Culture method for in-vitro induction of skin blood vessels

Abstract

The invention relates to the technical field of biology, and discloses a culture method for in-vitro induction of skin blood vessels, which comprises the following steps: S1, raw material preparation: centrifuging human umbilical vein endothelial cells, inoculating into a culture bottle, culturing with a serum-containing culture medium, and replacing with a serum-free culture medium after starvation culture; s2, inoculation preparation: melting the substrate glue at a low temperature, spreading the melted substrate glue on a 24-pore plate, and solidifying the substrate glue in an incubator; s3, digesting and inoculating, namely cleaning the human umbilical vein endothelial cells, digesting with trypsin, adding a culture medium to terminate digestion reaction, centrifuging, counting, inoculating to the surface of the solidified substrate glue, and culturing; and S4, after induced molding, starvation treatment and counting, preparing the substrate glue again, inoculating the cells, and adding an inducer for culture. Blood vessels are induced through human umbilical vein endothelial cells, the function of a 3D skin model is improved, application limitation caused by lack of blood vessels is solved, and support is provided for clinical skin repair.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a culture method for inducing skin blood vessels in vitro. Background Art

[0002] Artificial skin models are widely used in the pharmaceutical and cosmetic industries to predict the irritation and corrosiveness of specific substances to the skin. Artificial skin models are very similar to human skin in structure and function. At present, the application of commercialized artificial skin models is very common in many countries. However, commercialized skin has no blood vessels. This type of skin without blood vessels can only be used in the cosmetics industry and cannot be used in skin clinical trials, such as burn transplantation, because this type of skin faces a problem. This type of skin tissue has no blood vessels and cannot be fully integrated with the host, and will eventually necrotize. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides a method for culturing skin blood vessels in vitro, which solves the problem that the existing commercial artificial skin model cannot be applied to skin clinical trials due to the lack of vascular structure.

[0004] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for culturing skin blood vessels in vitro, comprising the following steps:

[0005] S1. Raw material preparation:

[0006] The human umbilical vein endothelial cells are placed in a culture medium and centrifuged, and then inoculated into a culture bottle, and serum is added to the culture bottle for culture, and starvation culture is performed after the culture is completed. After the starvation culture is completed, all the human umbilical vein culture medium containing serum is aspirated and replaced with serum-free human umbilical vein culture medium;

[0007] S2. Preparation for vaccination:

[0008] Thaw the base gel in an ice box. When the base gel is completely melted, spread it on a 24-well plate. After completion, place the 24-well plate in an incubator to allow the base gel to solidify.

[0009] S3, digestion and inoculation:

[0010] Digestion: The human umbilical vein endothelial cells obtained in S1 were washed, and then trypsin was added to digest the human umbilical vein endothelial cells. After the digestion, human umbilical vein endothelial cell culture medium containing serum was added to terminate the digestion reaction, and then the cell suspension was transferred to a centrifuge tube and centrifuged again. After the centrifugation, the supernatant was discarded, the cells were resuspended, and the cells were counted;

[0011] Inoculation: The human umbilical vein endothelial cells obtained after digestion are inoculated onto the solidified basement gel surface obtained in S2. After the inoculation is completed, serum-free human umbilical vein endothelial cell culture medium is added to the basement gel surface for culture;

[0012] S4, Induction molding:

[0013] Preliminary preparation: Human umbilical vein endothelial cells cultured in S3 were starved, then digested and counted for later use;

[0014] Preparation of base gel: Prepare base gel again based on S2 for later use;

[0015] Cell inoculation: inoculate the prepared human umbilical vein endothelial cells into the prepared basement gel, and then place them in an incubator for culture;

[0016] Induction culture: After the basement gel solidifies, add inducer to the basement gel for culture.

[0017] Preferably, the step S1 specifically includes:

[0018] Culture steps: After taking out the human umbilical vein endothelial cells from the liquid nitrogen tank, resuscitate them in a 36-38°C water bath, add human umbilical vein endothelial cell culture medium, and centrifuge them in a centrifuge at a ratio of culture medium to cell suspension of (8.5-9.5): (0.9-1.1), with centrifugation parameters of 290-310G and a centrifugation time of 4.5-5.5min;

[0019] Serum culture: Add 9-11% serum human umbilical vein endothelial cell culture medium to the culture bottle and culture at 36-38°C and 4.5-5.5% CO2 for 3 days;

[0020] Starvation culture: Continue starvation culture for 24 hours at 36-38°C and CO2 concentration of 4.5-5.5%.

[0021] Preferably, the step S2 specifically includes:

[0022] The thawing temperature of the basement gel is 4-5°C, the amount of basement gel used in each well of a 24-well plate is 95-105 μl, and the incubator parameters are a temperature of 36-38°C, a CO2 concentration of 4.5-5.5%, and a culture time of 14-16 minutes.

[0023] Preferably, the step S3 specifically includes:

[0024] Digestion: The human umbilical vein endothelial cells obtained in S1 were washed with PBS buffer, and 0.015-0.025% trypsin was added to digest the human umbilical vein endothelial cells, and then digested at 36-38°C for 0.9-1.5 minutes. After digestion, human umbilical vein endothelial cell culture medium containing 9-11% serum was added to terminate the trypsin digestion, and then the cell suspension in the culture bottle was pipetted into a centrifuge tube, and centrifuged at 380-420G for 9.5-11 minutes to separate the cells and count them;

[0025] Inoculation: The inoculation concentration is controlled at 1×10 5 / well, and culture at 36-38°C and 4.5-5.5% CO2 for 5.9-6.1h after inoculation.

[0026] Preferably, the step S4 specifically includes:

[0027] Preliminary preparation: Same as the digestion step in step S3;

[0028] Preparation of base gel: same as step S2;

[0029] Cell inoculation: Human umbilical vein endothelial cells were seeded at 2×10 6 ~4×10 6 The cells were inoculated onto the surface of the base gel at a density of 10 cells / well, and 95-105 μl of serum-free human umbilical vein endothelial cell culture medium was added to each well after inoculation; the cells were then placed in an incubator at a temperature of 36-38°C and a CO2 concentration of 4.5-5.5% for 28-32 minutes;

[0030] Induction culture: After the base gel solidifies, add induction medium to each well. The specific formula of the induction medium is:

[0031] Human umbilical vein endothelial cell culture medium: 58-62%

[0032] 154 culture medium: 38-42%

[0033] EGF: 20ng / ml, i.e. 2μg per 100ml culture medium

[0034] IL-10: 20 ng / ml, i.e., add 2 μg per 100 ml of culture medium

[0035] During the induction process, the amount of inducer added was 145-150 μl / well.

[0036] Preferably, the requirement for changing the induction medium is: changing the medium every day from day 1 to day 3, and changing the medium every 3 days from day 4 to day 30.

[0037] The present invention provides a method for culturing skin blood vessels in vitro, which has the following beneficial effects:

[0038] 1. The present invention successfully constructed a 3D skin model with blood vessels by introducing human umbilical vein endothelial cells and inducing the formation of vascular structures in basement gel. Compared with traditional 3D skin models, the present invention achieves the generation of blood vessels, making the skin tissue have a structure and function closer to natural skin, solving the technical problem that the existing 3D skin models on the market are limited in application due to the lack of blood vessels.

[0039] 2. The present invention uses an innovative culture method to successfully construct a vascular network in vitro in a 3D skin model, significantly improving the survival rate of skin tissue, the efficiency of oxygen and nutrient delivery, and the ability of tissue regeneration. The vascularized 3D skin model can better integrate with the host tissue, avoid the problem of necrosis after transplantation, and is suitable for clinical treatment of burns, trauma, etc. It overcomes the limitation that traditional 3D skin models cannot be used clinically, and provides important support for the fields of tissue engineering and regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a flow chart of the culture method of the present invention;

[0041] Figure 2 This is a schematic diagram of the tubular structure of human umbilical vein endothelial cells of the present invention;

[0042] Figure 3 This is a schematic diagram of live cell staining of human umbilical vein endothelial cells of the present invention;

[0043] Figure 4 This is a schematic diagram of blood vessel formation observed under HE staining microscope of the present invention;

[0044] Figure 5 This is a schematic diagram of the angiogenesis protein expression captured by immunofluorescence staining and confocal microscopy of the present invention. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] In order to better understand the present invention, the above contents are described in detail below in conjunction with specific embodiments.

[0047] Please see attached Figure 1 -Attached Figure 5The embodiment of the present invention provides a method for culturing skin blood vessels in vitro, comprising the following steps:

[0048] S1. Raw material preparation:

[0049] The human umbilical vein endothelial cells are placed in a culture medium and centrifuged, and then inoculated into a culture bottle, and serum is added to the culture bottle for culture, and starvation culture is performed after the culture is completed. After the starvation culture is completed, all the human umbilical vein culture medium containing serum is aspirated and replaced with serum-free human umbilical vein culture medium;

[0050] S2. Preparation for vaccination:

[0051] Thaw the base gel in an ice box. When the base gel is completely melted, spread it on a 24-well plate. After completion, place the 24-well plate in an incubator to allow the base gel to solidify.

[0052] S3, digestion and inoculation:

[0053] Digestion: The human umbilical vein endothelial cells obtained in S1 were washed, and then trypsin was added to digest the human umbilical vein endothelial cells. After the digestion, human umbilical vein endothelial cell culture medium containing serum was added to terminate the digestion reaction, and then the cell suspension was transferred to a centrifuge tube and centrifuged again. After the centrifugation, the supernatant was discarded, the cells were resuspended, and the cells were counted;

[0054] Inoculation: The human umbilical vein endothelial cells obtained after digestion are inoculated onto the solidified basement gel surface obtained in S2. After the inoculation is completed, serum-free human umbilical vein endothelial cell culture medium is added to the basement gel surface for culture;

[0055] S4, Induction molding:

[0056] Preliminary preparation: Human umbilical vein endothelial cells cultured in S3 were starved, then digested and counted for later use;

[0057] Preparation of base gel: Prepare base gel again based on S2 for later use;

[0058] Cell inoculation: inoculate the prepared human umbilical vein endothelial cells into the prepared basement gel, and then place them in an incubator for culture;

[0059] Induction culture: After the basement gel solidifies, add inducer to the basement gel for culture.

[0060] Preferably, step S1 specifically includes:

[0061] Culture steps: After taking out the human umbilical vein endothelial cells from the liquid nitrogen tank, resuscitate them in a 36-38°C water bath, add human umbilical vein endothelial cell culture medium, and centrifuge them in a centrifuge at a ratio of culture medium to cell suspension of (8.5-9.5): (0.9-1.1), with centrifugation parameters of 290-310G and a centrifugation time of 4.5-5.5min;

[0062] Serum culture: Add 9-11% serum human umbilical vein endothelial cell culture medium to the culture bottle and culture at 36-38°C and 4.5-5.5% CO2 for 3 days;

[0063] Starvation culture: Continue starvation culture for 24 hours at 36-38°C and CO2 concentration of 4.5-5.5%.

[0064] Preferably, step S2 specifically includes:

[0065] The thawing temperature of the basement gel is 4-5°C, the amount of basement gel used in each well of a 24-well plate is 95-105 μl, and the incubator parameters are a temperature of 36-38°C, a CO2 concentration of 4.5-5.5%, and a culture time of 14-16 minutes.

[0066] Preferably, step S3 specifically includes:

[0067] Digestion: The human umbilical vein endothelial cells obtained in S1 were washed with PBS buffer, and 0.015-0.025% trypsin was added to digest the human umbilical vein endothelial cells, and then digested at 36-38°C for 0.9-1.5 minutes. After digestion, human umbilical vein endothelial cell culture medium containing 9-11% serum was added to terminate the trypsin digestion, and then the cell suspension in the culture bottle was pipetted into a centrifuge tube, and centrifuged at 380-420G for 9.5-11 minutes to separate the cells and count them;

[0068] Inoculation: The inoculation concentration is controlled at 1×10 5 / well, and culture at 36-38°C and 4.5-5.5% CO2 for 5.9-6.1h after inoculation.

[0069] Preferably, step S4 specifically includes:

[0070] Preliminary preparation: Same as the digestion step in step S3;

[0071] Preparation of base gel: same as step S2;

[0072] Cell inoculation: Human umbilical vein endothelial cells were seeded at 2×10 6 ~4×10 6The cells were inoculated onto the surface of the base gel at a density of 10 cells / well, and 95-105 μl of serum-free human umbilical vein endothelial cell culture medium was added to each well after inoculation; the cells were then placed in an incubator at a temperature of 36-38°C and a CO2 concentration of 4.5-5.5% for 28-32 minutes;

[0073] Induction culture: After the base gel solidifies, add induction medium to each well. The specific formula of the induction medium is:

[0074] Human umbilical vein endothelial cell culture medium: 58-62%

[0075] 154 culture medium: 38-42%

[0076] EGF: 20ng / ml, i.e. 2μg per 100ml culture medium

[0077] IL-10: 20 ng / ml, i.e., add 2 μg per 100 ml of culture medium

[0078] During the induction process, the amount of inducer added was 145-150 μl / well.

[0079] Preferably, the requirement for changing the induction medium is: changing the medium every day from day 1 to day 3, and changing the medium every 3 days from day 4 to day 30.

[0080] Example 1: Cultivation experiment of human umbilical vein endothelial cells inducing skin angiogenesis

[0081] Experimental grouping and design

[0082] Three experimental groups were set up, including:

[0083] Experimental Group 1:

[0084] S1: Raw material preparation:

[0085] Cultivation steps:

[0086] After taking out the human umbilical vein endothelial cells from the liquid nitrogen tank, they were revived in a 37°C water bath;

[0087] Centrifuge at a ratio of 9:1 between culture medium and cell suspension (centrifugation parameters: 300G, 5 minutes)

[0088] Serum culture:

[0089] Use human umbilical vein endothelial cell culture medium containing 10% serum;

[0090] The cells were cultured at 37°C and 5% CO2 for 3 days.

[0091] Hungry training:

[0092] Aspirate the serum-containing medium and replace it with serum-free medium;

[0093] Starvation culture was continued at 37°C, 5% CO2 for 24 hours.

[0094] S2: Inoculation preparation:

[0095] Preparation of Matrigel:

[0096] Thawing temperature of base gel: 4°C;

[0097] Amount of base gel per well: 100 μl;

[0098] Place in an incubator (37°C, 5% CO2) to coagulate for 15 minutes

[0099] S3: Digestion and inoculation:

[0100] Digestion:

[0101] Digest with 0.02% trypsin at 37°C for 1 min;

[0102] Digestion was terminated by adding medium containing 10% serum;

[0103] Centrifugation conditions: 400G, 10 minutes.

[0104] Vaccination:

[0105] Seeding density: 1×10 5 / hole;

[0106] Incubate for 6 hours after inoculation.

[0107] S4: Induction molding:

[0108] Cell seeding:

[0109] Cell density: 3×10 6 / hole;

[0110] The amount of serum-free medium added to each well: 100 μl.

[0111] Induction medium:

[0112] Human umbilical vein endothelial cell culture medium: 60%;

[0113] 154 culture medium: 40%;

[0114] EGF: 20 ng / ml (2 μg per 100 ml culture medium);

[0115] IL-10: 20 ng / ml (2 μg added per 100 ml of culture medium).

[0116] Liquid volume: 150 μl per well;

[0117] Fluid change frequency:

[0118] Change the medium daily on days 1-3;

[0119] From day 4 to day 30, the medium was changed every 3 days.

[0120] Experimental Group 2:

[0121] S1: Raw material preparation:

[0122] Cultivation steps:

[0123] Water bath temperature: 36°C;

[0124] Ratio of culture medium to cell suspension: 8.5:1.5;

[0125] Centrifugation conditions: 290G, 5 minutes.

[0126] Serum culture:

[0127] Serum concentration: 9%;

[0128] Culture conditions: 36°C, 4.5% CO2, for 3 days.

[0129] Hungry training:

[0130] Replace serum-free medium and starve the cells for 24 hours at 36°C and 4.5% CO2.

[0131] S2: Inoculation preparation:

[0132] Preparation of Matrigel:

[0133] Thawing temperature: 3.5℃;

[0134] Amount of base gel: 95 μl;

[0135] Setting time: 14 minutes.

[0136] S3: Digestion and inoculation:

[0137] Digestion:

[0138] Trypsin concentration: 0.015%;

[0139] Digestion time: 0.9 minutes;

[0140] Centrifugation conditions: 380G, 9.5 minutes.

[0141] Vaccination:

[0142] Seeding density: 8×10 4 / hole;

[0143] The culture time was 5.9 hours after inoculation.

[0144] S4: Induction molding:

[0145] Cell seeding:

[0146] Cell density: 2×10 6 / hole;

[0147] The amount of serum-free medium added to each well: 95 μl.

[0148] Induction medium:

[0149] Human umbilical vein endothelial cell culture medium: 58%;

[0150] 154 culture medium: 38%;

[0151] EGF: 18 ng / ml;

[0152] IL-10: 18 ng / ml.

[0153] Liquid volume: 145 μl per well;

[0154] Fluid change frequency:

[0155] Change the medium daily on days 1-3;

[0156] From day 4 to day 30, the medium was changed every 3 days.

[0157] Experimental group three:

[0158] S1: Raw material preparation:

[0159] Cultivation steps:

[0160] Water bath temperature: 38°C;

[0161] Ratio of culture medium to cell suspension: 9.5:0.5;

[0162] Centrifugation conditions: 310G, 5.5 minutes.

[0163] Serum culture:

[0164] Serum concentration: 11%;

[0165] Culture conditions: 38°C, 5.5% CO2, for 3 days.

[0166] Hungry training:

[0167] The serum-free medium was replaced and the cells were starved and cultured at 38°C and 5.5% CO2 for 24 hours.

[0168] S2: Inoculation preparation:

[0169] Preparation of Matrigel:

[0170] Thawing temperature: 5℃;

[0171] Amount of base gel: 105 μl;

[0172] Setting time: 16 minutes.

[0173] S3: Digestion and inoculation:

[0174] Digestion:

[0175] Trypsin concentration: 0.025%;

[0176] Digestion time: 1.5 minutes;

[0177] Centrifugation conditions: 420G, 11 minutes.

[0178] Vaccination:

[0179] Seeding density: 1.2×10 5 / hole;

[0180] The culture time was 6.1 hours after inoculation.

[0181] S4: Induction molding:

[0182] Cell seeding:

[0183] Cell density: 4×10 6 / hole;

[0184] The amount of serum-free medium added to each well: 105 μl.

[0185] Induction medium:

[0186] Human umbilical vein endothelial cell culture medium: 62%;

[0187] 154 culture medium: 42%;

[0188] EGF: 22 ng / ml;

[0189] IL-10: 22ng / ml.

[0190] Liquid volume: 155 μl per well;

[0191] Fluid change frequency:

[0192] Change the medium daily on days 1-3;

[0193] From day 4 to day 30, the medium was changed every 3 days.

[0194] Determination method

[0195] HE staining method: After HE staining, the cultured basement gel sections are sliced ​​and the structure and quantity of blood vessels formed are observed under a microscope, and the formation rate is calculated.

[0196] Immunofluorescence staining: Confocal microscopy was used to detect the expression levels of angiogenesis-related proteins (such as CD31) to evaluate the quality of angiogenesis.

[0197] The experimental results and data comparison table are shown in Table 1:

[0198] Table 1:

[0199]

[0200] in:

[0201] The angiogenesis rate (92%) and CD31 protein expression level (95%) of experimental group 1 were higher than those of other groups, showing the best experimental effect, proving that the parameter setting was reasonable.

[0202] In the experimental group 2, due to insufficient culture conditions, the angiogenesis rate (68%) and protein expression level (72%) were significantly decreased, and the vascular structure was incomplete.

[0203] Although experimental group 3 had a high formation rate (80%) and protein expression level (85%), there was uneven vascular structure and some culture conditions were too high, causing metabolic stress on the cells.

[0204] By comparing the experimental groups, it was proved that the best combination of operating conditions was within the range (experimental group 1).

[0205] Example 2: Comparative experiment between the culture method of the present invention and the traditional artificial skin model

[0206] Experimental background and purpose

[0207] Traditional artificial skin models are widely used in drug testing and cosmetic research, but they lack vascular structure and cannot be applied to clinical skin repair.

[0208] The purpose of this experiment is to evaluate the advantages of the method of the present invention in terms of angiogenesis ability, tissue survival rate, oxygen and nutrient transfer efficiency, etc. by comparing the performance of the culture method of the present invention (experimental group 1 in Example 1) and the traditional artificial skin model.

[0209] Experimental design

[0210] Experimental groups:

[0211] Experimental group: The culture method of the present invention (experimental group 1 in Example 1) was used to construct a 3D skin model with a vascular structure.

[0212] Control group: traditional artificial skin model without vascular structure.

[0213] Evaluation Metrics:

[0214] Blood vessel formation rate: HE staining and immunofluorescence staining were used to evaluate the number of blood vessels and the expression level of vascular protein (CD31) in the tissue.

[0215] Tissue survival rate: MTT was used to detect cell survival rate and evaluate the biological activity of the skin model.

[0216] Oxygen and nutrient transfer efficiency: The oxygen and nutrient utilization capacity of the skin model was evaluated by measuring the oxygen concentration and glucose consumption rate in the tissue.

[0217] Repair ability: In a simulated wound repair model, the ability of the skin model to promote wound repair was evaluated by measuring the healing area.

[0218] Experimental process:

[0219] Experimental group preparation: A 3D skin model with a vascular structure was cultured according to the method of Experimental Group 1 in Example 1.

[0220] Preparation of the control group: An artificial skin model without a vascular structure was prepared according to the traditional method, using the same cell types (without adding human umbilical vein endothelial cells), and the culture medium did not contain EGF and IL-10.

[0221] Determination method:

[0222] HE staining: observe the presence and distribution of vascular structures in the two groups of skin models.

[0223] Immunofluorescence staining: CD31 protein expression was detected by confocal microscopy.

[0224] MTT assay: Active cells from the two groups of skin models were extracted separately, and the cell viability was detected using MTT reagent.

[0225] Oxygen and nutrient delivery: Oxygen concentration sensors and glucose measurement kits were used to monitor oxygen and nutrient consumption in both groups of tissues.

[0226] Wound repair experiment: Two groups of skin models were transplanted onto simulated wounds, the healing area was measured, and the repair ability was evaluated.

[0227] The experimental results and data analysis are shown in Table 2

[0228] Table 2:

[0229]

[0230] Results and Discussion

[0231] Angiogenesis rate and CD31 protein expression:

[0232] The method of the present invention (experimental group) successfully constructed a 3D skin model with a vascular structure. A clear vascular structure was observed under HE staining, and a high level of CD31 protein expression was detected under confocal microscopy, proving that the vascularization was successful.

[0233] The control group (traditional artificial skin model) had no vascular structure at all and the CD31 protein test was negative.

[0234] Tissue survival rate and oxygen and nutrient transfer efficiency:

[0235] The vascular structure in the experimental group significantly improved the efficiency of oxygen and nutrient delivery, allowing the cell survival rate in the tissue to reach 95%, significantly higher than the 60% in the control group.

[0236] The control group lacked blood vessels, and the deep tissues were unable to effectively obtain oxygen and nutrients, resulting in poor cell survival rate and functionality.

[0237] Wound repair ability:

[0238] The experimental group model showed superior repair ability in the simulated wound repair experiment, with the healing area reaching 85%, much higher than the 35% of the control group.

[0239] The control group had a slow wound repair rate and a small healing area due to the lack of vascular support.

[0240] Summarize

[0241] Through comparative experiments, the results fully demonstrate the significant advantages of the method of the present invention:

[0242] The 3D skin model with blood vessels can increase the efficiency of oxygen and nutrient delivery and significantly improve tissue activity.

[0243] The introduction of blood vessels greatly improves the functionality and clinical application value of the skin model. For example, the wound repair effect is far better than that of traditional artificial skin models.

[0244] The method of the present invention shows obvious advantages in terms of angiogenesis, tissue function and repair ability, and is suitable for application in skin tissue engineering and clinical treatment.

[0245] The experimental results further verified the effectiveness and superiority of the culture method of the present invention.

[0246] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for inducing skin blood vessels in vitro, characterized in that: The following steps are involved: S1. Raw material preparation: The human umbilical vein endothelial cells are placed in a culture medium and centrifuged, and then inoculated into a culture bottle, and serum is added to the culture bottle for culture, and starvation culture is performed after the culture is completed. After the starvation culture is completed, all the human umbilical vein culture medium containing serum is aspirated and replaced with serum-free human umbilical vein culture medium; S2. Preparation for vaccination: Thaw the base gel in an ice box. When the base gel is completely melted, spread it on a 24-well plate. After completion, place the 24-well plate in an incubator to allow the base gel to solidify. S3, digestion and inoculation: Digestion: The human umbilical vein endothelial cells obtained in S1 were washed, and then trypsin was added to digest the human umbilical vein endothelial cells. After the digestion, human umbilical vein endothelial cell culture medium containing serum was added to terminate the digestion reaction, and then the cell suspension was transferred to a centrifuge tube and centrifuged again. After the centrifugation, the supernatant was discarded, the cells were resuspended, and the cells were counted; Inoculation: The human umbilical vein endothelial cells obtained after digestion are inoculated onto the solidified basement gel surface obtained in S2. After the inoculation is completed, serum-free human umbilical vein endothelial cell culture medium is added to the basement gel surface for culture; S4, Induction molding: Preliminary preparation: Human umbilical vein endothelial cells cultured in S3 were starved, then digested and counted for later use; Preparation of base gel: Prepare base gel again based on S2 for later use; Cell inoculation: inoculate the prepared human umbilical vein endothelial cells into the prepared basement gel, and then place them in an incubator for culture; Induction culture: After the basement gel solidifies, add inducer to the basement gel for culture.

2. The method for inducing skin blood vessels in vitro according to claim 1, characterized in that: The step S1 specifically includes: Culture steps: After taking out the human umbilical vein endothelial cells from the liquid nitrogen tank, resuscitate them in a 36-38°C water bath, add human umbilical vein endothelial cell culture medium, and centrifuge them in a centrifuge at a ratio of culture medium to cell suspension of (8.5-9.5): (0.9-1.1), with centrifugation parameters of 290-310G and a centrifugation time of 4.5-5.5min; Serum culture: add 9-11% serum human umbilical vein endothelial cell culture medium to the culture bottle and culture at 36-38°C and 4.5-5.5% CO2 for 3 days; Starvation culture: Continue starvation culture for 24 hours at 36-38°C and CO2 concentration of 4.5-5.5%.

3. The method for inducing skin blood vessels in vitro according to claim 1, characterized in that: The step S2 specifically includes: The thawing temperature of the basement gel is 4-5°C, the amount of basement gel used in each well of a 24-well plate is 95-105 μl, and the incubator parameters are a temperature of 36-38°C, a CO2 concentration of 4.5-5.5%, and a culture time of 14-16 minutes.

4. The method for inducing skin blood vessels in vitro according to claim 1, characterized in that: The step S3 specifically includes: Digestion: The human umbilical vein endothelial cells obtained in S1 were washed with PBS buffer, and 0.015-0.025% trypsin was added to digest the human umbilical vein endothelial cells, and then digested at 36-38°C for 0.9-1.5 minutes. After digestion, human umbilical vein endothelial cell culture medium containing 9-11% serum was added to terminate the trypsin digestion, and then the cell suspension in the culture bottle was pipetted into a centrifuge tube, and centrifuged at 380-420G for 9.5-11 minutes to separate the cells and count them; Inoculation: The inoculation concentration is controlled at 1×10 5 / well, and culture at 36-38°C and 4.5-5.5% CO2 for 5.9-6.1h after inoculation.

5. The method for inducing skin blood vessels in vitro according to claim 1, characterized in that: The step S4 specifically includes: Preliminary preparation: Same as the digestion step in step S3; Preparation of base gel: same as step S2; Cell inoculation: Human umbilical vein endothelial cells were seeded at 2×10 6 ~4×10 6 The cells were inoculated onto the surface of the base gel at a density of 10 cells / well, and 95-105 μl of serum-free human umbilical vein endothelial cell culture medium was added to each well after inoculation; the cells were then placed in an incubator at a temperature of 36-38°C and a CO2 concentration of 4.5-5.5% for 28-32 minutes; Induction culture: After the base gel solidifies, add induction medium to each well. The specific formula of the induction medium is: Human umbilical vein endothelial cell culture medium: 58-62% 154 culture medium: 38-42% EGF: 20ng / ml, i.e. 2μg per 100ml culture medium IL-10: 20 ng / ml, i.e., add 2 μg per 100 ml of culture medium During the induction process, the amount of inducer added was 145-150 μl / well.

6. The method for inducing skin blood vessels in vitro according to claim 5, characterized in that: The requirements for changing the induction medium are: changing the medium every day from day 1 to day 3, and changing the medium every 3 days from day 4 to day 30.

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