Methods for determining the level of immune rejection between allogeneic cells, tissues or organs

Abstract

The present disclosure provides a method for judging the level of immune rejection between heterologous cells, tissues or organs, and relates to the field of biological medicine. The method provided by the present disclosure can quickly and objectively compare the interspecies relative phagocytosis rate of phagocytes in a single experiment, and provides a new way for evaluating and researching the level of immune rejection between heterologous cells, tissues or organs.

Description

Technical Field

[0001] This disclosure relates to the field of biomedicine, and in particular, to a method for determining the level of immune rejection between heterologous cells, tissues or organs. Background Technology

[0002] Immune rejection (or immune denial) refers to the process by which the recipient's immune system recognizes the transplanted organ, tissue, or cell as a "foreign object" after receiving a transplant from another individual, thus activating a series of immune responses to attack and eliminate the transplant. Immune rejection is a significant challenge in xenotransplantation of cells, tissues, and organs, as donor cells first face phagocytosis and clearance by the host's innate immune system. For example, even in rodents like Rag1... - / - Rag2 - / - Il2rg - / - In other immunodeficient strains, the innate immune system remains highly active. Therefore, before conducting transplantation therapy or in vivo experiments, it is necessary to establish a preliminary experimental system capable of reproducing phagocytic-mediated immune rejection responses in vitro.

[0003] Current methods for evaluating immune rejection have many shortcomings. For example, evidence from traditional phagocytosis experiments mostly comes from independent cultures, resulting in significant batch-to-batch variations and making direct comparisons impossible; existing studies often report "single-specific heterologous phagocytosis rates," making cross-sectional comparisons under different experimental conditions difficult; methods using slides / microscopic imaging require fixation and staining, which are cumbersome and have low throughput; methods requiring in vivo models are time-consuming and mouse-intensive; existing protocols are mostly customized for single species and single cell types; and traditional endpoint measurements cannot capture phagocytic rates, etc. Therefore, developing a new method for assessing or determining interspecies immune rejection is crucial for addressing these issues. Summary of the Invention

[0004] Technical problems to be solved:

[0005] The first aspect of this disclosure is to provide a method for determining the level of immune rejection between heterologous cells, tissues, or organs, addressing the aforementioned shortcomings in the prior art.

[0006] Technical solution:

[0007] Methods for determining the level of immune rejection between heterologous cells, tissues, or organs include the following steps:

[0008] Step 1) Obtain phagocytes from individual A;

[0009] Step 2) Obtain the test cells for individual B;

[0010] Step 3) Obtain the analyte for individual A, wherein the analyte for individual A is the test cell or detectable particle of individual A;

[0011] Step 4) Simultaneously bring the test sample from individual A and the test cells from individual B into contact with the phagocytes of individual A in a 1:1:1 ratio;

[0012] Step 5) Detect the number of test cells in individual B after contact, and calculate the heterologous phagocytosis rate according to the formula: number of test cells in individual B after contact / number of phagocytes in individual A.

[0013] Step 6) Detect the number of test samples in individual A after contact, and calculate the homologous phagocytosis rate according to the formula: number of test samples in individual A after contact / number of phagocytes in individual A;

[0014] Step 7) Using the heterophagocytic rate and homophagocytic rate obtained in steps 5) and 6), calculate the relative phagocytic rate according to the formula heterophagocytic rate / homogeneous phagocytic rate. When the relative phagocytic rate is less than or equal to 1, it indicates that there is no immune rejection reaction between heterogeneous cells.

[0015] In some embodiments, the phagocytes may be at least one selected from macrophages, neutrophils, or dendritic cells. When the phagocytes are a mixture of macrophages and neutrophils, a mixture of macrophages and dendritic cells, a mixture of neutrophils and dendritic cells, or a mixture of macrophages, neutrophils, and dendritic cells, the mixing ratio can be arbitrarily set as needed. In a more specific embodiment, the phagocytes may be macrophages.

[0016] In some embodiments, the test cells can be at least one selected from xenograft cells, xenograft tumor cells, apoptotic or necrotic xenograft cells, artificial or experimental xenograft cells, and pathogenic microorganisms. When two or more of the test cells are used in combination, the mixing ratio can be arbitrarily set as needed. In a more specific embodiment, the test cells can be xenograft cells. In another more specific embodiment, the test cells can be red blood cells.

[0017] In some embodiments, the analyte of individual A and the test cells of individual B can be detected using methods known in the art. In other embodiments, the analyte of individual A and the test cells of individual B may carry distinguishable detection markers. In a more specific embodiment, the distinguishable detection marker may be a fluorescent marker. In other embodiments, the detection may be performed using flow cytometry.

[0018] In some embodiments, the contact can be performed in the same space or in different spaces, as long as the start time of the contact is the same. However, in order to better achieve the purpose of this disclosure, in other embodiments, the contact is performed within the same container.

[0019] In some embodiments, the contact time is at least about 10 minutes to about 300 minutes. For example, at least about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, about 180 minutes, about 190 minutes, about 200 minutes, about 210 minutes, about 220 minutes, about 230 minutes, about 240 minutes, about 250 minutes, about 260 minutes, about 270 minutes, about 280 minutes, about 290 minutes, or about 300 minutes. In some specific embodiments, the contact time is about 120 to 300 minutes. In a more specific embodiment, the contact time is about 120 minutes. In other embodiments, the contact time can be longer, for example, including but not limited to 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, and 30 hours.

[0020] The second aspect of this disclosure is to provide an application of relative throughput in a scenario, wherein the relative throughput is obtained by the above method, and the scenario is:

[0021] (1) Validation of gene editing effectiveness;

[0022] (2) Batch quality control of cell / organoid therapy;

[0023] (3) Screening of phagocytic inhibitor drugs;

[0024] (4) Pre-assessment of humanized animal models;

[0025] (5) Screening for gene modification pathways in xenotransplantation; or

[0026] (6) Evaluation of CAR-Macrophage functional specificity.

[0027] A second aspect of this disclosure is the application of a rapid relative phagocytosis rate assay kit in determining the level of immune rejection between heterologous cells, tissues, or organs, the rapid relative phagocytosis rate assay kit comprising:

[0028] i) Markers or detectable particles in the test cells of individual A; and

[0029] ii) Markers for the cells to be tested in individual B.

[0030] A third aspect of this disclosure is the application of a rapid relative phagocytosis rate assay kit in a specific scenario, the rapid relative phagocytosis rate assay kit comprising:

[0031] i) Markers or detectable particles in the test cells of individual A; and

[0032] ii) Markers for the test cells of individual B,

[0033] The scenario is as follows:

[0034] (1) Validation of gene editing effectiveness;

[0035] (2) Batch quality control of cell / organoid therapy;

[0036] (3) Screening of phagocytic inhibitor drugs;

[0037] (4) Pre-assessment of humanized animal models;

[0038] (5) Screening for gene modification pathways in xenotransplantation; or

[0039] (6) Evaluation of CAR-Macrophage functional specificity.

[0040] The fourth aspect of this disclosure is to provide a method for constructing a xenograft animal model, comprising the following steps:

[0041] Step 1) Use the above methods to determine the level of immune rejection between heterologous cells, tissues, or organs;

[0042] Step 2) When the level of the above-mentioned immune rejection response is within a reasonable range, construct the xenograft animal model.

[0043] In some more specific embodiments, the above-mentioned xenograft animal model is a humanized rat model, and the construction method includes the following steps:

[0044] Step 1) Use the above method to determine the level of immune rejection between human cells and rat macrophages;

[0045] Step 2) When the above-mentioned immune rejection level is not greater than 1, the human SIRPA, SLAMF3 or SLAMF4 sequence is knocked into the rat genome to construct the humanized rat model.

[0046] Beneficial effects:

[0047] This disclosure presents a method for detecting and reducing the level of immune rejection between xenogeneic cells, tissues, or organs, which has significant advantages such as high versatility, simple operation, and quantifiable results. This method provides important technical support for the construction and optimization of cross-species transplantation models and also provides an in vitro evaluation platform for the screening and validation of novel immune modulation strategies. Specifically,

[0048] 1) "One well with two or more labels" internal control system: The method in this disclosure adds homologous and heterologous test cells or test substances to the same phagocyte population at one time. The phagocytes themselves are the "internal control", eliminating batch differences and reducing the experimental coefficient of variation. A single experiment can yield highly comparable data.

[0049] 2) Normalization index: The method in this disclosure provides a real-time calibration baseline through homologous test cells or detectable particles. The calculated ratio can offset the differences in injection concentration and staining efficiency, which can improve the reproducibility of results and facilitate direct comparison between different laboratories or different animal strains.

[0050] 3) Rapid high-throughput flow cytometry plate reading: The method in this disclosure can be used for flow cytometry detection, which can complete the detection of 50,000 to 100,000 cells per minute, significantly improving the speed.

[0051] 4) In vitro prediction to in vivo validation closed loop: In the pre-experimental system, the method of this disclosure can be used to screen out high-risk protocols in vitro and advance only the low-phagocytic group to treatment or experiment.

[0052] 5) Universal and scalable platform: The method in this disclosure can test different donor cells (e.g., iPSCs, organoids or tumor cells) and different recipient phagocytes simply by changing the fluorescent probe of the labeled cells.

[0053] 6) Real-time dynamics are scalable: The method in this disclosure can set multiple time points for on-machine readings and can obtain a complete phagocytic dynamics curve for output. Attached Figure Description

[0054] Figure 1 Example of a gating strategy for streaming analysis in this disclosure. Figure 1 ;

[0055] Figure 2 Example of a gating strategy for streaming analysis in this disclosure. Figure 2 ;

[0056] Figure 3 Example of a gating strategy for streaming analysis in this disclosure. Figure 3 ;

[0057] Figure 4 This is a diagram showing the identification results of the Sirpa gene on rat chromosome 3 in the embodiments of this disclosure;

[0058] Figure 5 A flowchart illustrating the construction of the "Don't Eat Me" rat model (humanized macrophage rat model) in this disclosure embodiment;

[0059] Figure 6 This is a diagram showing the results of qPCR detection of rat macrophages in an embodiment of this disclosure;

[0060] Figure 7 This is a graph showing the statistical analysis results of offspring genotypes in the embodiments of this disclosure;

[0061] Figure 8 This is a diagram showing the results of an in vitro macrophage phagocytosis experiment in an embodiment of this disclosure. Detailed Implementation

[0062] This disclosure provides a method for determining the level of immune rejection between heterologous cells, tissues, or organs. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0063] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The term "a," "an," and "the" includes plural indicators. The term "a plurality of" means two or more. The terms "such as," "for example," etc., are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure.

[0064] In this disclosure, when a range of values ​​is provided, it should be understood that, unless the context otherwise explicitly indicates otherwise, the range includes endpoints and each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range and any value within a smaller range between specified values.

[0065] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0066] In this disclosure, terms such as "one embodiment," "an example," "some embodiments," "a particular embodiment," "related embodiment," "a certain embodiment," "some embodiments," "additional embodiment," or "further embodiment," "further implementation," or "another embodiment," "some other embodiments," mean that at least one feature or characteristic description is included in relation to the embodiment. Therefore, throughout this disclosure, the above phrases do not necessarily refer to the same embodiment. Furthermore, specific features may be combined in any suitable manner in one or more embodiments.

[0067] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common molecular biology terms can be found in Lewin's *GENES*, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Jones & Bartlett Learning. Definitions of common biochemistry terms can be found in Lehninger's *Principles of Biochemistry*, Eighth Edition, David L. Nelson, Michael M. Cox, WHFreeman. Definitions of common cell biology terms can be found in *Molecular Biology of the Cell*, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Garland Science. Definitions of common genetics terms can be found in *Genetics: Analysis of Genes and Genomes*, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Jones & Bartlett Learning.

[0068] Unless otherwise specified, the experimental techniques used in this paper employ standard techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as *Molecular Cloning: A Laboratory Manual* and *Cell Biology: A Laboratory Handbook*.

[0069] definition :

[0070] The term "heterologous" in this disclosure refers to a foreign and biologically different cell, tissue, or organ relative to a specific cell, tissue, or organ. In some embodiments, it can be a xenogeneic or allogeneic cell, tissue, or organ. For example, a human cell is a heterologous cell relative to a mouse cell; a hematopoietic stem cell from a healthy donor A is a heterologous cell relative to the hematopoietic stem cells of recipient patient B. In some embodiments, the heterologous cell can be a xenograft cell, a xenograft tumor cell, apoptotic or necrotic xenograft cell, artificial or experimental xenograft cell, or pathogenic microorganism; in other embodiments, the heterologous cell can also be a mixture of the above-mentioned cell types. Examples of xenograft cells include, but are not limited to, hematopoietic stem cells, mesenchymal stem cells, immune cells, pancreatic islet cells, neural progenitor cells, retinal pigment epithelial cells, cardiac progenitor cells, hepatocytes, etc. Examples of heterologous tumor cells include, but are not limited to, skin cancer cells, breast cancer cells, brain cancer cells, cervical cancer cells, testicular cancer cells, head and neck cancer cells, lung cancer cells, mediastinal cancer cells, gastrointestinal cancer cells, genitourinary cancer cells, gynecological cancer cells, endocrine cancer cells, cancer cells of unknown primary origin or metastatic cancer cells, soft tissue and osteosarcoma cells, mesothelioma cells, melanoma cells, central nervous system tumor cells, lymphoma cells, leukemia cells, peritoneal cancer cells, immunosuppression-related malignant tumors and / or metastatic cancer cells, etc. In some embodiments, the aforementioned heterologous cells may be naturally occurring, unmodified cells obtained through isolation, enrichment, or culture; they may also be engineered cells. For example, in other embodiments, the aforementioned heterologous cells may be induced pluripotent stem cell-derived heterologous cells (iPSC-Derived), synthetically designed heterologous cells, humanized animal chimeric cells, chimeric antigen receptor T cells, or chimeric antigen receptor NK cells, etc. The term "individual" as used in this disclosure refers to the aforementioned heterologous or homologous individuals.

[0071] The term "immune rejection" or "immune rejection reaction" in this disclosure refers to the pathological process in which, after an allogeneic or xenograft (such as an organ, tissue, or cell) is implanted or introduced into a recipient (host), the recipient's immune system recognizes the antigens expressed by the graft (such as the major histocompatibility complex antigen) as non-self, thereby activating a specific immune response and producing effector T lymphocytes (such as cytotoxic T cells), natural killer cells, and / or antibodies (such as anti-HLA antibodies) against the graft. Through mechanisms such as cytotoxicity, release of inflammatory factors, and complement activation, the graft suffers from inflammatory infiltration, parenchymal cell damage, vascular destruction, and loss of function.

[0072] The term "phagocyte" in this disclosure refers to key effector cells in the immune rejection response, which participate in the attack on the graft through direct phagocytosis, release of inflammatory mediators, and presentation of antigens. In this disclosure, the level of the immune rejection response is measured by the relative phagocytic rate of phagocytes on xenograft cells. The phagocytes involved in this disclosure are macrophages, neutrophils, or dendritic cells, as well as combinations thereof. Macrophages are the core cells, primarily involved in hyperacute rejection, acute rejection, and chronic rejection, mediating the immune rejection response through direct phagocytosis, antigen presentation, release of inflammatory mediators, causing tissue damage, and promoting fibrosis. Neutrophils primarily participate in hyperacute rejection and early acute rejection, mediating the immune rejection response through the release of proteases (such as elastase), ROS, and neutrophil extracellular traps (NETs), and the secretion of chemokines such as IL-8. Dendritic cells primarily participate in early acute rejection. To better achieve the objectives of this disclosure, in one specific embodiment, macrophages are used as the phagocytes.

[0073] The term "distinguished detection marker" in this disclosure refers to at least one marker capable of generating a detectable signal directly or indirectly. In some embodiments, examples of such markers include, but are not limited to, enzymes and luminescent substances that generate detectable signals by, for example, colorimetry or fluorescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent dyes. Detectable groups with electron density, detected by electron microscopy or by their electrical properties (e.g., conductivity, amperometry, voltammetry, impedance), such as groups whose molecules are large enough to cause detectable modifications in their physical and / or chemical properties, can be detected by optical methods (e.g., diffraction, surface plasmon resonance, surface variation, contact angle variation) or physical methods (e.g., atomic force spectroscopy, tunneling effect) or radioactive molecules (e.g.,...). 32 P, 35 S, 89 Zr or 125 I). The aforementioned detectable markers can be qualitatively and / or quantitatively detected using suitable methods in the prior art. For example, spectrophotometry or flow cytometry. In one specific embodiment, the marker used is a fluorescent marker, and the detection method used is flow cytometry.

[0074] The term "contact" as used in this disclosure refers to the permission for two or more substances to interact, for example through chemical interactions, including ionic, nonionic, polar, hydrophobic, or hydrophilic interactions, or through physical contact as accepted in the art. In some embodiments, the analyte of individual A and the test cells of individual B are simultaneously in contact with the phagocytes of individual A. This contact causes an interaction between the analyte of individual A and the phagocytes of individual A, and between the test cells of individual B and the phagocytes of individual A. This interaction can occur in a medium, such as, for example, cell culture medium, buffer solution, etc. In some embodiments, the contact can occur in the same space / container. In other embodiments, the contact can occur in different spaces / containers. The aforementioned space / container can be any suitable space / container capable of holding cells, particles, liquids, etc., such as, but not limited to, cell culture dishes, perforated cell culture plates, etc. For better realization of the purposes of this disclosure, in one specific embodiment, the contact is conducted in the same cell culture well.

[0075] Example:

[0076] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions of this disclosure will be further described in detail below with reference to specific embodiments.

[0077] In this embodiment, human erythrocytes and rat macrophages are used as two different heterologous cells to evaluate the level of immune rejection between them. It is foreseeable that, based on the core idea of ​​this embodiment, those skilled in the art can adjust the method used according to specific circumstances. For example, this includes, but is not limited to, replacing the aforementioned human erythrocytes with other heterologous cells or combinations of heterologous cells; replacing the aforementioned macrophages with other phagocytes or combinations of phagocytes; replacing the aforementioned rats with cells from other species; using different labeling strategies and corresponding detection methods, etc.

[0078] The core idea of ​​this embodiment is:

[0079] In a completely uniform culture environment, macrophages are exposed to both "indigenous" and "exotic" organisms simultaneously, and then the frequency of these two interactions is read out using flow cytometry. This minimizes experimental error and transforms "alien rejection strength" into a mathematically quantifiable ratio.

[0080] Macrophage origin and adherence purification:

[0081] Directly comparing phagocytic behavior among different rats would inevitably amplify experimental noise due to differences in genetic background. Therefore, to further address this issue, this embodiment first used peritoneal lavage fluid from the same rat as the initial cell pool. After erythrocyte lysis, all cells were briefly placed in RPMI-1640 medium for adhesion; macrophages exhibited sufficient adhesion ability and firmly attached themselves within 2 hours, while the remaining cells were gently washed away. This step is not merely about "obtaining macrophages," but more importantly, it establishes a uniform host innate immune background, providing a stable reference frame for subsequent comparisons.

[0082] The necessity and concentration design of dual fluorescent labeling:

[0083] If homologous and heterologous erythrocytes are incubated separately, any time difference in sample loading, minor fluctuations in cell count, or even batch differences in culture wells will be amplified in the final reading. Therefore, to further address these issues, this embodiment places both types of erythrocytes in the same well. To enable flow cytometry to simultaneously identify their origin in a single event, BV421 and Deep Red, with minimal spectral overlap, were selected for staining in this embodiment. The antibody concentration was not arbitrarily chosen but confirmed through preliminary titration experiments.

[0084] The spatiotemporal setting of "one hole, two standards" co-cultivation:

[0085] Macrophage recognition and phagocytosis of target cells require the simultaneous fulfillment of two dynamic processes: receptor-ligand binding and membrane rearrangement. If the incubation time is too short, the readings are insufficient to distinguish between modified and unmodified cells; if it is too long, secondary phagocytosis or exocytosis will blur the differences. Therefore, to further address these issues, this embodiment employs a gradient experiment ranging from 120 to 300 minutes. The 120-minute time point, balancing signal-to-noise ratio and operational convenience, was selected as the standard time. Furthermore, to reduce the random initiation differences in phagocytic rates, macrophages undergo an additional 2-hour serum-free "starvation" treatment before the addition of red blood cells to synchronize their endogenous phagocytic activity. Precise control of time and pretreatment is key to compressing biological fluctuations within a statistically controllable range.

[0086] Gating strategies for streaming analytics (e.g.) Figure 1 , Figure 2 , Figure 3 (as shown)

[0087] During flow cytometry collection, cell adhesion was first excluded using FSC-H / FSC-A, followed by staining for negative CD163 cells with Propidium Iodide. +The (FITC) phylum delineates the macrophage population. Only events that enter this phylum and are positive for BV421 or Deep Red, or both, are counted as "phagocytosis." This three-step screening ensures that: ① the object is indeed a macrophage and not red blood cells adhering to its surface; ② the fluorescence comes from the phagocytoid itself and not from free dyes; ③ whether the same cell can simultaneously phagocytose two types of red blood cells is immediately apparent. Using two-dimensional gating with area and length, the gating strategy progresses layer by layer, clearly separating "phagocytosis" from "adhesion."

[0088] Statistical significance of the normalized index:

[0089] In this embodiment, the final metric obtained is not the heterologous phagocytosis rate, but rather the calculated relative phagocytosis rate (heterologous phagocytosis rate / homogeneous phagocytosis rate). The reasoning behind this is that the total number of macrophages in the same well, the culture medium environment, and residual dye all affect the numerator and denominator simultaneously, thus canceling each other out in the ratio. Ratio-ratio processing eliminates all "non-differential noise," making the experimental results highly comparable and reproducible.

[0090] Application scenario example:

[0091] (1) Validation of gene editing effectiveness:

[0092] When constructing gene-edited animals such as SIRPα or CD47, there is an urgent need to quickly confirm whether the modification truly reduces macrophage phagocytosis. The technical solution in this embodiment uses a "one-well dual-label" flow cytometry method to provide a quantitative indicator of "heterologous phagocytosis rate ÷ homologous phagocytosis rate" within 24 hours, helping researchers decide whether to proceed with the next in vivo experiment.

[0093] (2) Batch quality control (QC) for cell / organoid therapy:

[0094] Cell therapy companies often face the problem of significant compatibility differences between different production batches in their GMP manufacturing facilities. Co-culturing each batch of iPSC-derived cells or organoids with standardized host macrophages can quickly screen out phagocytosis-sensitive batches, serving as a basis for release or rework and ensuring product consistency.

[0095] (3) High-throughput screening of phagocytic inhibitor drugs:

[0096] Innovative pharmaceutical companies and CROs need to discover molecules that can inhibit macrophage phagocytosis at an early stage. The technical solution in this embodiment employs a 96-well flow cytometry format, enabling the IC50 analysis of hundreds of compounds within a single day. 50 Measurement and dynamic curve plotting significantly reduce the time and cost required for high-content imaging.

[0097] (4) Pre-assessment of humanized animal models:

[0098] In humanized mouse / rat projects, direct in vivo modeling is expensive and time-consuming. By first screening for "low phagocytic" recipient-donor combinations using in vitro phagocytic indicators, and then carrying out humanized modeling, 60-80% of the number of animals and the time required can be saved.

[0099] (5) Screening for gene modification routes in xenotransplantation:

[0100] When companies developing transgenic pig or primate organs for human transplantation, they need to verify the innate immune compatibility of organ cells with human macrophages. By simply replacing red blood cells with fragments of liver cells or pancreatic islet cells, multiple gene-editing strategies can be compared in parallel, allowing for rapid selection of the optimal approach.

[0101] (6) Evaluation of CAR-Macrophage functional specificity:

[0102] When developing CAR-Mac, immunotherapy companies need to distinguish between "targeted phagocytosis" and "background phagocytosis." By simultaneously adding target cells (red) and non-target cells (green), the ratio of increased specificity of CAR-Mac can be quantified, providing data support for structural optimization.

[0103] Example 1:

[0104] 1. Macrophage collection.

[0105] (1) Prepare the rats required for the experiment, specimen bottles for anesthesia, beakers, anesthetic isoflurane, etc.; a pre-cooled 4℃ centrifuge;

[0106] (2) Anesthetize and kill rats: Place the rats in a specimen bottle, add 2-3 mL of isoflurane, and the rats will suffocate and die after 6-7 minutes;

[0107] (3) Place the euthanized rat into a beaker, add an appropriate amount of 75% alcohol until the entire rat is submerged, and soak for 10 minutes.

[0108] (4) Disinfect the abdomen and inject 50ml of pre-cooled sterile PBS into the abdominal cavity along the midline using a syringe. Shake from side to side while simultaneously massaging the peritoneal wall with your fingers from both sides to allow the fluid to flow fully within the abdominal cavity. Massage for 2-3 minutes;

[0109] (5) Make a small incision in the abdominal wall, tilt the animal's body slightly to one side, and use a syringe to draw out the liquid;

[0110] (6) Centrifuge at 4℃ for 500g for 5 minutes, then discard the supernatant;

[0111] (7) Add 3-5 times the volume of red blood cell lysis buffer, gently pipette to mix, and lyse for 1-2 minutes. For example, if the volume of the cell pellet is 1 ml, add 3-5 ml of red blood cell lysis buffer. Operate at room temperature; centrifuge at 400-500g for 5 minutes and discard the red supernatant. Centrifugation at 4℃ yields better results; if incomplete red blood cell lysis is found, the above steps can be repeated once. Usually, trace amounts of red blood cells will not affect some subsequent tests. If there are few red blood cells in the cell pellet, this step can be omitted.

[0112] (8) Wash the cells twice with about 10 ml of pre-cooled RPMI 1640 culture medium, centrifuge at 500g at 4℃ for 5 minutes each time, and discard the supernatant;

[0113] (9) Resuspend cells in pre-cooled RPMI 1640-10% FBS-1% PS medium, count cells and determine cell viability using trypan blue staining;

[0114] (10) Arrange the cells at a density of 1×10⁶ cells per well. 6 One macrophage was seeded into a 12-well plate, and 1 mL of RPMI 1640 + 10% FBS + 1% PS was added. The plate was then incubated for 2 hours.

[0115] 2. Macrophage starvation pretreatment.

[0116] (1) After discarding the culture medium, wash away the suspended cells with RPMI 1640 and discard the liquid;

[0117] (2) Starve adherent macrophages with RPMI 1640 (serum-free) for 2 hours;

[0118] 3. Co-cultivation and phagocytosis.

[0119] (1) The separated human red blood cells and rat red blood cells were stained with hCD235a-BV421 and Deep red respectively in advance;

[0120] (2) After starvation treatment, the stained phagocytosed cells were mixed with human red blood cells: mouse red blood cells: macrophages in a ratio of 1:1:1 and added to the adherent macrophages. The mixture was then phagocytosed at 37°C and 30 rpm for 2 hours.

[0121] 4. Staining.

[0122] (1) Digest the cells with Trypsin-EDTA and gently tap the culture plate. Add 5 mL of RPMI 1640 + 10% FBS + 1% PS to neutralize. After blowing the cells off, transfer them to a 15 mL centrifuge tube and filter them through a 40 μm filter. Centrifuge at 400 g for 5 minutes at 4°C. Resuspend the cells in 1 mL of FACS Buffer and centrifuge again.

[0123] (2) After discarding the supernatant, stain with anti-rat CD163-FITC, incubate on ice for 30 min, wash away excess antibody, and then perform flow cytometry. Cell viability is determined by PI.

[0124] 5. Data processing and results.

[0125] (1) By removing attached cells, only single cells in suspension are retained for subsequent analysis. Figure 1 ).

[0126] (2) In flow cytometry, a population of cells that are negative for PI staining and positive for CD163-FITC is selected and defined as an active macrophage population. Figure 2 ).

[0127] (3) In this population, BV421 positive cells represent macrophages that have engulfed human erythrocytes, reflecting their heterophagocytic capacity; Deep Red positive cells represent macrophages that have engulfed rat erythrocytes, indicating their allophagetic capacity. Figure 3 ).

[0128] (4) The formula for calculating the relative phagocytic rate is: Relative phagocytic rate = Heterogeneous phagocytic rate / Homogeneous phagocytic rate. The calculated relative phagocytic rate of macrophages from wild-type rats in vitro was 3.1902±2.0419.

[0129] Example 2: Variation of the method in Example 1

[0130] The experiment was conducted using the same method as in Example 1, except that ① human red blood cells were treated with pHrodo TM ① Perform single staining with Red; ② Replace rat red blood cells with 2 µm green fluorescent microbeads that will not be phagocytosed as a sampling reference (no staining required).

[0131] In this embodiment, pHrodo TM Upon entering the acidic phagosome, the macrophages emit spontaneous light, naturally distinguishing between "already phagocytosed" and "unalready phagocytosed" cells; therefore, only a single-color dye needs to be applied to the xenogeneic erythrocytes. After co-culturing for 120 min, the normalized index is obtained by flow cytometry as "pHrodo⁺ macrophage count ÷ microbead count". This method involves one-step staining and single-fluorescence detection, minimizing instrument channel usage; pHrodo⁺ TM Automatically eliminate "adhesive false positives".

[0132] Example 3: Variation of the method in Example 1

[0133] The experiment was conducted using the same method as in Example 1, except that: ① both homologous and heterologous RBCs were stained with FITC at the same concentration; ② after co-culturing, 0.2% Trypan Blue was added for 5 min and the cells were immediately loaded onto the instrument; ③ F4 / 80–APC macrophages were circled, and FITC⁺ was considered a phagocytic event; ④ APC-Cy7 microbeads were added to the wells to calibrate the collection volume.

[0134] In this embodiment, Trypan Blue can quench extracellular FITCs but cannot penetrate the intact cell membrane; phagocytosed RBCs are located inside the phagosome, thus preserving the FITC signal. This method requires only two channels: FITC and APC, and can be implemented with any mid-range flow cytometer; it is also low-cost.

[0135] Example 4: Variation of the method in Example 1

[0136] The experiment was conducted using the same method as in Example 1, except that: ① macrophages were seeded into 96-well optical plates; ② human RBCs were stained red and rat RBCs were stained green; ③ co-culture with PI to exclude dead cells; ④ nuclei were stained with DAPI, and HCS was scanned and the ratio of "red / green area ÷ number of nuclei" was output.

[0137] In this embodiment, HCS software is used to automatically identify the outline of macrophages and the area of ​​intracellular fluorescent targets. Then, DAPI is used to count the macrophage nuclei as the denominator to obtain advanced parameters such as spatial distribution and cluster size, supporting real-time dynamic imaging.

[0138] Example 4: Variation of the method in Example 1

[0139] The experiment was conducted using the same method as in Example 1, except that homologous and heterologous RBCs were stained with pHrodoGreen / pHrodoRed respectively, and samples were continuously taken within 15 to 240 min using the difference in acidification rate to generate phagocytic kinetic curves.

[0140] The method described in this embodiment can obtain three types of information—rate, maximum phagocytosis, and saturation time—within a single experiment, making it suitable for pharmacokinetic studies.

[0141] Example 5: Construction of a humanized rat model of macrophages

[0142] 1. An SD rat model with the Kozak-HumanSIRPACDS-SV40latepA insert fragment was created at the rat Sirpa locus using CRISPR / Cas9-mediated genome engineering. (Example:) Figure 4As shown, the Sirpa gene, located on rat chromosome 3, was identified with 8 exons, with the ATG start codon in exon 1 and the TGA stop codon in exon 8. Exon 1 will be selected as the target site. In the KI rat model, the ATG start codon will be replaced by Kozak-Human SIRPACDS-SV40 late pA; therefore, the expression of Human SIRPA will be controlled by regulatory elements of the rat Sirpa gene. Figure 5 The study demonstrated that Cas9 mRNA, sgRNA, and a donor vector, transcribed in vitro, were co-injected into fertilized eggs using DNA microinjection combined with CRISPR / Cas9 gene editing technology. These eggs were then implanted into pseudopregnant mice. The success rate of the knock-in was confirmed by PCR and DNA sequencing in the F0 offspring (first-generation mice). Subsequently, the successfully knocked-in first-generation mice were mated with wild-type rats to produce F1 offspring, verifying the heritability of the knock-in gene. A population of successfully heritable F1 offspring was then crossbred and expanded for subsequent experiments.

[0143] 2. The genotypes of the constructed F1 offspring rats and subsequent newborn rats were identified by PCR amplification of specific gene fragments and agarose gel electrophoresis to determine the presence of these gene fragments.

[0144] 3. For example Figure 6 As shown, qPCR detection of macrophages from homozygous (HOMO), heterozygous (HETER), and wild-type (WT) rats revealed that they expressed the human SIRPA gene in a manner similar to that in human cells.

[0145] 4. Statistical analysis of offspring genotypes, from Figure 7 As can be seen from the figure, there are three genotypes in the offspring of double heterozygotes. It can also be seen from the figure that all the offspring of double homozygotes are homozygous. Furthermore, human SIRPA is not lost as the number of generations increases. These results prove that human SIRPA can be stably inherited in SD rats.

[0146] 5. In vitro macrophage phagocytosis experiments, as described above, showed that the relative phagocytic rate was significantly lower than that of wild-type macrophages (e.g., Figure 8 (As shown). The relative phagocytosis rates were 0.7766±0.4119 for homozygotes and 1.2338±0.6545 for heterozygotes.

[0147] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining the level of immune rejection between heterologous cells, tissues, or organs, characterized in that, Includes the following steps: Step 1) Obtain phagocytes from individual A; Step 2) Obtain the test cells for individual B; Step 3) Obtain the analyte for individual A, wherein the analyte for individual A is the test cell or detectable particle of individual A; Step 4) Simultaneously bring the test sample from individual A and the test cells from individual B into contact with the phagocytes of individual A in a 1:1:1 ratio; Step 5) Detect the number of test cells in individual B after contact, and calculate the heterologous phagocytosis rate according to the formula: number of test cells in individual B after contact / number of phagocytes in individual A. Step 6) Detect the number of test samples in individual A after contact, and calculate the homologous phagocytosis rate according to the formula: number of test samples in individual A after contact / number of phagocytes in individual A. Step 7) Using the heterophagocytic rate and homophagocytic rate obtained in steps 5) and 6), calculate the relative phagocytic rate according to the formula heterophagocytic rate / homophagocytic rate. When the relative phagocytic rate is less than or equal to 1, it indicates that there is no immune rejection reaction between heterologous cells.

2. The method according to claim 1, characterized in that, The phagocytes are selected from at least one of macrophages, neutrophils, or dendritic cells.

3. The method according to claim 2, characterized in that, The phagocytes are macrophages.

4. The method according to claim 1, characterized in that, The cells to be tested are selected from at least one of the following: xenografted cells, xenografted tumor cells, apoptotic or necrotic xenografted cells, artificial or experimental xenografted cells, and pathogenic microorganisms.

5. The method according to claim 4, characterized in that, The xenografted cells are red blood cells.

6. The method according to claim 1, characterized in that, The test sample of individual A and the test cells of individual B carry distinguishable detection markers.

7. The method according to claim 6, characterized in that, The distinguishable detection marker is a fluorescent marker.

8. The method according to claim 1 or 6, characterized in that, The detection was performed using flow cytometry.

9. The method according to claim 1, characterized in that, The contact takes place within the same container.

10. The method according to claim 1 or 9, characterized in that, The contact time is from 10 minutes to 24 hours.

11. The method according to claim 10, characterized in that, The contact time is 120 minutes.

12. The application of relative swallowing rate in a scenario, characterized in that, The relative phasing rate is obtained by the method described in any one of claims 1 to 11, and the scenario is as follows: (1) Validation of gene editing effectiveness; (2) Batch quality control of cell / organoid therapy; (3) Screening of phagocytic inhibitor drugs; (4) Pre-assessment of humanized animal models; (5) Screening for gene modification pathways in xenotransplantation; or (6) Evaluation of CAR-Macrophage functional specificity.

13. The application of a rapid relative phagocytosis rate assay kit in determining the level of immune rejection between heterologous cells, tissues, or organs, characterized in that... The rapid relative phagocytosis rate assay kit includes: i) Markers or detectable particles in the test cells of individual A; and ii) Markers for the cells to be tested in individual B.

14. The application of a rapid relative phagocytosis rate assay kit in a specific scenario, characterized in that... The rapid relative phagocytosis rate assay kit includes: i) Markers or detectable particles in the test cells of individual A; and ii) Markers for the test cells of individual B, The scenario is as follows: (1) Validation of gene editing effectiveness; (2) Batch quality control of cell / organoid therapy; (3) Screening of phagocytic inhibitor drugs; (4) Pre-assessment of humanized animal models; (5) Screening for gene modification pathways in xenotransplantation; or (6) Evaluation of CAR-Macrophage functional specificity.

15. A method for constructing an allogeneic transplant animal model, characterized in that, Includes the following steps: Step 1) Determine the level of immune rejection between heterologous cells, tissues, or organs using the method described in any one of claims 1 to 11; Step 2) When the level of the above-mentioned immune rejection response is within a reasonable range, construct the xenograft animal model.

16. The construction method according to claim 15, characterized in that, The xenograft animal model is a humanized rat model of macrophages, and the construction method includes the following steps: Step 1) Determine the level of immune rejection between human cells and rat macrophages using the method described in any one of claims 1 to 11; Step 2) When the above-mentioned immune rejection level is not greater than 1, the human SIRPA, SLAMF3 or SLAMF4 sequence is knocked into the rat genome to construct the humanized rat model.

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