Extraction method and culture method of primary cardiac vascular endothelial cells of newborn mice

Abstract

The invention relates to the technical field of cell culture, in particular to an extraction method and a culture method of primary cardiac vascular endothelial cells of newborn mice. The extraction method comprises the following steps: S1, preparing a heart single-cell suspension; primary magnetic labeling sorting: mixing the heart single-cell suspension with a non-endothelial cell magnetic labeling antibody, incubating at 0-10 DEG C for 10-20 minutes, adding into a magnetic cell classifier for sorting, collecting effluent, and centrifuging to obtain a cell precipitate A; and S3, secondary magnetic labeling separation: mixing the cell precipitate with an endothelial cell magnetic labeling antibody, incubating at 0-10 DEG C for 10-20 minutes, adding into a magnetic cell separator for separation, and collecting endothelial cells in a magnetic column. According to the method, the primary cardiac endothelial cells of the newborn mice are specifically extracted through an immunomagnetic column method, and the extraction rate of the primary cardiac endothelial cells of the newborn mice is remarkably increased.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly relates to a method for extracting and culturing primary cardiac vascular endothelial cells of neonatal mice. Background Art

[0002] For research at the cellular and molecular levels, primary vascular endothelial cells have irreplaceable advantages. First, primary vascular endothelial cells can survive in an artificial culture environment, which avoids the interference of humoral, neural, and endocrine factors. Second, primary vascular endothelial cells maintain the genetic type, phenotype, and biomarkers of the original animal in vivo, which is conducive to the study of the cell cycle, cell signal transduction, and metabolic pathways. In addition, primary vascular endothelial cells can clarify the pathophysiological characteristics of heart diseases through various experimental techniques (such as flow cytometry, confocal microscopy, fluorescence, and isotope labeling, etc.). Therefore, primary vascular endothelial cells are an important tool for studying the pathogenesis of blood vessels.

[0003] Currently, the extraction and culture protocols for primary vascular endothelial cells of rats have been greatly optimized and popularized. However, in recent years, transgenic mouse models have begun to be widely used in cardiology research, which has led to the emergence of a need for a protocol for extracting and culturing primary vascular endothelial cells from neonatal mice. Compared with the hearts of neonatal rats, the hearts of neonatal mice are smaller, more fragile, and more prone to over-digestion, resulting in a decrease in cell yield and viability. Therefore, there is an urgent need for a more effective method to extract and purify primary endothelial cells of neonatal mice.

[0004] The following methods are mostly used for the extraction of primary vascular endothelial cells in the prior art: (1) enzyme digestion + cell sieve filtration method; (2) enzyme digestion + differential adhesion method; (3) enzyme digestion + tissue block adhesion method. However, currently, most of these methods use two digestive enzymes, collagenase and trypsin, for co-digestion, resulting in over-digestion, and the hearts of neonatal mice are originally very small, leading to a significant reduction in cell yield and cell viability; the methods for extracting primary vascular endothelial cells by these methods currently are all non-specific cell screening, and the adhesion times of endothelial cells and fibroblasts are similar, so there is a problem of low purity of the extracted endothelial cells. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for extracting and culturing primary cardiac vascular endothelial cells of neonatal mice. The technical problems to be solved by the present invention are: (1) solving the problem that neonatal mice are easily over-digested, resulting in a significant reduction in cell yield and cell viability; (2) solving the problem of low purity of the extracted primary vascular endothelial cells.

[0006] The present invention solves the technical problems by adopting the following technical solutions:

[0007] In the first aspect of the present invention, a method for extracting primary cardiac vascular endothelial cells from neonatal mice is provided, comprising the following steps:

[0008] S1 Preparation of a single-cell suspension of the heart

[0009] Digest the hearts of 1- to 10-day-old suckling mice with a digestive enzyme solution, pipette and resuspend, add erythrocyte lysate, and then centrifuge and resuspend to obtain a single-cell suspension of the heart;

[0010] S2 First magnetic labeling and sorting

[0011] Mix the single-cell suspension of the heart with a non-endothelial cell magnetic labeling antibody, incubate at 0-10 °C for 10-20 min, add to a magnetic cell sorter for sorting, collect the effluent, and centrifuge to obtain cell pellet A;

[0012] S3 Second magnetic labeling and sorting

[0013] Mix cell pellet A with an endothelial cell magnetic labeling antibody, incubate at 0-10 °C for 10-20 min, add to a magnetic cell sorter for sorting, and collect the endothelial cells in the magnetic column to obtain the desired product.

[0014] In some embodiments of the present invention, the S1 preparation of a single-cell suspension of the heart includes:

[0015] S11 Digest the hearts of 1- to 10-day-old suckling mice with a digestive enzyme solution to obtain cell suspension A;

[0016] S12 Incubate cell suspension A at 35-40 °C for 10-20 min and pipette; repeat step S12 2-5 times; to obtain cell suspension B. S13 Filter cell suspension B to terminate digestion, mix cell suspension B with erythrocyte lysate at a volume ratio of 1:(5-20), centrifuge and resuspend to obtain a single-cell suspension of the heart, and perform cell counting on the single-cell suspension of the heart to obtain the total number of cells.

[0017] In some embodiments of the present invention, the material-liquid ratio of type II collagenase powder to PBS in the digestive enzyme solution is 10 mg:

[0018] (1-3) ml; the volume ratio of cell suspension B to erythrocyte lysate is 1:(8-12).

[0019] In some embodiments of the present invention, the digestive enzyme solution is prepared by mixing type II collagenase powder and PBS at a material-liquid ratio of 10 mg:(1.5-2.5) ml.

[0020] In some embodiments of the present invention, the particle size filtered in the step S13 is 70 μm. The common pore sizes of the filter meshes used for filtering cell suspensions in cell experiments include 70 μm, 30 μm, and 40 μm. The main reasons for choosing a 70-μm pore size are as follows:

[0021] 1. Removal of cell clumps and tissue debris: The 70-μm pore size filter mesh can effectively remove undispersed cell clumps, tissue masses, or impurities, etc. Since specific sorting using immunomagnetic beads will be carried out later, it is not necessary to screen out non-target cells at this step. As long as cell clumps and tissue debris are removed.

[0022] 2. Ensure cell integrity: The 70-μm pore size is suitable for most cells to pass through, while avoiding physical damage to the cells and maintaining the cell activity and function. If a too small pore size is selected, it may cause physical damage to some cells, resulting in the loss of cell activity. If the cells lose their activity, they cannot specifically adhere to the immunomagnetic beads in subsequent experiments, and thus specific sorting cannot be carried out.

[0023] In some embodiments of the present invention, the first magnetic labeling sorting in the step S2 includes:

[0024] S21 Mix the single-cell suspension of the heart with the non-endothelial cell magnetic labeling antibody, incubate at 0-10 °C for 10-20 min, and adjust the total cell number concentration to be lower than 1×10 7 cells / ml to obtain cell suspension C;

[0025] S22 Place the LD column into the magnetic cell sorter, add cell suspension C to the magnetic cell sorter for sorting, collect the effluent, and centrifuge to obtain cell pellet A.

[0026] In some embodiments of the present invention, the concentration of the non-endothelial cell magnetic labeling antibody in the cell suspension C is 30 μl / ml to 40 μl / ml.

[0027] In some embodiments of the present invention, the second magnetic labeling sorting in the step S3 includes:

[0028] S31 Mix the cell pellet with the endothelial cell magnetic labeling antibody, incubate at 0-10 °C for 10-20 min, and adjust the total cell number concentration to be lower than 1×10 7 cells / ml to obtain cell suspension D;

[0029] S32 Place the MS column into the magnetic cell sorter, add cell suspension D to the magnetic cell sorter for sorting, add it to the magnetic cell sorter for sorting, and collect the endothelial cells in the magnetic column. That's it.

[0030] In some embodiments of the present invention, the concentration of the endothelial cell magnetic labeling antibody in the cell suspension D is 30 ul / ml to 40 ul / ml.

[0031] In a second aspect of the present invention, there is provided a method for culturing primary cardiac vascular endothelial cells of neonatal mice, comprising the following steps:

[0032] Step 1: Add the above-mentioned primary cardiac vascular endothelial cells of neonatal mice to a culture medium, and culture them under the conditions of 35-40 °C and 4-6% CO2, and change the medium every 20-30 h;

[0033] Step 2: Continue to culture for 1-3 generations, and that's it.

[0034] The present invention has the following beneficial effects: By only using a method of digesting neonatal mouse heart tissue with a digestive enzyme at time intervals, over-digestion is prevented; and then primary cardiac endothelial cells of neonatal mice are specifically extracted by the immunomagnetic column method, significantly improving the extraction rate of primary cardiac endothelial cells of neonatal mice. Brief Description of the Drawings

[0035] Figure 1 It shows the cell morphology and cell density at different time points of primary cardiac vascular endothelial cells of neonatal mice observed under a microscope (4×).

[0036] Figure 2 It shows the cell morphology of primary cardiac vascular endothelial cells of neonatal mice from the 0th generation (P0) to the 5th generation (P5) observed under a microscope (10×).

[0037] Figure 3 It shows the tube formation image of primary cardiac vascular endothelial cells of neonatal mice observed under a microscope (10×).

[0038] Figure 4 It shows the CD31 antibody immunofluorescence identification image of primary cardiac vascular endothelial cells of neonatal mice under a confocal microscope (40×).

[0039] Figure 5 It shows the comparative experimental results of different digestion methods in Example 1 and Comparative Example 1.

[0040] Figure 6 It shows the comparative experimental results of different sorting methods in Example 1 and Comparative Example 2.

[0041] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0042] As used in the present invention, the term "prevention" refers to preventing the occurrence of a disease and / or preventing the recurrence of a disease. Detailed Description of the Invention

[0043] The description of the present invention has described the specific implementation embodiments in detail. Those skilled in the art should recognize that the following implementation embodiments are exemplary and should not be construed as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the protection scope of the claims of the present invention. The beneficial effects of the present invention are specifically described below through examples.

[0044] Item preparation

[0045] 1. Reagents and tools

[0046] Neonatal mouse cardiac endothelial cell isolation kit (Miltenyi Biotec: 130-104-183), and there are non-endothelial cell magnetic-labeled antibodies and endothelial cell magnetic-labeled antibodies in the neonatal mouse cardiac endothelial cell isolation kit.

[0047] Magnetic cell sorter (Miltenyi Biotec: 130-042-301&130-090-312), type II collagenase powder, bovine serum albumin powder (BSA), disposable syringe (5 ml), sterilizing filter (0.22 um), filter screen (70 um), DMEM basal medium, 10% FBS DMEM complete medium, 10% FBS ECM complete medium, PBS solution, 10 cm culture dish, 6 cm culture dish, 75% alcohol, EP tube (1.5 ml), centrifuge tube (15 ml & 50 ml), LD magnetic column (Miltenyi Biotec: 130-042-901) and MS magnetic column (Miltenyi Biotec: 130-042-201), frozen ice box, instrument box, pipette tips and pipette, etc.

[0048] 2. Preparation and pretreatment

[0049] a. Digestive enzyme solution A: 20 mg of type II collagenase powder + 4 ml of PBS (filtered through a sterilizing filter and placed in a 37 °C incubator for preheating), digestive enzyme solution B: 10 mg of type II collagenase powder + 10 mg of trypsin powder + 4 ml of PBS (filtered through a sterilizing filter and placed in a 37 °C incubator for preheating).

[0050] b. Buffer solution (0.5% BSA): 100 mg of BSA + 20 ml of PBS (filtered through a sterilizing filter and placed in a 4 °C refrigerator for pre-cooling)

[0051] c. Heart tissue holding solution: 5 ml of DMEM basal medium (placed in a 4 °C refrigerator for pre-cooling)

[0052] d. Termination solution: 10% FBS DMEM complete medium (placed in a 37 °C incubator for preheating)

[0053] e.PBS: 15 ml (pre-cooled in a 4 °C refrigerator)

[0054] Example 1

[0055] A method for extracting and culturing primary cardiac vascular endothelial cells from neonatal mice, comprising the following steps:

[0056] S1 Preparation of single-cell suspension of neonatal mouse hearts

[0057] a. Twenty C57 neonatal mice (1 - 3 days old), regardless of gender, were immersed in 75% alcohol for about 10 s (about 4 mice each time). The neonatal mice were fixed in the left palm. The chest and abdominal skin of the mice were disinfected 3 times with a sterile cotton swab dipped in 75% ethanol. After opening the chest slightly to the left of the midline at the xiphoid process with an ophthalmic iris scissors (starting from the abdomen), the right edge of the sternum was pressed with scissors to make the heart jump out naturally. The heart was hooked at the root of the cardiac apex with a curved forceps and removed, and placed in the pre-cooled cardiac tissue holding solution. The heartbeat of the heart was still visible after removal. After all the neonatal mouse hearts were removed, connective tissue, fat and blood vessels on the hearts were removed, and the hearts were washed 3 times with pre-cooled PBS to remove blood stains (using three 6-cm culture dishes). The hearts were transferred to an EP tube containing 1 ml of digestive enzyme solution A, and the cardiac tissue was cut into pieces with a sterile scissors.

[0058] b. The mixture in the EP tube was transferred to a 15-ml centrifuge tube, and the remaining residue was rinsed with 500 μl of digestive enzyme solution A and aspirated into the 15-ml centrifuge tube. The centrifuge tube was transferred to a 37 °C incubator and waited for 15 min. After the time was up, the centrifuge tube was taken out, and the volume was adjusted to 500 μl with a pipette, and the cell suspension was pipetted. This pipetting step was repeated 3 - 4 times. During the process, the digestion state of the cell mass was observed in real time. If it was observed that the cell precipitate at the bottom of the centrifuge tube began to become clear during digestion, digestion could be stopped. The total digestion time should not exceed 60 min.

[0059] c. A 70-μm filter was rinsed with 2 ml of buffer, the cell suspension was filtered, and a termination solution was added to stop digestion (the volume of the termination solution was twice the volume of the cell suspension), obtaining cell suspension A.

[0060] d. The volume ratio of cell suspension A to red blood cell lysate was 1:10, vortexed for 5 s, and incubated at room temperature for 2 min. Centrifuged at 1200 rpm for 10 min, the supernatant was aspirated and discarded, and 1 ml of buffer was added to resuspend, obtaining cell suspension B and performing cell counting. The cell suspension at this time contained all the cells digested from the cardiac tissue, and the cell count was the total cell count.

[0061] S2 Magnetic-labeled antibody binding and sorting

[0062] The amounts of the non - endothelial cell magnetic - labeled antibody and the endothelial cell magnetic - labeled antibody added are both calculated based on the number of all cells initially digested from the heart (starting cell number). Therefore, the cell number here refers to the number of all cells counted initially (the "cell counting" in step d of the operation procedure).

[0063] S21 First magnetic - labeled sorting:

[0064] a. According to the total cell number, add 160 μl of buffer and 40 μl of non - endothelial cell magnetic - labeled antibody for every 1×10 7 cells, mix well, and incubate in the refrigerator (2 - 8 °C) for 15 min.

[0065] b. Adjust the volume of cell suspension B to 500 μl with buffer. The resuspended cell concentration should be less than 1×10 7 cell / ml. Without centrifugation, directly perform magnetic column sorting.

[0066] c. Place the LD column into the magnetic cell sorter (Miltenyi Biotec: 130 - 042 - 301, magnetic sorting parameter: for LD column, use MidiMACS Separator, Catalog no.: 130 - 042 - 302), rinse the magnetic column with 2 ml of buffer to prepare, and collect the effluent with a 15 - ml centrifuge tube.

[0067] d. After the buffer in the magnetic column runs out, add cell suspension B, and let it flow out slowly automatically. At this time, under the magnetic field of the sorter, the non - endothelial cells specifically bound to the magnetic beads will be adsorbed on the LD magnetic column. Therefore, the cell suspension C rich in endothelial cells flows out from the magnetic column, and continue to collect the effluent.

[0068] e. Wait until the cell suspension in the magnetic column runs out, add 1 ml of buffer to rinse the magnetic column (2 times), and continue to collect the effluent.

[0069] f. Centrifuge the collected effluent rich in endothelial cells at 1200 rpm for 10 min, aspirate and discard the supernatant to obtain cell pellet A.

[0070] S22 Second magnetic - labeled sorting:

[0071] a. In the centrifuge tube of the cell pellet A rich in endothelial cells after centrifugation, according to the total cell number, add 160 μl of buffer and 40 μl of endothelial cell magnetic - labeled antibody for every 1×10 7 cells, mix well, and incubate in the refrigerator (2 - 8 °C) for 15 min to obtain cell suspension D.

[0072] b. According to the total cell number, for every 1×10 7Resuspend cell suspension D with 2 - 4 ml of buffer (gently pipette to mix evenly), centrifuge at 1200 rpm for 5 min, aspirate and discard the supernatant. Adjust the volume of the magnetically labeled endothelial cell suspension to 500 μl with buffer to obtain cell suspension E. The resuspended cell concentration should be lower than 1×10 7 cells / ml.

[0073] d. Place the MS column into the magnetic cell sorter (Miltenyi Biotec: 130 - 090 - 312, use MiniMACS Separator for the MS column, Catalog no.: 130 - 042 - 102), prepare by rinsing the magnetic column with 500 μl of buffer, and collect the flowing - out buffer with a 15 - ml centrifuge tube.

[0074] e. After the buffer in the magnetic column has drained, add the magnetically labeled cell suspension E and let it flow out slowly by itself. At this time, under the magnetic field of the sorter, the endothelial cells specifically bound to the magnetic beads will be adsorbed on the MS magnetic column, and the flowing - out is the non - magnetically labeled cell suspension (without endothelial cells). Continue to collect the flowing - out liquid.

[0075] f. After the cell suspension in the magnetic column has drained completely, add 500 μl of buffer to rinse the magnetic column 3 times (to wash away the non - endothelial cells that are not adsorbed on the magnetic column and improve the purity of endothelial cells), and continue to collect the flowing - out liquid (without endothelial cells).

[0076] g. After the cell suspension in the magnetic column has drained completely, remove the magnetic column from the sorter (out of the magnetic field environment), place it into a new 15 - ml centrifuge tube, add 1 ml of buffer to the magnetic column, and flush out the suspension rich in magnetically labeled endothelial cells with the plunger. At this time, the obtained cell suspension F is a primary endothelial cell suspension rich in endothelial cells and with a relatively high purity.

[0077] S3 Endothelial cell adhesion and culture:

[0078] a. Add cell suspension F to a 6 - cm culture dish, make up the total volume in the culture dish to 4 ml with 10% FBS ECM complete medium, and place it in an incubator at 37°C with 5% CO2 for 24 h.

[0079] b. Observe the cell morphology and adhesion situation the next day. Generally, the first medium change can be carried out after 24 h.

[0080] c. The endothelial cells obtained by this method have good cell morphology and normal viability from P0 - P3, and are suitable for intervention experiments.

[0081] Figure 1The cell morphology and cell density of primary cardiac vascular endothelial cells of neonatal mice at different time points observed under a microscope (4×) are shown. (a) 24 h, (b) 48 h, (c) 72 h. The results show that the primary endothelial cells extracted by this method have normal morphology and size, good survival conditions, can adhere and grow normally, and proliferate. The present invention effectively solves the problem that neonatal mice are easily over-digested and the significant reduction in cell yield and cell viability by using only one digestive enzyme to digest at time intervals, and the cells are in good growth state from 24 to 72 hours.

[0082] Figure 2 The cell morphology of primary cardiac vascular endothelial cells of neonatal mice from passage 0 (P0) to passage 5 (P5) observed under a microscope (10×) is shown. The results show that during the culture of primary endothelial cells, starting from P4, significant cell morphological heterogeneity appears, manifested as diverse cell morphologies and disordered arrangements, and the loss of typical cell structures. This phenomenon suggests that the cells may have undergone phenotypic changes related to dedifferentiation or senescence. Based on these observations, it is recommended to use cells from P0 to P3 in experiments to ensure that the cells maintain stable endothelial cell characteristics and functions, thereby obtaining reliable experimental results.

[0083] Figure 3 The tube formation images of primary cardiac vascular endothelial cells of neonatal mice observed under a microscope (10×) are shown, and the tube formation peak is reached at 6 - 8 h. The results show that the angiogenesis function of the primary endothelial cells extracted by this method is normal.

[0084] Figure 4 The immunofluorescence identification image of CD31 antibody of primary cardiac vascular endothelial cells of neonatal mice under a confocal microscope (40×) is shown. The results show that the primary endothelial cells are identified by using the endothelial cell biomarker CD31 antibody for immunofluorescence staining and observing under an inverted fluorescence microscope: the red part is the specific staining of CD31 antibody, and the blue part is the nucleus stained with DAPI. The morphological structure of the cells can be clearly seen under a high-power microscope. The primary cells obtained by this method are indeed primary endothelial cells. By using the method of specifically binding magnetic beads for magnetic column sorting, the problem of low purity of primary endothelial cells is effectively solved. It can be seen that in the CD31 antibody immunofluorescence identification experiment, the positive rate of cells is as high as over 95%, see Figure 4 .

[0085] Comparative Example 1

[0086] A method for extracting and culturing primary cardiac vascular endothelial cells of neonatal mice includes the following steps:

[0087] S1 Preparation of single-cell suspension of neonatal mouse heart

[0088] a. Twenty C57 neonatal mice (1 - 3 days old), regardless of sex, were immersed in 75% alcohol for about 10 s (about 4 mice at a time). The neonatal mice were fixed in the left palm, and the chest and abdominal skin were disinfected 3 times with a sterile cotton swab dipped in 75% ethanol. After opening the chest slightly to the left of the midline at the xiphoid process (starting from the abdomen) with an ophthalmic iris scissors, the right edge of the sternum was pressed with scissors to allow the heart to jump out naturally. The heart was hooked at the root of the apex with a curved forceps and removed, then placed in pre-cooled cardiac tissue holding solution. The heart was still beating after removal. After all the neonatal mouse hearts were removed, connective tissue, fat, and blood vessels on the hearts were removed, and the hearts were washed 3 times with pre-cooled PBS to remove blood stains (using three 6-cm culture dishes). The hearts were transferred to an EP tube containing 1 ml of digestive enzyme solution B, and the cardiac tissue was minced with sterile scissors.

[0089] b. The mixture in the EP tube was transferred to a 15-ml centrifuge tube, and the remaining residue was rinsed with 500 μl of digestive enzyme solution B and aspirated into the 15-ml centrifuge tube. The centrifuge tube was transferred to a 37°C incubator and waited for 15 min. After the time was up, the centrifuge tube was taken out, and the volume was adjusted to 500 μl with a pipette, and the cell suspension was pipetted. This pipetting step was repeated 3 - 4 times. During the process, the digestion status of the cell clumps was observed in real time. If the cell precipitate at the bottom of the centrifuge tube was observed to start to become clear during digestion, digestion could be stopped, and the total digestion time did not exceed 60 min.

[0090] c. A 70-μm filter was rinsed with 2 ml of buffer, the cell suspension was filtered, and a stop solution was added to stop digestion (the volume of the stop solution was twice the volume of the cell suspension), obtaining cell suspension A.

[0091] d. The volume ratio of cell suspension A to red blood cell lysis solution was 1:10, vortexed for 5 s, and incubated at room temperature for 2 min. Centrifuged at 1200 rpm for 10 min, the supernatant was aspirated and discarded, and 1 ml of buffer was added to resuspend, obtaining cell suspension B. At this time, the cell suspension contained all the cells digested from the cardiac tissue, and the cell count was the total cell count.

[0092] S2 Magnetic Labeling Antibody Binding and Sorting

[0093] S21 First Magnetic Label Sorting:

[0094] a. According to the total cell count, add 160 μl of buffer and 40 μl of non-endothelial cell magnetic labeling antibody per 1×10 7 cells, mix well, and place in the refrigerator (2 - 8°C) for incubation for 15 min.

[0095] b. Adjust the volume of cell suspension B to 500 μl with buffer, and the resuspended cell concentration should be less than 1×10 7 cell / ml. Do not centrifuge and directly perform magnetic column sorting.

[0096] c. Place the LD column into a magnetic cell sorter (Miltenyi Biotec: 130 - 042 - 301), prepare by rinsing the column with 2 ml of buffer, and collect the effluent with a 15 ml centrifuge tube.

[0097] d. After the buffer in the column has drained, add cell suspension B and let it flow out slowly by itself. At this time, under the magnetic field of the sorter, non - endothelial cells specifically bound to the magnetic beads will be adsorbed on the LD column. Therefore, the cell suspension C rich in endothelial cells will flow out from the column, and continue to collect the effluent.

[0098] e. When the cell suspension in the column has drained completely, add 1 ml of buffer to rinse the column (twice), and continue to collect the effluent.

[0099] f. Centrifuge the collected effluent rich in endothelial cells at 1200 rpm for 10 min, aspirate and discard the supernatant to obtain cell pellet A.

[0100] S22 Second magnetic labeling sorting

[0101] a. In the centrifuge tube containing the cell pellet A rich in endothelial cells after centrifugation, add 160 ul of buffer and 40 ul of endothelial cell magnetic labeling antibody per 1×10 7 cells, mix well, and incubate in the refrigerator (2 - 8 °C) for 15 min to obtain cell suspension D.

[0102] b. Resuspend cell suspension D with 2 - 4 ml of buffer per 1×10 7 cells (gently pipette to mix), centrifuge at 1200 rpm for 5 min, aspirate and discard the supernatant. Adjust the volume of the magnetically labeled endothelial cell suspension to 500 ul with buffer to obtain cell suspension E, and the resuspended cell concentration should be less than 1×10 7 cells / ml.

[0103] d. Place the MS column into a magnetic cell sorter (Miltenyi Biotec: 130 - 090 - 312), prepare by rinsing the column with 500 ul of buffer, and collect the flowing - out buffer with a 15 ml centrifuge tube.

[0104] e. After the buffer in the column has drained, add the magnetically labeled cell suspension E and let it flow out slowly by itself. At this time, under the magnetic field of the sorter, endothelial cells specifically bound to the magnetic beads will be adsorbed on the MS column, and the flowing - out is the non - magnetically labeled cell suspension (without endothelial cells), and continue to collect the effluent.

[0105] f. After the cell suspension in the magnetic column has flowed out, add 500ul of buffer to rinse the magnetic column three times (to flush away non-endothelial cells that are not adsorbed on the magnetic column and improve the purity of endothelial cells), and continue to collect the outflow liquid (without endothelial cells).

[0106] g. After the cell suspension in the magnetic column has been drained, remove the magnetic column from the sorter (out of the magnetic field environment), place it in a new 15ml centrifuge tube, add 1ml of buffer to the magnetic column, and use the plunger to flush out the suspension rich in magnetically labeled endothelial cells. The cell suspension F obtained at this time is a primary endothelial cell suspension rich in endothelial cells and with a higher purity.

[0107] S3 endothelial cell attachment and culture:

[0108] a. Add cell suspension F to a 6 cm culture dish, add 10% FBS ECM complete medium to make up the total volume in the dish to 4 ml, and culture in a 37° 5% CO2 incubator for 24 h.

[0109] b. Observe the cell morphology and adhesion on the next day. Generally, the first medium change can be performed after 24 hours.

[0110] c. The endothelial cells P0-P3 obtained by this method have good morphology and normal vitality, and are suitable for intervention experiments.

[0111] The difference between Comparative Example 1 and Example 1 is that the digestive enzyme solution is different. Comparative Example 1 adopts a conventional digestive enzyme solution, that is, Comparative Example 1 adopts digestive enzyme solution B for digestion treatment.

[0112] Figure 5 The results of the comparative experiments under different digestion methods of Example 1 and Comparative Example 1. Comparative Example 1 uses a digestion enzyme solution obtained by mixing equal volumes of trypsin solution and type II collagenase solution in a ratio of 1:1 to digest the cells, while Example 1 uses type II collagenase solution to digest the cells. The primary endothelial cells extracted from Comparative Example 1 and Example 1 were planted in two culture dishes for observation, A and C are images (4× and 10×) of the cells of Comparative Example 1 observed under a microscope after 72 hours of culture, and B and D are images (4× and 10×) of the cells of Example 1 observed under a microscope after 72 hours of culture.

[0113] The results showed that the cells extracted from the Example 1 group were significantly more than those from the Comparative Example 1 group. The method of using only one digestive enzyme for digestion at intervals can effectively solve the problem that newborn mice are prone to over-digestion, resulting in a significant decrease in cell yield and cell viability.

[0114] Comparative Example 2

[0115] A method for extracting and culturing primary cardiac vascular endothelial cells of neonatal mice comprises the following steps:

[0116] Preparation of Single Cell Suspension from Hearts of Newborn S1 Mice

[0117] a. Twenty C57 newborn mice (1 - 3 days old), regardless of gender, were immersed in 75% alcohol for about 10 s (about 4 mice each time). The newborn mice were fixed in the left palm, and the chest and abdominal skin were disinfected 3 times with a sterile cotton swab dipped in 75% ethanol. After opening the chest slightly to the left of the midline at the xiphoid process (starting from the abdomen) with an ophthalmic iris scissors, the scissors were used to press on the right edge of the sternum to allow the heart to jump out naturally. The heart was hooked at the root of the apex with a curved forceps and removed, then placed in pre-cooled cardiac tissue holding solution. The heart was still beating after removal. After all the hearts of the newborn mice were removed, connective tissue, fat, and blood vessels on the hearts were removed, and the hearts were washed 3 times with pre-cooled PBS to remove blood stains (using three 6-cm culture dishes). The hearts were transferred to an EP tube containing 1 ml of digestive enzyme solution, and the cardiac tissue was minced with sterile scissors.

[0118] b. The mixture in the EP tube was transferred to a 15-ml centrifuge tube, and the remaining residue was rinsed with 500 μl of digestive enzyme solution A and then aspirated into the 15-ml centrifuge tube. The centrifuge tube was transferred to a 37 °C incubator and waited for 15 min. After the time was up, the centrifuge tube was taken out, and the pipette was adjusted to a volume of 500 μl to pipette the cell suspension. This pipetting step was repeated 3 - 4 times. During the process, the digestion status of the cell clumps was observed in real time. If it was observed that the cell precipitate at the bottom of the centrifuge tube began to become clear during digestion, digestion could be stopped. The total digestion time should not exceed 60 min.

[0119] c. A 70-μm filter was rinsed with 2 ml of buffer, and the cell suspension was filtered. A termination solution was added to stop digestion (the volume of the termination solution was twice the volume of the cell suspension) to obtain cell suspension A.

[0120] d. The volume ratio of cell suspension A to red blood cell lysate was 1:10. It was vortexed for 5 s and incubated at room temperature for 2 min. It was centrifuged at 1200 rpm for 10 min, the supernatant was aspirated and discarded, and 1 ml of buffer was added to resuspend it to obtain cell suspension B. At this time, the cell suspension contained all the cells digested from the cardiac tissue, and the cell count was the total cell count.

[0121] S2 Cell Sorting by Filtration Method

[0122] The cell suspension was filtered through a 200-mesh cell sieve and centrifuged at 1000 r / min for 5 min; the supernatant was discarded, 3 ml of PBS solution was added for rinsing, and after centrifuging at 1000 r / min for 5 min, the supernatant was discarded. The rinsing and centrifuging were repeated twice. The cell pellet was resuspended with 1 ml of 10% FBS ECM complete medium to obtain cell suspension F.

[0123] S3 Endothelial Cell Adhesion and Culture:

[0124] a. Add the cell suspension F into a 6-cm culture dish, make up the total volume in the culture dish to 4 ml with 10% FBS ECM complete medium, and place it in an incubator at 37°C with 5% CO2 for 24 h.

[0125] b. Observe the cell morphology and adhesion situation the next day. Generally, the first medium change can be carried out after 24 h.

[0126] c. The endothelial cells P0 - P3 obtained by this method have good cell morphology and normal viability, and are suitable for intervention experiments.

[0127] The difference between Comparative Example 1 and Example 1 lies in the different digestive enzyme solutions. Comparative Example 1 adopts a conventional digestive enzyme scheme, that is, Comparative Example 1 uses digestive enzyme solution B for digestion treatment.

[0128] Figure 6 This is the comparison experiment result under different sorting methods for Example 1 and Comparative Example 2. Comparative Example 2 uses the cell sieve filtration method, and Example 1 uses the magnetic column sorting method. The primary endothelial cells extracted from Example 1 and Comparative Example 2 are respectively seeded in two culture dishes. After the cells adhere, a CD31 antibody immunofluorescence identification experiment is carried out. Figure 6 A is the observation image of the cells in Comparative Example 2 under a confocal microscope (20×), Figure 6 B is the observation image of the cells in Example 1 under a confocal microscope (20×). The results show that the positive rate of the cells extracted in Example 1 is significantly higher than that in Comparative Example 2. By using the method of magnetic column sorting through specific binding of magnetic beads, the problem of low purity of primary cardiac endothelial cells extraction can be effectively solved. It can be seen from the CD31 antibody immunofluorescence identification experiment that the cell positive rate is as high as over 95%.

[0129] The specification of the present invention describes the specific implementation schemes in detail. Those skilled in the art should recognize that the above implementation schemes are exemplary and should not be construed as limitations on the present invention. For those skilled in the art, without departing from the principle of the present invention, through several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for extracting primary cardiac vascular endothelial cells from neonatal mice, characterized in that, It includes the following steps: S1 Prepare cardiac single cell suspension Digest the hearts of 1- to 10-day-old neonatal mice with a digestive enzyme solution, pipette and resuspend, add red blood cell lysate, and then centrifuge and resuspend to obtain the cardiac single cell suspension; S2 First magnetic labeling and sorting Mix the cardiac single cell suspension with non-endothelial cell magnetic labeling antibody, incubate at 0-10 °C for 10-20 min, add to a magnetic cell sorter for sorting, collect the effluent, and centrifuge to obtain cell pellet A; S3 Second magnetic labeling and sorting Mix cell pellet A with endothelial cell magnetic labeling antibody, incubate at 0-10 °C for 10-20 min, add to a magnetic cell sorter for sorting, and collect the endothelial cells in the magnetic column. That's it.

2. The extraction method of primary cardiac vascular endothelial cells of neonatal mice according to claim 1, characterized in that, The S1 for preparing the cardiac single cell suspension includes: S11 Digest the hearts of 1- to 10-day-old neonatal mice with a digestive enzyme solution to obtain cell suspension A; S12 Incubate cell suspension A at 35-40 °C for 10-20 min and pipette; repeat step S12 for 2-5 times to obtain cell suspension B S13 After filtering cell suspension B, terminate digestion. Mix cell suspension B with red blood cell lysate at a volume ratio of 1:(5-20), centrifuge and resuspend to obtain the cardiac single cell suspension, and perform cell counting on the cardiac single cell suspension to obtain the total cell number.

3. The extraction method of primary cardiac vascular endothelial cells of neonatal mice according to claim 1, characterized in that, In the digestive enzyme solution, the material ratio of type II collagenase powder to PBS is 10 mg:(1-3) m; the volume ratio of cell suspension B to red blood cell lysate is 1:(8-12).

4. The extraction method of primary cardiac vascular endothelial cells of neonatal mice according to claim 1, characterized in that, The digestive enzyme solution is prepared by mixing type II collagenase powder and PBS at a material ratio of 10 mg:(1.5-2.5) ml.

5. The extraction method of primary cardiac vascular endothelial cells of neonatal mice according to claim 1, characterized in that, The particle size of the filtration in step S13 is 70 μm.

6. The extraction method of primary cardiac vascular endothelial cells of neonatal mice according to claim 2, wherein, The S2 for the first magnetic labeling and sorting includes: S21 Mix the single-cell suspension of the heart with the non-endothelial cell magnetic labeling antibody, incubate at 0-10 °C for 10-20 min, and adjust the total cell number concentration to be lower than 1×10 7 cells / ml to obtain cell suspension C; S22 Place the LD column into the magnetic cell sorter, add cell suspension C to the magnetic cell sorter for sorting, collect the effluent, and centrifuge to obtain cell pellet A.

7. The extraction method of primary cardiac vascular endothelial cells of neonatal mice according to claim 6, characterized in that, The concentration of non-endothelial cell magnetic labeling antibody in cell suspension C is 30 μl / ml-40 μl / ml.

8. The extraction method of primary cardiac vascular endothelial cells of neonatal mice according to claim 2, wherein The S3 for the second magnetic labeling and sorting includes: S31 Mix the cell pellet with the endothelial cell magnetic labeling antibody, incubate at 0-10 °C for 10-20 min, and adjust the total cell number to less than 1×10 7 cell / ml to obtain cell suspension D; S32 Place the MS column into the magnetic cell sorter, add cell suspension D to the magnetic cell sorter for sorting, add to the magnetic cell sorter for sorting, and collect the endothelial cells in the magnetic column. That's it.

9. The extraction method of primary cardiac vascular endothelial cells of neonatal mice according to claim 8, wherein The concentration of endothelial cell magnetic labeling antibody in cell suspension D is 30 μl / ml-40 μl / ml.

10. A method for culturing primary cardiac vascular endothelial cells of neonatal mice, characterized in that, It includes the following steps: Step 1. Add the primary cardiac vascular endothelial cells of neonatal mice described in any one of claims 1-9 to a culture medium, culture under the conditions of 35-40 °C and 4-6% CO2, and change the medium every 20-30 h; Step 2. Continue to culture for 1-3 generations. That's it.

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