Method for extracting primary cardiac endothelial cells of mice

Abstract

The invention discloses a mouse primary cardiac endothelial cell extraction method, and belongs to the technical field of biological cell extraction. The method comprises the following steps: step 1, obtaining pure myocardial tissues; 2, the treated heart tissue is transferred into an intelligent tissue grinding system to be ground; step 3, transferring the ground heart tissue into a temperature-controlled enzymolysis reactor, automatically monitoring the digestion process, and terminating the reaction when a preset digestion degree is reached; step 4, carrying out primary cell separation, carrying out magnetic separation by using anti-CD31 magnetic beads, and specifically enriching heart endothelial cells; 5, transferring the purified endothelial cells to an intelligent microenvironment culture system, and automatically optimizing a culture medium replacement period to adapt to a cell metabolism state; and step 6, performing multi-dimensional quality evaluation on the endothelial cells, wherein the multi-dimensional quality evaluation comprises marker expression analysis, in-vitro lumen formation experiment, AI-assisted morphological analysis and digital quality scoring.

Description

Technical Field

[0001] The present application relates to the technical field of biological cell extraction, and more specifically, to a method for extracting primary mouse cardiac endothelial cells. Background Art

[0002] Mouse primary cardiac endothelial cells (CEs) hold significant application value in cardiovascular disease research. As a crucial component of the vascular wall, CEs play a key role in maintaining vascular permeability, regulating blood flow, endothelial function, and cardiovascular pathology. By obtaining high-quality CEs, researchers can better understand the mechanisms of cardiovascular disease, thereby advancing the development of novel drugs and therapeutic strategies.

[0003] However, current methods for extracting primary mouse cardiac endothelial cells generally have certain limitations, mainly manifested in problems such as cell damage, low purity, and cell dysfunction during the cell extraction process. Traditional cell extraction methods generally use methods such as manual separation, mechanical grinding, or enzymatic hydrolysis reactions. These methods are prone to cell damage during operation and make it difficult to ensure high cell purity and biological properties. In addition, existing cell culture environments are usually unable to adapt to the metabolic needs of cells in real time, resulting in unstable cell growth and further affecting the evaluation of cell function.

[0004] With the development of technology, the introduction of automated and intelligent systems has provided new directions for cell extraction and culture. Intelligent equipment can effectively improve the efficiency and quality of cell extraction and culture by precisely controlling environmental variables, monitoring cell growth status, and dynamically adjusting culture conditions during the cell extraction process. However, existing intelligent cell extraction systems still suffer from problems such as insufficient precision, complex operation, and high costs. Furthermore, they lack a complete technical system for multi-dimensional quality assessment, which cannot meet the needs of high-quality cell preparation.

[0005] In summary, how to achieve accurate and efficient extraction of primary mouse cardiac endothelial cells through intelligent technology and ensure the high purity, integrity and biological function of cells during the extraction process has become a technical problem that needs to be solved urgently. Summary of the Invention

[0006] In order to overcome the defects of the prior art, the present invention aims to provide a method for extracting primary mouse cardiac endothelial cells, comprising the following steps:

[0007] Step 1: obtaining pure myocardial tissue;

[0008] Step 2: transferring the processed heart tissue to the intelligent tissue grinding system for grinding;

[0009] Step 3: The ground heart tissue is transferred to a temperature-controlled enzymatic hydrolysis reactor, which automatically monitors the digestion process and terminates the reaction when the preset digestion level is reached.

[0010] Step 4: Perform preliminary cell separation using magnetic sorting with anti-CD31 magnetic beads to specifically enrich cardiac endothelial cells;

[0011] Step 5: Transfer the purified endothelial cells to the intelligent microenvironment culture system, which automatically optimizes the culture medium replacement cycle to adapt to the cell metabolic state;

[0012] In step 6, a multi-dimensional quality assessment of endothelial cells was performed, including marker expression analysis, in vitro lumen formation experiment, AI-assisted morphological analysis, and digital quality scoring.

[0013] Furthermore, step 1 includes the following steps:

[0014] Prepare a sterile operating table, disinfect the table and turn on the ultraviolet light for 30 minutes;

[0015] Pre-cool the D-Hanks buffer in a 4°C refrigerator and place it in an ice box to keep it low.

[0016] The anesthetized mouse was fixed on the operating table, the chest and abdominal skin was disinfected with 75% alcohol, the abdomen was incised and the chest cavity was opened, and the heart was quickly removed;

[0017] The removed heart was immediately placed in pre-cooled D-Hanks buffer and gently squeezed repeatedly to remove any remaining blood;

[0018] Under a dissecting microscope, use fine forceps to peel off the pericardium and remove attached fat tissue, connective tissue, and vascular stumps;

[0019] The processed heart was rinsed in new pre-cooled D-Hanks buffer to further remove residual tissue debris and obtain pure myocardial tissue.

[0020] Furthermore, step 2 includes the following steps:

[0021] Use sterile forceps to transfer the myocardial tissue sample to the grinding chamber of the intelligent tissue grinding system and ensure that the tissue is evenly spread;

[0022] The grinding gap was set to 0.5 mm to ensure uniform grinding of the tissue and to preserve its original structure and biological properties to the greatest extent possible;

[0023] Set the grinding speed to 30 rpm to avoid excessive shearing or thermal damage and ensure the integrity of cell components;

[0024] A mixture of 95% oxygen and 5% carbon dioxide is introduced into the grinding system to simulate physiological environmental conditions to maintain tissue metabolic activity;

[0025] The grinding process is monitored throughout to ensure that the tissue is fully exposed and no unexpected damage occurs, providing high-quality samples for subsequent analysis.

[0026] Furthermore, the intelligent tissue grinding system includes the following features: the inner wall of the grinding chamber is made of medical-grade titanium alloy material, and the surface has nano-level flatness; it is equipped with a micro temperature sensor to monitor the temperature changes during the grinding process in real time, and automatically starts cooling when the temperature exceeds 37.5°C; a microporous filter with a pore size of 200μm is provided at the bottom of the grinding chamber to separate large tissue fragments during the grinding process; it has an automatic cleaning function, and uses sterile water and 75% ethanol for three cycles of cleaning after each grinding.

[0027] Furthermore, step 3 includes the following steps:

[0028] Select a sterile, disinfected enzymatic hydrolysis reactor and evenly transfer the ground heart tissue to the center of the reactor;

[0029] Use digestion solution pre-equilibrated to 37°C to ensure stable liquid temperature and composition and optimize enzymatic hydrolysis reaction;

[0030] Adjust the reactor stirring rate to 10 rpm to promote full contact between the digestive fluid and the tissue and avoid tissue shear damage;

[0031] The determination value of reaction termination is obtained by detecting the degree of protein degradation, peptide chain length distribution and specific biomarkers;

[0032] When the reaction termination criteria are reached, the sample is quickly cooled and fixed to maximize the retention of key molecular information and biological characteristics during the enzymatic hydrolysis process.

[0033] Furthermore, the enzymatic hydrolysis reactor has the following features: the reactor is equipped with multiple pH microelectrodes to monitor the pH changes during the digestion process in real time and maintain it within the range of 7.2-7.4; it is equipped with an automatic rehydration system, which automatically adds digestion fluid preheated to 37°C when the liquid level is detected to be lowering; the bottom is equipped with a micro magnetic stirring device, and the stirring speed can be automatically adjusted within the range of 5-15rpm; it has a real-time image acquisition system, which uses computer vision algorithms to analyze changes in tissue block size and evaluate the digestion progress.

[0034] Furthermore, step 4 includes the following steps:

[0035] Accurately prepare Ficoll gradients with a density range of 1.077-1.119 g / ml;

[0036] Overlay the enzymatically digested tissue suspension on the top layer of the Ficoll gradient to avoid stirring the interface;

[0037] Centrifuge at 400-500 g for 20-30 minutes to precisely separate cells of different densities;

[0038] The mononuclear cell population located in the middle layer of the Ficoll gradient was collected and stored at 4°C until use;

[0039] Mix the pretreated anti-CD31 magnetic beads with the target cells according to the ratio and incubate for 30-45 minutes to ensure that the magnetic beads bind to the CD31-positive endothelial cells;

[0040] CD31-positive cells were captured by magnetic separation columns to purify and collect the target cardiac endothelial cell population.

[0041] Furthermore, the anti-CD31 magnetic beads have the following characteristics: the diameter of the magnetic beads is 2.8±0.2 microns; the concentration of the anti-CD31 antibody coated on the surface of the magnetic beads is 15-20 micrograms / ml; the mixing ratio of magnetic beads to cells is 20-50 magnetic beads / target cell; the magnetic beads are stored at 2-8°C and thoroughly mixed before use; the magnetic beads are superparamagnetic and stable for not less than 6 months.

[0042] Furthermore, step 5 includes the following steps:

[0043] Select a sterile and pre-treated microenvironment culture system to ensure that its internal surfaces are thoroughly disinfected to provide an ideal contamination-free environment for cell culture;

[0044] Using a precision pipette, the purified endothelial cells were evenly seeded on the pretreated culture surface, with the cell density precisely controlled between 3000 and 5000 cells / cm2;

[0045] The culture system temperature was set at 37°C and the temperature fluctuation was strictly controlled within ±0.5°C to simulate the physiological temperature conditions of the human body and ensure the stability of cell metabolic activity and growth state;

[0046] Adjust the carbon dioxide concentration in the culture system to 5% and maintain the humidity above 95% to simulate a microenvironment close to physiological conditions and promote normal cell growth and functional expression;

[0047] The system monitors the metabolic status, nutrient consumption, and metabolite accumulation of cells in real time, and intelligently adjusts the replacement frequency and composition of the culture medium based on changes in cell morphology, growth rate, and biomarkers to ensure that endothelial cells are always in optimal growth conditions.

[0048] Furthermore, step 6 includes the following steps:

[0049] Immunofluorescence combined with flow cytometry technology is used to accurately analyze the expression of key endothelial cell markers and evaluate cell purity and functional characteristics;

[0050] The cells were seeded on Matrigel to observe their ability to self-assemble into tubular structures, and their angiogenic potential was quantitatively assessed by measuring the length of the tubular lumen, the number of branches, and the number of junctions.

[0051] Capture high-resolution images of cells and automatically identify and quantify cell morphological characteristics, including cell area, perimeter, roundness, and shape complexity parameters;

[0052] Objective and quantitative quality assessment of endothelial cells is performed by integrating marker expression levels, lumen formation ability, and morphological characteristics data;

[0053] Conduct functional validation experiments, including endothelial cell migration and lumen repair experiments, to further assess the biological functional integrity of cells by tracking cell movement trajectories and wound healing rates;

[0054] The multi-dimensional evaluation results are presented through a visual interface to generate a detailed quality report, providing a comprehensive and accurate cell quality reference for subsequent experiments and applications.

[0055] Furthermore, functional verification experiments were conducted, including endothelial cell migration and lumen repair experiments. By tracking cell movement trajectories and wound healing rates, the biological functional integrity of the cells was further evaluated. The following steps were included:

[0056] Select appropriate culture medium and seed endothelial cells into culture plates at an appropriate density. Start the experiment after the cells adhere to the wall and grow to 80%-90% confluence.

[0057] Use a 200 μl sterile pipette tip to make a uniformly wide scratch on the cell monolayer and slowly wash with PBS three times to remove detached cells. Then, replace with fresh serum-free medium and take an initial image of the wound under a microscope as a starting reference for the experiment.

[0058] In a living cell workstation equipped with a carbon dioxide incubator, continuous imaging was performed to record the cell migration process under conditions maintained at 37°C and 5% carbon dioxide. Images were automatically collected every 2 hours for a period of 24-48 hours.

[0059] ImageJ software was used to analyze the wound area at different time points, calculate the wound healing rate, and simultaneously track the migration trajectory of individual cells to obtain cell migration speed and directionality parameters;

[0060] Endothelial cells were seeded into matrigel-coated culture plates, and the formation of tubular structures by the cells was observed and recorded. The number, length, and branch points of the tubular lumens were also counted.

[0061] Calculate the mean and standard deviation of each indicator, analyze the differences between different groups, and evaluate the overall biological function of cells.

[0062] Compared with the existing technology, this application has the following beneficial effects: This application integrates multiple automated steps, including intelligent tissue grinding, automatic monitoring of enzymatic reactions, magnetic sorting and microenvironment culture optimization, and combines multi-dimensional quality assessment methods to achieve efficient and accurate extraction and quality control of primary mouse cardiac endothelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a schematic diagram of the process of extracting primary mouse cardiac endothelial cells disclosed in the examples of this application. DETAILED DESCRIPTION

[0064] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Throughout the drawings, identical or similar reference numerals represent identical or similar elements or elements having identical or similar functions. The described embodiments are only some, not all, of the embodiments of the present invention.

[0065] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0066] The embodiments and directional terms described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0067] like Figure 1 As shown, a method for extracting primary mouse cardiac endothelial cells comprises the following steps:

[0068] Step 1: obtaining pure myocardial tissue;

[0069] Step 2: transferring the processed heart tissue to the intelligent tissue grinding system for grinding;

[0070] Step 3: The ground heart tissue is transferred to a temperature-controlled enzymatic hydrolysis reactor, which automatically monitors the digestion process and terminates the reaction when the preset digestion level is reached.

[0071] Step 4: Perform preliminary cell separation using magnetic sorting with anti-CD31 magnetic beads to specifically enrich cardiac endothelial cells;

[0072] Step 5: Transfer the purified endothelial cells to the intelligent microenvironment culture system, which automatically optimizes the culture medium replacement cycle to adapt to the cell metabolic state;

[0073] In step 6, a multi-dimensional quality assessment of endothelial cells was performed, including marker expression analysis, in vitro lumen formation experiment, AI-assisted morphological analysis, and digital quality scoring.

[0074] Step 1 is the basis of the entire cell extraction process and is crucial because the purity of the cardiac tissue directly affects the success rate and cell quality of subsequent cell extraction. First, during the mouse heart dissection process, it is necessary to ensure that the cardiac tissue is removed intact and to minimize contamination from surrounding tissues, such as fat and connective tissue. The tissue should be cut carefully to avoid excessive mechanical damage. After the tissue is collected, it needs to be stored quickly and at low temperatures to prevent degradation and denaturation of intracellular components. In order to improve the purity of the tissue, special disinfection methods and aseptic operations are recommended to ensure that the myocardial tissue is not affected by external sources of contamination. In addition, this process also requires precise processing based on the size and structure of the heart to ensure that each part of the heart tissue is fully collected to avoid missing any details that may affect cell extraction.

[0075] Step 2 is one of the key steps in cell separation. It uses mechanical and physical methods to break the heart tissue into small tissue fragments so that the cells can be better released. The advantage of using an intelligent tissue grinding system is that it can accurately control the speed and time of grinding to avoid excessive damage to tissue cells. The grinding program automatically adjusts the operating parameters according to different tissue types to ensure uniformity and fineness during the grinding process. In addition, the intelligent grinding system can also monitor the grinding process in real time, using built-in sensors to detect multiple parameters such as grinding temperature, humidity, particle size, etc. to ensure the accuracy and consistency of the grinding process. This automated and intelligent operation method greatly improves experimental efficiency, reduces human operation errors, and ensures that the ground heart tissue fragments can provide the best processing state for subsequent enzymatic hydrolysis reactions.

[0076] Step 3 is a key step in the extraction of cardiac endothelial cells. It effectively releases cells from cardiac tissue through temperature control and enzymatic hydrolysis. The reactor can accurately adjust the concentration, temperature and reaction time of the enzyme to control the degree of enzymatic hydrolysis. Different enzymes (such as collagenase, trypsin, etc.) can decompose the matrix in the tissue, allowing cardiac endothelial cells to be separated. The advantage of the temperature control system is that it can ensure that the enzymatic hydrolysis reaction is carried out at the most suitable temperature, avoiding damage to the cells caused by too high or too low temperatures. At the same time, the intelligent monitoring system in the reactor tracks the digestion process in real time, and dynamically adjusts the enzymatic hydrolysis conditions by sensing multiple indicators such as pH value and enzyme activity. Once the preset digestion degree is reached, the system will automatically terminate the reaction to ensure the optimization of cell quality. In addition, this system has a high degree of automation, which reduces errors caused by human intervention and ensures the reproducibility and consistency of the experiment.

[0077] After enzymatic hydrolysis, the cell suspension obtained in step 4 contains not only endothelial cells, but also other types of cells such as smooth muscle cells and fibroblasts. Therefore, magnetic sorting using anti-CD31 magnetic beads can specifically enrich cardiac endothelial cells. CD31 is a marker widely expressed on the surface of vascular endothelial cells. Its specificity enables the use of anti-CD31 magnetic beads for sorting to efficiently separate cardiac endothelial cells from a chaotic cell population. This magnetic sorting technology adds magnetic beads with anti-CD31 antibodies to the cell suspension, and the effect of the magnetic field enables the endothelial cells bound to the magnetic beads to be effectively separated from other cells. The advantage of this step is that it is efficient and rapid, and at the same time causes less damage to the cells, which helps to ensure the activity and function of the endothelial cells finally obtained.

[0078] After cell sorting, the purified cardiac endothelial cells are transferred to an intelligent microenvironment culture system for cultivation. This system automatically adjusts the composition and replacement cycle of the culture medium according to the cell growth requirements to adapt to the cell's metabolic state. Through built-in sensors and intelligent algorithms, this system can monitor the cell's growth status and metabolic needs in real time and automatically optimize culture conditions such as dissolved oxygen concentration, pH value, and temperature. Automatic replacement and addition of culture medium ensures that cells grow in optimal conditions, avoiding errors that may be caused by human operation. In addition, the system can dynamically adjust the cell growth environment, providing more precise and personalized culture conditions, thereby promoting the growth and proliferation of endothelial cells.

[0079] Finally, the quality assessment of endothelial cells is the last step to ensure the success of cell extraction. Quality assessment is carried out through multiple dimensions, including marker expression analysis, in vitro tube formation experiments, AI-assisted morphological analysis and digital quality scoring. First, the expression of endothelial cell surface markers, such as CD31 and VE-cadherin, is analyzed by flow cytometry and other technologies to confirm whether the cells are cardiac endothelial cells. Secondly, the in vitro tube formation experiment is an important means of evaluating endothelial cell function and can verify the angiogenesis ability of the cells. AI-assisted morphological analysis uses deep learning algorithms to evaluate the morphology of endothelial cells and further verify whether they have the biological characteristics of endothelial cells. The digital quality score comprehensively considers multiple factors such as cell purity, activity, and function to provide an objective and quantitative cell quality assessment. This multi-dimensional quality assessment system can comprehensively monitor the quality of endothelial cells and ensure that the cells finally extracted have good experimental application value.

[0080] In summary, this method for extracting primary mouse cardiac endothelial cells significantly improves the efficiency and precision of cell extraction by integrating intelligent equipment and automated control. Each step is optimized to address potential challenges encountered during the cell extraction process. From cardiac tissue acquisition to endothelial cell purification, and then to cell culture and quality assessment, the method demonstrates the advantages of high efficiency, precision, and controllability. This method is not only suitable for the extraction of cardiac endothelial cells but also provides a valuable framework for the isolation of other primary cell types.

[0081] Furthermore, step 1 includes the following steps:

[0082] Prepare a sterile operating table, disinfect the table top and turn on the ultraviolet light for 30 minutes. The use of a sterile operating table is to avoid possible microbial contamination in the external environment. During the table top disinfection process, 70% alcohol is usually used to thoroughly wipe the operating area to remove possible sources of contamination such as bacteria and fungi. Turning on the ultraviolet light further ensures that the microorganisms in the air in the experimental environment are effectively inactivated. Ultraviolet rays can kill bacteria and viruses in the air by destroying the DNA of microorganisms, reducing the risk of external contamination during anatomical operations. The 30-minute UV light exposure time is sufficient to ensure the cleanliness of the indoor environment. The implementation of this step not only protects the health of the operator, but also ensures the quality of the cells and reduces the chance of cell contamination.

[0083] Pre-cool the D-Hanks buffer in a 4°C refrigerator, and then put it in an ice box to keep it low. In this step, the D-Hanks buffer is first pre-cooled to 4°C. The purpose is to simulate the cooling environment of the cells in the body and reduce the metabolic stress and damage that may occur when the cells are exposed to high temperatures. The pre-cooling process is carried out by placing the buffer in a 4°C refrigerator to ensure that the buffer maintains a low temperature when it is taken out, which is crucial for maintaining cell activity. In the subsequent operation, the buffer is placed in an ice box to further maintain a low temperature state to ensure that the heart tissue and cells can be in a low-temperature protected state, thereby minimizing excessive cell metabolism or damage. The process of low-temperature preservation is also an effective means of preventing blood coagulation and cell death.

[0084] The anesthetized mouse is fixed on the operating table, the chest and abdominal skin is disinfected with 75% alcohol, the abdomen is cut open and the chest cavity is opened, and the heart is quickly removed. After the mouse is anesthetized with an appropriate anesthetic (such as isoflurane or ketamine), the mouse must first be fixed on the operating table to prevent it from having a violent reaction during the operation. In order to further prevent infection, the chest and abdominal skin of the mouse also needs to be disinfected before surgery. 75% alcohol is usually used for disinfection, which can quickly kill bacteria and eliminate surface contamination, ensuring that no external bacteria or viruses are introduced during the dissection process. Subsequently, a laparotomy is performed, the abdominal skin of the mouse is quickly and accurately cut open, and then the chest cavity is opened by dissection techniques to expose the heart. This process requires quick and accurate operation to avoid damage to the heart and other tissues caused by prolonged surgical operations.

[0085] The removed heart is immediately placed in pre-cooled D-Hanks buffer, and the remaining blood is removed by repeated gentle squeezing. Blood plays an important nutritional and protective role in cell extraction, but it also contains anticoagulants or other components, which will interfere with the subsequent cell extraction and separation process. Therefore, in this step, the heart is immediately placed in pre-cooled D-Hanks buffer after removal. The use of cooling fluid not only helps to protect cells, but also reduces the anticoagulant effect that may exist in the blood. After the heart is placed in the buffer, the blood remaining in the heart will be removed by gentle squeezing. The key to this step is to operate gently to avoid damage to the heart or endothelial cells due to excessive squeezing.

[0086] Under a dissecting microscope, fine forceps are used to peel away the pericardium, removing attached adipose tissue, connective tissue, and vascular stumps. Delicate manipulation under the dissecting microscope minimizes damage to cardiac cells and ensures effective removal of impurities from the heart's surface. The use of a dissecting microscope provides a magnified field of view, ensuring the operator can clearly see and remove the pericardium and other attached tissues, avoiding mishandling or omissions. Removal of these impurities leaves the heart's surface cleaner, and the purity of the extracted endothelial cells is correspondingly improved.

[0087] The processed heart is rinsed in new pre-cooled D-Hanks buffer to further remove residual tissue debris and obtain pure myocardial tissue. The purpose of this step is to further remove debris, cells and other impurities that may remain on the heart to ensure that pure myocardial tissue is obtained in the end. Rinsing in the buffer not only helps to remove physical debris, but also prevents cell residues from adversely affecting the cell separation process. Through sufficient rinsing, the final myocardial tissue will be cleaner and provide the best material for subsequent cell extraction and separation. Keeping the buffer at a low temperature is very important in this process because it helps to maintain the activity of the cells and prevents the cells from being damaged by environmental changes.

[0088] In summary, the above-mentioned operation process embodies the technical requirements of efficiency, precision, and compliance with biological principles. Every step has been meticulously designed and optimized, from the preparation of the sterile operating environment, to the precise manipulation during heart extraction, to the cooling and flushing of cells. All steps work together to ensure the final myocardial tissue is of high purity and viability. This series of operations not only minimizes the risk of cell damage but also improves the success rate of cell extraction, laying a solid foundation for subsequent cell isolation, culture, and experimental research.

[0089] Furthermore, step 2 includes the following steps:

[0090] Use sterile forceps to transfer the myocardial tissue sample to the grinding chamber of the intelligent tissue grinding system and ensure that the tissue is evenly spread;

[0091] The grinding gap was set to 0.5 mm to ensure uniform grinding of the tissue and to preserve its original structure and biological properties to the greatest extent possible;

[0092] Set the grinding speed to 30 rpm to avoid excessive shearing or thermal damage and ensure the integrity of cell components;

[0093] A mixture of 95% oxygen and 5% carbon dioxide is introduced into the grinding system to simulate physiological environmental conditions to maintain tissue metabolic activity;

[0094] The grinding process is monitored throughout to ensure that the tissue is fully exposed and no unexpected damage occurs, providing high-quality samples for subsequent analysis.

[0095] In summary, the operational details of the above steps demonstrate the important role of modern intelligent biological experimental equipment in precise control and monitoring. By reasonably setting the grinding gap, grinding speed, and simulating the gas conditions of the physiological environment, the intelligent tissue grinding system can ensure the uniformity of tissue grinding and the integrity of cellular components to the greatest extent. The application of these technical means not only ensures the integrity and biological characteristics of myocardial tissue during the grinding process, but also provides high-quality samples for subsequent cell separation and analysis. Real-time monitoring throughout the process provides additional protection for the grinding process and avoids any unexpected damage. Through this series of delicate operations, the laboratory can maximize the activity and availability of cells, laying a solid foundation for subsequent research and experiments.

[0096] Furthermore, the intelligent tissue grinding system includes the following features: the inner wall of the grinding chamber is made of medical-grade titanium alloy material, and the surface has nano-level flatness; it is equipped with a micro temperature sensor to monitor the temperature changes during the grinding process in real time, and automatically starts cooling when the temperature exceeds 37.5°C; a microporous filter with a pore size of 200μm is provided at the bottom of the grinding chamber to separate large tissue fragments during the grinding process; it has an automatic cleaning function, and uses sterile water and 75% ethanol for three cycles of cleaning after each grinding.

[0097] In summary, the intelligent tissue grinding system ensures the high efficiency of the tissue grinding process and the high quality of cell samples. The inner wall of the grinding chamber is made of medical-grade titanium alloy, which ensures the sterile processing of biological samples and improves the durability of the equipment; the micro-temperature sensor provides precise control of the temperature during the grinding process, avoiding thermal damage to cells caused by excessive temperatures; the microporous filter design effectively separates large tissue fragments, ensuring the purity of the sample; the automatic cleaning function ensures the cleanliness of the equipment after each grinding, reducing the risk of cross-contamination. Through these efficient technical means, the intelligent tissue grinding system provides a high-standard operating platform for the processing of biological samples such as myocardial tissue, which not only improves the accuracy of the grinding process, but also ensures the stability of sample quality, laying a solid foundation for subsequent cell separation and functional research.

[0098] Furthermore, step 3 includes the following steps:

[0099] Select a sterile, disinfected enzymatic hydrolysis reactor and evenly transfer the ground heart tissue to the center of the reactor;

[0100] Use digestion solution pre-equilibrated to 37°C to ensure stable liquid temperature and composition and optimize enzymatic hydrolysis reaction;

[0101] Adjust the reactor stirring rate to 10 rpm to promote full contact between the digestive fluid and the tissue and avoid tissue shear damage;

[0102] The determination value of reaction termination is obtained by detecting the degree of protein degradation, peptide chain length distribution and specific biomarkers;

[0103] When the reaction termination criteria are reached, the sample is quickly cooled and fixed to maximize the retention of key molecular information and biological characteristics during the enzymatic hydrolysis process.

[0104] In summary, enzymatic hydrolysis is one of the core steps in the extraction of cardiac endothelial cells, which directly affects the quality of the sample and the feasibility of subsequent experiments. By selecting a suitable reactor and ensuring a sterile and disinfected environment, sample contamination can be effectively prevented. The stable temperature and composition of the digestion solution and the appropriate stirring rate optimize the uniformity and effect of the enzymatic hydrolysis reaction. By detecting the degree of protein degradation, peptide chain length distribution and specific biomarkers, the termination timing of the reaction can be accurately assessed to avoid over-digestion or incomplete digestion. Finally, by rapidly cooling and fixing the sample, the maximum retention of biological characteristics during the enzymatic hydrolysis process is ensured. In summary, this process ensures the efficiency and accuracy of the enzymatic hydrolysis process, providing high-quality samples for subsequent cell separation and analysis.

[0105] Furthermore, the enzymatic hydrolysis reactor has the following features: Multiple pH microelectrodes are built into the reactor to monitor pH changes during digestion in real time, maintaining it within the range of 7.2-7.4. An automatic rehydration system automatically adds digestion fluid preheated to 37°C when the liquid level drops. A micro-magnetic stirring device is installed at the bottom of the reactor, and the stirring speed can be automatically adjusted within a range of 5-15 rpm. A real-time image acquisition system uses computer vision algorithms to analyze changes in tissue size and assess digestion progress.

[0106] In summary, the multiple technical features of the enzymatic hydrolysis reactor collectively ensure the efficiency and precision of the enzymatic hydrolysis process. The built-in pH microelectrode ensures that the enzymatic hydrolysis reaction takes place in an ideal acid-base environment by monitoring and adjusting the pH value in real time, thereby improving enzyme activity and digestion efficiency. The automatic rehydration system effectively prevents the impact of a drop in the digestive fluid level on the reaction, ensuring the stability of the liquid volume and temperature during the reaction. The automatic adjustment capability of the micro-magnetic stirring device ensures more uniform contact between the tissue and the digestive fluid, avoiding mechanical damage and uneven digestion. The combination of real-time image acquisition and computer vision algorithms provides visual and accurate feedback on the reaction process, helping to determine the progress of digestion and optimize the timing of reaction termination. In summary, these advanced designs and technologies have effectively improved the automation level and precision of the enzymatic hydrolysis reactor, providing a reliable guarantee for the extraction of high-quality cardiac endothelial cells.

[0107] Furthermore, the determination value of reaction termination is obtained by detecting the degree of protein degradation, peptide chain length distribution and specific biomarkers, including the following steps:

[0108] The mass change of protein degradation was detected by high performance liquid chromatography technology, and the percentage of the mass of degraded protein to the total mass of initial protein was calculated to obtain the degree of protein degradation D;

[0109] Use mass spectrometry to analyze the peptide chain length distribution and calculate the average peptide chain length L avg, to evaluate the shearing effect and reaction progress during enzymatic hydrolysis, the formula is: L avg =(1 / n)·∑ i＝1 n L i , where L i represents the length of the i-th peptide chain, and n is the total number of peptide chains.

[0110] Specific biomarkers are monitored in real time by immunoassays and their concentration percentage change B is calculated;

[0111] Combined with the degree of protein degradation D, the average peptide chain length Lavg and the biomarker concentration, the comprehensive evaluation value T of the reaction process is calculated. end , that is, the predicted result of reaction termination, which can be expressed as: T end =α·D+β·(L avg -L target )+γ·(1-B / B target ), where L target is the target peptide chain length. target is the marker concentration. α represents the weight of protein degradation degree D in the comprehensive evaluation, reflecting the importance of protein degradation in determining the timing of reaction termination. β represents the weight of peptide chain length distribution in the comprehensive evaluation, reflecting the influence of peptide chain length on determining the timing of reaction termination. γ represents the weight of marker concentration in the comprehensive evaluation, reflecting the influence of biomarker concentration on determining the timing of reaction termination.

[0112] In summary, through comprehensive analysis of the degree of protein degradation, peptide chain length distribution, and biomarker concentration, the termination timing of the enzymatic hydrolysis reaction can be scientifically predicted. High-performance liquid chromatography and mass spectrometry provide quantitative and qualitative analytical methods that can accurately monitor protein degradation and peptide chain changes during the enzymatic hydrolysis process. Immunoassays provide real-time feedback on changes in biomarker concentrations, further improving the accuracy of reaction termination. By calculating comprehensive evaluation values, the reactor can make accurate reaction termination decisions based on multiple factors, ensuring the efficiency of the enzymatic hydrolysis process and the maximum retention of biological properties. This multi-dimensional, multi-technology integrated reaction monitoring solution makes the control of enzymatic hydrolysis reactions more intelligent and precise, meeting the various needs of high-demand experiments.

[0113] Furthermore, step 4 includes the following steps:

[0114] Accurately prepare Ficoll gradients with a density range of 1.077-1.119 g / ml;

[0115] Overlay the enzymatically digested tissue suspension on the top layer of the Ficoll gradient to avoid stirring the interface;

[0116] Centrifuge at 400-500 g for 20-30 minutes to precisely separate cells of different densities;

[0117] The mononuclear cell population located in the middle layer of the Ficoll gradient was collected and stored at 4°C until use;

[0118] Mix the pretreated anti-CD31 magnetic beads with the target cells according to the ratio and incubate for 30-45 minutes to ensure that the magnetic beads bind to the CD31-positive endothelial cells;

[0119] CD31-positive cells were captured by magnetic separation columns to purify and collect the target cardiac endothelial cell population.

[0120] In summary, through the close coordination of the above steps, from the preparation of the Ficoll gradient to the magnetic separation and purification of CD31-positive cells, the entire cell separation process embodies efficient, precise, and meticulous technical requirements. Each step is closely linked to ensure the precise separation and purification of cells, thereby providing high-quality cell samples for subsequent experiments. Whether it is the precise stratification during gradient centrifugation or the specific capture during magnetic separation, each step plays a crucial role in the cell purification effect, ultimately ensuring the acquisition of the target cardiac endothelial cells, laying the foundation for related medical research and clinical applications.

[0121] Furthermore, the anti-CD31 magnetic beads have the following characteristics: the diameter of the magnetic beads is 2.8±0.2 microns; the concentration of the anti-CD31 antibody coated on the surface of the magnetic beads is 15-20 micrograms / ml; the mixing ratio of magnetic beads to cells is 20-50 magnetic beads / target cell; the magnetic beads are stored at 2-8°C and thoroughly mixed before use; the magnetic beads are superparamagnetic and stable for not less than 6 months.

[0122] In summary, anti-CD31 magnetic beads possess precise physical and chemical properties, making them an important tool for cell separation. Through optimized particle size design, antibody concentration, bead-to-cell mixing ratio, and storage conditions, anti-CD31 magnetic beads can provide efficient and specific separation during the cell separation process. Their superparamagnetic properties and excellent stability further enhance the beads' reliability and efficiency in long-term use. By rationally utilizing these properties, anti-CD31 magnetic beads can achieve precise separation of target cell populations, maximizing cell purity and activity, thereby providing strong support for cytological research and clinical applications.

[0123] Furthermore, step 5 includes the following steps:

[0124] Select a sterile and pre-treated microenvironment culture system to ensure that its internal surfaces are thoroughly disinfected to provide an ideal contamination-free environment for cell culture;

[0125] Using a precision pipette, the purified endothelial cells were evenly seeded on the pretreated culture surface, with the cell density precisely controlled between 3000 and 5000 cells / cm2;

[0126] The culture system temperature was set at 37°C and the temperature fluctuation was strictly controlled within ±0.5°C to simulate the physiological temperature conditions of the human body and ensure the stability of cell metabolic activity and growth state;

[0127] Adjust the carbon dioxide concentration in the culture system to 5% and maintain the humidity above 95% to simulate a microenvironment close to physiological conditions and promote normal cell growth and functional expression;

[0128] The system monitors the metabolic status, nutrient consumption, and metabolite accumulation of cells in real time, and intelligently adjusts the replacement frequency and composition of the culture medium based on changes in cell morphology, growth rate, and biomarkers to ensure that endothelial cells are always in optimal growth conditions.

[0129] In summary, the cell culture technology in step 5 ensures that endothelial cells are cultured under optimal growth conditions by precisely controlling culture conditions, including the provision of a sterile environment, precise control of cell seeding density, strict regulation of temperature, carbon dioxide concentration, and humidity, as well as real-time monitoring of cell metabolic status and intelligent adjustment of the culture medium. These technical measures not only improve the efficiency and stability of cell culture, but also ensure that the biological characteristics of the cells in the experiment are well maintained. Through these precise operations, a reliable cell model can be provided for subsequent cell function research, providing a solid foundation for cell therapy and clinical applications.

[0130] Furthermore, the metabolic status, nutrient consumption, and metabolite accumulation of cells are monitored in real time. Based on changes in cell morphology, growth rate, and biomarkers, the frequency and composition of culture medium replacement are intelligently adjusted to ensure that endothelial cells are always in optimal growth conditions. The following steps are included:

[0131] By real-time monitoring of the concentrations of key nutrients and metabolites in the culture medium, the metabolic state of the cells, M(t), is calculated using the following formula: M(t) = k1·N(t) - k2·P(t), where N(t) is the nutrient concentration in the culture medium; P(t) is the metabolite concentration; k1 and k2 represent the effects of nutrient consumption and metabolite accumulation on the metabolic state of the cells, respectively.

[0132] According to the dynamic behavior of cell growth, the growth rate R(t) is calculated by measuring the cell density C(t), which is expressed as: Where r is the maximum growth rate; K is the environmental carrying capacity; N max is the maximum nutrient concentration, which indicates the effect of nutrients in the culture medium on cell growth.

[0133] High-resolution imaging and immunofluorescence labeling techniques are used to monitor cell morphological changes and biomarker expression in real time, which can be expressed as follows: S(t) = g1(C(t), M(t)), B(t) = g2(C(t), M(t)), where S(t) is the cell morphological state, indicating how cell morphology changes over time and is used to assess the functional state of the cells; B(t) is the biomarker expression state, indicating how cell markers change over time and is used to assess the endothelial cell characteristics and functional state of the cells; g1(·) is a functional form used to describe the functional relationship between cell morphological change S(t) and cell growth rate R(t) and metabolic state M(t), indicating the effect of cell morphology on growth and metabolism; g2(·) is a functional form used to describe the functional relationship between biomarker expression B(t) and cell growth rate R(t) and metabolic state M(t), indicating the effect of cell markers on growth and metabolism.

[0134] Based on the real-time monitored cell growth rate R(t) and metabolic state M(t), the frequency of medium replacement is intelligently adjusted using the following formula: F(t) = k3·R(t)·(1+θ·M(t)), where F(t) is the intelligently adjusted medium replacement frequency, indicating that the medium replacement cycle is intelligently adjusted based on the cell growth rate R(t) and metabolic state M(t); k3 is a constant related to the cell growth rate, indicating the degree of influence of the growth rate on the adjustment of the medium replacement frequency; θ is the adjustment coefficient of the metabolic state on the medium replacement frequency, indicating the influence of changes in the cell metabolic state on the medium replacement frequency.

[0135] Based on the cell metabolic state M(t), morphological changes S(t) and marker expression B(t), the culture medium composition is intelligently adjusted according to the following formula: E(t) = μ·R(t)·(1+η·S(t)+δ·B(t)), where E(t) is the adjustment amount of the culture medium composition, indicating the change in the culture medium composition adjusted according to the cell growth and metabolic state; μ represents the degree of influence of the growth rate on the culture medium composition; η is the influence coefficient of cell morphological changes on the adjustment of the culture medium composition; and δ is the influence coefficient of cell marker expression on the adjustment of the culture medium composition.

[0136] In summary, by real-time monitoring of cell metabolic status, nutrient consumption, and metabolite accumulation, as well as intelligently adjusting the frequency and composition of culture medium replacement, the cell culture process can be precisely and dynamically regulated. Real-time monitoring and feedback of cell growth rate, metabolic status, morphological changes, and marker expression enable the culture environment to continuously adapt, ensuring that cells grow under optimal growth conditions. This intelligent, dynamic cell culture technology can significantly improve the efficiency, stability, and controllability of cell culture, and provide reliable data support and optimization methods for cell function research and applications.

[0137] Furthermore, step 6 includes the following steps:

[0138] Immunofluorescence combined with flow cytometry technology is used to accurately analyze the expression of key endothelial cell markers and evaluate cell purity and functional characteristics;

[0139] The cells were seeded on Matrigel to observe their ability to self-assemble into tubular structures, and their angiogenic potential was quantitatively assessed by measuring the length of the tubular lumen, the number of branches, and the number of junctions.

[0140] Capture high-resolution images of cells and automatically identify and quantify cell morphological characteristics, including cell area, perimeter, roundness, and shape complexity parameters;

[0141] Objective and quantitative quality assessment of endothelial cells is performed by integrating marker expression levels, lumen formation ability, and morphological characteristics data;

[0142] Conduct functional validation experiments, including endothelial cell migration and lumen repair experiments, to further assess the biological functional integrity of cells by tracking cell movement trajectories and wound healing rates;

[0143] The multi-dimensional evaluation results are presented through a visual interface to generate a detailed quality report, providing a comprehensive and accurate cell quality reference for subsequent experiments and applications.

[0144] In summary, the above steps constitute a comprehensive system for evaluating endothelial cell functionality and quality. Through immunofluorescence combined with flow cytometry, assessment of the cells' ability to self-assemble into tubular structures, high-resolution imaging and automated image analysis, comprehensive multidimensional quality assessment, cell migration and repair experiments, and visualization of multidimensional assessment results, the functional status, structural characteristics, and biological potential of endothelial cells are comprehensively evaluated. These techniques not only provide a scientific basis for the application of endothelial cells but also offer precise quality control methods and standardized operating procedures for cell biology research, disease treatment, and tissue engineering.

[0145] Furthermore, the cell area A is expressed as: A = ∑ i,j I(i,j), where I(i,j) is the pixel representing the cell area in the processed binary image;

[0146] The cell perimeter P is expressed as: P = ∑ 边界像素 d(i,j), where d(i,j) is the distance between adjacent pixels;

[0147] The cell roundness R is expressed as: R = 4πA / P 2 ;

[0148] The shape complexity C is expressed by the formula:

[0149] In summary, the morphological characteristics of cells are an important foundation for cell biology research. Parameters such as cell area, circumference, roundness, and shape complexity are quantified through mathematical formulas and image analysis methods, providing a powerful tool for the quantitative assessment of cell function. These quantitative methods not only accurately describe the morphology of cells, but also reveal the changing patterns of cell biological activities, thereby providing a scientific basis for fields such as cell research, disease diagnosis, and drug screening. By comprehensively analyzing these parameters, we can gain a deeper understanding of the behavior of cells under different physiological and pathological conditions and evaluate their functions such as growth, differentiation, and migration. The combined use of these morphological analysis techniques provides a comprehensive technical means for cell research and helps achieve more accurate and efficient cell quality control.

[0150] Furthermore, functional verification experiments were conducted, including endothelial cell migration and lumen repair experiments. By tracking cell movement trajectories and wound healing rates, the biological functional integrity of the cells was further evaluated. The following steps were included:

[0151] Select appropriate culture medium and seed endothelial cells into culture plates at an appropriate density. Start the experiment after the cells adhere to the wall and grow to 80%-90% confluence.

[0152] Use a 200 μl sterile pipette tip to make a uniformly wide scratch on the cell monolayer and slowly wash with PBS three times to remove detached cells. Then, replace with fresh serum-free medium and take an initial image of the wound under a microscope as a starting reference for the experiment.

[0153] In a living cell workstation equipped with a carbon dioxide incubator, continuous imaging was performed to record the cell migration process under conditions maintained at 37°C and 5% carbon dioxide. Images were automatically collected every 2 hours for a period of 24-48 hours.

[0154] ImageJ software was used to analyze the wound area at different time points, calculate the wound healing rate, and simultaneously track the migration trajectory of individual cells to obtain cell migration speed and directionality parameters;

[0155] Endothelial cells were seeded into matrigel-coated culture plates, and the formation of tubular structures by the cells was observed and recorded. The number, length, and branch points of the tubular lumens were also counted.

[0156] Calculate the mean and standard deviation of each indicator, analyze the differences between different groups, and evaluate the overall biological function of cells.

[0157] In summary, through systematic functional validation experiments, including endothelial cell migration and lumen repair assays, the researchers were able to comprehensively assess the biological integrity of endothelial cells. This experiment not only simulated the cell repair process through a wound model but also used image analysis software to precisely track cell migration trajectories and wound healing rates, further quantitatively assessing the cells' biological behavior. Furthermore, the lumen formation assay provided quantitative analysis of angiogenic potential, providing strong data support for the cells' application in tissue repair.

[0158] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art will appreciate that modifications may be made to the technical solutions described in the above embodiments, or that some of the technical features may be replaced with equivalents; such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for extracting primary mouse cardiac endothelial cells, characterized in that: The following steps are involved: Step 1: obtaining pure myocardial tissue; Step 2: transferring the processed heart tissue to the intelligent tissue grinding system for grinding; Step 3: The ground heart tissue is transferred to a temperature-controlled enzymatic hydrolysis reactor, which automatically monitors the digestion process and terminates the reaction when the preset digestion level is reached. Step 4: Perform preliminary cell separation using magnetic sorting with anti-CD31 magnetic beads to specifically enrich cardiac endothelial cells; Step 5: Transfer the purified endothelial cells to the intelligent microenvironment culture system, which automatically optimizes the culture medium replacement cycle to adapt to the cell metabolic state; In step 6, a multi-dimensional quality assessment of endothelial cells was performed, including marker expression analysis, in vitro lumen formation experiment, AI-assisted morphological analysis, and digital quality scoring.

2. A method for extracting primary mouse cardiac endothelial cells according to claim 1, characterized in that, Step 1 includes the following steps: Prepare a sterile operating table, disinfect the table and turn on the ultraviolet light for 30 minutes; Pre-cool the D-Hanks buffer in a 4°C refrigerator and place it in an ice box to keep it low. The anesthetized mouse was fixed on the operating table, the chest and abdominal skin was disinfected with 75% alcohol, the abdomen was incised and the chest cavity was opened, and the heart was quickly removed; The removed heart was immediately placed in pre-cooled D-Hanks buffer and gently squeezed repeatedly to remove any remaining blood; Under a dissecting microscope, use fine forceps to peel off the pericardium and remove attached fat tissue, connective tissue, and vascular stumps; The processed heart was rinsed in new pre-cooled D-Hanks buffer to further remove residual tissue debris and obtain pure myocardial tissue.

3. A method for extracting primary mouse cardiac endothelial cells according to claim 1, characterized in that, Step 2 includes the following steps: Use sterile forceps to transfer the myocardial tissue sample to the grinding chamber of the intelligent tissue grinding system and ensure that the tissue is evenly spread; The grinding gap was set to 0.5 mm to ensure uniform grinding of the tissue and to preserve its original structure and biological properties to the greatest extent possible; Set the grinding speed to 30 rpm to avoid excessive shearing or thermal damage and ensure the integrity of cell components; A mixture of 95% oxygen and 5% carbon dioxide is introduced into the grinding system to simulate physiological environmental conditions to maintain tissue metabolic activity; The grinding process is monitored throughout to ensure that the tissue is fully exposed and no unexpected damage occurs, providing high-quality samples for subsequent analysis.

4. A method for extracting primary mouse cardiac endothelial cells according to claim 3, characterized in that: The intelligent tissue grinding system includes the following features: the inner wall of the grinding chamber is made of medical-grade titanium alloy material, and the surface has nano-level flatness; it is equipped with a micro temperature sensor to monitor the temperature changes during the grinding process in real time, and automatically starts cooling when the temperature exceeds 37.5°C; a microporous filter with a pore size of 200μm is installed at the bottom of the grinding chamber to separate large tissue fragments during the grinding process; it has an automatic cleaning function, and uses sterile water and 75% ethanol for three cycles of cleaning after each grinding.

5. The method for extracting primary mouse cardiac endothelial cells according to claim 1, wherein Step 3 includes the following steps: Select a sterile, disinfected enzymatic hydrolysis reactor and evenly transfer the ground heart tissue to the center of the reactor; Use digestion solution pre-equilibrated to 37°C to ensure stable liquid temperature and composition and optimize enzymatic hydrolysis reaction; Adjust the reactor stirring rate to 10 rpm to promote full contact between the digestive fluid and the tissue and avoid tissue shear damage; The determination value of reaction termination is obtained by detecting the degree of protein degradation, peptide chain length distribution and specific biomarkers; When the reaction termination criteria are reached, the sample is quickly cooled and fixed to maximize the retention of key molecular information and biological characteristics during the enzymatic hydrolysis process.

6. The method for extracting primary mouse cardiac endothelial cells according to claim 5, wherein: The enzymatic hydrolysis reactor has the following features: the reactor is equipped with multiple pH microelectrodes to monitor the pH changes during the digestion process in real time and maintain it within the range of 7.2-7.4; it is equipped with an automatic rehydration system, which automatically adds digestion fluid preheated to 37°C when the liquid level is detected to be lowering; the bottom is equipped with a micro magnetic stirring device, and the stirring speed can be automatically adjusted within the range of 5-15rpm; it has a real-time image acquisition system, which uses computer vision algorithms to analyze changes in tissue block size and evaluate the digestion progress.

7. The method for extracting primary mouse cardiac endothelial cells according to claim 1, wherein Step 4 includes the following steps: Accurately prepare Ficoll gradients with a density range of 1.077-1.119 g / ml; Overlay the enzymatically digested tissue suspension on the top layer of the Ficoll gradient to avoid stirring the interface; Centrifuge at 400-500 g for 20-30 minutes to precisely separate cells of different densities; The mononuclear cell population located in the middle layer of the Ficoll gradient was collected and stored at 4°C until use; Mix the pretreated anti-CD31 magnetic beads with the target cells according to the ratio and incubate for 30-45 minutes to ensure that the magnetic beads bind to the CD31-positive endothelial cells; CD31-positive cells were captured by magnetic separation columns to purify and collect the target cardiac endothelial cell population.

8. The method for extracting primary mouse cardiac endothelial cells according to claim 7, wherein: Anti-CD31 magnetic beads have the following characteristics: a bead diameter of 2.8±0.2 μm; the concentration of anti-CD31 antibodies coated on the surface of the magnetic beads is 15-20 μg / ml; the mixing ratio of magnetic beads to cells is 20-50 magnetic beads per target cell; the magnetic beads are stored at 2-8°C and thoroughly mixed before use; the magnetic beads are superparamagnetic and stable for at least 6 months.

9. The method for extracting primary mouse cardiac endothelial cells according to claim 1, wherein Step 5 includes the following steps: Select a sterile and pre-treated microenvironment culture system to ensure that its internal surfaces are thoroughly disinfected to provide an ideal contamination-free environment for cell culture; Using a precision pipette, the purified endothelial cells were evenly seeded on the pretreated culture surface, with the cell density precisely controlled between 3000 and 5000 cells / cm2; The culture system temperature was set at 37°C and the temperature fluctuation was strictly controlled within ±0.5°C to simulate the physiological temperature conditions of the human body and ensure the stability of cell metabolic activity and growth state; Adjust the carbon dioxide concentration in the culture system to 5% and maintain the humidity above 95% to simulate a microenvironment close to physiological conditions and promote normal cell growth and functional expression; The system monitors the metabolic status, nutrient consumption, and metabolite accumulation of cells in real time, and intelligently adjusts the replacement frequency and composition of the culture medium based on changes in cell morphology, growth rate, and biomarkers to ensure that endothelial cells are always in optimal growth conditions.

10. The method for extracting primary mouse cardiac endothelial cells according to claim 1, wherein: Step 6 includes the following steps: Immunofluorescence combined with flow cytometry technology is used to accurately analyze the expression of key endothelial cell markers and evaluate cell purity and functional characteristics; The cells were seeded on Matrigel to observe their ability to self-assemble into tubular structures, and their angiogenic potential was quantitatively assessed by measuring the length of the tubular lumen, the number of branches, and the number of junctions. Capture high-resolution images of cells and automatically identify and quantify cell morphological characteristics, including cell area, perimeter, roundness, and shape complexity parameters; Objective and quantitative quality assessment of endothelial cells is performed by integrating marker expression levels, lumen formation ability, and morphological characteristics data; Conduct functional validation experiments, including endothelial cell migration and lumen repair experiments, to further assess the biological functional integrity of cells by tracking cell movement trajectories and wound healing rates; The multi-dimensional evaluation results are presented through a visual interface to generate a detailed quality report, providing a comprehensive and accurate cell quality reference for subsequent experiments and applications.

Images