A method for obtaining human venous endothelial cells from human umbilical cord veins
Patent Information
- Application Number
- CN202310760600.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-26
- Publication Date
- 2026-10-27
- Estimated Expiration
- 2043-06-26
AI Technical Summary
[0021]本发明与传统的内部灌注消化酶后结扎脐带两端的思路不同，传统结扎脐带的方法，单人操作不便，涉及的器械较多，增加了污染的机会。本发明最大的特点在于摆脱了脐带要结扎的思维定式，不结扎脐带而是撕开脐带外层的华通胶，让脐静脉暴露。且本发明在操作中只需两把有齿镊，沿脐静脉外的华通胶撕开脐带暴露部分脐静脉，一方面保障血管的生理活性，一方面因血管自身软塌的特点，可将注入的酶适度保存在血管中，且在可视状态下完成，可自行适量保留在血管中，保证无气泡，无须再结扎血管两端，避免因脐带过长注入I型胶原酶困难的问题，大大降低了因多操作步骤带来的污染。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of cell isolation and amplification technology, and in particular to a method for obtaining human venous endothelial cells from human umbilical veins. Background Technology
[0002] Vascular endothelial cells (VECs) are monolayered cells located on the inner wall of blood vessels, possessing diverse physiological functions. They produce and secrete various bioactive substances, playing a crucial role in vascular bioengineering, blood pressure regulation, anticoagulation, and maintaining the balance of the internal and external environments of the body. Damage and dysfunction of VECs are the initiating factors for many cardiovascular diseases, including hypertension, atherosclerosis, and coronary heart disease. Furthermore, VEC dysfunction is a target organ for cardiovascular disease risk factors; therefore, protecting VEC function has become a novel therapeutic target for cardiovascular diseases. In vitro culture of endothelial cells is an important method for studying VEC function. However, due to species differences, isolating VECs solely from animal blood vessels is far from meeting the growing demand for clinical translational research. The umbilical cord of newborns is readily available and abundant, representing one of the very few healthy organs / tissues that can be obtained from healthy humans under ethical conditions. The umbilical vein has thus become a primary material for in vitro experiments on VECs. While there are reports on the isolation and culture of human umbilical vein endothelial cells (HUVECs), traditional methods typically require assistants, and the isolation process is cumbersome, resulting in low cell yields and susceptibility to contamination.
[0003] Therefore, there is an urgent need to establish a simple, easy-to-operate, easily replicable, and highly efficient method for isolating and culturing HUVECs with minimal reagents and consumables, so as to lay the foundation for research on the pathogenesis of cardiovascular diseases. Summary of the Invention
[0004] The purpose of this invention is to provide a method for obtaining human venous endothelial cells from human umbilical veins, eliminating the need for ligating the umbilical cord and greatly reducing contamination caused by multiple operational steps.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides a method for obtaining human venous endothelial cells from human umbilical veins, comprising the following steps:
[0007] (1) Cut the umbilical cord into 3-5cm segments, rinse and set aside;
[0008] (2) After cutting the umbilical cord segment, expose the umbilical vein, remove the surrounding tissue and clean it until there is no blood before use;
[0009] (3) Use tweezers to extract one side of the umbilical vein opening after step (2) and infuse it with type I collagenase for digestion;
[0010] (4) Rinse the umbilical vein after digestion in step (3) with complete culture medium and collect the rinsing fluid;
[0011] (5) Centrifuge the rinsing solution, collect the cell pellet, and resuspend the cell pellet in complete culture medium to obtain a cell suspension;
[0012] (6) The cell suspension was cultured and passaged to obtain purified human venous endothelial cells.
[0013] Preferably, the rinsing is performed with PBS containing 1.0% (v / v) PS.
[0014] Preferably, the concentration of type I collagenase in step (3) is 1.0 mg / ml, and the amount of type I collagenase used is until the type I collagenase flows out from the other end of the umbilical vein.
[0015] Preferably, the digestion temperature in step (3) is 37°C; the digestion time is approximately 20 minutes.
[0016] Preferably, in step (4), each umbilical vein is rinsed 2-3 times with complete culture medium; the amount of complete culture medium used for each rinse is 2 mL.
[0017] Preferably, the centrifugation time in step (5) is 5 minutes; the centrifugation speed is 1000 rpm.
[0018] Preferably, in step (5), the amount of complete culture medium added to the resuspension is 6-10 cm of cells obtained from umbilical cord digestion, which is then resuspended in 4 ml of complete culture medium.
[0019] Preferably, the culture temperature in step (6) is 37°C; the CO2 concentration is 5%; the culture medium is changed once after 5-7 hours of culture, and once after 22-26 hours of culture, and the culture continues for 3-4 days.
[0020] Preferably, in step (6), the passage is performed when the cell fusion rate reaches 85-95%, and the cells are digested and passaged with 0.25% trypsin at a ratio of 1:4, and the passage is repeated 1-2 times to obtain human venous endothelial cells.
[0021] This invention differs from the traditional approach of ligating both ends of the umbilical cord after internal infusion of digestive enzymes. Traditional methods of ligating the umbilical cord are inconvenient for single-person operation, involve numerous instruments, and increase the risk of contamination. The biggest advantage of this invention is that it breaks free from the conventional thinking of ligating the umbilical cord. Instead of ligating it, the Wharton's jelly on the outer layer of the umbilical cord is torn open, exposing the umbilical vein. Furthermore, this invention requires only two toothed forceps to tear open the umbilical cord along the Wharton's jelly outside the vein, exposing part of the umbilical vein. This preserves the physiological activity of the blood vessel, and due to the soft and collapsible nature of the blood vessel, the injected enzyme can be appropriately preserved within the vessel. This process is performed under visual guidance, allowing the appropriate amount to be retained in the vessel, ensuring no air bubbles, eliminating the need to ligate both ends of the vessel, and avoiding the difficulty of injecting type I collagenase when the umbilical cord is too long. This significantly reduces the risk of contamination from multiple operational steps.
[0022] This invention aims to establish a simple, easy-to-operate, easily replicable, and highly efficient method for isolating and culturing HUVECs using minimal reagents and consumables. It also aims to identify the expression of endothelial cell markers using immunofluorescence cytochemical staining, laying the foundation for research on the pathogenesis of cardiovascular diseases. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0024] Figure 1 This is a diagram illustrating the procedure of flushing and injecting digestive enzymes into the exposed umbilical vein in Example 1.
[0025] Figure 2 The growth rate of primary human umbilical vein endothelial cells in Example 1 (A: 6 hours after isolation; B: 24 hours after isolation; C: 4 days after isolation);
[0026] Figure 3 The second-generation human umbilical vein endothelial cells were subjected to CD31 immunofluorescence staining in Example 1. Detailed Implementation
[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0028] Example 1
[0029] I. Materials
[0030] Two 1.20cm x 10cm trays
[0031] 2. Toothed tweezers (2 pairs, large size)
[0032] 3. Tissue scissors (1 pair)
[0033] 5 ml of 4.1 mg / ml type I collagenase (Worthington CAT#:LS004194, prepared with ECM medium for easy observation of type I collagenase injection into blood vessels)
[0034] 5. 100 ml of 1.0% penicillin-streptomycin solution (PS) (prepared with PBS)
[0035] 6. 10 ml of complete ECM culture medium (ScienceCellECM)
[0036] 7. 5 ml of ECM culture medium (ScienceCellECM)
[0037] 8. Four 10cm diameter petri dishes (large dishes)
[0038] 9. One 6cm diameter petri dish (medium-sized dish)
[0039] One 10.15ml centrifuge tube
[0040] 11.0.1% (volume fraction) gelatin 2ml
[0041] 12. One high-temperature sterilization bag
[0042] 13. Incubator
[0043] II. Steps
[0044] The tray, toothed forceps, and tissue scissors were placed in sterilization bags and sterilized at 121℃ and 103kPa for 20 min. The temperature of the incubator was then adjusted to 37℃. 0.1% gelatin was spread into 6cm diameter medium dishes and left to stand for 2 hours.
[0045] Place 20 ml of PBS containing 1.0% PS in a 10 cm diameter dish. Cut the newborn's umbilical cord into several 5 cm long segments with tissue scissors and place them horizontally in the dish. Use a 1 ml pipette tip to draw liquid from the dish to rinse the surface of the umbilical cord until the blood on the surface of the umbilical cord is rinsed off. Set aside.
[0046] Using toothed forceps, gradually tear open the Wharton's adhesive covering the umbilical vein from one end of the umbilical cord towards the other (the tear should be 1cm wide and 5cm long). Figure 1 (A, B) This allows the upward-facing portion of the umbilical vein in the horizontal cross-section to be exposed to air, while the downward-facing portion remains connected to the Wharton's adhesive. (e.g.) Figure 1As shown in B, the exposure area should be such that the injected type I collagenase can be seen from one end of the blood vessel to the other. Use tissue scissors to remove excess tissue around the umbilical vein, and turn the exposed umbilical vein face upwards. Figure 1 B) Reserved;
[0047] Place the exposed umbilical vein segment in a PBS dish containing 20 mL of 1.0% PS. Use toothed forceps to lift one side of the umbilical vein opening, and slowly inject PBS from the dish into the umbilical vein using a 1 mL pipette tip to flush out the blood. Figure 1 C) Place the rinsed umbilical cord segment into another large dish containing 20 mL of PBS with a volume fraction of 1.0% PS to rinse the umbilical vein. The rinsing method is the same as above. Repeat rinsing until no blood is visible to the naked eye in the umbilical vein. Set aside for later use.
[0048] After rinsing the umbilical cord segment, place it in another large dish without solution. Use toothed forceps to lift one side of the umbilical vein opening, and slowly perfuse the umbilical vein with a 1 mg / ml type I collagenase solution drawn from the tip of a 1 ml pipette. Figure 1 D) After type I collagenase flows out from the other end of the umbilical vein, place the umbilical vein horizontally. The umbilical vein with a length of 5cm requires 1ml of type I collagenase for digestion. After the umbilical vein is perfused, cover it with the culture dish lid and place it in a 37℃ incubator for 20min.
[0049] After the umbilical vein has settled, remove it and rinse it with complete culture medium. Use 2 mL of complete culture medium to rinse each segment of the umbilical vein. Transfer the rinse liquid (10 mL) to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. After aspirating the supernatant, add 4 mL of complete culture medium to the centrifuge tube and gently mix by pipetting to obtain a cell suspension. Place the cell suspension in a medium-sized dish and incubate in a cell culture incubator (37℃, 5% CO2). After 6 hours of culture, slowly aspirate the culture medium (at this point, the cells have adhered to the culture vessel). The cultured cells are as follows... Figure 2 As shown in Figure A, 4 mL of complete culture medium was added, and the cells were cultured for 24 hours. The medium was then changed again. The cells after 24 hours of culture are shown in Figure A. Figure 2 As shown in Figure B, after adding 4 mL of complete culture medium, continue culturing for 4 days, as... Figure 2 As shown in C. From Figure 2 The cells, after 4 days of culture, exhibited a cobblestone-like appearance and rapid growth. These cells were then passaged. When cell confluence reached 90%, 0.25% trypsin was added, and the cells were passaged twice at a 1:4 ratio to obtain human umbilical vein endothelial cells.
[0050] CD31 immunofluorescence staining of second-generation human umbilical vein endothelial cells yielded the following results: Figure 3 As shown: Under a fluorescence microscope, DAPI blue counterstaining represents the cell nucleus. Figure 3A) Endothelial cells widely express CD31 antigen (green fluorescence). Figure 3 B), distributed in sheets within the cytoplasm ( Figure 3 C) suggests that this method can obtain high-purity human umbilical vein endothelial cells.
[0051] Example 2
[0052] The materials used in this embodiment are the same as those in Embodiment 1.
[0053] The tray, toothed forceps, and tissue scissors were placed in sterilization bags and sterilized at 121℃ and 103kPa for 20 min. The temperature of the incubator was then adjusted to 36℃. 0.1% gelatin was spread into 6cm diameter medium dishes and left to stand for 2 hours.
[0054] Place 20 ml of PBS containing 0.5% PS in a 10 cm diameter dish. Cut the newborn's umbilical cord into several 3 cm long segments with tissue scissors and place them in the dish. Use a 1 ml pipette tip to draw liquid from the dish to rinse the surface of the umbilical cord until the blood on the surface of the umbilical cord is rinsed off. Set aside.
[0055] Using toothed forceps, gradually peel away the Wharton's adhesive covering the umbilical vein from one end of the umbilical cord towards the other (1cm wide, 3cm long). Figure 1 (A, B) Expose the upward-facing portion of the umbilical vein to air in the horizontal cross-section, while keeping the downward-facing portion connected to Wharton's jelly. The exposure size should be such that the injected type I collagenase can be seen from one end of the vessel to the other. Remove excess tissue around the umbilical vein with tissue scissors, and place the exposed umbilical vein face up for later use.
[0056] Place the exposed umbilical vein segment in a large dish containing 20 mL of PBS with a volume fraction of 0.5%. Use toothed forceps to lift one side of the umbilical vein opening, and slowly inject PBS from the dish into the umbilical vein with a 1 mL pipette tip to rinse the blood. Place the rinsed umbilical vein segment in another large dish containing 20 mL of PBS with a volume fraction of 0.5% to rinse the umbilical vein. Repeat the rinsing method as above until no blood is visible in the umbilical vein. Set aside for later use.
[0057] After rinsing the umbilical cord segment, place it in another large dish without solution. Use toothed forceps to lift one side of the umbilical vein opening and slowly perfuse the umbilical vein with 1mg / ml type I collagenase using a 1ml pipette tip until the type I collagenase flows out from the other end of the umbilical vein. Then place the umbilical vein horizontally and treat the other umbilical veins in the same way. After the umbilical veins have been perfused, cover the culture dish with the culture dish lid and place it in a 37℃ incubator for 20 minutes.
[0058] After the umbilical veins have been allowed to stand, remove them and rinse them with complete culture medium. Aspirate 8-12 ml of the rinsed liquid into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. After removing the supernatant, add 4 mL of complete culture medium to the centrifuge tube and gently mix. Place the cell suspension in a medium dish and incubate at 37°C and 5% CO2. After 6 h of culture, slowly aspirate the culture medium and add 4 mL of complete culture medium. After 24 h of culture, change the medium again, slowly aspirate the culture medium, and add 4 mL of complete culture medium. Continue culturing for 3 days. When the cell confluence reaches 85-95%, add 0.25% trypsin at a 1:4 ratio and passage once to obtain human venous endothelial cells.
[0059] Example 3
[0060] The materials used in this embodiment are the same as those in Embodiment 1.
[0061] The tray, toothed forceps, and tissue scissors were placed in sterilization bags and sterilized at 121℃ and 103kPa for 20 min. The temperature of the incubator was then adjusted to 37℃. 0.1% gelatin was spread into 6cm diameter medium dishes and left to stand for 2 hours.
[0062] Place 20 ml of PBS containing 1.0% PS in a 10 cm diameter dish. Cut the newborn's umbilical cord into several 3-5 cm long segments with tissue scissors and place them in the dish. Use a 1 ml pipette tip to draw liquid from the dish to rinse the surface of the umbilical cord until the blood on the surface of the umbilical cord is rinsed off. Set aside.
[0063] Using toothed forceps, gradually peel away the Wharton's adhesive (1cm wide, 4cm long) wrapping around the umbilical vein from one end of the umbilical cord towards the other. Figure 1 A, B), so that the part of the horizontal cross-section with the umbilical vein facing upward is exposed to air, while the part facing downward remains connected to Wharton glue;
[0064] Place the exposed umbilical vein segment in a large dish containing 20 mL of PBS with a volume fraction of 1.0%. Use toothed forceps to lift one side of the umbilical vein opening, and slowly inject PBS from the dish into the umbilical vein with a 1 mL pipette tip to rinse the blood. Place the rinsed umbilical vein segment in another large dish containing 20 mL of PBS with a volume fraction of 1.0% to rinse the umbilical vein. Repeat the rinsing method as above until no blood is visible in the umbilical vein. Set aside for later use.
[0065] After rinsing the umbilical cord segment, place it in another large dish without solution. Use toothed forceps to lift one side of the umbilical vein opening and slowly perfuse the umbilical vein with 1.0 mg / ml type I collagenase using a 1 ml pipette tip until the type I collagenase flows out from the other end of the umbilical vein. Then, place the umbilical vein horizontally and treat the other umbilical veins in the same way. After the umbilical veins have been perfused, cover the culture dish with the culture dish lid and place it in a 37°C incubator for 20 minutes.
[0066] After the umbilical veins have settled, remove them and rinse them with complete culture medium. Aspirate the rinse fluid into 15 mL centrifuge tubes and centrifuge at 1000 rpm for 5 min. After removing the supernatant, add 4 mL of complete culture medium to the centrifuge tubes and gently mix. Place the cell suspension in a medium-sized dish and incubate at 37°C and 5% CO2 for 6 h. Slowly aspirate the culture medium and add 4 mL of complete culture medium. After 24 h of incubation, change the medium again, slowly aspirating the medium and adding 4 mL of complete culture medium. Figure 2 B) Continue culturing for 4 days. When the cell confluence reaches 85-95%, add 0.25% trypsin and passage twice at a ratio of 1:4 to obtain human venous endothelial cells.
[0067] The human venous endothelial cells obtained using Example 1 of the present invention are simple to operate and the separation process is not prone to contamination.
[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for obtaining human venous endothelial cells from human umbilical veins, characterized in that, Includes the following steps: (1) Cut the umbilical cord into 3-5cm segments, rinse, and set aside; (2) After cutting the umbilical cord segment to expose the umbilical vein, remove the surrounding tissue and clean it until there is no blood before use; (3) Use tweezers to extract one side of the umbilical vein opening after step (2) and infuse it with type I collagenase for digestion; (4) Rinse the umbilical vein after digestion in step (3) with complete culture medium and collect the rinsing fluid; (5) Centrifuge the rinsing solution, collect the cell pellet, and resuspend the cell pellet in complete culture medium to obtain a cell suspension; (6) The cell suspension was cultured and passaged to obtain purified human venous endothelial cells; The concentration of type I collagenase in step (3) is 1.0 mg / ml, and the amount of type I collagenase used is until the type I collagenase flows out from the other end of the umbilical vein. The digestion temperature in step (3) is 37°C; the digestion time is approximately 20 minutes.
2. The method as described in claim 1, characterized in that, The rinsing was performed using PBS containing 1.0% (v / v) PS.
3. The method as described in claim 1, characterized in that, In step (4), each umbilical vein is rinsed 2-3 times with complete culture medium; the amount of complete culture medium used for each rinse is 2 mL.
4. The method as described in claim 1, characterized in that, The centrifugation time in step (5) is 5 minutes; the centrifugation speed is 1000 rpm.
5. The method as described in claim 1, characterized in that, In step (5), the amount of complete culture medium added to the resuspension is 6-10 cm of cells obtained from umbilical cord digestion, which are then resuspended in 4 ml of complete culture medium.
6. The method as described in claim 1, characterized in that, The culture temperature in step (6) is 37℃; the CO2 concentration is 5%; the medium is changed once after 5-7 hours of culture, and once after 22-26 hours of culture, and cultured for another 3-4 days.
7. The method as described in claim 1, characterized in that, The passage in step (6) involves digesting the cells with 0.25% trypsin at a ratio of 1:3 to 5 when the cell fusion rate reaches 85-95%, and passaged them 1-2 times to obtain human venous endothelial cells.