A method for isolating and culturing high-purity mouse kidney microvascular endothelial cells
By combining enzymatic digestion, multi-stage filtration, and immunomagnetic bead sorting, the problems of low purity and poor activity in the extraction of mouse kidney microvascular endothelial cells were solved, achieving high-purity and high-activity cell separation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
- Filing Date
- 2026-04-02
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies make it difficult to efficiently extract high-purity mouse kidney microvascular endothelial cells. Conventional methods are prone to introducing medullary components and epithelial cell contamination, resulting in low cell purity and poor activity.
A combined approach of enzymatic digestion, multi-stage filtration, differential adhesion, and immunomagnetic bead sorting was employed. This approach combined collagenase IV and deoxyribonuclease I digestion, utilizing the difference in adhesion speed between endothelial cells and fibroblasts. Initial purification was achieved by coating culture dishes with gelatin, followed by separation and purification using antibody-labeled magnetic beads.
It significantly improved the purity of mouse kidney microvascular endothelial cells to over 95%, maintained good biological activity, and effectively solved the problem of contamination by other cells.
Smart Images
Figure CN122146580A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell culture technology, specifically to a method for isolating and culturing high-purity mouse kidney microvascular endothelial cells. Background Technology
[0002] Renal microvascular endothelial cells (RMECs) are the fundamental structural and functional units of the renal microcirculation, widely distributed in the peritubular capillary network of the glomeruli and renal tubules. Studies have shown that RMECs not only act as a semipermeable barrier but also play a crucial role in the pathophysiological processes of various kidney diseases. For example, in glomerulonephritis and hypertensive nephropathy, endothelial cell damage and dysfunction are often the initiating factors leading to microvascular thinning and renal parenchymal sclerosis; in renal ischemia-reperfusion injury, endothelial cell activation is a key step in mediating inflammatory cell infiltration and acute kidney injury; furthermore, in kidney transplant rejection, endothelial cells, as the primary presenters of donor antigens, are the primary target of antibody-mediated rejection. Therefore, constructing a high-purity, biologically stable in vitro mouse renal endothelial cell model is of significant application value for elucidating the molecular mechanisms of these diseases, validating gene-edited mouse phenotypes, and screening targeted angiogenic protective drugs.
[0003] However, existing primary endothelial cell isolation techniques are mostly aimed at human umbilical vein endothelial cells (HUVECs) or rat aortic endothelial cells, while efficient extraction techniques for mouse kidney microvascular endothelial cells are relatively scarce. Due to the small size of mouse kidneys and the difficulty in accurately identifying the corticomedullary boundary with the naked eye, conventional tissue block adhesion methods or non-specific enzymatic digestion methods easily introduce medullary components, leading to excessive fibroblast proliferation. Furthermore, because renal tubular epithelial cells and endothelial cells overlap in density and adhesion characteristics, single physical separation methods are insufficient to completely remove epithelial cell contamination, resulting in low cell purity and phenotypic loss after passage. Currently, there is a lack of patent reports on an efficient extraction method that combines precise cortical sampling, gentle enzymatic digestion, and specific immunosorting.
[0004] In conclusion, developing a targeted, high-purity, and reproducible method for the extraction and identification of mouse renal microvascular endothelial cells is an urgent need to address the technical challenge of the scarcity of high-quality cell models in current basic research on nephrology. Summary of the Invention
[0005] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a method for isolating and culturing high-purity mouse renal microvascular endothelial cells, thereby solving the problems of low cell purity, severe contamination by other cells, and poor activity during the extraction of mouse renal microvascular endothelial cells.
[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for isolating and culturing high-purity mouse kidney microvascular endothelial cells is provided, comprising the following steps: (1) Enzymatic digestion: Digestive juice was added to a block of mouse renal cortex tissue, and a digestive mixture was obtained after digestion; (2) Multi-stage filtration to remove impurities: The digested mixture obtained in step (1) is filtered step by step, and the final filtrate is collected to obtain a single-cell suspension; then the single-cell suspension is centrifuged and the supernatant is discarded, and the cell precipitate is collected. (3) Differential adhesion initial purification: The cell pellet obtained in step (2) was resuspended in endothelial cell complete culture medium and then seeded into a culture dish pre-coated with gelatin and allowed to stand for culture. Then, the supernatant that did not adhere was aspirated and transferred to a culture flask pre-coated with gelatin. After replenishing with endothelial cell complete culture medium, amplification culture was carried out. (4) Immunomagnetic bead sorting: Add antibody to the single-cell suspension obtained in step (3) for incubation, wash and add magnetic beads for secondary incubation, then pass the single-cell suspension through a separation column placed in a magnetic field, collect the effluent, wash with buffer and collect the eluent, centrifuge and resuspend and inoculate into culture dishes pre-coated with gelatin for culture.
[0007] Furthermore, the digestive fluid in step (1) includes collagenase IV and deoxyribonuclease I.
[0008] Furthermore, in step (1), the digestion solution was prepared using RPMI 1640 basal medium as a solvent and fetal bovine serum was added.
[0009] Furthermore, the filter diameters of the step-by-step filtration in step (2) are 80 mesh, 120-160 mesh and 200 mesh respectively.
[0010] Furthermore, the conditions for static culture and amplification culture in step (3) are: 37℃, 5% CO2.
[0011] Furthermore, when the cell fusion rate of the cells cultured in step (3) reaches 75%-85%, step (4) immunomagnetic bead sorting is performed.
[0012] Furthermore, in step (4), the antibody is a biotinylated anti-mouse CD31 antibody.
[0013] Furthermore, in step (4), the magnetic beads are streptavidin-coupled magnetic beads.
[0014] This invention offers the following advantages: By combining an enzymatic digestion-differential adhesion-magnetic bead sorting strategy, particularly incorporating the differential adhesion step for fibroblast removal, this invention significantly improves the purity of mouse renal microvascular endothelial cells, which is crucial for obtaining high-quality primary cells. The mouse renal microvascular endothelial cells obtained using the isolation and culture method of this invention achieve a purity of over 95% and maintain good biological activity, effectively solving the problem of severe contamination by other cells in existing technologies. Attached Figure Description
[0015] Figure 1 A schematic diagram of the extraction process of mouse kidney microvascular endothelial cells; Figure 2 The light microscopic morphology of mouse kidney microvascular endothelial cells obtained after 30 minutes of enzymatic digestion; Figure 3 Immunofluorescence staining image of mouse renal microvascular endothelial cells obtained in Example 1; Figure 4 A diagram illustrating the lumen formation experiment of endothelial cells cultured at 37°C. Figure 5 A diagram illustrating the lumen formation experiment of endothelial cells cultured at 33°C. Figure 6 Morphological image of mouse kidney microvascular endothelial cells obtained after 45 minutes of enzymatic digestion; Figure 7 This is an immunofluorescence staining image of mouse kidney microvascular endothelial cells obtained in a comparative manner. Detailed Implementation
[0016] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0017] The 3-4 week old male C57BL / 6 mice used in the experiments of this invention were purchased from the Animal Experiment Center of Xi'an Jiaotong University and housed in a standard specific pathogen-free (SPF) laboratory with free access to water and food, and a 12-hour light / dark cycle. All experiments complied with the standards of the Animal Ethics Committee.
[0018] Example 1: A method for isolating and culturing high-purity mouse kidney microvascular endothelial cells; the extraction process flowchart is shown below. Figure 1 This includes the following steps: (1) Enzymatic digestion: Mice were euthanized by cervical dislocation and their entire bodies were immersed in 75% alcohol for 5 minutes for disinfection. The mice were then removed and fixed under aseptic conditions. The abdominal cavity was opened, and both kidneys, with intact capsules, were quickly removed and placed in pre-cooled complete culture medium containing 1% (v / v) penicillin / streptomycin antibiotics. In a laminar flow hood, the kidneys were immersed in 75% alcohol for 3 seconds, and immediately washed twice with copious amounts of PBS to remove the ethanol (this step is unnecessary if the dissection of the mice is performed under aseptic conditions). The renal capsule and connective tissue were removed, and the kidneys were cut along the longitudinal axis, separating and preserving the renal cortex, and removing the medulla and renal pelvis. The renal cortex was then cut into pieces approximately 1 mm in size. 3 The tissue blocks were added to a 5 mL digestion solution containing 0.75 mg / mL collagenase IV (Worthington Biotechnology) and 0.5 mg / mL DNase I (Solepro) (using RPMI 1640 basal medium (Gibco™, Thermo Fisher Scientific) as the solvent, with 5% (v / v) fetal bovine serum (FBS) added. The mixture was placed in a 37°C shaker and digested at 80 rpm for 30 minutes, vortexing for 30 seconds every 10 minutes until the tissue blocks were essentially dissolved. After digestion, the mixture was placed on ice to obtain the digested mixture.
[0019] (2) Multi-stage filtration to remove impurities: The digested mixture obtained in step (1) was filtered through an 80-mesh filter into a 15 mL centrifuge tube to remove undigested large tissue fragments; then the filtrate was filtered step by step (filter diameters of 80 mesh, 150 mesh and 200 mesh, respectively), and the final filtrate was collected into a 15 mL centrifuge tube to obtain a single-cell suspension. The filtrate was centrifuged at 1500 rpm for 5 minutes and the supernatant was discarded. If there were many precipitated red blood cells, 1 mL of red blood cell lysis buffer was added and incubated at room temperature for 2 minutes. After adding 10 mL of PBS to terminate the reaction, the precipitate was collected by centrifugation again.
[0020] (3) Differential Adhesion and Initial Purification: The cell pellet obtained in step (2) was resuspended in complete endothelial cell culture medium (purchased from ScienCell, prepared according to the instructions with the addition of matching endothelial cell growth supplement (ECGS), fetal bovine serum (FBS) and penicillin-streptomycin) and seeded into culture dishes pre-coated with gelatin. The culture was then placed in a 37°C, 5% CO2 incubator for 45 minutes. Taking advantage of the faster adhesion rate of fibroblasts compared to endothelial cells, after 45 minutes, the supernatant (containing endothelial cells) that had not adhered was carefully aspirated and transferred to a culture flask or dish pre-coated with gelatin, and fresh culture medium was added. When the cell confluence reached approximately 80%, the cells were digested and collected for subsequent sorting.
[0021] (4) Immunomagnetic bead sorting: The cells expanded in step (3) were made into a single-cell suspension and the concentration was adjusted to 1×10⁻⁶ with MojoSort™ buffer. 7 cells / 100μL. Per 1×10 7 10 μL of biotinylated anti-mouse CD31 antibody was added to each cell line, mixed, and incubated at 4°C for 15 minutes. After washing, 10 μL of streptavidin-conjugated magnetic beads were added, and the cells were incubated at 4°C for 15 minutes. The cell suspension was passed through a MojoSort™ separation column placed in a magnetic field, and the eluent was collected. This process was repeated three times, and the cells were washed with buffer to remove unbound negative cells (non-endothelial cells). The magnetic field was removed, and buffer was added to elute the positive cells (endothelial cells) adsorbed on the column. The eluent was collected, centrifuged, resuspended, and seeded into gelatin-coated culture dishes for culture. The biotinylated anti-mouse CD31 antibody and streptavidin magnetic beads were both MojoSort™ products purchased from BioLegend.
[0022] Example 2: Identification of renal microvascular endothelial cells Renal endothelial cells obtained in Example 1, passaged to the 4th generation, were subjected to morphological and immunofluorescence identification. The specific experimental steps for immunofluorescence identification are as follows: (1) The mouse primary renal microvascular endothelial cells extracted in this invention were cultured to the logarithmic growth phase and seeded at an appropriate density into culture wells pre-covered with coverslips. The cells were then cultured in a 37°C, 5% CO2 incubator. When the cells reached 70%-80% confluence, the cell culture wells were removed, the culture medium was discarded, and the cell slides were washed with pre-cooled PBS buffer to remove residual culture medium and impurities. An appropriate amount of 4% paraformaldehyde fixative was added to the culture wells, ensuring complete coverage of the cell slides. The cells were fixed at room temperature for 15 minutes. After fixation, the fixative was discarded, and the cell slides were washed three times with PBST. 5% BSA blocking solution was added to the culture wells to cover the cell slides, and the cells were sealed in a 37°C humidified chamber for 30 minutes.
[0023] (2) Primary antibody incubation: After blocking, discard the blocking solution and wash the cell slides with PBST; according to the primary antibody instructions, dilute vWF primary antibody (1:200) and α-SMA primary antibody (1:200) with 5% BSA and add them to the cell slides; place the cell slides in a humidified box and incubate overnight at 4°C in the dark.
[0024] (3) Secondary antibody incubation: After the primary antibody incubation is completed, remove the humidified box and place it at room temperature for 30 minutes to warm up; discard the primary antibody solution and wash the cell slides with PBST to remove unbound primary antibody; according to the fluorescent secondary antibody instructions, dilute the green fluorescent secondary antibody (corresponding to vWF primary antibody, 1:300) and the red fluorescent secondary antibody (corresponding to α-SMA primary antibody, 1:300) with 5% BSA and drop them onto the cell slides; place the cell slides in the humidified box and incubate at room temperature in the dark for 1 hour (avoid light throughout the process to prevent quenching of the fluorescent secondary antibody and affecting the experimental results).
[0025] (3) DAPI nuclear counterstaining: After the secondary antibody incubation, discard the secondary antibody solution and wash the cell slide with PBST to remove unbound fluorescent secondary antibody; dilute the DAPI staining solution with PBST to an appropriate concentration (1:1000) and add it to the cell slide, incubate at room temperature in the dark for 5-10 minutes to stain the cell nuclei; after the counterstaining, discard the DAPI staining solution and wash the cell slide with PBST.
[0026] (4) Mounting and microscopic examination: After washing, gently absorb the PBST liquid around the cell slide with absorbent paper; add 1-2 drops of anti-fluorescence quenching mounting solution to the center of the slide, and gently cover the cell slide (cell side down) on the mounting solution to avoid air bubbles; after the mounting solution solidifies, observe it under a fluorescence microscope: select the corresponding excitation light (green fluorescence corresponds to vWF, red fluorescence corresponds to α-SMA, and blue fluorescence corresponds to DAPI), observe and photograph the cell fluorescence signal, and record the experimental results.
[0027] (5) Fluorescent reagents (fluorescent secondary antibody, DAPI) must be stored and handled in the dark throughout the process to avoid fluorescence quenching and affecting experimental results; each step of washing must be thorough, especially after incubation of primary and secondary antibodies, unbound antibodies must be completely removed to reduce background fluorescence; the reagents used in the experiment must be freshly prepared to avoid contamination or pH changes affecting the experiment.
[0028] Depend on Figure 2 It can be seen that the cells exhibit a typical cobblestone arrangement and possess contact inhibition properties. From Figure 3 Cells were fixed with 4% paraformaldehyde and incubated with primary and fluorescent secondary antibodies. The endothelial cell marker von Willebrand factor (vWF) was positive (green fluorescence), while the mesenchymal / smooth muscle marker α-smooth muscle actin (α-SMA) was negative (red fluorescence). DAPI counterstaining revealed clearly visible cell nuclei. These results indicate that the cells extracted in this invention are high-purity mouse kidney microvascular endothelial cells with excellent purity, high viability, and few impurities. Only a small amount of mouse kidney is required, and a single extraction can yield a sufficient number of primary kidney microvascular endothelial cells for culture and identification.
[0029] Example 3: Validation of in vitro angiogenesis capacity To verify whether the endothelial cells obtained in Example 1 possess normal biological functions, a tube formation experiment was conducted. The specific experimental steps are as follows: The matrix gel (purchased from Corning) was thawed overnight at 4°C. A pre-cooled 24-well plate was used, and 30 μL of matrix gel was added to each well. The gel was spread evenly using a pipette tip, and the plate was incubated at 4°C for 6 hours. Afterward, the plate was removed and incubated at 37°C for 30 minutes to solidify. Cells extracted in Example 1 were digested and counted, resuspended in complete endothelial cell culture medium, and cultured at a ratio of 1 × 10⁻⁶. 5 The cells were seeded at a density of 1 cell / well on the solidified matrix gel surface and then incubated at 37°C in a 5% CO2 incubator for 2 to 4 hours.
[0030] The results are as follows Figure 4 As shown, the cells spontaneously migrated on the surface of the matrix gel and connected end to end, forming a typical, closed network lumen structure with obvious branching points, proving that the mouse kidney microvascular endothelial cells extracted in this invention maintained good angiogenesis capacity.
[0031] Example 4: Effects of different culture temperatures on cell growth The operation steps of this embodiment are the same as those of embodiment 1. The only difference is that in the subsequent cell culture process of differential adhesion initial purification in step (3) and immunomagnetic bead sorting in step (4), the temperature of the cell culture chamber is set to 33°C, and the other conditions remain unchanged.
[0032] The results showed that, when cultured at 33℃, the cell adhesion time, proliferation rate, and biological function were not significantly different from those in Example 1 (37℃), and the cells were in good condition. The results indicate that the cells extracted using this method have a certain degree of adaptability to culture temperature and can still maintain the endothelial cell phenotype at 33℃ (see...). Figure 5 ).
[0033] Example 5: Effect of different enzyme digestion times on cell yield The operation steps of this embodiment are basically the same as those of Embodiment 1. The only difference is that in step (1) enzymatic digestion, the digestion time of the mixture containing collagenase IV and DNase I is extended to 45 minutes.
[0034] The results showed that compared with Example 1 (digestion for 30 minutes), the total number of cells in the single-cell suspension obtained after digestion for 45 minutes increased by approximately 10-15%. After subsequent differential adhesion and magnetic bead sorting, a sufficient number of viable endothelial cells could still be obtained for subsequent experiments, and no abnormalities were observed in the growth status of the cells after adhesion (see...). Figure 2 and Figure 6 This indicates that the digestion system has good safety and can obtain a sufficient amount of target cells within a 30-45 minute timeframe.
[0035] Comparative example: This comparative example aims to demonstrate the necessity of differential adhesion initial purification in step (3) of the method of the present invention. The operation steps of this comparative example are the same as those of Example 1, except that step (3) is omitted. That is, the cell pellet obtained by multi-stage filtration in step (2) is directly resuspended in complete endothelial cell culture medium and all of them are seeded into culture flasks pre-coated with gelatin for culture. After the cells have grown to confluence, step (4) magnetic bead sorting is performed.
[0036] The results showed that, compared with Example 1, the vWF positivity rate in the cells obtained in this comparative example was significantly lower, and a large number of α-SMA-positive (red fluorescence) contaminating cells were present in the field of view (see Example 1). Figure 3 and Figure 7 ).
[0037] The results of the above embodiments and comparative examples show that the present invention, by combining the strategy of "enzymatic digestion-differential adhesion-magnetic bead sorting", especially the step of differential adhesion to remove fibroblasts, can significantly improve the purity of mouse kidney microvascular endothelial cells, which is the key to obtaining high-quality primary cells.
[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for isolating and culturing high-purity mouse kidney microvascular endothelial cells, characterized in that, Includes the following steps: (1) Enzymatic digestion: Digestive juice was added to a block of mouse renal cortex tissue, and a digestive mixture was obtained after digestion; (2) Multi-stage filtration to remove impurities: The digested mixture obtained in step (1) is filtered step by step, and the final filtrate is collected to obtain a single-cell suspension; then the single-cell suspension is centrifuged and the supernatant is discarded, and the cell precipitate is collected. (3) Differential adhesion initial purification: The cell pellet obtained in step (2) was resuspended in endothelial cell complete culture medium and then seeded into a culture dish pre-coated with gelatin and allowed to stand for culture. Then, the supernatant that did not adhere was aspirated and transferred to a culture flask pre-coated with gelatin. After replenishing with endothelial cell complete culture medium, amplification culture was carried out. (4) Immunomagnetic bead sorting: Add antibody to the single-cell suspension obtained in step (3) for incubation, wash and add magnetic beads for secondary incubation, then pass the single-cell suspension through a separation column placed in a magnetic field, collect the effluent, wash with buffer and collect the eluent, centrifuge and resuspend and inoculate into culture dishes pre-coated with gelatin for culture.
2. The isolation and culture method according to claim 1, characterized in that, The digestive fluid in step (1) includes collagenase IV and deoxyribonuclease I.
3. The isolation and culture method according to claim 2, characterized in that, The digestion solution described in step (1) was prepared using RPMI 1640 basal medium as a solvent and fetal bovine serum was added.
4. The isolation and culture method according to claim 1, characterized in that, The filter diameters of the step-by-step filtration in step (2) are 80 mesh, 120-160 mesh and 200 mesh.
5. The isolation and culture method according to claim 1, characterized in that, The conditions for static culture and amplification culture in step (3) are: 37℃, 5% CO2.
6. The isolation and culture method according to claim 1, characterized in that, When the cell fusion rate of the cells cultured in step (3) reaches 75%-85%, step (4) immunomagnetic bead sorting is performed.
7. The isolation and culture method according to claim 1, characterized in that, The antibody mentioned in step (4) is a biotinylated anti-mouse CD31 antibody.
8. The isolation and culture method according to claim 1, characterized in that, The magnetic beads mentioned in step (4) are streptavidin-coupled magnetic beads.