A pericyte and endothelial cell blood vessel model and methods of construction and use thereof

Abstract

The present application relates to the technical field of cell culture, and in particular to a pericyte and endothelial cell vascular model, a construction method thereof and application. The vascular model is a three-dimensional vascular structure formed by culturing dermal microvascular pericytes and endothelial cells on a matrigel. The construction method is as follows: obtaining dermal microvascular pericytes and preparing a pericyte cell suspension; forming a matrigel layer on an ibidi angiogenesis slide; adding the pericyte suspension to the matrigel layer and placing it in a culture box for culture, and discarding the culture medium; preparing an endothelial cell suspension by preparing endothelial cells, and adding the endothelial cell suspension to the ibidi angiogenesis slide for culture to form a vascular model. The present application innovatively proposes a three-dimensional vascular model at the cellular level, which can be constructed and obtained by cell culture, and has the technical advantages of being simple and easy to obtain, low in cost, and more in line with animal experiment ethics.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a pericyte and endothelial cell vascular model, its construction method, and its application. Background Technology

[0002] Human blood vessels are mainly composed of three parts: endothelial cells, the basement membrane, and pericytes. Pericytes and endothelial cells are not only closely related in anatomical structure, but also interact significantly through adjacent or paracrine signals. Studies have found that the occurrence and development of many diseases are related to changes in blood vessels, such as stroke, tumor formation, and coronary heart disease. Therefore, establishing a good in vitro model of human blood vessels is an urgent problem to be solved.

[0003] Pericytes, also known as Rouget cells, are distributed between endothelial cells and the basement membrane in various vascular systems throughout the body, and are an important structural component of blood vessels. Their cytoplasm often extends several large primary processes that span multiple endothelial cells, sometimes connecting with adjacent vascular branches. Pericytes and endothelial cells are not only closely related anatomically, but also interact significantly through adjacent or paracrine signaling. Pericytes play a crucial regulatory role in physiological and pathological processes such as angiogenesis, vascular leakage, and tumor formation. Exploring the activity and characteristics of pericytes has become an important direction in the study of vascular physiology and the impact of diseases on blood vessels.

[0004] Current in vitro studies of vascular-related diseases primarily involve extracting independent endothelial cells and culturing them in petri dishes. Alternatively, animal models can be used to directly study and observe animal blood vessels. However, blood vessels are three-dimensional tubular structures, and the two-dimensional environment created by petri dishes makes it impossible to observe the effects of altered experimental conditions on the tubular structure of the blood vessel, nor can it observe changes in the interaction between the main cells of the blood vessel, pericytes and endothelial cells. Using animal experiments is costly, does not comply with ethical requirements for protecting laboratory animals, and human blood vessels differ from those of other animals. Therefore, there is an urgent need to develop a cellular-level, three-dimensional vascular model.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a pericyte and endothelial cell vascular model, its construction method, and its application.

[0007] This invention provides a vascular model, which is a three-dimensional vascular-like structure formed by culturing pericytes and endothelial cells of dermal microvessels on a matrix gel.

[0008] Optionally, the vascular model is maintained for at least 16 hours after formation, preferably at least 24 hours.

[0009] Optionally, the vascular model uses human dermal microvascular pericytes as a framework, with human endothelial cells attached to the inner side of the human dermal microvascular pericytes; preferably, the ratio of dermal microvascular pericytes to endothelial cells is 1:1.

[0010] This invention provides a method for constructing a vascular model, comprising at least the following steps:

[0011] S1. Human dermal microvascular pericytes were obtained through extraction and identification, and a pericyte suspension was prepared.

[0012] S2. Form a matrix adhesive layer on the ibidi angiogenesis slide;

[0013] S3. Add the pericyte suspension to the matrix gel layer and incubate in an incubator for 0.5 to 1.5 hours. Human dermal microvascular pericytes form a tubular framework cell structure. Discard the culture medium.

[0014] S4. Prepare an endothelial cell suspension from human endothelial cells, add it to an ibidi angiogenesis slide, and incubate in an incubator for 0.5 to 1.5 hours to form a vascular model.

[0015] Optionally, in S1, the extraction includes:

[0016] S11. Take skin tissue and trim it to preserve the dermis;

[0017] S12. The trimmed skin tissue is hydrolyzed with neutral protease to separate the epidermis and dermis;

[0018] S13. Digest the dermis using collagenase;

[0019] S14. Filter the digested dermis, harvest the cells, culture the cells using pericyte culture medium, and use the cultured cells to prepare pericyte cell suspension.

[0020] The identification process included: simultaneous identification of cultured cells using CD31 antibody, PDGFR antibody, NG2 antibody, and α-SMA antibody. Cells that were negative for CD31 antibody and positive for PDGFR, NG2, and α-SMA antibodies were identified as perivascular cells of the dermis.

[0021] Optionally, in S12, the conditions for neutral protease hydrolysis are incubation at 0–5°C for 8–16 hours; the concentration of the neutral protease solution is 2 mg / mL.

[0022] In S13, the concentration of collagenase was 1.25 mg / mL, and the digestion conditions were 35℃~38℃ for 30~50 minutes;

[0023] In S14, the process of harvesting the cell suspension includes: first adding fetal bovine serum to terminate digestion, then filtering the dermal flap tissue and digestion solution obtained after digestion through a 40 μm filter, rinsing, and obtaining the cell suspension.

[0024] In S14, the centrifugation conditions are: time 4-6 min, speed 800-1200 r / min; the culture conditions are: place the culture dish in a cell culture incubator for incubation, and change the medium every 2-3 days.

[0025] Optionally, the concentration of pericytes in the pericyte suspension is 10. 5 ~10 6 Cells / mL, preferably 2×10 5 The concentration of human endothelial cells in the endothelial cell suspension was 10⁻⁶ cells / mL. 5 ~10 6 Cells / mL, preferably 2×10 5 Cells / mL; pericyte suspension was prepared using pericyte culture medium containing 2% fetal bovine serum, and endothelial cell suspension was prepared using 5% endothelial cell culture medium.

[0026] The composition of 5% endothelial cell culture medium (ECM) is: 500 mL ECM, 25 mL LFBS, 5 mL penicillin antibiotics, and 5 mL endothelial cell growth factor (VEGF).

[0027] Optionally, in S2, the matrix gel is melted, and 5–20 μL of matrix gel is added to each well of the ibidi angiogenesis slide. Then, the ibidi angiogenesis slide is placed in an incubator under humid conditions for 20–60 minutes. Preferably, 10 μL of matrix gel is added to each well of the ibidi angiogenesis slide. More preferably, the slide is placed in an incubator for 30–40 minutes.

[0028] Optionally, the substrate gel is dissolved under the following conditions: the substrate gel is placed at 1–5°C for 8–16 hours; preferably, the incubator conditions are: a temperature of 36–37°C and an atmosphere containing 5% CO2 by volume.

[0029] Optionally, in S3, the amount of pericyte cell suspension added is 10-100 μL, preferably 50 μL; the amount of endothelial cell suspension added is 10-100 μL, preferably 50 μL.

[0030] This invention provides the application of the above-described vascular model or the vascular model prepared by the above-described construction method in drug screening; preferably, the drugs include drugs for promoting angiogenesis and drugs for protecting blood vessels.

[0031] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0032] This invention innovatively proposes a three-dimensional vascular model at the cellular level, which can be constructed through cell culture and has the technical advantages of being simple, readily available, low-cost, and more in line with animal experiment ethics.

[0033] The vascular model of this invention is obtained by culturing pericytes and endothelial cells of dermal microvessels on Matricella. Unlike the planar cells obtained in existing technologies, the vascular model proposed in this invention more closely resembles the three-dimensional tubular physiological structure of blood vessels. Furthermore, the vascular model of this invention has the technical advantages of simple preparation method, short processing time, and long maintenance time. Attached Figure Description

[0034] Figure 1 This shows the growth of human dermal microvascular pericytes in vitro cultured in Example 1;

[0035] Figure 2 This shows the growth of human endothelial cells cultured in vitro in Example 1;

[0036] Figure 3 This is a schematic diagram of the positive identification results of human dermal microvascular pericytes in Example 2, using endothelial cells (EC) CD31+, NG2- and fibroblasts (FC) PDGFR-, α-SMA- as controls;

[0037] Figure 4 This is a photograph of the basic framework of the vascular model formed by pericytes of dermal microvessels in Example 3;

[0038] Figure 5 This is a photograph of endothelial cells just added to the basic framework in Example 3;

[0039] Figure 6 This is a photograph showing the state of endothelial cells after they adhered to pericytes of dermal microvessels in Example 3.

[0040] Figure 7 In the vascular model prepared for Example 3, dermal microvascular pericytes and endothelial cells can be clearly distinguished. The arrows on the outer side of the lumen represent dermal microvascular pericytes, and the arrows on the inner side represent endothelial cells.

[0041] Figure 8The image shown is from Example 4, which depicts the co-tube formation of dermal microvascular perivascular cells and endothelial cells. It is difficult to distinguish between dermal microvascular perivascular cells and endothelial cells.

[0042] Figure 9 This is a photograph showing the results of the tube formation experiment performed on fibroblasts in Example 5;

[0043] Figure 10 Photographs showing the results of tube formation experiments performed on endothelial cells and perivascular cells of dermal microvessels alone in Example 6;

[0044] Figure 11 This is a photograph showing the results of the tube forming experiment using a 96-well plate in Example 8. Detailed Implementation

[0045] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0046] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0047] This invention's embodiments reveal that both dermal microvascular pericytes and endothelial cells can form tubular structures in the 3D environment of Matricella. Furthermore, dermal microvascular pericytes exhibit rapid tube formation, excellent morphology, and structural stability, and they also recruit endothelial cells. Therefore, this invention proposes a three-dimensional vascular-like structure formed by culturing dermal microvascular pericytes and endothelial cells on Matricella. Specifically, using dermal microvascular pericytes as the basic framework, endothelial cells are attached to the inner side of the dermal microvascular pericytes, and the two cells co-form a three-dimensional vascular-like structure, creating a vascular model that more closely resembles the physiological structure of three-dimensional tubular vessels. This cell model not only facilitates the expansion of vascular research methods but also better complies with animal experimental ethics.

[0048] Mast gum is an extract from EHS tumors. EHS tumors are mouse tumors rich in extracellular matrix, and its extract composition is similar to that of the basement membrane, hence the name Mast gum. Basement membrane components include: main matrix components: laminin, type IV collagen, basement membrane proteoglycans, nestin, and endogenous proteins; proteases: matrix metalloproteinase-2 and tissue plasminogen activator; growth factors: transforming growth factor-β, fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor; and other proteins: amylase, transferrin, and stromalin. Mast gum is stored frozen, thaws into a liquid state at 4°C, and forms a gel within 30 minutes at 37°C. The gel cannot be dissolved again after cooling. It can promote the morphogenesis, differentiation, and tumor growth of various cells and is widely used in various studies on three-dimensional cell culture.

[0049] This invention, through long-term research on the characteristics and functions of dermal microvessels, has discovered that dermal microvascular pericytes possess a faster and stronger ability to form tubular structures than endothelial cells. Multiple cell experiments have confirmed that dermal microvascular pericytes can rapidly form lumen-like structures on a stable matrix gel, maintain these structures for a longer period, and have the ability to recruit endothelial cells. This model more closely resembles the structure and function of human blood vessels and can serve as an important basic model for vascular research. Using the vascular-like structures formed by dermal microvascular pericytes as the basic framework, endothelial cells grow attached to the inner side of the dermal microvascular pericytes. Compared to vascular models formed from a single dermal microvascular pericyte cell, this model can further extend the maintenance time after formation. In this invention, the maintenance time of the vascular model after formation is at least 16 hours, preferably at least 24 hours. The dermal microvascular pericytes used in this invention are mammalian-derived dermal microvascular pericytes, and human dermal microvascular pericytes are preferred for cell model preparation. The dermal microvascular pericytes used in this invention are mammalian-derived endothelial cells, and human endothelial cells are preferred for cell model preparation.

[0050] As an improvement to the technical solution of this invention, in order to clearly distinguish between endothelial cells and perivascular cells in the vascular model, the ratio of perivascular cells to endothelial cells in the vascular model is 1:1. If the proportion of one type of cell increases, only one type of cell can be observed in the vascular model, and the other type of cell is difficult to observe.

[0051] This invention also proposes a method for constructing this vascular model. A 3D environment is established using matrix gel, followed by the addition of pericyte suspension. After culturing in an incubator, dermal microvascular pericytes rapidly differentiate on the matrix gel, forming well-morphologically sound cellular structures that quickly develop into distinct tubular frameworks. Then, an endothelial cell suspension is added, utilizing the chemotaxis of dermal microvascular pericytes to endothelial cells to fix their position, forming a co-tube model where endothelial cells adhere to the inner wall of the dermal microvascular pericytes. This model allows for clear differentiation of dermal microvascular pericytes and endothelial cells under a microscope, and can be used to study the effects of various conditions on vascular structure and function, as well as the interaction between endothelial cells and dermal microvascular pericytes.

[0052] At least the following steps are included:

[0053] S1. After extraction and identification, perivascular cells of the dermal microvessels were obtained and prepared into a perivascular cell suspension.

[0054] S2. Form a matrix adhesive layer on the ibidi angiogenesis slide;

[0055] S3. Add the pericyte suspension to the matrix gel layer, incubate in an incubator for 0.5 to 1.5 hours, and discard the culture medium after the tubular framework cell structure formed by the rapid tube formation ability of pericytes is obtained.

[0056] S4. Endothelial cells are prepared into an endothelial cell suspension and added to the ibidi angiogenesis slide. Taking advantage of the rapid growth of perivascular cells into the matrix gel, the slide is placed in an incubator and cultured for 0.5 to 1.5 hours to form a vascular model.

[0057] The vascular model in this embodiment of the invention can be obtained through cell culture. During the research, it was found that the maintenance time of the vascular model is closely related to the activity and state of dermal microvascular pericytes. To further improve the maintenance time of the vascular model, this embodiment of the invention further proposes a method for the extraction and purification of dermal microvascular pericytes. Independent microvascular pericytes are obtained from excess skin tissue during surgery, and then their purity is identified. As a preferred embodiment of this invention, the extraction includes:

[0058] S11. Take skin tissue and trim it to preserve the dermis;

[0059] S12. The trimmed skin tissue is hydrolyzed with neutral protease to separate the epidermis and dermis;

[0060] S13. Digest the dermis using collagenase;

[0061] S14. Filter the digested dermis, harvest the cell suspension, centrifuge the cell suspension, and culture the cells using pericellular culture medium.

[0062] This invention utilizes the screening function of pericyte culture medium to conveniently and quickly extract pericytes resembling skin dermal microvessels.

[0063] As an improvement to the technical solution of this invention, step S11 includes: purchasing skin tissue with a size of 1.5–2.0 cm. 2 Place the tissue in a culture dish, add pre-cooled PBS containing penicillin and streptomycin (PBS to penicillin solution volume ratio of 100:1, preferably, the penicillin concentration in the solution is 10,000 U / mL and the streptomycin concentration is 10,000 μg / mL), and rinse off excess blood 2-4 times, 3-6 minutes each time. Transfer the skin tissue to a new culture dish, add an appropriate amount of PBS to fully cover the skin tissue. Remove the muscle and fat under the dermis, retaining the dermal tissue, and trim it into small pieces, preferably 0.2cm × 0.5cm × 0.5cm, to facilitate subsequent enzymatic digestion.

[0064] As an improvement to the technical solution of this invention, in S12, the trimmed skin tissue is added to a neutral protease solution and incubated at 0–5°C, preferably 4°C, for 8–16 hours, followed by separation of the epidermis and dermis. If the enzymatic hydrolysis time is too short, the epidermis and dermis are difficult to separate; if the enzymatic hydrolysis time is too long, it causes significant damage to the dermal tissue. This invention uses a low-temperature hydrolysis method with neutral protease, which allows for better separation of the epidermis and dermis. However, if the optimal hydrolysis temperature of 37°C for neutral protease is used, it accelerates the hydrolysis of the skin tissue, and the epidermis and dermis become difficult to separate.

[0065] As an improvement to the technical solution of this invention, the concentration of the neutral protease solution is 2 mg / mL.

[0066] As an improvement to the technical solution of this invention, in S13, the concentration of collagenase is 1.25 mg / mL, the digestion conditions are 35℃~38℃, preferably 37℃, and the time is 30~50 minutes. The collagenase incubation time should not be too long to avoid cell damage.

[0067] As an improvement to the technical solution of this invention, in S14, fetal bovine serum is first added to terminate digestion, and then the dermal flap tissue and digestion solution obtained after digestion are filtered and rinsed in a 40 μm filter to obtain a cell suspension; preferably, the volume ratio of fetal bovine serum to collagenase is 2:1; the centrifugation conditions are: time 4-6 min, speed 800-1200 r / min; after centrifugation, the supernatant is discarded, the cell pellet is retained, the cells are resuspended in pericyte culture medium, and cultured for a longer period; the culture dish is placed in a cell culture incubator for incubation, and the medium is changed every 2-3 days.

[0068] In a preferred embodiment of the present invention, cell purity is identified using a combination of multiple antibodies and immunofluorescence. Specifically, this includes simultaneously identifying cultured cells using CD31 antibody, PDGFR antibody, NG2 antibody, and α-SMA antibody. Cells that are negative for CD31 antibody but positive for PDGFR, NG2, and α-SMA antibodies are identified as dermal microvascular pericellular cells. Identification revealed that almost all of the prepared cells were dermal microvascular pericellular cells. In another preferred embodiment of the present invention, the concentration of dermal microvascular pericellular cells in the pericellular suspension is 10. 5 ~10 6 Cells / mL, preferably 1.8–2.2 × 10⁻⁶. 5 Cells / mL, more preferably 2×10⁻⁶ 5 Pericellular cells / mL. The pericellular suspension was prepared using pericellular culture medium containing 2% fetal bovine serum. If the concentration is too high, many pericellular cells will adhere directly together, resulting in fewer tubular structures; if the concentration is too low, it will be difficult to form complete tubular structures.

[0069] Endothelial cell suspensions were prepared using endothelial cell culture medium containing 5% ECM. The composition of the endothelial cell culture medium was: 5% ECM: 500 mL ECM, 25 mL FBS, 5 mL penicillin-drug antibody, and 5 mL VEGF endothelial cell growth factor.

[0070] In a preferred embodiment of the present invention, in step S2, the matrix gel is dissolved, and 5–20 μL of matrix gel, preferably 10 μL, is added to each well of the ibidi angiogenesis slide; then, the ibidi angiogenesis slide is incubated in a humid environment in an incubator for 20–60 minutes, preferably 30–40 minutes. Compared to 96-well plates, the ibidi angiogenesis slide has the technical advantages of requiring less reagent and producing better cell growth. If too much matrix gel is added to each well of the ibidi angiogenesis slide, the resulting matrix gel layer will be too thick, wasting reagents and causing cells to fall into different layers, making it difficult to focus and image, and hindering the observation of the tube formation model. Conversely, if too little matrix gel is added, the resulting cuticle layer will be too thin, potentially resulting in incomplete coverage, which also negatively impacts cell tube formation.

[0071] In a preferred embodiment of the present invention, the conditions for dissolving the matrix adhesive are as follows: placing the matrix adhesive at 1–5°C for 8–16 hours; the matrix adhesive is a substance that maintains its function at low temperatures. It solidifies below -10°C, and slowly thaws into a liquid as the temperature rises to around 0°C. Upon further temperature increases, the matrix adhesive polymerizes, and its morphology remains unchanged after polymerization. It must be used within two days, otherwise it will become ineffective. It is typically stored at -20°C to ensure that the substance does not undergo functional changes. It is slowly thawed at 1–5°C the night before the experiment to form a liquid state. Using higher temperatures for thawing will cause it to become ineffective immediately. Lower temperatures will require a longer thawing time.

[0072] The humidity level in a humid environment is 95%–98%.

[0073] The incubator conditions can be: a temperature of 36-37℃ and an atmosphere containing 5% CO2 by volume.

[0074] In a preferred embodiment of the present invention, in S3, the amount of pericyte cell suspension added is 10–100 μL, preferably 50 μL. If the amount of pericyte cells added is too small, it will be difficult to form a complete tubular structure. If the amount added is too large, more cells will adhere directly together, which will also reduce the number of tubular structures. The amount of endothelial cell suspension added is 10–100 μL, preferably 50 μL; if the amount of endothelial cells added is too small, it will also be difficult to form a complete tubular structure. If the amount added is too large, more cells will adhere directly together, which will also reduce the number of tubular structures.

[0075] This invention also relates to the application of the above-described vascular model or the vascular model prepared by the above-described construction method in drug screening, including drugs for promoting angiogenesis and drugs for protecting blood vessels. Using the vascular model of this invention, compared to obtaining blood vessels by dissecting animals, is more in line with animal experimental ethics. It can be used to study the effects of various conditions on vascular-like structures.

[0076] The cell model in this invention can also be used to detect the interaction between pericytes and endothelial cells.

[0077] The cell model in this invention can also be used to explore the effects of different pathological conditions on blood vessel damage, such as hypoxia, hyperxia, radiation, temperature, laser, and tension.

[0078] The reagents used in the following specific examples are from the following sources:

[0079] Neutral protease: Dispase II, catalog number D4693, Sigma-Aldrich, USA;

[0080] Collagenase: C0130, Sigma-Aldrich, USA;

[0081] Peripheral cell culture medium: 1201, ScienCell;

[0082] α-SMA: Product No. A1011, Manufacturer: ABclonal, Dilution Ratio: 1:100;

[0083] PDGFR-β: Product number A2180, manufacturer ABclonal, dilution ratio 1:100;

[0084] CD31 monoclonal antibody: Product number A18643, manufacturer ABclonal, dilution ratio 1:100;

[0085] NG2 antibody: Abclonal China Wuhan;

[0086] PBS solution: Product number P1020, manufacturer Solarbio;

[0087] Peripheral cell growth factor: Catalog number 1252, ScienCell;

[0088] Matrigel: Part No. 354248, Corning, USA;

[0089] Ibidi μ-Slide Angiogenesis Slide: Catalog No. 81506, Ibidi, Germany;

[0090] Preparation of pericyte culture medium: 500 mL of basal culture medium, with the addition of 10 mL of fetal bovine serum, 5 mL of pericyte growth factor and 5 mL of penicillin or streptomycin; the concentration of penicillin is 10,000 U / mL and the concentration of streptomycin is 10,000 μg / mL.

[0091] Example 1

[0092] Pericellular and endothelial cells of dermal microvessels were extracted.

[0093] I. Extraction of perivascular cells from dermal microvessels

[0094] 1. Acquisition and processing of human skin tissue: Purchase skin tissue; the flap size is approximately 1.5–2.0 cm. 2 Place the sample in a 60 mm culture dish, add pre-cooled PBS containing penicillin and streptomycin (PBS to penicillin solution volume ratio of 100:1, penicillin concentration of 10,000 U / mL, streptomycin concentration of 10,000 μg / mL), and rinse off excess blood 3 times for 5 minutes.

[0095] 2. Trim skin tissue: Trim the muscles and excess fascia under the skin flap, and finally trim it into a tissue strip of 0.5cm×0.5cm×0.1cm.

[0096] 3. Neutral protease digestion: Place the cleaned and trimmed skin flap tissue in a 35 mm dish, add 3 mL of 2 mg / mL neutral protease (Dispase II), and incubate overnight at 4°C.

[0097] 4. Separate the epidermis and dermis: Carefully separate the dermis and epidermis with tweezers. After discarding the epidermis, place the digested dermis in another 35mm dish and rinse 3 times with PBS.

[0098] 5. Collagenase digestion: Add 1 mL of collagenase I at a concentration of 1.25 mg / mL to a small dish, incubate at 37°C for 40 minutes, and then add 2 mL of fetal bovine serum (FBS) to terminate the reaction.

[0099] 6. Obtaining Dermal Microvascular Pericellular Cells: Using forceps, gently squeeze the skin flap and carefully filter the digested flap tissue and digestion solution through a 40μm filter. Place the filter in a 50mL centrifuge tube and rinse with an appropriate amount of PBS. Divide the filtrate into two 15mL centrifuge tubes and centrifuge at 1000 rpm for 5 minutes. Remove the supernatant from one centrifuge tube and resuspend the cell in 3mL of pericyte medium. Seed the resuspended cells in a 25T cell culture flask. Incubate at 37℃ with 5% CO2. Change the medium approximately every 2-3 days. After about one week, a significant number of pericytes will adhere and grow. The extracted pericytes can be identified using immunofluorescence with multiple antibodies. Cells exhibiting CD31-, PDGFR+, NG2+, and α-SMA+ are confirmed as dermal microvascular pericytes, indicating successful extraction.

[0100] Photos of the growth status in vitro are shown below. Figure 1 As shown.

[0101] II. Extraction of endothelial cells

[0102] In another 15 mL centrifuge tube, the supernatant was removed, and the cells were resuspended in ECM medium to a final volume of 1 mL. 40 μL of magnetic bead labeling solution containing CD31 was added, and the tube was incubated at 4°C for 15 minutes. Then, 500 μL of the cell suspension was passed through a magnetic rack (MS column) and washed with 1 mL of ECM medium. Human dermal microvascular endothelial cells were labeled and adsorbed onto the MS column. 2 mL of ECM medium was added, and the MS column was removed from the magnetic rack. Using a pusher, the ECM medium in the column was forcefully expelled into a 15 mL centrifuge tube at 1000 rpm for 4 minutes. The resulting cells were resuspended and seeded in 60 mm dishes. Magnetic bead sorting was performed twice to obtain endothelial cells with high purity.

[0103] Photos of the growth status in vitro are shown below. Figure 2 As shown in the image. Microscopic observation revealed that the cell purity was over 97%.

[0104] Example 2

[0105] This embodiment illustrates a method for identifying pericytes in dermal microvessels:

[0106] The extracted dermal microvascular pericytes were identified using a combination of multiple antibodies via immunofluorescence. The specific method is as follows:

[0107] 1. One to two days before the experiment, digest the cells and seed them into 12-well plates containing coverslips so that the cells have a density of 20%-50% on the day of the experiment.

[0108] 2. After preparing the cells, wash the coverslips three times with PBS and fix them with 4% paraformaldehyde at 25°C for 20 minutes.

[0109] 3. Then permeate the cell membrane with 0.3% Triton X-100 for 10 minutes;

[0110] 4. Incubate with 5% bovine serum albumin (BSA) for 1 hour to achieve non-specific binding blockade;

[0111] 5. Then incubate the coverslip with the primary antibody at 4°C for 24 hours;

[0112] 6. Wash cells with PBS for 15 minutes at room temperature, then incubate with secondary antibody (FITC with anti-mouse and Cy3 anti-rabbit immunoglobulin G; 1:500 dilution) for 1 hour.

[0113] 7. Finally, add 10 μL of anti-fluorescence quencher containing DAPI.

[0114] The results of the identification are as follows Figure 3 As shown.

[0115] Depend on Figure 3It was found that PDGFR+, NG2+, and α-SMA+ were expressed, while CD31 was not expressed, and the cells were identified as perivascular cells of the dermal microvessels.

[0116] In the immunofluorescence experiment, cell morphology was observed and counted, repeated three times, and the average value was calculated. The ratio of perivascular cells in the dermal microvessels was greater than or equal to 95%.

[0117] Example 3

[0118] This example illustrates the process of constructing a vascular model:

[0119] 1. The day before, place the matrix gel in an ice box and freeze at 4°C to allow it to slowly melt overnight. Simultaneously, cool the IBDI angiogenesis slides. Throughout the experiment, the matrix gel must remain in the ice box to prevent it from solidifying and becoming unusable due to high temperatures. Open the sterile packaging and remove the IBDI angiogenesis slides. Add 10 μL of matrix gel to each well. Note that when adding the matrix gel, the pipette tip should be perpendicular to the inner well to prevent matrix gel from flowing through the upper well and leaving residue. Cover the IBDI angiogenesis slides. Prepare a 10 cm culture dish and place a damp paper towel inside to create a humidified chamber. Place the IBDI angiogenesis slides into the culture dish and cover it. Place the entire culture dish in a 37°C, 5% CO2 incubator and let it stand for approximately 30 minutes to allow the gel to solidify. While waiting, digest the adherent pericytes and prepare 2 × 10⁶ cells. 5 Prepare a cell suspension of cells per mL and mix thoroughly.

[0120] 2. Remove the solidified ibidi angiogenesis slide from the humidified chamber. Gently add 50 μL of pericyte suspension, ensuring the pipette tip is perpendicular to the upper well and does not touch the gel in the lower well. Cap the slide, let it stand, and place it in an incubator. After a period of time, all cells will settle into the matrix gel. Approximately one hour later, normal dermal microvascular pericytes will form three-dimensional tubular structures. (See image below.) Figure 4 As shown.

[0121] 3. While waiting for the perivascular cells in the dermal microvessels to form tubular structures, the endothelial cells were digested and centrifuged to prepare 2×10⁻⁶ cells / years. 5 Add cell suspension at a density of 1 cell / mL and mix thoroughly. Once the perivascular cells of the dermal microvessels have formed well-developed tubular structures, aspirate the upper layer of culture medium from the angiogenesis slide. At this point, the perivascular cells of the dermal microvessels have firmly grown on the matrix gel. Then, add 50 μL of endothelial cell suspension to each well. Continue culturing in an incubator. After approximately 1 hour, the endothelial cells will adhere to the inner side of the perivascular cells of the dermal microvessels, thus forming a well-developed perivascular / endothelial cell co-forming vessel model. A photograph of the vessel immediately after adding the endothelial cells is shown below. Figure 5As shown in the image, this is a photograph of the state of endothelial cells after they have adhered to the pericytes of dermal microvessels. Figure 6 As shown, Figure 7 This is a schematic diagram showing how dermal microvascular pericytes and endothelial cells can be clearly distinguished in a vascular model. The arrows on the outer side of the lumen represent dermal microvascular pericytes, and the arrows on the inner side represent endothelial cells.

[0122] The experimental results above show that the model can be maintained for about 24 hours and can distinguish dermal microvascular pericytes and endothelial cells under a microscope.

[0123] Example 4

[0124] This example illustrates the impact of cell addition order on vascular model construction:

[0125] Simultaneously, dermal microvascular pericytes and endothelial cells were added to the matrix gel, resulting in the photograph shown below. Figure 8 As shown, by Figure 8 It is evident that it is difficult to distinguish between the two types of cells, making it inconvenient to observe the performance of the two types of cells during experiments using this model.

[0126] Example 5

[0127] This example illustrates the influence of cell type on vascular model construction: Fibroblasts were used for tube formation experiments. Specific operating conditions were the same as for perivascularization of dermal microvessels, except that the cells were fibroblasts and the culture medium was 20% DMEM. The 20% DMEM medium consisted of: 500 mL high-glucose DMEM, 100 mL LBS, and 5 mL of penicillin-streptomycin solution (containing 10,000 U / mL penicillin and 10,000 μg / mL streptomycin).

[0128] Experiments have shown that fibroblasts can only form dendritic structures and cannot form well-defined tubular structures, specifically as follows: Figure 9 As shown, the formation of tubular structures by perivascular cells in the dermal microvessels is one of the characteristic features of this cell type.

[0129] Example 6

[0130] This example illustrates the impact of cell type on vascular model construction:

[0131] 1. Tube formation experiments were performed using pericytes from dermal microvessels alone. The specific operating conditions were as follows:

[0132] The day before, place the matrix gel in an ice box and freeze at 4°C to allow it to slowly melt overnight. Simultaneously, cool the IBDI angiogenesis slides. Throughout the experiment, the matrix gel must remain in the ice box to prevent it from solidifying and becoming unusable due to high temperatures. Open the sterile packaging and remove the IBDI angiogenesis slides. Add 10 μL of matrix gel to each well. Note that when adding the matrix gel, the pipette tip should be perpendicular to the inner well to prevent matrix gel from flowing through the upper well and leaving residue. Cover the IBDI angiogenesis slides. Prepare a 10 cm culture dish and place a damp paper towel inside to create a humidified chamber. Place the IBDI angiogenesis slides into the culture dish and cover it. Place the entire culture dish in a 37°C, 5% CO2 incubator and let it stand for 30 minutes to allow the gel to solidify. While waiting, digest the adherent dermal microvascular pericytes and prepare 2×10⁻⁶ cells. 5 Prepare a cell suspension of cells per mL and mix thoroughly.

[0133] Remove the solidified ibidi angiogenesis slide from the humidified chamber. Gently add 50 μL of pericyte suspension, ensuring the pipette tip is perpendicular to the upper well and does not touch the gel in the lower well. Cap the slide, let it stand, and place it in an incubator. After a period of time, all cells will settle into the matrix gel. Approximately one hour later, normal dermal microvascular pericytes will form a three-dimensional vascular-like structure, which will remain for about eight hours.

[0134] 2. The same method and concentration of endothelial cells were used to perform the tube formation experiment.

[0135] Photos were taken at 1, 4, 8, and 16 hours after the start of the tube forming experiment, and the experimental results are as follows: Figure 10 As shown.

[0136] according to Figure 10 It was found that pericytes of dermal microvessels can form tubular structures within 1 hour, while endothelial cells only begin to form lumen-like structures after 4 hours and take 8 hours to fully form tubules. Furthermore, the number of tubules formed by pericytes of dermal microvessels is far greater than that of endothelial cells, typically 2 to 3 times the number of endothelial cells. Endothelial cell tubule formation can only be maintained for about 4 hours, while pericyte tubule formation of dermal microvessels can be maintained for more than 8 hours. However, both are shorter than the tubule formation maintenance time of the vascular model of this invention.

[0137] Example 7

[0138] This example illustrates the effect of pericyte suspension concentration on the construction of a vascular model:

[0139] A vascular model was constructed using the method described in Example 3, the difference being that the concentration of the pericyte suspension was changed sequentially to 1.5 × 10⁻⁶. 5 2×10 5 2.5×105 :

[0140] The results showed that the cell concentration during that week was 1.5 × 10⁻⁶. 5 At that time, the number of cells was too small to form a complete tubular structure. However, when the weekly cell concentration was 2.5 × 10⁻⁶, the cell count was insufficient. 5 At a cell density of 2 × 10⁻⁶, many cells adhere directly together, resulting in fewer tubular structures. However, when the cell count is 2 × 10⁻⁶, the density is too high, causing many cells to adhere together and forming fewer tubular structures. 5 At times, pericytes can form numerous tubular structures with excellent morphology, making them a good structural model for scientific research. Therefore, the pericyte density is 2 × 10⁻⁶. 5 At that time, it was a better experimental condition and concentration.

[0141] Example 8

[0142] This example illustrates the impact of the container used in modeling on the construction of the blood vessel model:

[0143] The vascular model was constructed using 96-well plates, requiring 100 μL of matrix gel. The experimental results are as follows: Figure 11 As shown.

[0144] Experiments using 96-well plates require at least 100 μL of matrix gel, which is costly. Furthermore, due to the greater depth of the wells and the thicker matrix gel, cells tend to fall into different layers, making it difficult to focus them on a single plane, resulting in blurry images and hindering observation of tubular structures. Additionally, the larger pore size makes uniformity of cells more likely, leading to the formation of black air bubbles.

[0145] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A vascular model, characterized in that, The vascular model is a three-dimensional vascular-like structure formed by first using human dermal microvascular pericytes and then adding human endothelial cells, and then culturing them on a matrix gel. The vascular model uses the human dermal microvascular pericytes as a framework, with the human endothelial cells attached to the inner side of the human dermal microvascular pericytes; The ratio of perivascular cells in the dermal microvessels to the endothelial cells is 1:1; The vascular model is maintained for at least 24 hours after its formation.

2. The method for constructing a vascular model as described in claim 1, characterized in that, At least the following steps are included: S1. Human dermal microvascular pericytes were obtained through extraction and identification, and a pericyte suspension was prepared. S2. Form a matrix adhesive layer on the ibidi angiogenesis slide; S3. Add the pericyte suspension to the matrix gel layer, and incubate in an incubator for 0.5 to 1.5 hours. The human dermal microvascular pericytes form a tubular framework cell structure, and the culture medium is discarded. S4. Prepare an endothelial cell suspension from human endothelial cells, add it to an ibidi angiogenesis slide, and incubate in an incubator for 0.5 to 1.5 hours to form the vascular model.

3. The construction method according to claim 2, characterized in that, In S1, The extraction includes: S11. Take skin tissue and trim it to preserve the dermis; S12. The trimmed skin tissue is hydrolyzed with neutral protease to separate the epidermis and dermis; S13. Digest the dermis using collagenase; S14. Filter the digested dermis, harvest the cell suspension, centrifuge the cell suspension, culture the cells using pericellular culture medium, and use the cultured cells to prepare pericellular suspension. The identification process includes: simultaneously identifying cultured cells using CD31 antibody, PDGFR antibody, NG2 antibody, and α-SMA antibody. Cells that are negative for CD31 antibody and positive for PDGFR, NG2, and α-SMA antibodies are identified as perivascular cells of the dermis.

4. The construction method according to claim 3, characterized in that, In S12, the conditions for hydrolysis of the neutral protease are incubation at 0–5°C for 8–16 hours; the concentration of the neutral protease solution is 2 mg / mL. In S13, the concentration of collagenase is 1.25 mg / mL, and the digestion conditions are 35℃~38℃ for 30~50 minutes; In S14, the harvested cell suspension includes: first adding fetal bovine serum to terminate digestion, then filtering the digested dermal flap tissue and digestion solution through a 40 μm filter, rinsing, and obtaining a cell suspension. In S14, the centrifugation conditions are: time 4-6 min, speed 800-1200 r / min; the culture conditions are: place the culture dish in a cell culture incubator for incubation, and change the medium every 2-3 days.

5. The construction method according to claim 2, characterized in that, The concentration of dermal microvascular pericytes in the pericyte suspension was 10. 5 ~10 6 cells / mL; The concentration of human endothelial cells in the endothelial cell suspension was 10. 5 ~10 6 cells / mL; The pericyte suspension was prepared using pericyte culture medium containing 2% fetal bovine serum, and the endothelial cell suspension was prepared using endothelial cell culture medium containing 5% ECM.

6. The construction method according to claim 5, characterized in that, The concentration of dermal microvascular pericytes in the pericyte suspension was 2 × 10⁻⁶. 5 per mL.

7. The construction method according to claim 5, characterized in that, The concentration of human endothelial cells in the endothelial cell suspension was 2 × 10⁻⁶. 5 per mL.

8. The construction method according to claim 2, characterized in that, In S2, the matrix gel is melted, and 5-20 μL of matrix gel is added to each well of the ibidi angiogenesis slide. The ibidi angiogenesis slide is then placed in an incubator under humid conditions for 20-60 minutes.

9. The construction method according to claim 8, characterized in that, In S2, 10 μL of matrix gel is added to each well of the ibidi angiogenesis slide.

10. The construction method according to claim 8, characterized in that, In S2, the ibidi angiogenesis slide is placed in an incubator under humid conditions for 30-40 minutes.

11. The construction method according to claim 8, characterized in that, The conditions for dissolving the matrix gel are: place the matrix gel at 1-5°C for 8-16 hours.

12. The construction method according to claim 8, characterized in that, The conditions of the incubator are: a temperature of 36-37°C and an atmosphere containing 5% CO2 by volume.

13. The construction method according to claim 2, characterized in that, In S3, the amount of pericyte suspension added is 10–100 μL; The amount of endothelial cell suspension added is 10–100 μL.

14. The construction method according to claim 13, characterized in that, In S3, the amount of pericyte suspension added is 50 μL.

15. The construction method according to claim 13, characterized in that, In S3, the amount of endothelial cell suspension added is 50 μL.

16. The application of the vascular model as described in claim 1 or the vascular model prepared by the construction method as described in any one of claims 2 to 15 in drug screening.

17. The application as described in claim 16, characterized in that, The drugs mentioned include drugs for promoting angiogenesis and drugs for protecting blood vessels.

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