rpm for 5 min. Wash the primary cells once with preheated serum-containing culture medium (DMEM + 10% FBS).

[0046] Example 1

[0047] Method for constructing a large-scale liver cancer model

[0048] Step 1: Mix the supporting hydrogel (3 ml) with primary liver cancer cells until the final concentration of primary liver cancer cells is 5×10 6 / mL, and refrigerated at 4°C for 20 min to obtain a hydrogel containing liver cancer cells.

[0049] Step 2: Use an extrusion nozzle with a specification of 23G-27G, adjust the print nozzle temperature to 10-15°C, and the print platform temperature to 5-10°C. Use 1-2mm 3 The hydrogel containing liver cancer cells was printed in a 12-well plate at an extrusion speed of / s to obtain a liver cancer print body.

[0050] Step 3: Place the printed liver cancer body in a 6 cm dish (final concentration of calcium chloride solution is) 100 mM calcium chloride solution to make it cross-linked quickly, then use DMEM + 10% FBS culture medium (5 ml) for culture (the culture medium completely covers the print body), replace fresh culture medium every two days, culture for 5 days, and obtain a large-scale liver cancer model.

[0051] In order to characterize the ability of liver cancer cells to proliferate stably in the culture model, a plasmid containing GFP was transfected into primary mouse liver cancer cells using lentivirus, and the cells were tested on day 1, 3, 5, 7, and 14. The results showed that GFP fluorescence gradually increased over time, indicating that the mouse liver cancer cells in the printed body could proliferate normally ( Figure 1 ). Similarly, in human liver cancer cells, the plasmid expressing GFP was transfected for detection, and the human liver cancer cells in the printed body were also able to proliferate normally ( Figure 2 ).

[0052] Step 4: Isolation and activation of immune cells:

[0053] Separation: The mouse spleen was ground using a grinding rod, and after passing through a 100uM filter, single cells were collected by centrifugation.

[0054] Activation: Splenocytes were centrifuged, washed, and cultured at 2 × 10 6 The cells were resuspended at a density of 10 cells / ml in RPMI-1640 medium containing 10% FBS and 10 ng / ml mouse recombinant IL-2. The culture dish was pre-coated with mouse anti-CD3 and anti-CD28 antibodies, and the immune cells were cultured for 5 to 7 days. + T cell separation kit was used to sort and collect activated CD8 + T cells, used for co-culture. Figure 3 As shown, after successful activation, CD8 + T cells appear in clusters.

[0055] (Or, human peripheral blood mononuclear cells (1-2×10 6 PBMCs were cultured in RPMI-1640 medium (2 ml) containing 10% FBS, 1% antibiotics and 10 ng / ml human IL-2. The culture dishes were pre-coated with human anti-CD3 and anti-CD28 antibodies, and the cells were cultured for 5-7 days. + T cell isolation kit (Miltenyi Biotec) for CD8 + T cells are sorted.

[0056] Step 5: Mouse CD8 + T cells were co-cultured with a large-scale mouse liver cancer model constructed by 3D printing (the culture medium was DMEM + 10% FBS): the activated CD8 + After T cells and printed bodies were co-cultured for 48 hours, a large-scale liver cancer model was obtained. Figure 4 As shown, different proportions of mouse CD8 + There are differences in the degree of damage to the overall structure of the printed body caused by T cells. + When the ratio of T cells to liver cancer cells in the liver cancer print was 5:1, the print structure was almost completely destroyed. This shows that the model of the present invention can directly reflect the CD8 + The killing ability of T cells on liver cancer cells.

[0057] In order to further reflect the degree of killing, the established liver cancer model was tested using the Calcein / PI cell activity and cytotoxicity detection kit. Figure 5 As shown, the hepatoma cells in the printed body are expressed with mouse CD8 + The increase in the proportion of T cells represents the increasing proportion of apoptotic PI-positive cells, further indicating the successful establishment of the co-culture model.

[0058] In order to further illustrate that the co-cultured large-scale liver cancer model established by the present invention can reflect the degree of response to immunotherapy, PD1 antibody was added to the model. Figure 6 As shown in Figure 2, after adding PD-1, the PI-positive proportion of tumor cells in the printed body increased significantly, indicating that PD-1 antibodies can enhance the expression of mouse CD8 + The ability of T cells to kill tumor cells.

[0059] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0060] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a large-scale liver cancer model co-cultured with immune cells, characterized in that the process include: Step 1: preparing primary liver cancer cells from ex vivo liver cancer tumor tissue; Step 2: Mix the supporting hydrogel with primary liver cancer cells until the final concentration of primary liver cancer cells is 5×10 6 / mL, refrigerate to obtain a hydrogel containing liver cancer cells; then print it in a 12-well plate to obtain a liver cancer print body; Step 3: Place the liver cancer print in a 100 mM calcium chloride solution to allow rapid cross-linking, and then culture it in a DMEM+10% FBS culture medium, replace the culture medium with fresh one every 2 days, and culture for 5 days to obtain a large-scale liver cancer model; Step 4: Isolation of CD8 + T cells; Step 5: Mix the activated CD8 + T cells were co-cultured with the large-scale liver cancer model for 48 hours to obtain a large-scale liver cancer model co-cultured with immune cells.

2. The method for constructing a large-scale liver cancer model co-cultured with immune cells according to claim 1, characterized in that: The process of preparing primary liver cancer cells in step 1 is: 1) Digest the ex vivo liver cancer tumor tissue in 1 mg / mL collagenase IV solution for 40 min to obtain a cell suspension; filter the cell suspension with a 100 μm filter membrane, and centrifuge at 4°C and 1000 rpm for 5 min to collect the primary cells digested from the tissue; 2) Wash the primary cells twice with 10× Wash Buffer. Aspirate the supernatant after centrifugation each time, re-add 10× Wash Buffer to resuspend the cells, centrifuge at 4°C, 1000 rpm for 5 min, and then wash the primary cells once with preheated serum-containing culture medium.

3. The method for constructing a large-scale liver cancer model co-cultured with immune cells according to claim 1, characterized in that: In step 2, the supporting hydrogel is prepared by mixing 3-6% gelatin and 0.05-0.1% sodium alginate in a volume ratio of 2:

1.

4. The method for constructing a large-scale liver cancer model co-cultured with immune cells according to claim 1, characterized in that: In step 2, the refrigeration is at 4° C. for 20 min.

5. The method for constructing a large-scale liver cancer model co-cultured with immune cells according to claim 1, characterized in that: In step 2, the printing is performed using an extrusion nozzle with a specification of 23G-27G, the printing nozzle temperature is adjusted to 10-15°C, the printing platform temperature is adjusted to 5-10°C, and the printing temperature is adjusted to 1-2 mm. 3 The hydrogel containing liver cancer cells was printed in a 12-well plate at an extrusion speed of / s.

6. The method for constructing a large-scale liver cancer model co-cultured with immune cells according to claim 1, characterized in that: In step 4, the CD8 + T cells are human CD8 + T cells or mouse CD8 + T cells.

7. The method for constructing a large-scale liver cancer model co-cultured with immune cells according to claim 6, characterized in that: Human CD8 + T cells were cultured from human peripheral blood mononuclear cells.

8. The method for constructing a large-scale liver cancer model co-cultured with immune cells according to claim 6, characterized in that: Mouse CD8 + T cells were isolated from mouse spleen.

9. The method for constructing a large-scale liver cancer model co-cultured with immune cells according to claim 1, characterized in that: In step 4, the activated CD8 + T cells were co-cultured with the large-scale liver cancer model for 48 hours to obtain a large-scale liver cancer model co-cultured with immune cells.

10. A large-scale liver cancer model prepared by the method according to any one of claims 1 to 9.

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