A pericyte vascular model, its construction method and application

Abstract

This invention relates to the field of cell culture technology, and more particularly to a perivascular model, its construction method, and its application. The vascular model is a three-dimensional vascular-like structure formed by culturing dermal microvascular perivascular cells on a matrix gel. The construction method of the vascular model includes: extracting and identifying dermal microvascular perivascular cells and preparing a cell suspension; forming a matrix gel layer on an IBDI angiogenesis slide; adding the cell suspension to the matrix gel layer; and culturing in an incubator for 0.5–1.5 hours to form the vascular model. This invention innovatively proposes a three-dimensional vascular model at the cellular level, which can be constructed through cell culture, offering advantages such as simplicity, low cost, and greater compliance with animal experimental ethics.

Description

Technical Field

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

[0002] 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.

[0003] Current in vitro studies of pericytes mainly involve extracting individual pericytes and culturing them in culture dishes. Alternatively, animal models are used to directly study and observe pericytes from animal blood vessels. However, blood vessels are three-dimensional tubular structures, and the two-dimensional environment created by culture dishes makes it impossible to observe the effects of altered experimental conditions on the pericyte tubular structure. Using animal experiments, on the other hand, requires significant costs and does not comply with ethical requirements for protecting laboratory animals. Therefore, there is an urgent need to develop a cellular-level blood vessel model with a three-dimensional structure.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a pericyte vascular model, its construction method, and its application.

[0006] This invention provides a vascular model, which is a three-dimensional vascular-like structure formed by culturing perivascular cells of dermal microvessels on a matrix gel.

[0007] Optionally, the vascular model is maintained for at least 5 hours after formation, preferably at least 8 hours.

[0008] This invention provides a method for constructing this vascular model, comprising at least the following steps:

[0009] S1. Dermal microvascular perivascular cells were obtained through extraction and identification, and then prepared into a cell suspension.

[0010] S2. Form a matrix adhesive layer on the ibidi angiogenesis slide;

[0011] S3. Add the cell suspension to the matrix gel layer and incubate in an incubator for 0.5 to 1.5 hours to form the vascular model.

[0012] Optionally, in S1, the extraction includes:

[0013] S11. Take skin tissue and trim it to preserve the dermis;

[0014] S12. The trimmed skin tissue is hydrolyzed with neutral protease to separate the epidermis and dermis;

[0015] S13. Digest the dermis using collagenase;

[0016] S14. Filter the digested dermis, harvest the cells, culture the cells using pericyte culture medium, and use the cultured cells to prepare cell suspensions.

[0017] Optionally, in S1, the identification 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 perivascular cells of the dermal microvessels.

[0018] Optionally, in S12, the neutral protease hydrolysis conditions are incubation at 0–5℃ for 8–16 hours; the concentration of the neutral protease solution is 2 mg / mL; in S13, the collagenase concentration is 1.25 mg / mL, and the digestion conditions are 35℃–38℃ for 30–50 minutes; in S14, harvesting the 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 the cell suspension; in S14, the centrifugation conditions are: time 4–6 min, speed 800–1200 r / min; the culture conditions are: placing the culture dish in a cell culture incubator for incubation, changing the medium every 2–3 days.

[0019] Optionally, the concentration of perivascular cells in the cell suspension is 10. 5 ~10 6 Cells / mL, preferably 2×10⁻⁶ 5 Cells / mL; the cell suspension was prepared using pericyte culture medium containing 2% fetal bovine serum.

[0020] 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.

[0021] 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.

[0022] Optionally, in S3, the amount of cell suspension added is 10 to 100 μL, preferably 50 μL.

[0023] This invention provides the application of the vascular model or the vascular model prepared by the above construction method in identifying perivascular cells of dermal microvessels, detecting perivascular cell activity of dermal microvessels, or in drug screening.

[0024] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0025] 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.

[0026] The vascular model of this invention is obtained by culturing perivascular cells of dermal microvessels on Matricella, which differs from 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

[0027] Figure 1 Photographs of primary in vitro cultured human dermal microvascular pericytes extracted;

[0028] Figure 2 Photograph of human dermal microvascular perivascular cells after third-generation passage in vitro culture.

[0029] Figure 3 This is a schematic diagram of the positive identification results of pericytes in human dermal microvessels, using endothelial cells (EC) CD31+, NG2- and fibroblasts (FC) PDGFR-, α-SMA- as controls;

[0030] Figure 4 A photograph showing the formation of three-dimensional vascular-like structures by perivascular cells in the human dermis on a matrix gel after 1 hour.

[0031] Figure 5 A photograph showing the formation of three-dimensional vascular-like structures by perivascular cells in human dermal microvessels on a matrix gel after 4 hours.

[0032] Figure 6A photograph showing the formation of three-dimensional vascular-like structures by perivascular cells in the human dermis on a matrix gel after 8 hours.

[0033] Figure 7 Photographs showing the results of tube formation experiments on fibroblasts;

[0034] Figure 8 Photographs showing the results of a tube-forming experiment on endothelial cells;

[0035] Figure 9 The concentration of the cell suspension was 1.5 × 10⁻⁶. 5 Photographs showing the results of the tube forming experiment; Figure 10 The concentration of the cell suspension was 2 × 10⁻⁶. 5 Photographs showing the results of the tube forming experiment;

[0036] Figure 11 The concentration of the cell suspension was 2.5 × 10⁻⁶. 5 Photographs showing the results of the tube forming experiment;

[0037] Figure 12 Photographs showing the results of tube forming experiments using a 96-well plate. Detailed Implementation

[0038] 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.

[0039] 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.

[0040] This invention, through long-term research on the characteristics and functions of dermal microvascular pericellular cells, has discovered that dermal microvascular pericellular cells possess a faster and stronger ability to form tubular structures than endothelial cells. Extensive experiments have confirmed that dermal microvascular pericellular cells can form lumen-like structures on stable Matrigel and maintain them for a relatively long time. Therefore, a cell-level vascular model is proposed, specifically a three-dimensional vascular-like structure formed by culturing dermal microvascular pericellular cells on Matrigel. The vascular model of this invention maintains its tubular structure for at least 5 hours, preferably at least 8 hours. The dermal microvascular pericellular cells used in this invention are mammalian-derived dermal microvascular pericellular cells, and human dermal microvascular pericellular cells are preferred for preparing the cell model.

[0041] The matrix gel is an extract of EHS tumor. EHS tumor is a mouse tumor rich in extracellular matrix, and its extract has a composition similar to the basement membrane, hence the name matrix gel. Basement membrane components include: main matrix: laminin, type IV collagen, basement membrane proteoglycans, nestin, endogenin; proteases: matrix metalloproteinase-2, tissue plasminogen activator; growth factors: transforming growth factor-β, fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor; other proteins: amylase, transferrin, clustering proteins, etc. The matrix gel is 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 of three-dimensional cell culture. This invention also proposes a method for constructing this vascular model, which includes at least the following steps:

[0042] S1. Dermal microvascular perivascular cells were obtained through extraction and identification, and then prepared into a cell suspension.

[0043] S2. Form a matrix adhesive layer on the ibidi angiogenesis slide;

[0044] S3. Add the cell suspension to the matrix gel layer and incubate in an incubator for 0.5 to 1.5 hours to form a blood vessel-like structure, which is the blood vessel model of this embodiment of the invention.

[0045] The vascular model in this embodiment of the invention can be obtained by culturing dermal microvascular pericytes. During the research, it was found that the maintenance time of the vascular model is closely related to the activity state of the 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:

[0046] S11. Take skin tissue and trim it to preserve the dermis;

[0047] S12. The trimmed skin tissue is hydrolyzed with neutral protease to separate the epidermis and dermis;

[0048] S13. Digest the dermis using collagenase;

[0049] S14. Filter the digested dermis, harvest the cell suspension, centrifuge the cell suspension, and culture the cells using pericellular culture medium.

[0050] This invention utilizes the screening function of pericyte culture medium to conveniently and quickly extract skin-like dermal microvascular pericytes.

[0051] 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.

[0052] 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.

[0053] As an improvement to the technical solution of this invention, the concentration of the neutral protease solution is 2 mg / mL.

[0054] 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.

[0055] 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.

[0056] 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. The identification revealed that almost all of the prepared cells were dermal microvascular pericellular cells.

[0057] In a preferred embodiment of the present invention, the concentration of perivascular cells in the cell suspension used for modeling is 10. 5 ~10 6 Cells / mL, preferably 1.8–2.2 × 10⁻⁶. 5 Cells / mL, more preferably 2×10⁻⁶ 5 Cells / mL. The cell suspension was prepared using pericyte culture medium containing 2% fetal bovine serum. If the concentration is too high, too many cells will adhere together directly, resulting in fewer tubular structures; if the concentration is too low, it will be difficult to form complete tubular structures.

[0058] 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 placed in an incubator under humid conditions 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 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. In a preferred embodiment of the present invention, the matrix gel is dissolved under the following conditions: the matrix gel is placed at 1–5°C for 8–16 hours; matrix gel is a substance that maintains its function at low temperatures. It solidifies below -10℃ and slowly thaws into a liquid as the temperature rises to around 0℃. Upon further temperature increases, the matrix gel polymerizes, and once polymerized, its form remains unchanged. It must be used within two days, otherwise it will become ineffective. It is typically stored at -20℃ to prevent functional changes. It should be slowly thawed at 1–5℃ the night before the experiment to form a liquid state. Thawing at higher temperatures will cause it to become ineffective immediately. Lower temperatures will require a longer thawing time.

[0059] The humidity level in a humid environment is 95%–98%.

[0060] The incubator can be set to a temperature of 36–37°C and an atmosphere containing 5% CO2 by volume.

[0061] In a preferred embodiment of the present invention, in step S3, the amount of cell suspension added is 10–100 μL, preferably 50 μL. If the amount of cells added is too small, there will be too few cells, making it difficult to form a complete tubular structure. If the amount added is too large, too many cells will adhere together directly, which will also reduce the number of tubular structures.

[0062] 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, which can be used to study the effects of various conditions on pericytes. Using the vascular model of this invention, compared to obtaining blood vessels by dissecting animals, is more in line with animal experimental ethics.

[0063] The cell model in this invention can also be used to detect the activity and function of pericytes, expanding the research methods for dermal microvascular pericytes. In this invention, independent dermal microvascular pericytes are obtained from skin tissue. After purity identification, a suspension of dermal microvascular pericytes is added to a matrix gel and cultured in an incubator, confirming that dermal microvascular pericytes can form obvious vascular-like structures. This characteristic can be used for various applications. For example, the tube-forming property can be used to identify cells and determine whether they are dermal microvascular pericytes; the tube-forming ability and maintenance time can be used to determine and compare the activity of dermal microvascular pericytes; and the effects of various conditions on the tube-forming structure, tube-forming ability, and tube-forming maintenance time of dermal microvascular pericytes can be determined to assess the impact of these conditions on blood vessels. These conditions include drug effects or other conditions, such as hypoxia, hyperxia, radiation, temperature, laser, and tension.

[0064] The reagents used in the following specific examples are from the following sources:

[0065] Neutral protease: Dispase II, catalog number D4693, Sigma-Aldrich, USA;

[0066] Collagenase: C0130, Sigma-Aldrich, USA;

[0067] Peripheral cell culture medium: 1201, ScienCell;

[0068] α-SMA: Manufacturer: ABclonal, Product No.: A1011, Dilution Ratio: 1:100;

[0069] PDGFR-β: Manufacturer: ABclonal, Product No.: A2180, Dilution Ratio: 1:100;

[0070] CD31 monoclonal antibody: Manufacturer: ABclonal, Product No. A18643, Dilution ratio: 1:100;

[0071] NG2 antibody: Abclonal China Wuhan;

[0072] PBS solution: Manufacturer: Solarbio, Product No. P1020;

[0073] Periperocyte growth factor: ScienCell, catalog number 1252;

[0074] Matrigel: Part No. 354248, Corning, USA;

[0075] Ibidi μ-Slide Angiogenesis Slide: Product No. 81506, Manufacturer: Ibidi, Germany.

[0076] 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.

[0077] Example 1

[0078] This embodiment illustrates a specific method for isolating pericytes from human dermal microvessels:

[0079] 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, 5 minutes each time.

[0080] 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.

[0081] 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, and incubate overnight at 4°C.

[0082] 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.

[0083] 5. Collagenase digestion: Add 1 mL of collagenase I at a concentration of 1.25 mg / mL to a small dish, then incubate in an incubator (37℃, 95% air) for 40 minutes, and add 2 mL of fetal bovine serum (FBS) to terminate the reaction.

[0084] 6. Using forceps, gently squeeze the skin flap, then carefully filter the digested flap tissue and digestion solution through a 40μm filter. Place the filter in a 50mL centrifuge tube and rinse the filter with an appropriate amount of PBS. Centrifuge the 50mL tube at 1000 rpm for 5 minutes. Remove the supernatant and resuspend the tissue in 3mL of pericyte medium. Seed the resuspended tissue in 25T cell culture flasks. Incubate at 37℃ in a 5% CO2 incubator. Change the medium approximately every 2–3 days. After about one week, a significant number of dermal microvascular pericytes will adhere and grow. Cell extraction results are shown below. Figure 1 and Figure 2 As shown.

[0085] according to Figure 1 It can be seen that the cells are growing well, and the number of dead cells is less than 1%. For example... Figure 2 It can be seen that after three passages, the cells still grew well, and the number of dead cells was less than 3%.

[0086] Example 2

[0087] This embodiment illustrates a method for identifying dermal microvascular pericytes: Extracted dermal microvascular pericytes are identified using a combination of multiple antibodies via immunofluorescence. The specific method is as follows:

[0088] 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.

[0089] 2. After preparing the cells, wash the coverslips three times with PBS and fix them with 4% paraformaldehyde at 25°C for 20 minutes.

[0090] 3. Then permeate the cell membrane with 0.3% Triton X-100 for 10 minutes;

[0091] 4. Incubate with 5% bovine serum albumin (BSA) for 1 hour to achieve non-specific binding blockade;

[0092] 5. Then incubate the coverslip with the primary antibody at 4°C for 24 hours;

[0093] 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.

[0094] 7. Finally, add 10 μL of anti-fluorescence quencher containing DAPI.

[0095] The results of the identification are as follows Figure 3 As shown.

[0096] Depend on Figure 3 It 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.

[0097] 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%.

[0098] Example 3

[0099] This example illustrates the process of constructing a vascular model:

[0100] 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.

[0101] 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 incubate 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, thus successfully establishing a human dermal microvascular pericyte vascular model. Experimental results are as follows: Figures 4-6 .like Figures 4-6 As shown, the density or number of blood vessels formed can reach approximately 1.5 to 2.5 times that of endothelial cell tube formation.

[0102] Example 4

[0103] This example illustrates the impact of cell type on vascular model construction:

[0104] This embodiment uses fibroblasts for tube formation experiments. The specific operating conditions are as follows: except that the cells are fibroblasts and the culture medium is 20% DMEM, everything else is the same as the pericyte tube formation procedure. The composition of the 20% DMEM culture medium is: 500 mL high-glucose DMEM, 100 mL FBS, and 5 mL of penicillin-streptomycin solution (the penicillin-streptomycin solution contains a concentration of 10,000 U / mL and a concentration of 10,000 μg / mL).

[0105] Experiments have shown that fibroblasts can only form dendritic structures and cannot form well-defined tubular structures, specifically as follows: Figure 7 As shown, the formation of tubular structures in pericytes is one of the characteristic features of this cell type.

[0106] Example 5

[0107] This example illustrates the impact of cell type on vascular model construction:

[0108] Endothelial cells of the same concentration were used for tube formation experiments. The specific operating conditions were the same as those for pericyte tube formation, except that the cells were fibroblasts and the culture medium was 20% DMEM. The composition of the 20% DMEM culture medium was: 500 mL high-glucose DMEM, 100 mL FBS, and 5 mL of penicillin-streptomycin solution (containing 10,000 U / mL penicillin and 10,000 μg / mL streptomycin).

[0109] Simultaneously, human dermal microvascular perivascular cells of the same density were used for comparison, and images were taken at 1, 4, 8, and 16 hours after the start of the tube formation experiment. The experimental results are as follows: Figure 8 As shown.

[0110] according to Figure 8 It was found that pericytes of dermal microvessels can form tubular structures within 1 hour, while endothelial cells take 4 hours to begin forming lumen-like structures and 8 hours to fully form tubules. Furthermore, the number of tubules formed by pericytes of dermal microvessels is significantly greater than that of endothelial cells, typically 2-3 times more. Endothelial cell tubule formation only lasts about 4 hours, while pericyte tubule formation can last for more than 8 hours, meaning that pericytes of dermal microvessels maintain tubule formation for a longer period.

[0111] Example 6

[0112] This example illustrates the effect of cell suspension concentration on the construction of a vascular model:

[0113] A vascular model was constructed using the method described in Example 3, the difference being that the concentration of the cell suspension was changed sequentially to 1.5 × 10⁻⁶. 5 2×105 2.5×10 5 The experimental results obtained are as follows: Figures 9-11 As shown:

[0114] The results showed that when the cell concentration 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 cell concentration was 2.5 × 10⁻⁶, the number of cells was too low to form a complete tubular structure. 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 Pericytes can form numerous tubular structures with excellent morphology, making them a good structural model for scientific research. Currently, we believe the pericyte density is 2 × 10⁻⁶. 5 At that time, it was a good experimental condition and concentration, and can be used as the experimental condition for this experiment.

[0115] Example 6

[0116] This example illustrates the impact of the container used in modeling on the construction of the blood vessel model:

[0117] The vascular model was constructed using 96-well plates, requiring 100 μL of matrix gel. The experimental results are as follows: Figure 12 As shown.

[0118] 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.

[0119] 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 method for constructing a vascular model, characterized in that, The vascular model is a three-dimensional vascular-like structure formed by culturing pericytes of dermal microvessels on a matrix gel. The construction method includes at least the following steps: S1. Dermal microvascular pericytes were obtained through extraction and identification, and prepared into a concentration of 2×10⁶ cells using a pericyte culture medium containing 2% fetal bovine serum. 5 Cells / mL cell suspension; S2. Melt the matrix gel at 1~5℃ for 8~16 hours. Add 10 μL of matrix gel to each well of the ibidi angiogenesis slide. Then, place the ibidi angiogenesis slide in a humid environment in an incubator at 36~37℃ with a CO2 atmosphere containing 5% CO2 by volume for 20~60 minutes to form a matrix gel layer. S3. Add 50 μL of the cell suspension to the matrix gel layer and incubate in an incubator for 0.5 to 1.5 hours to form the vascular model; The vascular model is maintained for at least 8 hours after it is formed.

2. The construction method according to claim 1, 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 pericyte culture medium, and use the cultured cells to prepare cell 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.

3. The construction method according to claim 2, characterized in that, In S12, the neutral protease hydrolysis is performed under the condition of incubation at 0-5°C for 8-16 hours; the concentration of the neutral protease solution used 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.

4. The construction method according to claim 1, characterized in that, In S2, the ibidi angiogenesis slide is placed in an incubator under humid conditions for 30 to 40 minutes.

5. The application of the vascular model prepared by the construction method according to any one of claims 1 to 4 in identifying dermal microvascular pericytes, detecting the activity of dermal microvascular pericytes, or in drug screening.

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