Biological immune model and construction method thereof, culture medium composition, application, and evaluation method for cell therapy effect

Abstract

The present disclosure proposes a biological immune model and its construction method, culture medium composition, application, and evaluation method for cell therapy effect, which belongs to the field of biomedical technology. The construction method includes: adding extracellular matrix and endothelial cells on the first side of the culture chip, forming an endothelial layer through adherent culture; inoculating vascularized spheroids or fibroblasts and organoids on the second side of the culture chip, forming a three-dimensional cell structure through three-dimensional culture; adding a first mixed culture medium containing an activation factor to the first side of the culture chip for flow culture to activate endothelial cells; adding an immune cell suspension to the first side of the culture chip for flow culture to obtain a biological immune model. The model has a tumor microenvironment, which can simulate the process of immune cell recruitment and killing in a complex environment, and can also simulate the microenvironment formed by CAF in a complex environment of an organism and the influence of sites in the microenvironment on immune killing, and the influence of blood vessels generated by vascular endothelium entering the tumor on immune killing.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine technology, and specifically relates to a biological immune model and a construction method thereof, a culture medium composition, an application thereof, and a method for evaluating the cell therapy effect. Background Art

[0002] Cancer is a highly structured, complex ecosystem that includes tumor cells and a large number of non-tumor cells embedded in an extracellular matrix. The tumor microenvironment (TME) includes a variety of immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, and a variety of other tissue-resident cells. This complex environment directly or indirectly affects tumor cell phenotypes through complex reciprocal signaling mechanisms mediated by soluble factors, cell-cell contacts, and extracellular matrix (ECM) remodeling, resulting in a high degree of tumor heterogeneity. TME cells are involved in every stage of cancer progression, influencing tumor initiation, progression, invasion, intravasation, metastatic spread, and growth. TME cells and the molecules they secrete are now believed to play a key role in the pathogenesis of cancer and may represent attractive therapeutic targets. The complex interactions between tumor cells and their mediators are essential for the rational development of effective anticancer drugs.

[0003] In addition, cancer heterogeneity greatly contributes to different patient treatment responses, including treatment resistance, and this complexity has become a considerable obstacle to discovering specific mechanisms of treatment failure. Organotypic models of patient-specific tumors are revolutionizing our understanding of cancer heterogeneity and its impact on personalized medicine. These advances are due in part to the ability of organoid models to stably preserve the genetic, proteomic, morphological, and drug characteristics of parent tumors in vitro, while also providing unprecedented genomic and environmental manipulation. However, it is difficult for tumor organoid models to fully characterize the original tissue because they often lack a complete microenvironment. Tumor organoids and tumor microenvironment can be likened to "seeds and soil". Constructing tumor organoid models with microenvironment for drug testing will be closer to the real response of the human body to drug effects.

[0004] In recent years, various emerging cell therapies have brought a qualitative leap in the field of tumor treatment, especially chimeric antigen receptor T cell (CAR-T cell) therapy has a significant effect in hematological tumors. However, in the field of solid tumors, which accounts for more than 90%, cell therapy has made slow progress. The main reason is that there are many difficulties in cell therapy for solid tumors, such as the large heterogeneity of different types of solid tumors, the lack of unique tumor-associated antigens as CAR-T targets, the inability of T cells to effectively home to the tumor site, the insufficient persistence of CAR-T cells, and the complex microenvironment in the tumor that has an inhibitory effect on immunity. In addition, traditional two-dimensional (2D) models are difficult to evaluate the recruitment efficiency of immune cells (such as CAR-T cells). They can neither simulate the spatial structure and heterogeneity of tumors in vivo, nor reflect the interactions between tumor cells and the tumor microenvironment. Patient-derived three-dimensional (3D) organoid cultures have been developed, which consist of complex multicellular structures grown by epithelial tumor cells isolated from tumor tissues, usually grown in extracellular matrices such as Matrigel. However, due to the lack of immune components in organoids, patient-specific immunotherapy responses cannot be determined.

[0005] In response to this, a system of co-culturing organoids and immune cells has been developed to construct an immune microenvironment of patient-derived organoids. However, the immune microenvironment constructed by this method cannot simulate the complex microenvironment with blood vessels, fibroblasts, and cell-cell interactions, and cannot simulate the process of cell recruitment and killing in a complex environment. Summary of the invention

[0006] The present disclosure aims to solve at least one of the technical problems existing in the prior art, and provides a biological immune model and a construction method thereof, a culture medium composition, an application, and a method for evaluating the effect of cell therapy.

[0007] In one aspect of the present disclosure, a method for constructing a biological immune model is provided, wherein

[0008] The build methods include:

[0009] Adding extracellular matrix and endothelial cells to the first side of the culture chip to form an endothelial layer through adherent culture;

[0010] Vascularized spheroids, or fibroblasts and organoids are seeded on the second side of the culture chip to form a three-dimensional cell structure through three-dimensional culture;

[0011] Adding a first mixed culture medium containing activation factors to the first side of the culture chip for flow culture to activate endothelial cells;

[0012] An immune cell suspension is added to the first side of the culture chip for flow culture to obtain a biological immune model; wherein the immune cells can pass through the endothelial layer from the first side of the culture chip into the three-dimensional cell structure on the second side, and the first mixed culture medium is an organoid culture medium and an endothelial cell culture medium.

[0013] Optionally, when the vascularized spheroids are seeded on the second side of the culture chip, the three-dimensional culture process includes:

[0014] Vascularized spheroids are inoculated on the second side of the culture chip, and then the first mixed culture medium is added and cultured in an incubator.

[0015] Alternatively, the vascularized aggregates can be tumor spheres.

[0016] Optionally, when fibroblasts and organoids are seeded on the second side of the culture chip, the three-dimensional culture process includes:

[0017] Inoculating a fibroblast suspension on the second side of the culture chip and culturing the cells in an incubator;

[0018] The culture medium on the second side of the culture chip is removed, the organoid suspension is inoculated onto the second side of the culture chip, the first mixed culture medium is added, and the culture is carried out in an incubator.

[0019] Optionally, the organoid can be a tumor organoid or a normal tissue organoid.

[0020] Optionally, the first mixed culture medium includes organoid culture medium and endothelial cell culture medium.

[0021] Optionally, the volume ratio of the organoid culture medium to the endothelial cell culture medium is (1-2):1.

[0022] Optionally, a first mixed culture medium containing inflammatory factors is added to the first side of the culture chip, and the flow culture time is 2-24 hours; and / or,

[0023] The inflammatory factor is any one of TNFα, TGFβ, and LPS; and / or,

[0024] The concentration of the inflammatory factor is 1-50 ng / mL.

[0025] Optionally, adding an immune cell suspension to the first side of the culture chip for flow culture to obtain a biological immune model, including:

[0026] For the immune cells to be detected, adding immune cell culture medium to resuspend them to obtain immune cell suspension;

[0027] removing all the culture medium on the first side of the culture chip, and adding the immune cell suspension to the first side of the culture chip;

[0028] All the culture medium on the second side of the culture chip is removed, and a second mixed culture medium is added for flow culture for 24-48 hours to obtain a biological immune model; the second mixed culture medium includes an immune cell culture medium and an organoid culture medium or a spheroid culture medium.

[0029] Optionally, the immune cell is any one of CAR-T, CAR-NK, TIL, TCR-T, and DC cells; and / or,

[0030] When the vascularized spheroids are inoculated on the second side of the culture chip, the number of the immune cells added is (100-1000) cells / 100 μm;

[0031] When inoculating fibroblasts and organoids on the second side of the culture chip, the ratio of the number of immune cells added to the total number of organoid cells is 1:(2-10); and / or,

[0032] In the second mixed culture medium, the volume ratio of the immune cell culture medium to the organoid culture medium or the spheroid culture medium is (1-3):1.

[0033] Optionally, the flow culture adopts a non-perfusion rocking dynamic culture method.

[0034] Another aspect of the present disclosure provides a biological immune model, which is constructed using the construction method described above.

[0035] In another aspect of the present disclosure, a culture medium composition for culturing the biological immune model described above is provided, wherein the culture medium composition comprises: a first mixed culture medium for culturing before adding immune cells, and / or a second mixed culture medium for culturing after adding immune cells;

[0036] The first mixed culture medium comprises an organoid culture medium and an endothelial cell culture medium in a volume ratio of (1-2):1;

[0037] The second mixed culture medium includes immune cell culture medium and organoid culture medium or spheroid culture medium in a volume ratio of (1-3):1.

[0038] Another aspect of the present disclosure provides an application of a biological immune model, wherein the biological immune model described above is applied to any one of immunotherapy analysis, immune killer cell screening, and cytotoxicity detection.

[0039] Another aspect of the present disclosure provides a method for evaluating the effect of cell therapy using the biological immune model described above, the evaluation method comprising:

[0040] Conducting at least one of tumor cell activity detection, tumor cell apoptosis detection, immune cell infiltration analysis, and inflammatory factor release analysis on the biological immune model;

[0041] Based on the test results, the effects of immune cells on tumors or non-tumors are obtained.

[0042] The present disclosure proposes a biological immune model and its construction method, culture medium composition, application, and evaluation method for cell therapy effect. Among them, the construction method of the biological immune model includes: adding extracellular matrix and endothelial cells on the first side of the culture chip, forming an endothelial layer through adherent culture; inoculating vascularized spheroids or fibroblasts and organoids on the second side of the culture chip, and forming a three-dimensional cell structure through three-dimensional culture; adding a first mixed culture medium containing an activation factor to the first side of the culture chip for flow culture to activate endothelial cells; adding an immune cell suspension to the first side of the culture chip for flow culture to obtain a biological immune model; wherein, immune cells can pass through the endothelial layer from the first side of the culture chip into the three-dimensional cell structure on the second side, and the first mixed culture medium is an organoid culture medium and an endothelial cell culture medium. This biological immune model is a biological immune model with a tumor microenvironment, with a high degree of simulation, which can simulate the process of immune cell recruitment and killing in a complex environment, and can also simulate the microenvironment formed by CAF in a complex environment of an organism and the influence of sites in the microenvironment on immune killing, as well as the influence of blood vessels generated by vascular endothelium entering the tumor on immune killing. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 A flowchart of a biological immune model construction method according to an embodiment of the present disclosure;

[0044] Figure 2 This is a schematic diagram of the structure of a membrane chip according to another embodiment of the present disclosure; wherein: Figure 2 (A) is the overall structure of the plug-in membrane chip. Figure 2 (B) is a detailed view of the plug-in membrane chip. Figure 2 (C) is a structural diagram of a plug-in type chamber in the middle hole of a plug-in type membrane chip;

[0045] Figure 3 A schematic diagram of a biological immune model constructed by a plug-in membrane chip according to another embodiment of the present disclosure;

[0046] Figure 4 A flowchart of a method for evaluating the effect of cell therapy using a biological immune model according to another embodiment of the present disclosure;

[0047] Figure 5A pancreatic cancer organoid and pancreatic cancer-related fibroblast co-culture immune model was constructed for the plug-in membrane chip of Example 1 of the present disclosure to detect the results of CAR-T cell killing effect; wherein, Figure 5 (A) is a schematic diagram of the killing results of the pancreatic cancer organoid and pancreatic cancer-associated fibroblast co-culture immune model. Figure 5 (B) shows the killing ratio results of the pancreatic cancer organoid and pancreatic cancer-associated fibroblast co-culture immune model;

[0048] Figure 6 The plug-in membrane chip of Example 2 of the present disclosure is used to construct an ovarian cancer tumor sphere immune model to detect the results of CAR-T cell killing effects; wherein, Figure 6 (A) is a schematic diagram of the killing results of the ovarian cancer tumor sphere immune model. Figure 6 (B) shows the killing ratio results of the ovarian cancer tumor sphere immune model. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the technical solution of the present disclosure, the present disclosure is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. It should be understood that the specific embodiments described herein are only used to explain the present disclosure and are not used to limit the disclosure. The described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present disclosure.

[0050] In some descriptions of the present disclosure, the terms "include" or "comprises" and the like neither limit the mentioned shapes, numbers, steps, actions, operations and / or groups thereof, nor exclude the presence or addition of one or more other different shapes, numbers, steps, actions, operations and / or groups thereof.

[0051] like Figure 1 As shown, in one aspect of the present disclosure, a method S100 for constructing a biological immune model is provided, which specifically includes the following steps S110 to S140:

[0052] S110, adding extracellular matrix and endothelial cells to the first side of the culture chip, and forming an endothelial layer through adherent culture.

[0053] It should be noted that the present embodiment does not specifically limit the type of culture chip and its first side and second side. The first side and second side of the culture chip need to be determined according to the type of culture chip used. The first side and second side can be the upper side and lower side of the culture chip, or the left side and right side. For example, a membrane chip is used, which is composed of a base film, a cover film and an intermediate membrane film to form a structure of upper and lower culture chambers. The upper and lower sides of the membrane of the membrane chip are respectively cultured with different functional tissue cells, that is, the first side of the membrane chip is the lower side of the culture chip, and the second side of the membrane chip is the upper side of the culture chip. For another example, a commercially available barrier culture chip is used, and the left and right sides of the meniscus formed by the barrier are respectively cultured with different functional tissue cells. Similarly, the first side of the chip is one side of the meniscus formed by the gel structure or other components in the culture chip, and the second side of the chip is the other side of the meniscus. Of course, in other embodiments, the first side of the culture chip can also be the lower side, and the second side of the culture chip can also be the upper side. In addition, chips with other structural designs can also be used.

[0054] The present invention is described by taking the culture chip using a plug-in membrane chip as an example.

[0055] For example, Figure 2 and Figure 3 As shown, the membrane culture chip 200 is a plug-in membrane chip, with a total of 6 culture units 210, each culture unit 210 includes a left sample loading hole 211, a plug-in chamber 212 set in the middle hole, and a right sample loading hole 213, and the bottoms of the three holes are connected to allow liquid to flow between the three holes. Among them, the membrane plug-in chamber in the middle hole can be taken out and inserted, and the chamber contains a layer of membrane, which is divided into two layers, namely, the upper membrane layer 212a and the lower membrane layer 212b. Of course, for those skilled in the art, other cell culture chips can also be selected, and this is not specifically limited.

[0056] It should be further explained that, in this embodiment, the plug-in membrane chip is taken as an example, the lower layer of the membrane plug-in membrane arranged in the middle hole serves as the first side of the culture chip, and the upper layer of the membrane plug-in membrane of the middle hole serves as the second side of the culture chip.

[0057] Specifically, combined with Figure 2 As shown, the middle hole membrane chip plug-in is taken out and turned upside down, 10 to 30 μL of extracellular matrix is ​​added above the lower membrane layer 212b in the plug-in chamber 212 to evenly cover the membrane, and then placed in a 37°C, 5% CO 2 After gelation, add 30-50 μL of endothelial cell suspension and place the membrane chip in a 37°C, 5% CO 2 Culture the cells in an incubator for 1 to 3 hours.

[0058] In some preferred embodiments, the vascular endothelial cell suspension is formed by the following method: endothelial cells are digested into single cells, resuspended in a first mixed culture medium, counted, and adjusted to a suitable density to form a vascular endothelial cell suspension.

[0059] In other preferred embodiments, the first mixed culture medium is a mixture of organoid culture medium and endothelial cell culture medium to meet the culture requirements of endothelial cells and organoids.

[0060] As a further preferred embodiment, in the first mixed culture medium, the volume ratio of the organoid culture medium to the endothelial cell culture medium is (1-2):1, for example, 1:1, 1.5:1, 2:1, etc.

[0061] It should be noted that the present embodiment does not specifically limit the extracellular matrix, for example, other hydrogel materials such as matrix glue, natural biomaterials, synthetic biomaterials and mixed materials thereof.

[0062] It should be noted that the present embodiment also does not impose any specific limitation on the gelling method. The gelling method varies according to the hydrogel material, including any gelling method such as temperature, light, and adjusting pH value. Of course, the gelling method may also be a combination of multiple methods.

[0063] S120. Inoculate vascularized spheroids or fibroblasts and organoids on the second side of the culture chip to form a three-dimensional cell structure through three-dimensional culture.

[0064] It should be understood that since the vascularized spheroids are formed by mixed culture of organoids and their associated fibroblasts, that is, the vascularized spheroids contain fibroblasts, they can be directly inoculated into the culture chip to simulate the complex microenvironment containing blood vessels, fibroblasts and cell-to-cell interactions, for example, the spheroid model disclosed by the applicant in the invention patent applications CN2023113769158 and CN202311377344X. When the organoids are inoculated into the second side of the culture chip, the organoid-related fibroblasts need to be inoculated into the culture chip at the same time to simulate the complex microenvironment containing blood vessels, fibroblasts and cell-to-cell interactions.

[0065] In some preferred embodiments, Figure 2As shown, when the vascularized spheroids are inoculated on the second side of the culture chip, the process of forming a three-dimensional cell structure includes: after the endothelial cells adhere to the wall, the membrane chip is placed upright, the plug-in chamber 212 is inserted into the middle hole, 2 to 5 mL of the first mixed culture medium is added from the left sample well 211 of the chip, and the culture medium flows through the left sample well 211 to the lower membrane layer 212b of the plug-in chamber 212 in the middle hole and the right sample well 213, and the culture medium in each hole is 800 to 1000 μL. The vascularized spheroids are inoculated on the upper membrane layer 212a of the middle hole of the membrane chip, one vascularized spheroid is inoculated in each hole, and 100 to 300 μL of the first mixed culture medium is added to the upper membrane layer 212a of the middle hole, and placed in a 37°C, 5% CO 2 Incubate the cells in a static incubator for 1 to 2 days.

[0066] It should be noted that the present embodiment does not impose any specific restrictions on the volume of the first mixed culture medium added and the content of the culture medium in each well, which should match the volume of the membrane chip culture chamber and can be adaptively adjusted according to the volume.

[0067] As a further preferred embodiment, the first mixed culture medium is a mixture of organoid culture medium and endothelial cell culture medium, which meets the culture requirements of both endothelial cells and organoids.

[0068] As a further preferred embodiment, in the first mixed culture medium, the volume ratio of the organoid culture medium to the endothelial cell culture medium is (1-2):1, for example, 1:1, 1.5:1, 2:1, etc.

[0069] As a further preferred embodiment, the vascularized spheroids may be tumor spheres.

[0070] As a further preferred embodiment, tumor spheres are formed by the following method:

[0071] 1) Select primary extracted or to-be-passaged organoids, collect and centrifuge to obtain tumor organoid precipitates; add TrypleE for enzymatic hydrolysis for 5-15 minutes, digest into single cells and centrifuge (1000 rpm, 5 minutes), remove the supernatant, add PBS and centrifuge again (1000 rpm, 5 minutes), remove excess enzyme solution, and obtain precipitates; add the first mixed culture medium (organoid culture medium and endothelial cell culture medium are mixed in a volume ratio of (1-2):1), count the cells, and obtain an organoid suspension.

[0072] Wherein, in step 1), the tumor organoids include any type of organoids of epithelial cell cancer such as lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, intestinal cancer, liver cancer, etc.

[0073] 2) The tumor-associated fibroblasts were digested into single cells, resuspended in the first mixed culture medium (organoid culture medium and endothelial cell culture medium were mixed in a volume ratio of (1-2):1), and counted.

[0074] Among them, in step 2), tumor-associated fibroblasts include primary fibroblasts extracted from corresponding tumor tissues, corresponding tumor tissue fibroblasts differentiated from stem cells, primary fibroblasts extracted from other induced or uninduced tumors or normal tissues, induced or uninduced fibroblast cell lines, etc.

[0075] 3) The endothelial cells were digested into single cells, resuspended in the first mixed culture medium (organoid culture medium and endothelial cell culture medium were mixed in a volume ratio of (1-2):1), and counted.

[0076] Among them, in step 3), the endothelial cells include primary vascular endothelial cells extracted from corresponding tumor tissues, corresponding tumor tissue vascular endothelial cells differentiated from stem cells, primary vascular endothelial cells extracted from other tumors or normal tissues, vascular endothelial cell lines, etc.

[0077] 4) Mix the organoids with tumor-associated fibroblasts and endothelial cells in a certain proportion; add 100-300 μL of the mixed suspension to a low-adsorption U-bottom plate or a culture container with a low-adsorption U-bottom structure, centrifuge at 1000 rpm for 5 min, and place in a 37°C, 5% CO 2 The cells were cultured in an incubator for 2-3 days to obtain tumor spheres.

[0078] In some other preferred embodiments, Figure 2 As shown, when the organoids and related fibroblasts are inoculated on the second side of the culture chip, the process of forming a three-dimensional cell structure includes: after the endothelial cells adhere to the wall, the membrane chip 200 is placed upright, the plug-in chamber 212 is inserted into the middle hole, and 2 to 5 mL of the first mixed culture medium is added from the left sample well 211 of the chip, so that the culture medium flows through the left sample well 211 to the lower membrane layer 212b of the plug-in chamber 212 in the middle hole and the right sample well 213, and the culture medium in each hole is 800 to 1000 μL. 100 to 300 μL of fibroblast suspension is inoculated into the upper membrane layer 212a of the plug-in chamber 212 in the middle hole of the membrane chip, and the membrane chip 200 is placed in a 37°C, 5% CO 2 After the fibroblasts adhere to the wall and culture for 2 hours, the membrane chip is taken out of the incubator, the culture medium in the upper layer 212a of the membrane is discarded, 100 to 300 μL of the organoid suspension is inoculated into the upper layer 212a of the plug-in chamber 212 in the middle hole of the membrane chip, and the membrane chip 200 is placed in a 37°C, 5% CO 2 Incubate the cells in a static incubator for 1 to 2 days.

[0079] As a further preferred embodiment, the first mixed culture medium is also a mixture of organoid culture medium and endothelial cell culture medium to meet the culture requirements of endothelial cells and organoids. Similarly, in the first mixed culture medium, the volume ratio of organoid culture medium to endothelial cell culture medium is preferably (1-2):1, for example, 1:1, 1.5:1, 2:1, etc.

[0080] As a further preferred embodiment, the organoid suspension is formed by the following method: select primary extracted or to-be-passaged tumor organoids, collect and centrifuge to obtain a tumor organoid precipitate; add Tryple E for enzymatic hydrolysis for 5-15 minutes, digest into single cells and then centrifuge (1000rpm, 5min), remove the supernatant, add PBS and centrifuge again (1000rpm, 5min), remove excess enzyme solution, and obtain a precipitate; add the first mixed culture medium (organoid culture medium and endothelial culture medium (1-2): 1) to resuspend, count the cells, obtain an organoid suspension, and adjust to an appropriate density.

[0081] It should be noted that the organoids in the above preferred embodiments may be tumor organoids or non-tumor organoids, and are not specifically limited thereto. Among them, the tumor organoids may be lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, intestinal cancer, liver cancer, etc.

[0082] As a further preferred embodiment, the organoid-associated fibroblast suspension is formed by the following method: tumor-associated fibroblasts are digested into single cells, resuspended in a first mixed culture medium (organoid culture medium and endothelial culture medium in a volume ratio of (1-2):1), counted, and adjusted to an appropriate density.

[0083] It should be noted that the tumor-associated fibroblasts in the above-mentioned preferred embodiments include primary fibroblasts extracted from corresponding tumor tissues, corresponding tumor tissue fibroblasts differentiated from stem cells, primary fibroblasts extracted from other induced or uninduced tumors or normal tissues, induced or uninduced fibroblast cell lines, etc.

[0084] In this embodiment, based on inoculating tumor spheres or inoculating organoids and fibroblasts in a culture chip, a microenvironment with tumor-associated fibroblasts (CAFs) is constructed after culture, which can simulate blood vessels, fibroblasts and cell-cell interactions.

[0085] S130, adding a first mixed culture medium containing activation factors to the first side of the culture chip for flow culture to activate endothelial cells.

[0086] Specifically, combined with Figure 2As shown, all the culture medium in the culture chamber is removed from the left sample well 211 and the right sample well 213 of the chip, and 2-3 mL of the first mixed culture medium containing inflammatory factors is added from the left sample well 211 of the chip. The first mixed culture medium flows through the left sample well 211 to the lower layer 212b of the middle hole membrane and the right sample well 213. Each well contains 800-1000 μL of culture medium. The culture medium is placed in a 37°C, 5% CO 2 The cells were cultured in a flow incubator for 2-24 hours to activate the endothelial cells.

[0087] It should be noted that the first mixed culture medium in step S130 also includes organoid culture medium and endothelial cell culture medium, and the volume ratio of the two is also preferably (1-2):1.

[0088] It should be further explained that, in this embodiment, the volume content of the first mixed culture medium containing inflammatory factors is also related to the volume of the culture chamber, and different contents of culture medium can be selected for different culture chamber volumes.

[0089] In some preferred embodiments, the inflammatory factor is any one of TNFα, TGFβ, and LPS, and the endothelial cells are activated by using endothelial activating factors, and the activation time is 2-24 hours, for example, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, etc.

[0090] In other preferred embodiments, the concentration of the inflammatory factor is 1 ng / mL to 50 ng / mL, for example, 1 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL.

[0091] In other preferred embodiments, the flow culture time is 2 h, 3 h, 5 h, 10 h, 15 h, 17 h, 20 h, 24 h, etc., and the flow culture time is also the activation time of the inflammatory factors.

[0092] S140, adding immune cell suspension to the first side of the culture chip for flow culture to obtain a biological immune model.

[0093] It should be understood that in the biological immune model constructed in this embodiment, immune cells can pass through the endothelial layer from the first side of the culture chip into the three-dimensional cell structure on the second side, and can simulate the immune cell recruitment process in a complex environment.

[0094] Specifically, combined with Figure 2As shown, step S140 includes: taking the immune cells to be detected, counting them, and resuspending them with immune cell culture medium, adjusting the density of immune cells, and obtaining immune cell suspension. Afterwards, remove all the culture medium in the culture chamber from the sample well 211 on the left side of the chip and the sample well 213 on the right side, add 2 to 3 mL of immune cell suspension from the sample well 211 on the left side of the chip, and allow the immune cells to flow through the sample well 211 on the left side to the lower layer 212b of the middle hole membrane and the sample well 213 on the right side. Each hole contains 800 to 1000 μL of culture medium containing immune cells. Afterwards, remove the culture medium from the upper layer 212a of the middle hole membrane, add 100 to 300 μL of the second mixed culture medium, and place the membrane chip 200 in a 37°C, 5% CO 2 After culturing in an incubator for 24-48 hours, a biological immune model is obtained for immune cell killing detection.

[0095] In some preferred embodiments, the immune cell is any one of CAR-T, CAR-NK, TIL, TCR-T, and DC cells.

[0096] It should be noted that the number of immune cells is determined by the type of cells inoculated. For example, when vascularized spheroids are inoculated into a culture chip, the number of immune cells is selected according to the size of the vascularized spheroids, and the specific number is (100-1000) cells / 100 μm, where 100 μm is the size of the vascularized spheroids. For another example, when organoids and fibroblasts are inoculated into a culture chip, the ratio of the number of immune cells to the total number of organoid cells is 1:(2-10).

[0097] As a further preferred embodiment, the ratio of the number of immune cells to the total number of organoid cells is 1:2, 1:3, 1:5, 1:7, 1:8, 1:10, etc.

[0098] It should be further explained that the type of the second mixed culture medium also needs to be determined according to the cells to be inoculated. For example, when vascularized spheroids are inoculated, the second mixed culture medium is an immune cell culture medium and a spheroid culture medium. For another example, when organoids and fibroblasts are inoculated, the second mixed culture medium is an immune cell culture medium and an organoid culture medium.

[0099] In some preferred embodiments, in the second mixed culture medium, the volume ratio of the immune cell culture medium to the organoid culture medium or the spheroid culture medium is (1-3):1, for example, 1:1, 2:1, 3:1.

[0100] It should be noted that the present embodiment does not specifically limit the flow culture method, for example, a perfusion culture method or a non-perfusion but dynamic culture method may be used.

[0101] In some preferred embodiments, flow culture is preferably a dynamic culture method without perfusion, for example, a self-gravity membrane chip is used and cultured in a rocking perfusion instrument (item number: AvatarFlow) at 7° / 30min. In this way, the culture medium flows under the action of the rocking force to simulate the cell recruitment and killing process in a complex environment.

[0102] The bioimmune model constructed in the present disclosure can simulate a complex microenvironment including blood vessels, fibroblasts, and cell-cell interactions, and can simulate the process of cell recruitment and killing in a complex environment, and can further be applied to evaluate the efficacy of cell therapy. For example, vascularized spheroids are inoculated in a culture chip, and a CAF environment is formed by culture. After endothelial activation factors are used to activate endothelial cells, and then the immune cells to be tested are introduced for culture, it can be observed that immune cells are recruited, and immune cells pass through the endothelial cell layer below the porous membrane to the inside of the vascularized tumor sphere for killing. For another example, in a culture chip, organoids + fibroblasts are inoculated, and a CAF environment is also formed by culture. After endothelial activation factors are used to activate endothelial cells, and then the immune cells to be tested are introduced for culture, it can be observed that immune cells are recruited, and immune cells cross the endothelial cell layer below the porous membrane, and the fibroblast layer above the porous membrane to kill organoids.

[0103] Another aspect of the present disclosure provides a biological immune model, which is constructed using the construction method described above. For the specific process, please refer to the above description and will not be repeated here.

[0104] The immune model constructed in this embodiment has a complex microenvironment of blood vessels, fibroblasts and cell-cell interactions, and can simulate the process of immune cell recruitment and killing in a complex environment. For example, Figure 5 and Figure 6 , which can show the infiltration and killing of tumor organoids by immune cells after they pass through the endothelium and migrate into the tumor organoids.

[0105] In this embodiment, the biological immune model can be a tumor model for detecting the killing of tumors by immune cells, or a non-tumor model for detecting off-target effects.

[0106] Another aspect of the present disclosure is a culture medium composition for culturing the biological immune model described above, the culture medium composition comprising: a first mixed culture medium for culturing before adding immune cells, and / or a second mixed culture medium for culturing after adding immune cells; the first mixed culture medium comprises an organoid culture medium and an endothelial cell culture medium in a volume ratio of (1 to 2):1; the second mixed culture medium comprises an immune cell culture medium and an organoid culture medium or a spheroid culture medium in a volume ratio of (1 to 3):1.

[0107] It should be understood that the culture process before adding immune cells includes: forming an endothelial layer, three-dimensional culture to form a three-dimensional cell structure, and activating endothelial cells, that is, in the process of forming the endothelial layer, the culture medium composition used to resuspend the endothelial cells is a first mixed culture medium; and, in the process of forming a three-dimensional cell structure, the culture medium composition used to culture spheroids or organoids, and for resuspending organoids and related fibroblasts are both the first mixed culture medium; and, in the process of activating endothelial cells, the culture medium composition used for flow culture is also the first mixed culture medium, and a certain amount of inflammatory factors needs to be added thereto to activate the endothelial cells.

[0108] It should also be understood that the process of culturing after adding immune cells includes: adding immune cell suspension for flow culture, that is, in this process, the culture medium composition used for flow culture is a second mixed culture medium. It should be noted that the second mixed culture medium needs to be selected according to the three-dimensional cell structure to be cultured. For example, when the three-dimensional cell structure is a vascularized spheroid, the culture medium composition here should be an immune cell culture medium and a spheroid culture medium. When the three-dimensional cell structure is an organoid, the culture medium composition here should be an immune cell culture medium and an organoid culture medium.

[0109] Another aspect of the present disclosure provides an application of a biological immune model, wherein the biological immune model described above is applied to any one of immunotherapy analysis, immune killer cell screening, and cytotoxicity detection.

[0110] Specifically, the response of biological immune models to immunotherapy is evaluated by detecting cell activity, cell apoptosis, cytokine release, immune cell infiltration, inflammatory factor detection, marker detection, and image analysis, which is suitable for precision medicine, drug development and other fields.

[0111] like Figure 4 As shown, another aspect of the present disclosure provides a method S300 for evaluating the effect of cell therapy using the biological immune model described above. The evaluation method includes the following processes S310 to S320:

[0112] S310, performing at least one of tumor cell activity detection, tumor cell apoptosis detection, immune cell infiltration analysis, and inflammatory factor release analysis on the biological immune model.

[0113] In some preferred embodiments, a cell activity detection method is used to evaluate cell activity, and the detection process includes:

[0114] Take out the biological immune model to be tested and equilibrate it to room temperature. Discard all the culture medium from the sample wells on the left and right sides of the chip, and at the same time, discard the culture medium on the middle hole membrane of the chip. Prepare the lysis solution: PBS buffer and CTG stock solution 1:1. Add 200μL of lysis solution to the upper layer of the middle hole membrane of each culture unit of the chip, mix for 5 minutes, and shake for 25 minutes. Then take out all the lysis solution, transfer it to a black 96-well plate, read it using a chemiluminescence microplate reader, and statistically analyze the cell activity.

[0115] In other preferred embodiments, a cell apoptosis detection method is used to evaluate the killing effect of immune cells, and the detection process includes:

[0116] Take out the biological immune model to be tested, add 10 μL of real-time apoptosis detection reagent (such as Annexin V or Cleaved Caspase-3) directly from the upper layer of the middle hole membrane of the chip, and place it in a 37°C, 5% CO 2 A rocking perfusion apparatus (Cat. No.: AvatarFlow) was used in the incubator for flow culture at 7° / 30min. After 1 hour, a fluorescence microscope or confocal microscope was used to take pictures to observe the killing effect of immune cells.

[0117] In other preferred embodiments, an immune cell infiltration analysis method is used to evaluate the infiltration effect of immune cells, and the analysis process includes:

[0118] When analyzing the infiltration of immune cells, live cell tracers (such as Cell tracker dyes, fluorescent probes, etc.) can be used to mark immune cells, tumor organoids, and endothelial cells in advance when constructing the biological immune model, and then the biological immune model is constructed. After that, the biological immune model to be tested is taken out, and a confocal microscope is used to take pictures to observe the infiltration effect of immune cells.

[0119] In other preferred embodiments, a cytokine release analysis method is used to assess the secretion level of inflammatory factors, and the analysis process includes:

[0120] When analyzing the release of cytokines, at different time points, such as 0 hours, 24 hours, and 48 hours of co-culture with immune cells, take an appropriate volume of culture medium from the sample well on the right side of the chip, centrifuge (12000rpm, 15min), and collect the supernatant. ELISA is used to detect the secretion levels of inflammatory factors such as TNFα, IL-2, and IL-6.

[0121] It is worth noting that when the culture medium is removed from the sample wells on the right side of the chip, the corresponding volume of fresh culture medium should be added from the sample wells on the left side of the chip.

[0122] S320. Obtain the effect of immune cells on tumors or non-tumors based on the detection results.

[0123] Specifically, the results of the effect of immune cells on tumors or non-tumors are obtained based on at least one of the results of tumor cell activity, tumor cell killing, immune cell infiltration, and inflammatory factor release. In other words, the biological immune model can be a tumor model for detecting the killing of tumors by immune cells, and the biological immune model can also be a non-tumor model for detecting the off-target effects of immune cells on non-tumors.

[0124] The present disclosure can evaluate the therapeutic effect of immune cells based on the biological immune model, and further realize accurate analysis of personalized immunotherapy, screening of immune killer cells and toxicity detection.

[0125] The following will further illustrate the construction method and application of the biological immune model in conjunction with several specific examples:

[0126] Example 1

[0127] This example uses the construction of a pancreatic cancer organoid and pancreatic cancer-associated fibroblast co-culture immune model in a plug-in membrane chip and the CAR-T cell killing test as an example to illustrate.

[0128] Combined Figure 3 As shown, the preparation of the pancreatic cancer organoid and pancreatic cancer-associated fibroblast co-culture immune model specifically includes the following steps:

[0129] S1. Inoculation of endothelial cells

[0130] (1) Take the plug-in membrane chip 200, remove the plug-in chamber 212 in the middle hole membrane chip and turn it upside down, add 20 μL of Matrigel to the upper layer 212a of the membrane, evenly cover the membrane, and place it at 37°C and 5% CO 2 Let it stand in the incubator for 30 minutes to form a gel.

[0131] (2) HUVEC cells were obtained, digested into single cells, resuspended in the first mixed culture medium (pancreatic cancer organoid culture medium and endothelial cell culture medium 1:1), counted, and adjusted to an appropriate density. After gelation, 40 μL of HUVEC cell suspension (100,000 HUVEC cells per well) was added and placed in a 37°C, 5% CO 2 The cells were cultured in an incubator for 2 hours.

[0132] (3) After the endothelial cells adhere to the wall, the membrane chip is placed upright, the plug-in chamber 212 is inserted into the middle hole, and 2.7 mL of the first mixed culture medium (a 1:1 mixture of pancreatic cancer organoid culture medium and endothelial cell culture medium) is added from the left side loading hole 211 of the chip. The culture medium is allowed to flow through the left side loading hole 211 to the lower membrane layer 212b of the middle hole and the right side loading hole 213. The culture medium in each hole is 900 μL.

[0133] S2. Inoculation of pancreatic cancer organoids and pancreatic cancer-associated fibroblasts

[0134] (1) Take pancreatic cancer-associated fibroblasts, digest them into single cells, resuspend them in the first mixed culture medium (a mixture of pancreatic cancer organoid culture medium and endothelial cell culture medium in a ratio of 1:1), count them, and adjust them to an appropriate density; inoculate 200 μL of the tumor-associated fibroblast suspension into the upper membrane layer 212a of the plug-in chamber 212 in the middle hole of the membrane chip (100,000 pancreatic cancer-associated fibroblasts per hole). Place in a 37°C, 5% CO 2 The cells were cultured in an incubator for 2 hours.

[0135] (2) Select pancreatic cancer organoids that are extracted from the primary generation or to be passaged, collect and centrifuge to obtain pancreatic cancer organoid precipitates; add Tryple E for 10 minutes, digest into single cells and centrifuge (1000rpm, 5min), remove the supernatant, add PBS and centrifuge again (1000rpm, 5min), remove excess enzyme solution, and obtain precipitates (pre-label pancreatic cancer organoids with blue cell tracker). Add the first mixed culture medium (pancreatic cancer organoid culture medium and endothelial cell culture medium mixed in a ratio of 1:1) and resuspend, count the cells, obtain pancreatic cancer organoid suspension, and adjust to the appropriate density.

[0136] (3) After the fibroblasts were cultured for 2 hours, the membrane chip was taken out of the incubator and the culture medium in the upper layer 212a of the middle hole membrane was discarded. 200 μL of the pancreatic cancer organoid suspension was inoculated into the upper layer 212a of the membrane of the plug-in chamber 212 in the middle hole of the membrane chip (50,000 pancreatic cancer organoids per hole).

[0137] (4) Place in a 37°C, 5% CO 2 Incubate the cells in a static incubator for 1 day.

[0138] S3. Add immune cells for co-culture

[0139] (1) After static culture for 1 day, immune cell co-culture can be performed. Activate endothelial cells: remove all the culture medium in the culture chamber from the left sample well 211 and the right sample well 213 of the chip, add 2.7 mL of the first mixed culture medium (pancreatic cancer organoid culture medium and endothelial culture medium 1:1) containing 2 ng / mL TNFα from the left sample well 211 of the chip, and allow the culture medium to flow through the left sample well 211 to the lower membrane layer 212b of the middle well and the right sample well 213, with 900 μL of culture medium in each well.

[0140] (2) Place in a 37°C, 5% CO 2 The cells were cultured using a rocking perfusion apparatus (Cat. No.: AvatarFlow) in an incubator at 7° / 30 min for 2 h.

[0141] (3) After 2 hours of dynamic culture, immune cells (CAR-T or T) are added for co-culture, and the T cells here serve as the control group. Take the CAR-T cells to be tested and the control T cells (immune cells pre-labeled with green cell tracker), count them, resuspend them in T cell culture medium, and adjust the density of CAR-T cells and control T cells.

[0142] (4) All the culture medium in the culture chamber was removed from the sample wells 211 and 213 on the left and right sides of the chip, and 2.7 mL of CAR-T cell suspension or control T cell suspension was added from the sample wells 211 on the left side of the chip, so that the CAR-T cells or T cells flowed through the sample wells on the left side to the lower membrane layer 212b of the middle hole and the sample wells on the right side 213. The amount of culture medium containing immune cells in each well was 900 μL. The ratio of the number of CAR-T cells or T cells to the total number of pancreatic cancer organoid cells was 1:5.

[0143] (5) Aspirate the culture medium 212a on the upper layer of the membrane in the middle hole of the membrane, and add 200 μL of the second mixed culture medium (the volume ratio of CAR-T cell culture medium or T cell culture medium to pancreatic cancer organoid culture medium is 1:1).

[0144] (6) Place in a 37°C, 5% CO 2 The cells were cultured using a rocking perfusion apparatus (Article No.: AvatarFlow) in an incubator at 7° / 30 min for 24 h to obtain a biological immune model.

[0145] It should be understood that in the immune model of co-culture of tumor organoids and tumor organoid-associated fibroblasts, endothelial cells were added to the lower layer of the membrane chip, tumor-associated fibroblasts and tumor organoids were added to the upper layer of the membrane, and immune cells were added to the lower layer of the membrane. In addition, since the bottoms of the left-side loading well, the middle well, and the right-side loading well are all connected, the lower layers of the membranes of the left and right loading wells and the middle well are all immune cells.

[0146] Furthermore, the biological immune model is subjected to cell apoptosis detection and cell activity detection, including:

[0147] (1) After culturing the biological immune model given above for 23 hours, 10 μL of real-time apoptosis detection reagent Annexin V (with red fluorescence) was added from the upper layer of the sample well membrane in the middle of the chip. After adding the apoptosis detection reagent, the chip was placed in a 37°C, 5% CO 2 A rocking perfusion apparatus (Cat. No.: AvatarFlow) in the incubator was used for flow culture at 7° / 30min.

[0148] (2) After 1 hour, take photos using a confocal microscope to observe and count the total number of apoptotic cells and analyze the immune killing effect of CAR-T cells.

[0149] (3) Discard all the culture medium from the sample wells on the left and right sides of the chip, and at the same time, discard the culture medium on the membrane of the middle hole of the chip. Prepare the lysis solution: PBS buffer and CTG stock solution mixed in a ratio of 1:1. Add 200 μL of lysis solution to the upper layer of the middle hole membrane of each culture unit of the chip, mix for 5 minutes, and shake for 25 minutes. Then remove all the lysis solutions, transfer them to a black 96-well plate, read them using a chemiluminescence microplate reader, and statistically analyze the cell activity.

[0150] Figure 5 It shows that immune cells (Car-T) pass through the endothelium, migrate to tumor organoids, infiltrate and kill tumor organoids. Immune cells (T cells) pass through the endothelium, migrate to tumor organoids, partially infiltrate tumor organoids, and kill less.

[0151] like Figure 5 As shown in (A), the constructed pancreatic cancer organoids and pancreatic cancer-associated fibroblasts co-culture immune model, red represents apoptotic organoids, green represents CAR-T cells or T cells, and blue represents unkilled pancreatic cancer organoids. It can be seen that in the immune model with the addition of CAR-T cells to be tested, almost all of them are red. In the control group with T cells, there is less red and more blue and green, which indicates that the immune cell CAR-T has a strong killing effect on pancreatic cancer.

[0152] like Figure 5 As shown in (B), in the immune model with the addition of the CAR-T cells to be tested, the killing rate is far above 70%, and in the control group with the addition of T cells, the killing rate is 20%, indicating that the CAR-T cells to be tested have a significant killing effect on pancreatic cancer. It further illustrates that the pancreatic cancer organoid and pancreatic cancer-related fibroblast co-culture immune model constructed in this example can detect the killing effect of CAR-T cells.

[0153] Example 2

[0154] This example uses the construction of an ovarian cancer tumor sphere immune model in a plug-in membrane chip and the CAR-T cell killing test as an example.

[0155] Combined Figure 3 As shown, the preparation of the ovarian cancer tumor sphere immune model specifically includes the following steps:

[0156] S1. Preparation of ovarian cancer tumor spheres

[0157] (1) Selecting primary extracted or to-be-passaged ovarian cancer organoids, collecting and centrifuging to obtain ovarian cancer organoid precipitates; adding Tryple E for 15 min, digesting into single cells and centrifuging (1000 rpm, 5 min), removing the supernatant, adding PBS and centrifuging again (1000 rpm, 5 min), removing excess enzyme solution, and obtaining precipitates; adding tumor sphere culture medium (ovarian cancer organoid culture medium and endothelial cell culture medium mixed in a ratio of 2:1), counting cells, and obtaining ovarian cancer organoid suspensions;

[0158] (2) Ovarian cancer-associated fibroblasts were obtained, digested into single cells, resuspended in tumor sphere culture medium (a mixture of ovarian cancer organoid culture medium and endothelial cell culture medium in a ratio of 2:1), and counted;

[0159] (3) HUVEC cells were taken, digested into single cells, resuspended in tumor sphere culture medium (ovarian cancer organoid culture medium and endothelial cell culture medium mixed in a ratio of 2:1), and counted;

[0160] (4) Ovarian cancer organoids were mixed with ovarian cancer-associated fibroblasts and HUVEC cells at a ratio of 5:1:1 (the total number of cells per well was 10,000, the number of ovarian cancer organoid cells was 7,143 / well, the number of ovarian cancer-associated fibroblasts was 1,428 / well, and the number of HUVEC cells was 1,428 / well);

[0161] (5) Add the above mixed suspension to a low-adsorption U-bottom 96-well plate, 200 μL per well, and centrifuge at 1000 rpm for 5 min;

[0162] (6) Place in a 37°C, 5% CO 2 The cells were cultured in an incubator for 2 days.

[0163] S2. Inoculation of endothelial cells

[0164] (1) Take the plug-in membrane chip 200, remove the plug-in chamber 212 in the middle hole membrane chip and turn it upside down, add 20 μL of Matrigel to the upper layer 212a of the membrane, evenly cover the membrane, and place it at 37°C and 5% CO 2 Place in an incubator for 30 min to form a gel;

[0165] (2) HUVEC cells were obtained, digested into single cells, resuspended in the first mixed culture medium (ovarian cancer organoid culture medium and endothelial cell culture medium 2:1), counted, and adjusted to an appropriate density. After gelation, 40 μL of HUVEC cell suspension (100,000 HUVEC cells per well) was added and placed in a 37°C, 5% CO 2 The cells were cultured in an incubator for 2 hours.

[0166] S3. Inoculation of ovarian cancer tumor spheres

[0167] (1) After the endothelial cells adhere to the wall, the membrane chip is placed upright, the plug-in chamber 212 is inserted into the middle hole, and 2.7 mL of the first mixed culture medium (ovarian cancer organoid culture medium and endothelial cell culture medium mixed in a ratio of 2:1) is added from the left side sample well 211 of the chip. The culture medium is allowed to flow through the left side sample well 211 to the lower membrane layer 212b of the middle hole and the right side sample well 213. The culture medium in each well is 900 μL.

[0168] (2) The ovarian cancer tumor spheres were transferred to the upper membrane layer 212a of the plug-in chamber 212 in the middle hole of the membrane chip, and one tumor sphere was inoculated in each hole. 200 μL of the first mixed culture medium (a mixture of ovarian cancer organoid culture medium and endothelial cell culture medium in a ratio of 2:1) was added to the upper membrane layer 212a of the middle hole.

[0169] (3) Place in a 37°C, 5% CO 2 Incubate the cells in a static incubator for 1 day.

[0170] S4. Immune Cell Co-culture

[0171] (1) After static culture for 1 day, immune cell co-culture can be performed to activate endothelial cells: all the culture medium in the culture chamber is aspirated from the left sample well 211 and the right sample well 213 of the chip, and 2.7 mL of the first mixed culture medium (ovarian cancer organoid culture medium and endothelial cell culture medium mixed in a ratio of 2:1) containing 20 ng / mL TNFα is added to the left sample well 211 of the chip. The culture medium is allowed to flow through the left sample well 211 to the lower membrane layer 212b of the middle well and the right sample well 213. The culture medium in each well is 900 μL.

[0172] (2) Place in a 37°C, 5% CO 2 The cells were cultured using a rocking perfusion apparatus (Cat. No.: AvatarFlow) in an incubator at 7° / 30 min for 24 h.

[0173] (3) After 24 hours of dynamic culture, immune cells (CAR-T or T cells) are added for co-culture, and the T cells here serve as the control group. Take the CAR-T cells to be tested and the control T cells, count them, resuspend them in T cell culture medium, and adjust the density of CAR-T cells and control T cells.

[0174] (4) All the culture medium in the culture chamber was removed from the sample wells 211 and 213 on the left and right sides of the chip, and 2.7 mL of CAR-T cell suspension or control T cell suspension was added from the sample wells 211 on the left side of the chip, so that the CAR-T cells or T cells flowed through the sample wells 211 on the left side to the lower membrane layer 212b of the middle hole and the sample wells 213 on the right side. Each well contained 900 μL of culture medium containing immune cells. 1,000 CAR-T cells or T cells were added to each 100 μm ovarian cancer tumor sphere.

[0175] (5) Aspirate the culture medium on the upper layer of the middle hole of the membrane and add 200 μL of the second mixed culture medium (the volume ratio of CAR-T cell culture medium or T cell culture medium to tumor sphere culture medium is 3:1).

[0176] (6) Place in a 37°C, 5% CO 2 The cells were cultured in a rocking perfusion apparatus (Cat. No.: AvatarFlow) in an incubator at 7° / 30 min for 48 h.

[0177] (7) Testing can be performed after 48 hours of incubation.

[0178] It should be understood that in the immune model of tumor sphere co-culture, endothelial cells were added to the lower layer of the membrane of the membrane chip, and tumor spheres were added to the upper layer of the membrane. In addition, since the bottoms of the left sample well, the middle well, and the right sample well are all connected, the lower layer of the membrane of the left and right sample wells and the middle well are all immune cells.

[0179] Furthermore, the ovarian cancer tumor sphere immune model was subjected to cell apoptosis detection and cell activity detection, including:

[0180] (1) After the biological immune model was cultured for 23 hours, 10 μL of real-time apoptosis detection reagent Cleaved Caspase-3 (green fluorescence) was added from the upper layer of the sample well in the middle of the chip. After adding the apoptosis detection reagent, the chip was placed in a 37°C, 5% CO 2 A rocking perfusion apparatus (Cat. No.: AvatarFlow) in the incubator was used for flow culture at 7° / 30min.

[0181] (2) Take photos 1 hour later using a confocal microscope to observe the immune killing effect of CAR-T cells.

[0182] (3) Discard all the culture medium from the sample wells on the left and right sides of the chip, and at the same time, discard the culture medium on the membrane of the middle hole of the chip. Prepare the lysis solution: PBS buffer and CTG stock solution mixed in a ratio of 1:1. For each unit chip, add 200 μL of lysis solution to the upper layer of the middle hole membrane, mix for 5 minutes, and shake for 25 minutes. Then remove all the lysis solutions, transfer them to a black 96-well plate, read them using a chemiluminescence microplate reader, and statistically analyze the cell activity.

[0183] like Figure 6 As shown in (A), the constructed ovarian cancer multi-tumor sphere immune model, green represents apoptotic ovarian cancer tumor spheres. It can be seen that in the immune model with the addition of CAR-T cells to be tested, there is a clear green color, and in the control group with T cells, the green color is lighter, which indicates that the immune cell CAR-T has a strong killing effect on ovarian cancer tumor spheres.

[0184] like Figure 6 As shown in (B), in the immune model with the CAR-T cells to be tested, the average fluorescence intensity is about 7500, and in the control group with T cells, the average fluorescence intensity is less than 5000, indicating that the CAR-T cells to be tested have a significant killing effect on ovarian cancer tumor spheres. This further illustrates that the ovarian cancer tumor sphere immune model constructed in this example can detect the killing effect of CAR-T cells.

[0185] The present disclosure proposes a biological immune model and its construction method, culture medium composition, application, and evaluation method for cell therapy effect, which has the following beneficial effects compared with the prior art: the biological immune model constructed by the present disclosure is a biological model with a tumor microenvironment, with a high degree of simulation, and can simulate a complex microenvironment including blood vessels, fibroblasts, and cell-to-cell interactions, and, in a complex environment, can simulate the recruitment and killing of immune cells in the process of immune cells killing solid tumors. Furthermore, based on the biological immune model, it can be used to accurately evaluate the recruitment efficiency and killing effectiveness of immune cells, so as to achieve the evaluation of cell therapy effect.

[0186] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present disclosure, but the present disclosure is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and substance of the present disclosure, and these modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A method for constructing a biological immune model, characterized in that: The construction method comprises: Adding extracellular matrix and endothelial cells to the first side of the culture chip to form an endothelial layer through adherent culture; Vascularized spheroids, or fibroblasts and organoids are seeded on the second side of the culture chip to form a three-dimensional cell structure through three-dimensional culture; Adding a first mixed culture medium containing activation factors to the first side of the culture chip for flow culture to activate endothelial cells; An immune cell suspension is added to the first side of the culture chip for flow culture to obtain a biological immune model; wherein the immune cells can pass through the endothelial layer from the first side of the culture chip into the three-dimensional cell structure on the second side, and the first mixed culture medium is an organoid culture medium and an endothelial cell culture medium.

2. The method for constructing a biological immune model according to claim 1, characterized in that: When vascularized spheroids are seeded on the second side of the culture chip, the three-dimensional culture process includes: Vascularized spheroids are inoculated on the second side of the culture chip, and then the first mixed culture medium is added and cultured in an incubator.

3. The method for constructing a biological immune model according to claim 1, characterized in that: When seeding fibroblasts and organoids on the second side of the culture chip, the 3D culture process includes: Inoculating a fibroblast suspension on the second side of the culture chip and culturing the cells in an incubator; The culture medium on the second side of the culture chip is removed, the organoid suspension is inoculated onto the second side of the culture chip, the first mixed culture medium is added, and the culture is carried out in an incubator.

4. The method for constructing a biological immune model according to claim 1, characterized in that: The volume ratio of the organoid culture medium to the endothelial cell culture medium is (1-2):

1.

5. The method for constructing a biological immune model according to any one of claims 1 to 4, characterized in that: A first mixed culture medium containing inflammatory factors is added to the first side of the culture chip for a flow culture time of 2-24 hours; and / or, The inflammatory factor is any one of TNFα, TGFβ, and LPS; and / or, The concentration of the inflammatory factor is 1-50 ng / mL.

6. The method for constructing a biological immune model according to any one of claims 1 to 4, characterized in that: Add immune cell suspension to the first side of the culture chip for flow culture to obtain a biological immune model, including: For the immune cells to be detected, adding immune cell culture medium to resuspend them to obtain immune cell suspension; removing all the culture medium on the first side of the culture chip, and adding the immune cell suspension to the first side of the culture chip; All the culture medium on the second side of the culture chip is removed, and a second mixed culture medium is added for flow culture for 24-48 hours to obtain a biological immune model; the second mixed culture medium includes an immune cell culture medium and an organoid culture medium or a spheroid culture medium.

7. The method for constructing a biological immune model according to claim 6, characterized in that: The immune cell is any one of CAR-T, CAR-NK, TIL, TCR-T, and DC cells; and / or, When the vascularized spheroids are inoculated on the second side of the culture chip, the number of the immune cells added is (100-1000) cells / 100 μm; When inoculating fibroblasts and organoids on the second side of the culture chip, the ratio of the number of immune cells added to the total number of organoid cells is 1:(2-10); and / or, In the second mixed culture medium, the volume ratio of the immune cell culture medium to the organoid culture medium or the spheroid culture medium is (1-3):

1.

8. The method for constructing a biological immune model according to any one of claims 1 to 4, characterized in that: The flow culture adopts a non-perfusion shaking dynamic culture mode.

9. A biological immune model, characterized in that: The biological immune model is constructed by the construction method described in any one of claims 1 to 8.

10. A culture medium composition for culturing the biological immune model according to claim 9, characterized in that: The culture medium composition comprises: a first mixed culture medium for culturing before adding immune cells, and / or a second mixed culture medium for culturing after adding immune cells; The first mixed culture medium comprises an organoid culture medium and an endothelial cell culture medium in a volume ratio of (1-2):1; The second mixed culture medium includes immune cell culture medium and organoid culture medium or spheroid culture medium in a volume ratio of (1-3):

1.

11. An application of a biological immune model, characterized in that: The biological immune model described in claim 9 is used in any one of immunotherapy analysis, immune killer cell screening and cytotoxicity detection.

12. A method for evaluating the effect of cell therapy using the biological immune model of claim 9, characterized in that: The evaluation method includes: Conducting at least one of tumor cell activity detection, tumor cell apoptosis detection, immune cell infiltration analysis, and inflammatory factor release analysis on the biological immune model; Based on the test results, the effects of immune cells on tumors or non-tumors are obtained.

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