Method for judging level of immunological rejection among heterologous cells, tissues or organs

Abstract

The invention provides a method for judging the level of immunological rejection among heterologous cells, tissues or organs, and relates to the field of biological medicine. According to the method provided by the invention, the interspecific relative phagocytic rates of the phagocytes can be quickly and objectively compared in a single experiment, and a new way is provided for evaluating and researching the immunological rejection level among heterologous cells, tissues or organs.

Description

Technical Field

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

[0002] Immune rejection (or immune rejection reaction) refers to the process in which the recipient's immune system identifies the transplant as "foreign" after an individual receives an organ, tissue, or cell transplant from another individual, thereby activating a series of immune responses in an attempt to attack and eliminate the transplant. Immune rejection is a huge challenge faced by allogeneic cell, tissue, and organ transplantation. The first thing donor cells face is phagocytosis and elimination by the host's innate immune system. Taking rodents as an example, even in Rag1 - / - 、Rag2 - / - 、Il2rg - / - In immunodeficient strains, innate immunity is still very active. Therefore, before conducting transplantation therapy or in vivo experiments, it is necessary to establish a pilot experimental system that can reproduce the phagocyte-mediated immune rejection reaction in vitro.

[0003] Current traditional methods for evaluating immune rejection have numerous shortcomings. For example, evidence from traditional phagocytosis experiments is largely derived from independent cultures, resulting in significant batch-to-batch variability and inability to directly compare. Existing studies often report "single-source allogeneic phagocytosis rates," making cross-sectional comparisons across different experimental conditions difficult. Sectioning / microscopy requires fixation and staining, making it cumbersome and low-throughput. Methods that pre-create in vivo models are time-consuming and mouse-intensive. Existing protocols are often customized for a single species or cell type. Traditional endpoint assays cannot capture phagocytosis rates, among other issues. Consequently, developing novel methods for assessing or diagnosing interspecies immune rejection has become an important approach to addressing these challenges. Summary of the Invention

[0004] Technical issues solved:

[0005] The first aspect of the present disclosure is to address the above-mentioned shortcomings in the prior art and provide a method for determining the level of immune rejection reaction between heterologous cells, tissues or organs.

[0006] Technical solution:

[0007] A method for determining the level of immune rejection reaction between heterologous cells, tissues or organs comprises the following steps:

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

[0009] Step 2) obtaining cells to be tested from individual B;

[0010] Step 3) obtaining an analyte of individual A, wherein the analyte of individual A is a cell or detectable particle to be tested of individual A;

[0011] Step 4) The analyte of individual A and the test cells of individual B are simultaneously contacted with the phagocytes of individual A at a ratio of 1:1:1;

[0012] Step 5) Detecting the number of cells to be tested in individual B after contact, and calculating the heterologous phagocytosis rate according to the formula: number of cells to be tested in individual B after contact / number of phagocytes in individual A;

[0013] Step 6) Detecting the amount of the analyte in individual A after contact, and calculating the homologous phagocytosis rate according to the formula: amount of the analyte in individual A after contact / number of phagocytes in individual A;

[0014] Step 7) Using the heterologous phagocytosis rate and homologous phagocytosis rate obtained in steps 5) and 6), a relative phagocytosis rate is calculated according to the formula: heterologous phagocytosis rate / homologous phagocytosis rate. When the relative phagocytosis rate is less than or equal to 1, it indicates that there is no immune rejection reaction between the heterologous 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 ratio of the mixture 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 the group consisting of allogeneic transplanted cells, allogeneic tumor cells, apoptotic or necrotic allogeneic cells, artificial or experimental allogeneic cells, and pathogenic microorganisms. When two or more of the above test cells are mixed, the mixing ratio can be arbitrarily set as needed. In a more specific embodiment, the test cells can be allogeneic transplanted 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 cells to be tested of individual B can be detected, using methods known in the art. In other embodiments, the analyte of individual A and the cells to be tested of individual B can carry distinguishable detection markers. In a more specific embodiment, the distinguishable detection marker can be a fluorescent marker. In other embodiments, the detection can be performed using flow cytometry.

[0018] In some embodiments, the above-mentioned contacting can be carried out in the same space or in different spaces, as long as the starting time of the contacting is the same. However, in order to better achieve the purpose of the present disclosure, in other embodiments, the above-mentioned contacting is carried out in the same container.

[0019] In some embodiments, the contact time is at least about 10 min to about 300 min. For example, at least about 10 min, about 20 min, about 30 min, about 40 min, about 50 min, about 60 min, about 70 min, about 80 min, about 90 min, about 100 min, about 110 min, about 120 min, about 130 min, about 140 min, about 150 min, about 160 min, about 170 min, about 180 min, about 190 min, about 200 min, about 210 min, about 220 min, about 230 min, about 240 min, about 250 min, about 260 min, about 270 min, about 280 min, about 290 min or about 300 min. In some specific embodiments, the contact time is about 120 to 300 min. In a more specific embodiment, the contact time is about 120 min. In other embodiments, the contact time can be longer, 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] A second aspect of the present disclosure provides an application of a relative phagocytosis rate in a scenario where:

[0021] (1) Verification of the effectiveness of gene editing;

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

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

[0024] (4) Preliminary evaluation of humanized animal models;

[0025] (5) Screening of genetic modification routes for xenotransplantation; or

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

[0027] A second aspect of the present disclosure provides a rapid relative phagocytosis rate determination kit for use in determining the level of immune rejection between heterologous cells, tissues, or organs. The rapid relative phagocytosis rate determination kit comprises:

[0028] i) a marker or detectable particle of the cells to be tested in individual A; and

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

[0030] The third aspect of the present disclosure provides an application of a rapid relative phagocytosis rate determination kit in a scenario, wherein the rapid relative phagocytosis rate determination kit comprises:

[0031] i) a marker or detectable particle of the cells to be tested in individual A; and

[0032] ii) markers of cells to be tested in individual B,

[0033] The scenario is:

[0034] (1) Verification of the effectiveness of gene editing;

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

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

[0037] (4) Preliminary evaluation of humanized animal models;

[0038] (5) Screening of genetic modification routes for xenotransplantation; or

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

[0040] The fourth aspect of the present disclosure provides a method for constructing a heterologous transplantation animal model, comprising the following steps:

[0041] Step 1) using the above method to determine the level of immune rejection reaction between allogeneic cells, tissues or organs;

[0042] Step 2) When the above immune rejection reaction level is within a reasonable range, constructing the xenotransplantation animal model.

[0043] In some more specific embodiments, the xenotransplantation animal model is a humanized rat model, and the construction method comprises the following steps:

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

[0045] Step 2) When the above immune rejection level is no 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 paper designs and establishes a method for detecting and reducing the level of immune rejection between heterologous cells, tissues or organs, which has significant advantages such as strong 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 verification of new immune regulation strategies. Specifically,

[0048] 1) "One-well dual-label or multi-label" internal control system: The method disclosed herein adds homologous and heterologous test cells or analytes to the same phagocyte population at the same time. The phagocytes themselves serve as "internal controls," eliminating inter-batch differences and reducing the coefficient of variation. A single experiment can produce highly comparable data.

[0049] 2) Normalization index: The method disclosed in the present invention provides a real-time calibration baseline by using homologous test cells or detectable particles. The calculated ratio can offset the differences in sample concentration and staining efficiency, thereby improving the reproducibility of the results and facilitating direct comparison between different laboratories or different animal strains.

[0050] 3) Fast high-throughput flow plate reading: The method disclosed herein can be performed using flow cytometry, which can detect 50,000 to 100,000 cells per minute, significantly increasing the speed.

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

[0052] 5) Universal and scalable platform: The method disclosed herein can be used to test different donor cells (e.g., iPSCs, organoids, or tumor cells) and different recipient phagocytes simply by replacing the fluorescent probes used to label the cells.

[0053] 6) Real-time kinetics is scalable: The method disclosed in the present invention can set multiple time points for on-machine reading, and can obtain and output a complete phagocytic kinetic curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 An example of a gating strategy for flow cytometry analysis in an embodiment of the present disclosure is shown in FIG. Figure 1 ;

[0055] Figure 2 An example of a gating strategy for flow cytometry analysis in an embodiment of the present disclosure is shown in FIG. Figure 2 ;

[0056] Figure 3 An example of a gating strategy for flow cytometry analysis in an embodiment of the present disclosure is shown in FIG. 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 the present disclosure;

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

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

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

[0061] Figure 8 This is a diagram showing the results of an in vitro macrophage phagocytosis experiment in an embodiment of the present disclosure. DETAILED DESCRIPTION

[0062] The present disclosure provides a method for determining the level of immune rejection reaction between heterologous cells, tissues or organs. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. It is obvious that relevant persons can modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0063] In this disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "comprise" or its variations such as "comprises" or "comprising" will be understood to include the elements or components stated without excluding other elements or other components. The terms "a", "an", and "the" include plural referents. The term "multiple" refers to 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 is understood that the endpoints are included in the range and that each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the stated range and any smaller range between the specified values ​​are encompassed unless the context clearly dictates otherwise.

[0065] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below the specified value, for example, 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 the specified value.

[0066] Throughout this disclosure, references to "one embodiment," "an example," "some embodiments," "specific embodiments," "related embodiments," "an example," "some examples," "additional embodiments," or "further embodiments," "further implementations," or "another embodiment," "other examples" mean that at least one feature or characteristic description is included in connection with an embodiment. Thus, references to these phrases in various places throughout this disclosure are not necessarily referring to the same embodiment. Furthermore, particular features may be combined in any suitable manner in one or more embodiments.

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

[0068] Unless otherwise specified, the experimental techniques herein employ conventional techniques of 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; Cell Biology: A Laboratory Handbook, etc.

[0069] definition :

[0070] The term "heterologous" in this disclosure refers to cells, tissues, or organs that are foreign and biologically different from a specific cell, tissue, or organ. In some embodiments, they can be cells, tissues, or organs from a xenogeneic or allogeneic source. For example, human cells are heterologous cells relative to mouse cells; and hematopoietic stem cells from a normal donor, A, are heterologous cells relative to hematopoietic stem cells from recipient patient B. In some embodiments, heterologous cells can be allogeneic transplant cells, allogeneic tumor cells, apoptotic or necrotic allogeneic cells, artificial or experimental allogeneic cells, or pathogenic microorganisms. In other embodiments, heterologous cells can also be a mixture of the above cells. Examples of allogeneic transplant 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, myocardial progenitor cells, hepatocytes, and the like. 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 system cancer cells, gynecological system cancer cells, endocrine system cancer cells, cancer cells of unknown primary site or metastatic cancer, soft tissue and bone sarcoma cells, mesothelioma cells, melanoma cells, central nervous system tumor cells, lymphoma cells, leukemia cells, peritoneal cancer cells, immunosuppression-related malignancies and / or metastatic cancer cells, etc. In some embodiments, the heterologous cells can be naturally unmodified cells obtained by isolation, enrichment, or culture; or they can be engineered cells. For example, in other embodiments, the heterologous cells can be induced pluripotent stem cell-derived heterologous cells (iPSC-derived), heterologous cells designed by synthetic biology, humanized animal chimeric cells, chimeric antigen receptor T cells, or chimeric antigen receptor NK cells, etc. The "individual" described in this disclosure refers to the above-mentioned heterologous individuals or homologous individuals.

[0071] The term "immune rejection" or "immune rejection reaction" as used herein refers to a pathological process in which, after an allogeneic or xenogeneic transplant (e.g., organ, tissue, cell) is implanted or transfused into a recipient (host), the recipient's immune system recognizes the antigens (e.g., major histocompatibility complex antigens) expressed by the transplant as non-self, thereby activating a specific immune response and producing effector T lymphocytes (e.g., cytotoxic T cells), natural killer cells, and / or antibodies (e.g., anti-HLA antibodies) against the transplant. This pathological process results in inflammatory infiltration, parenchymal cell damage, vascular destruction, and functional loss of the transplant through mechanisms such as cytotoxicity, release of inflammatory factors, and complement activation.

[0072] The term "phagocytes" as used herein refers to key effector cells in immune rejection reactions. They participate in the attack on transplants through direct phagocytosis, release of inflammatory mediators, and antigen presentation. In this disclosure, the level of immune rejection is measured by the relative phagocytic rate of allogeneic cells by phagocytes. The phagocytes referred to herein include macrophages, neutrophils, dendritic cells, and combinations thereof. Macrophages are core cells that primarily participate in hyperacute, acute, and chronic rejection, mediating immune rejection reactions through direct phagocytosis, antigen presentation, release of inflammatory mediators, tissue damage, and promotion of fibrosis. Neutrophils primarily participate in hyperacute and early acute rejection, mediating immune rejection reactions 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, the phagocytes used are macrophages.

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

[0074] The term "contact" in this disclosure refers to allowing two or more substances to interact, for example, through chemical interactions, including ionic, non-ionic, polar, hydrophobic, or hydrophilic interactions, or through physical contact as recognized in the art. In some embodiments, the analyte of individual A and the test cells of individual B are simultaneously contacted with the phagocytes of individual A. Such contact causes 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 a cell culture medium, a buffer, 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 space / container can be any suitable space / container capable of holding cells, microparticles, liquids, etc., including, but not limited to, a cell culture dish, a cell culture plate with holes, etc. To better achieve the purposes of this disclosure, in a specific embodiment, the contact occurs in the same cell culture well.

[0075] Example:

[0076] In order to enable those skilled in the art to better understand the technical solutions of the present disclosure, the technical solutions of the present disclosure are further described in detail below in conjunction with specific embodiments.

[0077] In this example, human red blood cells and rat macrophages were used as two different heterologous cells to evaluate the level of immune rejection between them. It is foreseeable that, based on the core concept of this example, 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 human red blood cells with other heterologous cells or combinations of heterologous cells; replacing the macrophages with other phagocytes or combinations of phagocytes; replacing the rats with cells from other species; using different labeling strategies and corresponding detection methods, etc.

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

[0079] By allowing macrophages to simultaneously encounter both "internal" and "foreign" cells in a completely identical culture environment, and then using flow cytometry to measure the frequency of these two interactions, experimental errors can be minimized and the "strength of xenogeneic rejection" can be converted into a mathematically quantifiable ratio.

[0080] Source and adherent purification of macrophages:

[0081] If phagocytic behavior is directly compared between different rats, differences in genetic background will inevitably amplify experimental noise. Therefore, to further address the above issues, in this example, peritoneal lavage fluid from the same rat was first selected as the original cell pool. After red blood cell lysis, all cells were placed in RPMI-1640 culture medium for short-term adherence; macrophages have sufficient adhesion ability to firmly adhere within 2 hours, and the remaining cells are gently washed away. This step is not just about "obtaining macrophages", but more importantly, it constructs a unified host innate immune background, providing a solid reference system for subsequent comparisons.

[0082] Necessity and concentration design of dual fluorescent labeling:

[0083] If homologous and heterologous RBCs are incubated separately, any differences in sample loading time, slight fluctuations in cell count, or even differences in well batches will be amplified in the final reading. Therefore, to further address these issues, this example places both types of RBCs in the same well. To enable the flow cytometer to simultaneously identify their origins in a single event, the staining methods used in this example are BV421 and Deep Red, respectively, which have minimal spectral overlap. Antibody concentrations are not arbitrarily chosen but confirmed through preliminary titration.

[0084] Time and space settings for "one-well dual-label" co-culture:

[0085] The recognition and phagocytosis of target cells by macrophages require the simultaneous fulfillment of two kinetic processes: receptor-ligand binding and membrane rearrangement. If the incubation time is too short, the readings will not be sufficient to distinguish between transformation and non-transformation; if it is too long, secondary phagocytosis or exocytosis will blur the difference. Therefore, to further address the above issues, in this example, after a 120-300 min gradient experiment, the 120 min time point was selected as the standard time because it took into account both signal-to-noise ratio and operational convenience. Furthermore, to reduce the random start-up differences in phagocytic rate, macrophages underwent an additional 2 h of serum-free "starvation" treatment before the addition of red blood cells to synchronize their endogenous phagocytic activity. Refined control of time and pretreatment is the key to compressing biological fluctuations into a statistically controllable range.

[0086] Gating strategies for flow cytometry analysis (e.g. Figure 1 、 Figure 2 、 Figure 3 shown):

[0087] When collecting flow cytometry data, first use FSC-H / FSC-A to exclude cell adhesion, then use Propidium Iodide to stain negative CD163 +The FITC group demarcates the macrophage population. Only events that enter this gate and test positive for BV421, Deep Red, or both are counted as "phagocytosed." This three-step screening ensures: ① the subject is indeed a macrophage, not an RBC adhered to its surface; ② the fluorescence originates within the phagosome, not free dye; and ③ it is clear whether the same cell can simultaneously phagocytize both types of RBCs. Using two-dimensional gating by area and length, a progressive gating strategy can clearly separate "phagocytosis" from "adhesion."

[0088] Statistical significance of normalized indicators:

[0089] The final metric obtained in this example is not the heterologous phagocytosis rate, but rather the calculated relative phagocytosis rate (heterologous phagocytosis rate / homologous phagocytosis rate). This is based on the assumption that the total number of macrophages in a well, the culture medium environment, and the residual dye influence both the numerator and denominator, thereby canceling them out in the ratio. This ratiometric process eliminates any "non-differential noise," making the experimental results highly comparable and reproducible.

[0090] Application scenario examples:

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

[0092] When creating gene-edited animals for genes like SIRPα or CD47, it's crucial to quickly confirm whether the modification truly reduces macrophage phagocytosis. The technical solution in this example uses a "one-well, dual-label" flow cytometry method to provide a quantitative metric, "heterologous phagocytosis rate divided by homologous phagocytosis rate," within 24 hours, helping researchers decide whether to proceed with in vivo experiments.

[0093] (2) Cell / Organoid Therapy Batch Quality Control (QC):

[0094] GMP production facilities at cell therapy companies often face significant compatibility differences between production batches. Co-culturing each batch of iPSC-derived cells or organoids with standardized host macrophages allows for rapid screening of phagocytosis-sensitive batches, which can be used as a basis for release or rework, ensuring product consistency.

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

[0096] Innovative drug companies and CROs need to discover molecules that can inhibit macrophage phagocytosis at an early stage. The technical solution in this embodiment uses a 96-well flow cytometry format, which can complete the IC of hundreds of compounds within a day. 50 Measurement and kinetic curve drawing significantly reduce the time and cost required for high-content imaging.

[0097] (4) Preliminary evaluation of humanized animal models:

[0098] In humanized mouse / rat projects, direct in vivo modeling is expensive and time-consuming. Using in vitro phagocytosis metrics to first identify recipient-donor combinations with "low phagocytosis" before proceeding with humanized modeling can save 60-80% of the number of animals and time.

[0099] (5) Screening of genetic modification routes for xenotransplantation:

[0100] When developing transgenic pig or primate organs for human transplantation, companies need to verify the innate immune compatibility of organ cells with human macrophages. Simply by replacing red blood cells with hepatocytes or pancreatic islet cell fragments, multiple gene editing strategies can be compared in parallel to quickly identify the optimal approach.

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

[0102] When developing CAR-Macs, immune cell therapy companies need to distinguish between "targeted phagocytosis" and "background phagocytosis." By simultaneously adding target cells (red) and non-target cells (green), the improved specificity ratio of CAR-Macs 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, beakers, anesthetic isoflurane, and other items for anesthesia; pre-cooled 4°C centrifuge;

[0106] (2) Anesthetize and kill the rat: Place the rat in a specimen bottle, add 2-3 mL of isoflurane, and wait for 6-7 minutes until the rat suffocates to death;

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

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

[0109] (5) Cut a small hole in the abdominal wall, tilt the animal slightly to one side, and aspirate the fluid with a syringe;

[0110] (6) Centrifuge at 500g for 5 minutes at 4°C and discard the supernatant;

[0111] (7) Add 3 to 5 times the volume of the cells to the red blood cell lysis buffer, gently blow to mix, and lyse for 1 to 2 minutes. For example, if the volume of the cell pellet is 1 ml, add 3 to 5 ml of red blood cell lysis buffer. Operate at room temperature; centrifuge at 400 to 500 g for 5 minutes and discard the red supernatant. Centrifugation at 4°C is more effective; if the red blood cell lysis is found to be incomplete, repeat the above steps once. Usually, a very small amount 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 4°C, 500g for 5 minutes each time, and discard the supernatant;

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

[0114] (10) Cells were plated at 1×10 6 Macrophages were seeded into 12-well plates, 1 mL of RPMI 1640 + 10% FBS + 1% PS was added, and cultured for 2 hours;

[0115] 2. Macrophage starvation preconditioning.

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

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

[0118] 3. Co-culture and phagocytosis.

[0119] (1) Separate human and rat erythrocytes in advance and stain them with hCD235a-BV421 and Deep Red, respectively;

[0120] (2) After starvation treatment, the stained phagocytic cells were mixed with human erythrocytes: mouse erythrocytes: macrophages at a ratio of 1:1:1 and added to the adherent macrophages. The cells were shaken at 37°C and 30 rpm for 2 hours.

[0121] 4. Dyeing.

[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 the cells. Blow off the cells and transfer them to a 15 mL centrifuge tube. Filter them with a 40 μm filter and centrifuge at 400 g for 5 minutes at 4°C. Resuspend the cells with 1 mL of FACS Buffer and centrifuge again.

[0123] (2) After discarding the supernatant, stain with anti-rat CD163-FITC and incubate on ice for 30 min. Wash off excess antibody and load on flow cytometry. Cell viability is assessed using 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, the cell population that is PI-negative and CD163-FITC-positive is selected and defined as the active macrophage population ( Figure 2 ).

[0127] (3) In this population, BV421-positive cells represent macrophages that phagocytize human red blood cells, reflecting their xenophagic phagocytic ability; Deep Red-positive cells represent macrophages that phagocytize rat red blood cells, indicating their allogeneic phagocytic ability ( Figure 3 ).

[0128] (4) The formula for calculating the relative phagocytic rate is: relative phagocytic rate = heterologous phagocytic rate / homologous phagocytic rate. The calculated relative phagocytic rate of wild-type rat macrophages in vitro was 3.1902 ± 2.0419.

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

[0130] The experiment was carried out in the same manner as in Example 1, except that ① human erythrocytes were treated with pHrodo TM ② The rat erythrocytes were replaced with 2 μm green fluorescent microbeads that would not be phagocytosed as a sampling reference (no staining required).

[0131] In this embodiment, pHrodo TM After entering the acidic phagosome, it spontaneously luminesces, naturally distinguishing between "engulfed" and "unengulfed", so only a single-color dye is needed to be applied to the heterologous red blood cells. After 120 minutes of co-culture, the "pHrodo⁺ macrophage number ÷ microbead number" is read out by flow cytometry as the normalized indicator. This method uses a one-step staining and single fluorescence detection method, which minimizes instrument channel usage; pHrodo TM Automatically eliminate "sticky false positives".

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

[0133] The experiment was performed using the same method as in Example 1, except that ① both homologous and heterologous RBCs were stained with the same concentration of FITC; ② 0.2% Trypan Blue was added for 5 minutes after co-culture and the cells were immediately loaded onto the instrument; ③ F4 / 80–APC circle macrophages were identified as phagocytic events by FITC⁺; and ④ APC-Cy7 microbeads were added to the wells to calibrate the collection volume.

[0134] In this example, Trypan Blue quenches extracellular FITC but cannot penetrate intact cell membranes; engulfed RBCs reside within the phagosome, preserving the FITC signal. This method requires only two channels, FITC and APC, and can be implemented on any mid-range flow cytometer, resulting in low cost.

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

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

[0137] In this example, HCS software is used to automatically identify macrophage outlines and intracellular fluorescent target areas, and DAPI is used to count macrophage nuclei as the denominator to obtain advanced parameters such as spatial distribution and cluster size, supporting real-time kinetic imaging.

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

[0139] The experiment was performed using the same method as in Example 1, except that homologous and heterologous RBCs were stained with pHrodoGreen / pHrodo Red, respectively. The phagocytosis kinetic curve was generated by continuously sampling within 15 to 240 minutes using the difference in acidification rate.

[0140] The method of this embodiment can obtain three types of information, namely, rate, maximum phagocytic amount and saturation time, in just one experiment; and is suitable for pharmacokinetic studies.

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

[0142] 1. Use CRISPR / Cas9-mediated genome engineering to create a SD rat model with a Kozak-HumanSIRPACDS-SV40latepA insert in the rat Sirpa locus. Figure 4As shown in the figure, the Sirpa gene located on rat chromosome 3 has 8 exons, with the ATG start codon located in exon 1 and the TGA stop codon located in exon 8. Exon 1 will be selected as the target site. For the KI rat model, the ATG start codon will be replaced by Kozak-Human SIRPACDS-SV40 late pA, so that the expression of Human SIRPA will be controlled by the rat Sirpa gene regulatory elements. Figure 5 The demonstration used DNA microinjection combined with CRISPR / Cas9 gene editing technology to co-inject in vitro-transcribed Cas9 mRNA, sgRNA, and a donor vector into fertilized eggs, which were then implanted into pseudopregnant female mice. F0 offspring (founder mice) were born, and the knock-in success rate was confirmed by PCR and DNA sequencing. Subsequently, the knock-in founder mice were mated with wild-type rats to produce F1 offspring, verifying the heritability of the knock-in gene. Successfully inherited F1 offspring were then mated with each other to expand the population for subsequent experiments.

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

[0144] 3. If Figure 6 As shown, qPCR detection of homozygous (HOMO), heterozygous (HETER) and wild-type (WT) rat macrophages showed that they had human SIRPA gene expression similar to that of 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 double heterozygous offspring. As can be seen from the figure, all the double homozygous offspring are homozygous, and human SIRPA is not lost with the increase of generations. 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 the wild type (e.g. Figure 8 The relative phagocytic rates of homozygotes were 0.7766±0.4119 and heterozygotes were 1.2338±0.6545.

[0147] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for determining the level of immune rejection reaction between heterologous cells, tissues or organs, characterized in that: The following steps are involved: Step 1) obtaining phagocytes from individual A; Step 2) obtaining cells to be tested from individual B; Step 3) obtaining an analyte of individual A, wherein the analyte of individual A is a cell or detectable particle to be tested of individual A; Step 4) The analyte of individual A and the test cells of individual B are simultaneously contacted with the phagocytes of individual A at a ratio of 1:1:1; Step 5) Detecting the number of cells to be tested in individual B after contact, and calculating the heterologous phagocytosis rate according to the formula: number of cells to be tested in individual B after contact / number of phagocytes in individual A; Step 6) Detecting the amount of the analyte in individual A after contact, and calculating the homologous phagocytosis rate according to the formula: amount of the analyte in individual A after contact / number of phagocytes in individual A; Step 7) Using the heterologous phagocytosis rate and homologous phagocytosis rate obtained in steps 5) and 6), the relative phagocytosis rate is calculated according to the formula: heterologous phagocytosis rate / homologous phagocytosis rate. When the relative phagocytosis rate is less than or equal to 1, it indicates that there is no immune rejection reaction between the heterologous cells.

2. The method according to claim 1, characterized in that The phagocytes are at least one selected from 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, wherein The cells to be tested are at least one selected from heterologous transplanted cells, heterologous tumor cells, apoptotic or necrotic heterologous cells, artificial or experimental heterologous cells, and pathogenic microorganisms.

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

6. The method according to claim 1, characterized in that The object to be tested of individual A and the cells to be tested of individual B carry distinguishable detection labels.

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

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 contacting is carried out in the same container.

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

11. The method according to claim 10, characterized in that The contact time was 120 min.

12. The application of relative phagocytosis rate in the scene is characterized by: The relative phagocytosis rate is obtained by the method according to any one of claims 1 to 11, wherein the scenario is: (1) Verification of the effectiveness of gene editing; (2) Batch quality control of cell / organoid therapies; (3) Screening of phagocytic inhibitor drugs; (4) Preliminary evaluation of humanized animal models; (5) Screening of genetic modification routes for xenotransplantation; or (6) Evaluation of CAR-Macrophage functional specificity.

13. Use 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 determination kit comprises: i) a marker or detectable particle of the cells to be tested in individual A; and ii) Markers of cells to be tested in individual B.

14. Application of a rapid relative phagocytosis rate determination kit in a scenario, characterized in that: The rapid relative phagocytosis rate determination kit comprises: i) a marker or detectable particle of the cells to be tested in individual A; and ii) markers of cells to be tested in individual B, The scenario is: (1) Verification of the effectiveness of gene editing; (2) Batch quality control of cell / organoid therapies; (3) Screening of phagocytic inhibitor drugs; (4) Preliminary evaluation of humanized animal models; (5) Screening of genetic modification routes for xenotransplantation; or (6) Evaluation of CAR-Macrophage functional specificity.

15. A method for constructing a heterologous transplantation animal model, characterized in that: The following steps are involved: Step 1) determining the level of immune rejection reaction between allogeneic cells, tissues or organs using the method according to any one of claims 1 to 11; Step 2) When the immune rejection reaction level is within a reasonable range, constructing the xenotransplantation animal model.

16. The construction method according to claim 15, characterized in that: The xenotransplantation animal model is a macrophage humanized rat model, and the construction method comprises the following steps: Step 1) determining the level of immune rejection between human cells and rat macrophages using the method according to any one of claims 1 to 11; Step 2) When the above immune rejection level is no 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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