Primary endocardial endothelial cell extraction method suitable for cardiovascular disease research

Abstract

The invention discloses a primary endocardial endothelial cell extraction method suitable for cardiovascular disease research. Non-specific components are removed through directional sampling, enzyme digestion and mechanical dispersion of an endocardium microscopic spatula and in combination with a CD31 positive selection and CD36 negative selection combined strategy; after culture, Npr3 backtracking, functional verification and passage limitation (less than or equal to P2) are used for controlling quality. According to the method, the cells with the purity of 80-90%, the motility rate of more than or equal to 85% and stable Npr < 3 + > proportion can be obtained, and compared with a traditional method, the method has the advantages in the aspects of material taking specificity, function fidelity and repeatability. The invention provides a reliable cell source for cardiovascular disease research and transformation application.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a method for extracting primary endocardial endothelial cells suitable for cardiovascular disease research. Background Technology

[0002] The heart maintains systemic perfusion through rhythmic contractions. Within a cardiac cycle, blood flow in the cardiac chambers exhibits alternating laminar and turbulent characteristics at different geometric locations, creating significant spatiotemporal differences. This flow pattern exists not only in adulthood but also plays a crucial role in cardiac morphogenesis during the embryonic period. Zebrafish and avian models show that even slight alterations in inflow / outflow patency can affect primitive cardiac tube rotation and interfere with valve patterning, suggesting that blood flow shear force is an important regulatory factor in cardiac morphogenesis. Therefore, accurate "sensory-translated" blood flow patterns are a prerequisite for the anatomical remodeling of the primitive cardiac tubes into a mature four-chamber heart.

[0003] It should be clarified first that cardiac endothelium is divided into two categories according to its anatomical location: endocardial endothelial cells (EEC) that line the inner surface of the heart chambers and vascular endothelium (coronary / microvascular EC, or C / MV-EC) distributed in the coronary arteries and myocardial microvessels. The two have systemic differences in their stress environment, molecular spectrum and functional focus. The term "endocardial endothelium" in this article refers to the former.

[0004] To adapt to and utilize the aforementioned hydrodynamic information, unique endocardial endothelial cells form on the inner surface of the cardiac chambers. Compared to vascular endothelium, endocardial endothelium differs in cytoskeleton morphology, stress direction, and basal matrix, and participates in ventricular trabeculation and the formation of primitive nutrient channels during the embryonic period. Multiple studies suggest that endocardial endothelial dysfunction is associated with pathological processes such as myocardial remodeling and heart failure, indicating that it is not merely a simple lining of the cardiac chambers, but rather an active regulator maintaining myocardial structure-function homeostasis. Recent ultrastructural evidence reinforces this understanding. A 2025 report in *Science* revealed that in the developing mammalian heart, nanotube-like (TNT-like) tunnel structures exist between the endocardial endothelium and cardiomyocytes, directly facilitating the communication of substances and signals; blocking these structures disrupts their interaction and leads to ventricular structural abnormalities. This discovery explains the anatomical and functional basis of "endocardial regulation of myocardium" at the nanoscale and provides a new target for intervention in ventricular remodeling.

[0005] However, related research has long been constrained by shortcomings in experimental materials and methodologies: First, there is a lack of standardized procedures for preparing primary endocardial endothelial cells. Mainstream commercial channels primarily provide coronary artery endothelium or cardiac microvascular endothelium, but there are no standardized commercial cells named "endocardial endothelium" and validated by a specific labeling system, which can easily lead to systematic errors in basic research. Second, the common "endothelial separation" protocol is geared towards vascular endothelium and lacks key indicators that can distinguish between endocardial and coronary origins during the separation process. Third, the aforementioned commercial cells lack an in vitro quality control system based on cardiac single-cell atlases, causing in vitro cells to often deviate from their in vivo identity: their response to shear stress is weakened or abnormal, tending towards "coronary-like" characteristics. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide a method for extracting primary endocardial endothelial cells through "anatomical localization-enzymatic digestion-flow cytometry sorting-functional verification": starting with precise sampling from the inner surface of the cardiac chambers, using CD45... － / CD31 ＋ / CD36 － The FACS fine sorting technology, which targets phenotypes, yields a high-purity population. The Npr3 immune retrospective and KLF2 / eNOS laminar flow response are used as dual indicators for identity and functional quality control, achieving standardization of the entire process from tissue to in vitro. This eliminates coronary / microvascular contamination and phenotypic deviation, providing cardiovascular disease research with a clearly identified, functionally accurate, and reproducible endocardial endothelial cell model, filling a gap in existing technologies.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] On the one hand, single-cell sequencing provides a molecular basis for targeted separation. Our previous mouse single-cell data showed that endocardial endothelium and coronary / microvascular endothelium can be clearly distinguished on cluster maps: although both express classic endothelial markers such as CD31, they are enriched with specific molecules—endocardial endothelium characteristically highly expresses Npr3, while coronary / microvascular endothelium highly expresses fatty acid transport-related molecules (such as CD36). This difference has been verified by in situ immunofluorescence staining (see...). Figure 1 Based on this, we identified reliable targets for distinguishing between the endocardium and the coronary / microvascular endothelium.

[0009] Specifically, we analyzed single-cell heart data from eight mice (a total of 90,875 cells) and identified endocardial endothelium and coronary / microvascular endothelium as two independent cell populations. Figure 1 (A) Although both cell populations expressed the common endothelial marker Pecam1, differential gene analysis revealed unique molecular characteristics: endocardial endothelium (shown by the white dashed line) specifically expressed Npr3, while coronary / microvascular endothelium highly expressed CD36 and FABP4 (…). Figure 1 (B in the text). Immunofluorescence staining further confirmed that endocardial endothelium specifically expressed Npr3 (…). Figure 1 C in the diagram (shown by the white dashed line), while coronary artery / microvascular endothelium specifically expresses CD36 ( Figure 1 (D in the middle).

[0010] On the other hand, the present invention provides a method for extracting primary endothelial cells suitable for cardiovascular disease research, comprising the following steps:

[0011] (1) Sampling and endocardial enrichment: Mice were anesthetized and heparinized. After the heart was lavaged to remove blood, pericardial fat and adventitia were removed under a stereomicroscope. The free walls of the left and right ventricles and the interventricular septum were precisely removed along the aortic / pulmonary artery inlet. The endocardial tissue was enriched by gently scraping the cavitary surface with a sterile soft scraper, avoiding deep myocardial dissection; or a thin slice of tissue 0.5-3.0 mm (preferably 1.0-2.0 mm) close to the endocardium was carefully cut as a digestion sample.

[0012] (2) Tissue digestion and single-cell formation: The enriched endocardial tissue blocks were cut into small pieces, digested with a mixed enzyme solution, and then mechanically dispersed by grinding through a filter to terminate digestion. The enzyme digestion and mechanical dispersion steps were repeated. The cell pellet was then collected by centrifugation, and the red blood cells were removed with red blood cell lysis buffer. Finally, the cell suspension was obtained by repeated low-speed centrifugation and resuspending and washing with PBB medium.

[0013] (3) FACS fine sorting

[0014] S1. Staining: Add anti-CD45 antibody, anti-CD31 antibody, anti-CD36 antibody, and live / dead cell dye DAPI to the cell suspension. Gently mix the mixture and incubate in the dark for 20-30 minutes.

[0015] S2, Control Settings:

[0016] FMO control: Prepare single-color controls missing CD36 and CD31 respectively, and set a 99% confidence threshold for differentiation during subsequent gating;

[0017] Isotype control: Use isotype antibodies of the same species to assess nonspecific binding;

[0018] Monochrome compensation beads: Special compensation beads are used to compensate the spectrum of the fluorescence channel to ensure the accuracy of multicolor detection;

[0019] S3. Washing and loading: After staining, add an appropriate amount of PBS to each tube, centrifuge, remove the supernatant, repeat washing once to remove unbound antibodies, resuspend the washed cells in an appropriate amount of PBS, and transfer them to flow cytometer sample tubes.

[0020] S4, Gating Strategy:

[0021] Cell population → Single cell: Plot a two-parameter graph on FSC-A and FSC-H, excluding aggregated cells and retaining single cell populations; → Live cells: Set a threshold on the DAPI channel and select DAPI... － Negative cells, excluding dead cells; → CD45 － Gate: Set a threshold on the CD45 channel based on the FMO reference to exclude CD45. ＋ White blood cell population; → CD31 ＋ Gate: Set the threshold on channel CD31 according to the FMO reference, and select CD31. ＋ Endothelial cells; →CD36 － Gate: Set a threshold on channel CD36 based on the FMO reference to exclude CD36. ＋ Cells, retaining CD36 － Target endothelial cells;

[0022] After each gating step, the corresponding cell proportion is recorded, and the compensation matrix and gating statistics are exported from the instrument software.

[0023] S5. Cell collection: Based on the final gate CD45 － CD31 ＋ CD36 － The target cells were sorted into collection tubes containing pre-cooled PBS solution containing FBS;

[0024] (4) Adherent culture and short-term amplification

[0025] The sorted target cells were seeded into gelatin-coated culture dishes and cultured using EGM-2 / EC maintenance medium;

[0026] (5) Quality control indicators and judgment

[0027] 1) Quality control indicators

[0028] ①Phenotype / Molecular

[0029] Flow cytometry: CD31 ＋ Cell percentage ≥85%, and CD36 low / − Cell percentage ≥80%;

[0030] Immunofluorescence / staining: ≥70% of cells were positive for Npr3 in the membrane / cytoplasm;

[0031] ② Functional identification

[0032] Shear response: Cells respond to simulated blood flow shear forces of 5-10 dyn / cm 2 After 12 hours of culture on an orbital shaker, KLF2 and eNOS mRNA were upregulated by ≥2-fold.

[0033] ③ Negative exclusion

[0034] CD45 + ;

[0035] Coronary artery vascularization risk markers: simultaneous elevation of CD36, FABP4, and VWF with a decrease in Npr3 → considered "off-target" endothelial sorting, requiring exclusion;

[0036] ④ Survival rate and pollution

[0037] Cell viability ≥85%;

[0038] Bacteria / Mycoplasma negative;

[0039] 2) Judgment criteria

[0040] CD31 ＋ ≥85%, Npr3 ＋ ≥70%, survival rate ≥85%, contamination negative; any two functions must meet the standards.

[0041] Preferably, the (2) tissue digestion and single-cell formation specifically includes the following steps:

[0042] S1. Pretreatment: The enriched endocardial tissue blocks were placed in PBS containing double antibodies, cut into small pieces, and placed on ice to allow the tissue blocks to settle. The supernatant was then removed and the tissue blocks were washed.

[0043] S2, Enzyme Digestion I: Digest the tissue precipitate for 30-40 minutes using an enzyme mixture containing Collagenase II, Collagenase I, Collagenase IV and DNase I;

[0044] S3. Mechanical dispersion: Place the digested mixture in a sterile filter, grind it using a syringe plunger, and add an equal volume of PBB culture medium to rinse and terminate digestion.

[0045] S4. Repeat digestion: Centrifuge the cell suspension, remove the supernatant, add enzyme digestion solution again, and continue digestion for 5-8 minutes. Then repeat the mechanical dispersion step in S3, centrifuge again, and collect the cell pellet.

[0046] S5. Remove red blood cells: Add twice the volume of red blood cell lysis buffer to the cell pellet and treat for 1-2 minutes to remove red blood cells. Stop the lysis with PBS.

[0047] S6. Low-speed washing: Centrifuge the lysate, remove the supernatant and resuspend in PBB medium. Repeat twice to complete the washing and obtain a cell suspension.

[0048] Preferably, the enzyme mixture comprises: Collagenase II 1-2 mg / mL, Collagenase I 1-2 mg / mL, Collagenase IV 0.5-1 mg / mL, and DNase I 50-100 μg / mL, using Ca-free... 2+ / Mg 2+ Prepare with PBS.

[0049] Alternatively, the enzyme mixture comprises: Collagenase II 1 mg / mL, Collagenase I 1 mg / mL, Collagenase IV 1 mg / mL, and DNase I 50 μg / mL, using Ca-free... 2+ / Mg 2+ Prepare with PBS.

[0050] Preferably, in step (2), the PBB culture medium is a PBS solution containing 2% v / v FBS and 0.2% m / v BSA.

[0051] Preferably, in step (2), the total duration of enzyme digestion is 30-40 min.

[0052] Preferably, in step (2), the centrifugation conditions are: 4°C, 300-350×g centrifugation for 5 minutes.

[0053] Preferably, in step (4), the composition of the EGM-2 / EC maintenance medium is: Endothelial Cell Medium (ECM, Cat. #1001) basal + 1% Endothelial Cell Growth Supplement (ECGS, Cat. #1052) + 2%-5% FBS (Cat. #0025) + 1% penicillin / streptomycin solution (P / S, Cat. #0503).

[0054] Preferably, in step (4), the cell seeding density is 1.0-2.5 × 10⁻⁶. 4 cells / cm 2 .

[0055] Preferably, in step (4), the culture conditions are: 37°C, 5% CO2 culture for 24 h followed by medium change, and then medium change every 48 h thereafter.

[0056] Preferably, in step (5), P0-P2 generation cells are used for quality control testing.

[0057] In preliminary experiments, this invention attempted to replace the FACS fine sorting method with a two-step MACS sorting method of "CD31 positive selection + CD36 negative platelet removal". The results showed that the purity of target cells obtained by the two-step MACS sorting was significantly lower than that obtained by the FACS fine sorting method. Therefore, this invention ultimately adopted FACS fine sorting as the standardized protocol for this study. Specifically, the two-step MACS sorting method includes the following steps:

[0058] S1, CD31 ＋ Positive selection: Cells expressing CD31 were captured in a magnetic column using anti-CD31 magnetic beads, and enriched CD31 was obtained by demagnetization and elution. ＋ cell;

[0059] S2, CD36 － Yin selection: Enriched CD31 ＋ Cells were negatively removed using anti-CD36 magnetic beads. ＋ The cells were retained in the column and discarded; the eluent was collected to obtain CD31. ＋ CD36 － Target cells.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] Endocardial endothelial cells play a unique role in cardiac development and myocardial homeostasis, but existing methods struggle to distinguish them from coronary / microvascular endothelial cells, leading to insufficient purity and phenotypic bias. This invention identifies "CD31" based on single-cell sequencing. ＋ CD36 － "To achieve stable separation and combination, and with Npr3 retrospective verification, combined with targeted endometrial sampling, enzymatic digestion, and precise sorting by flow cytometry, [the following results can be obtained]..."

[0062] CD31 ＋ ≥85%, Npr3 ＋ The cell population is ≥80%. The resulting cells maintain the KLF2 / eNOS shear response, avoiding "coronary vascularization," and exhibit significantly better purity and functional fidelity than existing methods, providing a reliable model for cardiac research. Attached Figure Description

[0063] Figure 1 Single-cell atlas of mouse heart identified differences in molecular characteristics between the endocardium and coronary / microvascular endothelium.

[0064] Figure 2 Flow cytometry was used to verify the specificity of endocardial endothelium.

[0065] Figure 3 Based on a flow cytometry sorting strategy that only selects CD31 positive cells and does not select CD36 negative cells, the endothelial cell acquisition rate is extremely low. Detailed Implementation

[0066] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0067] 1. Definition of Terminology and Objects

[0068] Endocardial endothelial cells (EEC): Endothelial cells lining the inner surface of the heart chambers, distinct from the coronary / myocardial microvascular endothelium.

[0069] Target sorting phenotype: CD45 － / CD31 ＋ / CD36 － And during culture / staining verification, Npr3 was stained. ＋ verify.

[0070] Applicable species: mice.

[0071] 2. Main Reagents and Equipment

[0072] (1) Tissue digestion and separation

[0073] Collagenase II (1-2 mg / mL working solution, preferably 1 mg / mL);

[0074] Collagenase I (1-2 mg / mL working solution, preferably 1 mg / mL);

[0075] Collagenase IV (0.5-1 mg / mL working solution, preferably 1 mg / mL);

[0076] DNase I (50-100 μg / mL, preferably 50 μg / mL);

[0077] Red blood cell lysis buffer;

[0078] Trypsin-EDTA digestion solution: 0.25% Trypsin-EDTA (for short-term adhesion removal);

[0079] The filter is 100μm or 70μm, preferably 70μm;

[0080] Antibody / Magnetic Beads / Dye (FACS / MACS): Anti-CD31 (PECAM1)-PE;

[0081] Anti-CD36-APC (negative option);

[0082] Anti-CD45 (excluding immune cells);

[0083] Viable / dead cell staining: DAPI;

[0084] (2) Culture and substrate

[0085] EGM-2 / EC maintenance medium: Endothelial Cell Medium (ECM, Cat. #1001) basal + 1% Endothelial Cell Growth Supplement (ECGS, Cat. #1052) + 2%-5% FBS (Cat. #0025) + 1% penicillin / streptomycin solution (P / S, Cat. #0503);

[0086] Vascular endothelial growth factor (VEGF-A 20 ng / mL);

[0087] 0.1% gelatin coating;

[0088] Antibiotics (penicillin or streptomycin or broad-spectrum alternatives);

[0089] (3) Functional / phenotypic testing

[0090] Anti-Npr3, KLF2 / 4 antibodies;

[0091] (4) Equipment

[0092] Sterile dissection instruments (3 pairs of 10cm curved forceps, 2 pairs of 10cm straight-tipped tissue scissors, 2 pairs of 12cm microscopic scissors), stereomicroscope;

[0093] Vortex oscillator, 37℃ constant temperature water bath or shaking table;

[0094] Tabletop centrifuge (4℃), ice pack;

[0095] Flow cytometer (BD FACSAriaFusion);

[0096] Inverted confocal microscope;

[0097] Microfluidic or oscillating shear culture device (optional).

[0098] Example 1

[0099] 1. Material Sampling and Endometrial Enrichment

[0100] (1) Animal treatment: Mice were anesthetized and heparinized (100-200 U / kg), and their hearts were lavaged with pre-cooled 1% penicillin-antibody (P / S) + PBS solution to remove blood.

[0101] (2) Directional dissection: Under stereoscopic guidance, use sterilized 10 cm straight-tipped tissue scissors to remove as much pericardial fat and pericardial tissue as possible. Use the aorta as the entry point to separate the free wall of the left ventricle and the interventricular septum; use the pulmonary artery as the entry point to separate the right ventricle and the interventricular septum, and fully expose the free wall of the ventricle, the inner surface of the interventricular septum, the surface of the papillary muscles, etc.

[0102] (3) Endometrial enrichment: Gently scrape the luminal surface (including the free wall of the ventricle, the inner surface of the interventricular septum, and the surface of the papillary muscles) with a sterile soft scraper or a sterile plastic sheet, using gentle force to avoid deep myocardial dissection; or carefully cut a thin tissue 1.0-2.0 mm close to the endothelium as a digestion sample.

[0103] Objective: To increase EEC content and reduce coronary / microvascular components at the pre-digestive histological level.

[0104] 2. Tissue digestion and single-cell formation

[0105] (1) Pretreatment: The enriched endocardial tissue blocks were placed in 1% penicillin-peptide (P / S) + PBS and minced into 1mm pieces using 10cm straight-tipped tissue scissors. 3 Take small pieces and let them stand on ice for 1 minute. After the tissue blocks settle, remove the supernatant and wash twice.

[0106] (2) Enzyme digestion I: Collagenase II (1-2 mg / mL working solution, preferably 1 mg / mL) + DNase I (50-100 μg / mL, preferably 50 μg / mL) + Collagenase I (1-2 mg / mL working solution, preferably 1 mg / mL) + Collagenase IV (0.5-1 mg / mL working solution, preferably 1 mg / mL), place in a 37°C constant temperature water bath for 30-40 min, and thoroughly mix by pipetting with a sterile pipette tip every 5 minutes.

[0107] (3) Mechanical dispersion: Place a sterile 70μm filter in a 50mL centrifuge tube, add the digestion system obtained in the above steps to the filter, grind with a 1mL syringe plunger, add an equal volume of PBB medium (PBS solution containing 2% v / v FBS and 0.2% w / v BSA) to rinse the filter and terminate digestion.

[0108] (4) Repeat digestion: Centrifuge at 300-350×g for 5 minutes using a 4℃ benchtop centrifuge, remove the supernatant, add digestion solution again and place in a 37℃ constant temperature water bath to digest the undissociated tissue for 5-8 minutes. Repeat the above mechanical dispersion steps, and centrifuge at 300-350×g for 5 minutes using a 4℃ benchtop centrifuge again to obtain cell pellet.

[0109] (5) Remove red blood cells: Add 2 times the volume of red blood cell lysis buffer and lyse for 1-2 min, then terminate with PBS.

[0110] (6) Low-speed washing: Centrifuge at 300-350×g for 5 minutes using a benchtop centrifuge at 4℃, remove the supernatant and resuspend in PBB medium, repeat twice.

[0111] Key parameters: Total digestion time 30-40 min; over-digestion can damage surface epitopes (CD31, Npr3), leading to false negatives, while under-digestion will reduce yield and increase aggregation. A viability rate ≥85% and a clumping rate <5% are considered acceptable.

[0112] 3. FACS Fine Sorting

[0113] (1) Staining (protected from light, 4℃, 20-30 min):

[0114] Anti-CD45 antibody (negative category)

[0115] Anti-CD31 antibody (positive category)

[0116] Anti-CD36 antibody (negative threshold, excluding coronary / microvascular endothelium)

[0117] DAPI (Dye for Live / Dead Cells)

[0118] (2) Reference settings:

[0119] FMO control (Fluorescence Minus One, one tube each of CD36-APC and CD31-PE missing).

[0120] Isotype control (same subtype and same fluorescence as CD36 and CD31);

[0121] Monochrome compensation bead (used for instrument compensation adjustment).

[0122] (3) Washing and loading: After staining, add an appropriate amount of PBS to each tube, centrifuge, remove the supernatant, repeat the washing once to remove unbound antibodies, resuspend the washed cells in an appropriate amount of PBS, and transfer them to the flow cytometer sample tube.

[0123] (4) Gating strategy:

[0124] Cell population → Single cell: Plot a two-parameter graph on FSC-A and FSC-H, excluding aggregated cells and retaining single cell populations; → Live cells: Set a threshold on the DAPI channel and select DAPI... － Negative cells, excluding dead cells; → CD45 － Gate: Set a threshold on the CD45 channel based on the FMO reference to exclude CD45. ＋White blood cell population; → CD31 ＋ Gate: Set the threshold on channel CD31 according to the FMO reference, and select CD31. ＋ Endothelial cells; →CD36 － Gate: Set a threshold on channel CD36 based on the FMO reference to exclude CD36. ＋ Cells, retaining CD36 － The target endothelial cells.

[0125] After each gating step, the corresponding cell proportion is recorded, and the compensation matrix and gating statistics are exported from the instrument software.

[0126] (5) Cell collection: based on the final gate CD45 － CD31 ＋ CD36 － The target cells were collected in PBS solution containing 20% ​​FBS and maintained at 4°C.

[0127] Note: If FACS is not available, a two-step MACS process of "CD31 positive selection + CD36 negative platelet ablation" can be used; however, the purity is usually lower than that of FACS.

[0128] Specifically, the two-step MACS process of "CD31 positive selection + CD36 negative platelet ablation" is as follows:

[0129] (1) CD31 ＋ Positive selection: Cells expressing CD31 were captured in a magnetic column using anti-CD31 magnetic beads, and enriched CD31 was obtained by demagnetization and elution. ＋ cell;

[0130] (2) CD36 － Yin selection: Enriched CD31 ＋ Cells were negatively removed using anti-CD36 magnetic beads. ＋ The cells were retained in the column and discarded; the eluent was collected to obtain CD31. ＋ CD36 － Target cells.

[0131] Because CD36 is highly expressed on platelets, negative selection (negative sorting) can remove platelets from CD31. ＋ Removed from cells.

[0132] 4. Adherent culture and short-term amplification

[0133] Matrix coating: 0.1% gelatin (room temperature for 30 minutes).

[0134] Inoculation density: 1.0-2.5×10 4 cells / cm 2(High density on the first day is beneficial for adhesion to the wall).

[0135] Culture conditions: EGM-2 / EC maintenance medium, 37℃, 5% CO2; change the medium after 24 h, and then every 48 h thereafter.

[0136] Identification of phenotype maintenance:

[0137] Laminar flow stimulation: set at 5 dyn / cm², 1 Hz, to simulate intracardiac blood flow pattern, KLF2 / eNOS upregulation can be induced after 12 hours.

[0138] Passage limitation: The experiment was completed within P0-P2, and a "coronary artery-like" tendency gradually appeared after P3 (CD36 increased, Npr3 decreased).

[0139] 5. Cryopreservation and thawing

[0140] Cryopreservation solution: 90% FBS + 10% DMSO; density 1-2 × 10⁻⁶ 6 Cells / mL; the temperature was programmed to drop to -80°C and then transferred to liquid nitrogen.

[0141] Resuscitation: Thaw rapidly at 37℃, dilute with 2× volume of culture medium, centrifuge at 300×g for 5 min to remove DMSO, and inoculate as usual.

[0142] 6. Quality Control Indicators and Judgments

[0143] (1) Phenotype / Molecular

[0144] Flow cytometry: CD31 ＋ Cell percentage ≥85%, and CD36 low / − Cell percentage ≥80%;

[0145] Immunofluorescence / staining: Npr3 was positive in ≥70% of cells in the membrane / cytoplasm.

[0146] (2) Functional identification

[0147] Shear response: 5-10 dyn / cm on a track-shaking table 2 After culturing for 12 hours (oscillating at 100-180 rpm), KLF2 and eNOS mRNA were upregulated by ≥2-fold.

[0148] (3) Negative exclusion

[0149] CD45 + ;

[0150] Coronary artery vascularization risk markers: simultaneous increase in CD36, FABP4, and VWF with decrease in Npr3 → considered "off-target" endothelial sorting, requiring exclusion.

[0151] (4) Survival rate and contamination

[0152] Cell viability ≥85% (7-AAD or DAPI rejection method, dye negative means live cells); bacteria / mycoplasma negative.

[0153] (5) Release criteria

[0154] CD31 ＋ ≥85%, Npr3 ＋ ≥70%, survival rate ≥85%, contamination negative; any two functions must meet the standards.

[0155] Example 2

[0156] EEC extraction from newborn mice (newborn mice aged 3 to 5 days after birth) (using FACS sorting technology):

[0157] Due to the softening of the myocardium, the digestion time is shortened to 6-8 minutes per round, for a total of 2 rounds.

[0158] After cell adhesion, CD31 and Npr3 were co-stained.

[0159] Yield 0.8-2.0×10 5 Cells / heart; Npr3 positivity rate is higher (>80%).

[0160] like Figure 2 As shown, for the flow sorting (CD45) － CD31 ＋ CD36 － Immunofluorescence staining of the cells showed that most cells specifically expressed Npr3 (positive rate >80%), confirming that the cell population obtained by this sorting strategy was endocardial endothelium.

[0161] Comparative Example

[0162] Comparative Example A: CD31 only ＋ Selected, no CD36 － Negative sorting (using FACS sorting technology).

[0163] Purity: The endothelial cell yield was extremely low, and Npr3 positivity was significantly reduced.

[0164] like Figure 3 As shown, for the flow sorting (CD45) － CD31 ＋ Immunofluorescence staining of cells showed that only a small number of cells specifically expressed Npr3 (positive rate <10%), indicating that the endocardial endothelial cell acquisition rate was extremely low based on a flow cytometry sorting strategy that only selected CD31 positive cells and did not select CD36 negative cells.

[0165] Conclusion: CD36 negative gating is a necessary condition for distinguishing EEC from coronary / microvascular endothelium.

[0166] Comparative Example B: Whole myocardial digestion without endothelial enrichment (using FACS sorting technology).

[0167] High yield but low EEC ratio (Npr3) ＋ <40%), and subsequent culture showed "coronary artery-like" characteristics.

[0168] Conclusion: Directional sampling / scraping of the endometrium can significantly increase the initiation rate of EEC.

[0169] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. A method for extracting primary endocardial endothelial cells suitable for cardiovascular disease research, characterized in that, Includes the following steps: (1) Sampling and endocardial enrichment: Mice were anesthetized and heparinized. After the heart was lavaged to remove blood, pericardial fat and adventitia were removed under a stereomicroscope. The free walls of the left and right ventricles and the interventricular septum were precisely removed along the aortic / pulmonary artery inlet. The endocardial tissue was enriched by gently scraping the cavitary surface with a sterile soft scraper, avoiding deep myocardial dissection; or a thin slice of tissue close to the endocardium of 0.5-3.0 mm was carefully cut as a digestion sample. (2) Tissue digestion and single-cell formation: The enriched endocardial tissue blocks were cut into small pieces, digested with a mixed enzyme solution, and then mechanically dispersed by grinding through a filter to terminate digestion. The enzyme digestion and mechanical dispersion steps were repeated. The cell pellet was then collected by centrifugation, and the red blood cells were removed with red blood cell lysis buffer. Finally, the cell suspension was obtained by repeated low-speed centrifugation and resuspending and washing with PBB medium. (3) FACS fine sorting: S1. Staining: Add anti-CD45 antibody, anti-CD31 antibody, anti-CD36 antibody, and live / dead cell dye DAPI to the cell suspension. Gently mix the mixture and incubate in the dark for 20-30 minutes. S2, Control Settings: FMO control: Prepare single-color controls missing CD36 and CD31 respectively, and set a 99% confidence threshold for differentiation during subsequent gating; Isotype control: Use isotype antibodies of the same species to assess nonspecific binding; Monochrome compensation beads: Special compensation beads are used to compensate the spectrum of the fluorescence channel to ensure the accuracy of multicolor detection; S3. Washing and loading: After staining, add an appropriate amount of PBS to each tube, centrifuge, remove the supernatant, repeat washing once to remove unbound antibodies, resuspend the washed cells in an appropriate amount of PBS, and transfer them to flow cytometer sample tubes. S4, Gating Strategy: Cell population → Single cell: Plot a two-parameter graph on FSC-A and FSC-H, excluding aggregated cells and retaining single cell populations; → Live cells: Set a threshold on the DAPI channel and select DAPI... － Negative cells, excluding dead cells; →CD45 － Gate: Set a threshold on the CD45 channel based on the FMO reference to exclude CD45. ＋ White blood cell population; → CD31 ＋ Gate: Set the threshold on channel CD31 according to the FMO reference, and select CD31. ＋ Endothelial cells; →CD36 － Gate: Set a threshold on channel CD36 based on the FMO reference to exclude CD36. ＋ Cells, retaining CD36 － Target endothelial cells; After each gating step, the corresponding cell proportion is recorded, and the compensation matrix and gating statistics are exported from the instrument software. S5. Cell collection: Based on the final gate CD45 － CD31 ＋ CD36 － The target cells were sorted into collection tubes containing pre-cooled PBS solution containing FBS; (4) Adherent culture and short-term amplification The sorted target cells were seeded into gelatin-coated culture dishes and cultured using EGM-2 / EC maintenance medium; (5) Quality control indicators and judgment 1) Quality control indicators ①Phenotype / Molecular Flow cytometry: CD31 ＋ Cell percentage ≥85%, and CD36 low / − Cell percentage ≥80%; Immunofluorescence / staining: ≥70% of cells were positive for Npr3 in the membrane / cytoplasm; ② Functional identification Shear response: Cells respond to simulated blood flow shear forces of 5-10 dyn / cm 2 After 12 hours of culture on an orbital shaker, KLF2 and eNOS mRNA were upregulated by ≥2-fold. ③ Negative exclusion CD45 + ; Coronary artery vascularization risk markers: simultaneous elevation of CD36, FABP4, and VWF with a decrease in Npr3 → considered "off-target" endothelial sorting, requiring exclusion; ④ Survival rate and pollution Cell viability ≥85%; Bacteria / Mycoplasma negative; 2) Judgment criteria CD31 ＋ ≥85%, Npr3 ＋ ≥70%, survival rate ≥85%, contamination negative; any two functions must meet the standards.

2. The method according to claim 1, characterized in that, The (2) tissue digestion and single-cell formation specifically includes the following steps: S1. Pretreatment: The enriched endocardial tissue blocks were placed in PBS containing double antibodies, cut into small pieces, and placed on ice to allow the tissue blocks to settle. The supernatant was then removed and the tissue blocks were washed. S2, Enzyme Digestion I: Digest the tissue precipitate for 30-40 minutes using an enzyme mixture containing Collagenase II, Collagenase I, Collagenase IV and DNase I; S3. Mechanical dispersion: Place the digested mixture in a sterile filter, grind it using a syringe plunger, and add an equal volume of PBB culture medium to rinse and terminate digestion. S4. Repeat digestion: Centrifuge the cell suspension, remove the supernatant, add enzyme digestion solution again, and continue digestion for 5-8 minutes. Then repeat the mechanical dispersion step in S3, centrifuge again, and collect the cell pellet. S5. Remove red blood cells: Add twice the volume of red blood cell lysis buffer to the cell pellet and treat for 1-2 minutes to remove red blood cells. Stop the lysis with PBS. S6. Low-speed washing: Centrifuge the lysate, remove the supernatant and resuspend in PBB medium. Repeat twice to complete the washing and obtain a cell suspension.

3. The method according to claim 2, characterized in that, The enzyme mixture consisted of: Collagenase II 1-2 mg / mL, Collagenase I 1-2 mg / mL, Collagenase IV 0.5-1 mg / mL, and DNase I 50-100 μg / mL, using Ca-free... 2+ / Mg 2+ Prepare with PBS.

4. The method according to claim 1, characterized in that, In step (2), the PBB culture medium is a PBS solution containing 2% v / v FBS and 0.2% m / v BSA.

5. The method according to claim 1, characterized in that, In step (2), the total digestion time of the enzyme is 30-40 min.

6. The method according to claim 1, characterized in that, In step (2), the centrifugation conditions are: 4°C, 300-350×g for 5 minutes.

7. The method according to claim 1, characterized in that, In step (4), the EGM-2 / EC maintenance medium consists of: Endothelial Cell Medium base + 1% Endothelial Cell Growth Supplement + 2%-5% FBS + 1% penicillin / streptomycin solution.

8. The method according to claim 1, characterized in that, In step (4), the seeding density of the cells is 1.0-2.5 × 10⁻⁶. 4 cells / cm 2 .

9. The method according to claim 1, characterized in that, In step (4), the culture conditions are: 37°C, 5% CO2 culture for 24 hours, then change the medium, and then change the medium every 48 hours thereafter.

10. The method according to claim 1, characterized in that, In step (5), P0-P2 generation cells are used for quality control testing.

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