Vascularized skin chip, vascularized skin model and application thereof

Abstract

The invention discloses a vascularized skin chip, a vascularized skin model and application of the vascularized skin chip and the vascularized skin model. The material of the vascularized skin chip does not contain polydimethylsiloxane; the vascularized skin chip comprises a dermal culture chip layer and an epidermal culture chip layer from bottom to top, wherein a vertically through culture cavity is formed between the dermal culture chip layer and the epidermal culture chip layer; the dermal culture chip layer is provided with a capillary channel, and the capillary channel is used for constructing a capillary in the dermal layer. According to the vascularized skin chip, dermis and epidermis can make direct contact, interaction between cells is truly simulated, the activity and functions of the cells are kept, good mechanical support is provided for the epidermis and the dermis layer, and cell growth and tissue form maturation are promoted. The microvessel channel can simulate microvessels of a corium layer and is used for simulating supply of nutrient substances and excretion of metabolic waste, a dynamic fluid environment is provided, and the skin structure and the microenvironment are restored more truly.

Description

Technical Field

[0001] The present application relates to the field of microfluidic chips, and specifically to a vascularized skin chip, a vascularized skin model and applications thereof. Background Art

[0002] The skin is mainly composed of three layers: epidermis, dermis and subcutaneous tissue. The dermis contains a large number of blood vessels, which provide oxygen and nutrients to the skin, discharge metabolic waste, regulate body temperature, immune defense, and repair and regenerate functions. Therefore, constructing a skin model including a vascular system is of great significance for regenerative medicine, drug testing and tissue engineering.

[0003] At present, skin models usually use simple epidermal or dermal cell culture, lacking vascular structure and dynamic fluid environment, or constructing the epidermis and dermis in layers, without achieving direct contact between the dermis and the epidermis, which has great limitations in simulating the functions of real skin. These models cannot effectively reproduce the nutrient exchange, gas supply, signal transmission and waste discharge between the epidermis and dermis, making it difficult to maintain cell vitality for a long time.

[0004] Therefore, how to provide a skin model that is similar or identical to real skin structure becomes a technical problem that needs to be solved in the art. Summary of the invention

[0005] In view of this, the present application proposes a vascularized skin chip, a vascularized skin model and applications thereof.

[0006] According to the first aspect of the present application, a vascularized skin chip is proposed, wherein the material of the vascularized skin chip does not include polydimethylsiloxane; the vascularized skin chip comprises, from bottom to top: a dermis culture chip layer and an epidermis culture chip layer; wherein a culture cavity which passes through from top to bottom is provided between the dermis culture chip layer and the epidermis culture chip layer; the dermis culture chip layer is provided with a microvascular channel, which is used to construct microvessels in the dermis layer.

[0007] Specifically, the dermis culture chip layer includes: a carrier layer, which is arranged at the bottom of the vascularized skin chip and is made of transparent material; a dermis culture layer, which is arranged inside the dermis culture connection layer, and a first culture cavity penetrating the dermis culture layer is arranged in the middle of the dermis culture layer for culturing the dermis layer; a dermis culture connection layer, which is used to connect the carrier layer and the dermis culture layer, and a culture cavity hole is arranged on the dermis culture connection layer.

[0008] Specifically, the microvascular channel is disposed in the dermis culture layer of the dermis culture chip layer, and the microvascular channel is communicated with the culture cavity channel and is parallel to the radial direction of the culture cavity.

[0009] Specifically, the thickness of the carrier layer is 1.0 mm-1.3 mm; the thickness of the dermis culture connection layer is 0.2 mm-0.3 mm; the thickness of the dermis culture layer is 3.3 mm-3.6 mm; and the diameter of the first culture cavity and the culture cavity hole is 4.5 mm-4.9 mm.

[0010] Specifically, the outer diameter of the microvascular channel is 1.5 mm-1.8 mm, and the inner diameter is 0.8 mm-0.9 mm.

[0011] Specifically, the epidermal culture chip layer includes an epidermal culture layer and an upper chip; the epidermal culture layer is arranged below the upper chip, and a second culture cavity and a culture medium channel are arranged in the middle, and the culture medium channel is radially connected to the second culture cavity for providing nutrients to the epidermal layer.

[0012] Specifically, a third culture cavity, a culture medium inlet and a culture medium outlet are provided in the middle of the upper chip. The culture medium inlet and the culture medium outlet penetrate the upper chip and are respectively connected to the two ends of the culture medium channel to provide culture medium for the culture medium channel.

[0013] Specifically, the thickness of the epidermal culture layer is 0.2 mm-0.3 mm, the thickness of the upper chip is 5.2 mm-5.5 mm; the length of the culture medium channel is 21.0 mm-22.0 mm, the width of the culture medium channel is 1.4 mm-1.6 mm, and the diameter of the second culture cavity is 4.5 mm-4.9 mm.

[0014] Specifically, the diameter of the third culture chamber is 4.5 mm-4.9 mm.

[0015] Specifically, a culture cavity plug is provided on the top of the vascularized skin chip. The culture cavity plug is made of a transparent material, and has a diameter of 4.9 mm-5.2 mm and a thickness of 5.0 mm-5.3 mm.

[0016] Specifically, the material of the vascularized skin chip does not contain polydimethylsiloxane.

[0017] According to the second aspect of the present application, a vascularized skin model is proposed, which is achieved by inserting a microneedle into a microvascular channel of a vascularized skin chip, perfusing a skin equivalent in a culture chamber, removing the microneedle after the skin equivalent has solidified, and perfusing endothelial cells in the microvascular channel to form a microvascular channel in the culture chamber; the pore diameter of the microneedle is 0.2mm-0.4mm; the vascularized skin chip is the above-mentioned vascularized skin chip.

[0018] Specifically, epidermal cell culture medium was perfused at the culture medium inlet and endothelial culture medium was perfused at the microvascular channel using a syringe pump to form a vascularized skin model.

[0019] According to the third aspect of the present application, an application of a vascularized skin model is proposed, which is the application of the vascularized skin model in in vitro non-therapeutic scientific research, drug testing, drug screening, drug metabolism, toxicity research or disease model.

[0020] The vascularized skin chip proposed in this application has a culture cavity that runs through the top and bottom of the chip, which allows the dermis cultured in the chip to be in direct contact with the epidermis, which can more realistically simulate cell-to-cell interactions and promote the efficient exchange of nutrients and signal molecules, thereby maintaining the activity and function of the cells. It also enhances the degree of reduction of the barrier function, allowing epidermal cells to better differentiate and form a stratum corneum. At the same time, direct contact provides better mechanical support for the epidermis and dermis, promoting cell growth and maturation of tissue morphology. Secondly, the microvascular channels of the dermis culture chip layer can simulate the vascular network in the dermis, which can be used to simulate the supply of nutrients and the excretion of metabolic waste. It can also provide a dynamic fluid environment to more realistically restore the structure and microenvironment of the real dermis.

[0021] Other features and advantages of the present application will be described in detail in the subsequent detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings constituting a part of the present application are used to provide a further understanding of the present application, and the schematic implementation modes and descriptions of the present application are used to explain the present application. In the accompanying drawings: Figure 1 This is an exploded view of a vascularized skin chip according to a preferred embodiment of the present application.

[0023] Figure 2 This is a top view of a vascularized skin chip according to a preferred embodiment of the present application.

[0024] Figure 3 This is a diagram of a culture device for a vascularized skin chip according to a preferred embodiment of the present application.

[0025] Figure 4 It is a schematic diagram of the side structure of the vascularized skin model according to the preferred embodiment of the present application.

[0026] Figure 5 It is a top view of the vascularized skin model according to a preferred embodiment of the present application.

[0027] Figure 6 This is an identification diagram of a vascularized skin model according to a preferred embodiment of the present application.

[0028] Explanation of the numbers in the accompanying drawings: 1-carrier layer; 2-dermal culture connection layer; 3-dermal culture layer; 4-epidermal culture layer; 5-upper chip; 6-culture chamber plug; 21-culture chamber hole; 31-first culture chamber; 41-second culture chamber; 51-third culture chamber; 7-microvascular channel; 71-microvascular channel entrance; 72-microvascular channel exit; 82-culture medium channel entrance; 81-culture medium channel exit. DETAILED DESCRIPTION

[0029] The technical solution of the present application will be described in detail below with reference to the accompanying drawings and in combination with the implementation modes.

[0030] In the human or animal body, the epidermis and dermis in the skin are naturally connected, and the natural transfer of nutrients, oxygen and cytokines between the dermis and the epidermis is allowed; the dermis also promotes the differentiation of the epidermis to form structures such as the stratum corneum. In addition, the dermis is also rich in vascular networks, which provide sufficient nutrients and oxygen to the cells of the epidermis and dermis and take away metabolic waste. Skin models usually use simple epidermal or dermal cell culture, lack vascular structure and dynamic fluid environment, or construct the epidermis and dermis in layers, and do not achieve direct contact between the dermis and the epidermis, which makes it have great limitations in simulating the function of real skin. Therefore, the present application proposes a vascularized skin chip, a vascularized skin model and its application.

[0031] According to the first aspect of the present application, a vascularized skin chip is proposed, wherein the material of the vascularized skin chip does not include polydimethylsiloxane; the vascularized skin chip comprises, from bottom to top: a dermis culture chip layer and an epidermis culture chip layer; wherein a culture cavity which passes through from top to bottom is provided between the dermis culture chip layer and the epidermis culture chip layer; the dermis culture chip layer is provided with a microvascular channel, which is used to construct microvessels in the dermis layer.

[0032] In order to avoid the material of the vascularized chip affecting the vascularized skin model, the material of the vascularized skin chip does not contain polydimethylsiloxane (PDMS). PDMS easily absorbs small molecules and interferes with drug delivery. Not using PDMS ensures that the vascularized skin model can more realistically simulate the in vivo environment. It can promote the natural growth and differentiation of cells while avoiding the deviation of experimental results caused by material interference, ensuring the physiological relevance and reliability of the model. It lays the foundation for a precise vascularized skin model and provides more favorable conditions for subsequent research and application.

[0033] The vascularized skin chip proposed in this application has a culture cavity that runs through the top and bottom of the chip, which allows the dermis cultured in the chip to be in direct contact with the epidermis, which can more realistically simulate cell-to-cell interactions and promote the efficient exchange of nutrients and signal molecules, thereby maintaining the activity and function of the cells. It also enhances the degree of reduction of the barrier function, allowing epidermal cells to better differentiate and form a stratum corneum. At the same time, direct contact provides better mechanical support for the epidermis and dermis, promoting cell growth and maturation of tissue morphology. Secondly, the microvascular channels of the dermis culture chip layer can simulate the vascular network in the dermis, which can be used to simulate the supply of nutrients and the excretion of metabolic waste. It can also provide a dynamic fluid environment to more realistically restore the structure and microenvironment of the real dermis.

[0034] In order to construct dermal tissue. Specifically, the dermal culture chip layer may include: a carrier layer 1, which is arranged at the bottom of the vascularized skin chip, and the carrier layer 1 is a transparent material; a dermal culture layer 3, which is arranged inside the dermal culture connection layer 2, and a first culture cavity 31 penetrating the dermal culture layer 3 is arranged in the middle of the dermal culture layer 3 for culturing the dermal layer; a dermal culture connection layer 2, which is used to connect the carrier layer 1 with the dermal culture layer 3, and a culture cavity 21 is arranged on the dermal culture connection layer 2. The transparent material of the above-mentioned carrier layer 1 can be one of polycarbonate (PC), glass, polystyrene, polymethyl methacrylate (PMMA) or polyethylene terephthalate (PET), preferably glass. The glass carrier layer 1 has excellent transparency, chemical stability and biocompatibility, and the culture state of the cells inside the chip can be clearly observed, and it can withstand high temperature sterilization and a variety of chemical reagents to ensure the stability of the culture environment. In addition, the high mechanical strength and low gas permeability of glass make it suitable for long-term culture experiments, providing good attachment and growth conditions for cells, and is particularly suitable for high-precision and complex cell model construction. The material of the above-mentioned dermal culture connection layer 2 can be polyethylene terephthalate (PET), polyvinyl chloride (PVC), preferably polyimide (PI). Polyimide has high biocompatibility and chemical stability and is easy to process. The surface of the above-mentioned dermal culture connection layer 2 is also provided with double-sided tape, which can enhance the connectivity with the carrier layer 1 and the dermal culture layer 3, ensure its stability, simplify the assembly process, and the sealing of the double-sided tape is also good. The above-mentioned double-sided tape is a transparent double-sided tape, which can be made of an appropriate material. In the present application, the material of the transparent double-sided tape is the same as that of the dermal culture connection layer 2, and the culture cavity hole is die-cut on the dermal culture connection layer and the transparent double-sided tape. The carrier layer 1 and the dermis culture layer 3 are detachably connected by using double-sided adhesive tape; the dermis culture layer 3 may be made of polymethyl methacrylate (PMMA), which has high biocompatibility, transparency, mechanical strength and stability, and is easy to process. The first culture cavity 31 and the culture cavity hole 21 are connected vertically, and the dermis model is directly constructed on the carrier layer 1.

[0035] In order to construct blood vessels in the dermis, specifically, the microvascular channel 7 can be arranged in the dermis culture layer 3 of the dermis culture chip layer, and the microvascular channel 7 is connected to the culture cavity channel and is parallel to the radial direction of the culture cavity. It can promote the interaction between cells, simulate the real biological environment in the body, provide dynamic culture conditions, and enhance the growth and functional performance of cells. It provides a more realistic and effective platform for cell culture. The above-mentioned microvascular channel 7 is arranged at both ends of the first culture cavity 31 of the above-mentioned dermis culture layer 3, and is not arranged through the first culture cavity 31 in the chip structure; the inlet 71 and outlet 72 of the above-mentioned microvascular channel can be arranged above the vascularized skin chip, or on the side of the above-mentioned dermis culture layer 3.

[0036] In order to truly simulate the thickness structure of the dermis layer of the skin, specifically, the thickness of the carrier layer 1 can be 1.0mm-1.3mm, preferably 1.1mm; within this thickness range, the above-mentioned carrier layer 1 can provide better mechanical support to ensure the stability of the chip; the thickness of the dermis culture connection layer 2 can be 0.2mm-0.3mm, preferably 0.3mm; the thickness of the dermis culture layer 3 can be 3.3mm-3.6 mm, preferably 3.5mm; the thickness of the above-mentioned dermis culture connection layer 2 and the dermis culture layer 3 is within this range, which provides sufficient space to support cell growth and development and enhances the interaction between cells; secondly, this thickness range is closer to the actual thickness of the dermis layer in the skin, which increases the authenticity of constructing the skin model; the diameter of the first culture cavity 31 and the culture cavity hole 21 can be 4.5mm-4.9mm, preferably 4.8mm, so that the cells in the dermis layer have sufficient space to grow and migrate; using the above-mentioned chip to construct an in vitro skin model can more realistically restore the dermis layer.

[0037] In order to ensure that the liquid flows smoothly in the microvascular channel 7, specifically, the outer diameter of the microvascular channel 7 can be 1.5mm-1.8mm, preferably 1.8mm, and the inner diameter can be 0.8mm-0.9mm, preferably 0.8mm. The microvascular channel 7 can be compatible with conventional experimental equipment, which is convenient for operation and monitoring. The larger inner diameter also helps to reduce the resistance in the flow of liquid, reduce the risk of blockage of the microvascular channel 7, and ensure the smooth progress of the experiment.

[0038] In order to construct a skin chip with an epidermal layer. Specifically, the epidermal culture chip layer includes an epidermal culture layer 4 and an upper chip 5; the epidermal culture layer 4 is arranged below the upper chip 5, and a second culture cavity 41 and a culture medium channel are arranged in the middle, and the culture medium channel is radially connected to the second culture cavity 41 to provide nutrients to the epidermal layer. The above-mentioned surface culture layer is used to culture the epidermal layer. The thickness of the epidermal culture layer 4 can be 0.2mm-0.3mm, preferably 0.3mm. On the one hand, a certain thickness is provided for the epidermal culture layer 4, so that the epidermal layer is close to the thickness of the human epidermis, and the structure and function of the epidermis can be simulated more realistically. It provides space for culturing different epidermal cells in the epidermal culture layer 4, which can form a tissue structure closer to real skin; it can also make the epidermal layer have a certain mechanical strength, reduce the risk of model damage, and make the accuracy and repeatability of experimental data higher; on the other hand, it provides a certain space for the culture medium above the epidermal layer. The culture medium channel arranged in the epidermal culture layer 4 provides nutrients to the cells in the second culture chamber 41, thereby promoting the formation of the epidermal layer.

[0039] The epidermal culture layer 4 and the upper chip 5 can be made of appropriate materials. The material of the epidermal culture layer 4 can be polyimide (PI), polyethylene terephthalate (PET) or polyvinyl chloride (PVC), preferably polyimide (PI). Double-sided adhesive tapes are respectively provided on the upper and lower sides of the epidermal culture layer 4 to connect the epidermal culture layer 4 with the dermal culture layer 3 and the upper chip 5, and to ensure sealing and stability, so that the epidermal culture layer 4 and the adjacent chip layer can be detachably connected; the double-sided adhesive tape is a transparent double-sided adhesive tape, which can be made of appropriate materials. In the present application, the material of the transparent double-sided adhesive tape is the same as that of the epidermal culture layer 4. The material of the upper chip 5 can be an appropriate material, for example, polymethyl methacrylate (PMMA), which has high light transmittance, resistance to ultraviolet radiation, not easy to turn yellow, light weight, good processability and chemical resistance.

[0040] In order to provide nutrients to the epidermis, specifically, a third culture chamber 51, a culture medium inlet 82 and a culture medium outlet 81 may be provided in the middle of the upper chip 5. The culture medium inlet 82 and the culture medium outlet 81 penetrate the upper chip 5 and are respectively connected to the two ends of the culture medium channel to provide culture medium for the culture medium channel. The two ends of the culture medium channel are respectively connected to the culture medium inlet 82 and the outlet on the upper chip 5. The culture medium inlet 82 and the culture medium outlet 81 may be provided at appropriate positions, for example, on the side or top surface of the upper chip 5, to facilitate the flow and supply of culture medium. The upper chip 5 provides additional structural support for the vascularized skin chip, and also helps to guide the fluid between the culture medium channel and the second culture chamber 41, so that the culture medium is more evenly distributed on the surface of the epidermis, ensuring that the cells are adequately supplied with nutrients. The upper chip 5 may also prevent internal contamination. The epidermis is not cultured inside the third culture chamber 51, and isolation protection is provided for the epidermis culture layer 4, making the vascularized skin chip more stable, reliable and adaptable to experiments.

[0041] In order to facilitate the growth of the epidermis, specifically, the thickness of the upper chip 5 can be 5.2mm-5.5mm, preferably 5.3mm; the length of the culture medium channel can be 21.0mm-22.0mm, preferably 21.8mm, the width of the culture medium channel can be 1.4mm-1.6 mm, preferably 1.4mm, and the diameter of the second culture chamber 41 can be 4.5mm-4.9mm, preferably 4.8mm. The upper chip 5 with moderate thickness can maintain good stability during operation, avoid cracking or deformation, and ensure smooth flow of the culture medium. The appropriate length and width design of the culture medium channel can effectively promote the transfer of nutrients, reduce flow resistance, and thus reduce the risk of blockage. In addition, the diameter of the second culture chamber 41 provides sufficient space for the flow of the culture medium, ensuring that the nutrients can be evenly distributed to the epidermis, supporting the healthy growth and metabolism of cells. The overall design not only provides a stable growth environment for cells and simulates physiological conditions in vivo, but also simplifies the operation and maintenance process of the experiment, and facilitates the replacement and cleaning of the culture medium. The flexibility and adaptability of this structure enables it to meet a variety of experimental needs, including drug screening and cell behavior studies, providing ideal experimental conditions, thereby improving the reliability and effectiveness of the research.

[0042] In order to correspond to the diameters of the second culture chamber 41, the first culture chamber 31 and the culture chamber hole 21, specifically, the diameter of the third culture chamber 51 is 4.5mm-4.9mm, preferably 4.8mm. The third culture chamber 51 is used to perfuse skin equivalents. The diameter of the third culture chamber 51 corresponds to the diameter of the chip culture chamber of the chip below. The fluid connection and gas exchange between the culture chambers are smoother, which helps to maintain a stable culture environment. By keeping the diameters of these cavities consistent, the flow of culture medium can be optimized so that nutrients and oxygen can be effectively delivered to various areas, thereby supporting the healthy growth of cells. In addition, the uniform diameter also helps to simplify the design and operation of the system, reduce potential blockage risks, and ensure reliability and effectiveness during cell culture and experiments. This design not only improves the functionality of the entire culture system, but also enhances the repeatability of the experiment, providing a more solid foundation for subsequent research.

[0043] In order to prevent internal contamination of the chip, specifically, a culture cavity plug 6 can be provided on the top of the vascularized skin chip. The culture cavity plug 6 is made of a transparent material, and the diameter of the culture cavity plug is 4.9 mm-5.2 mm, preferably 5 mm. The thickness is 5.0 mm-5.3 mm, preferably 5.3 mm. The culture cavity plug is used to seal the third culture cavity 51 to prevent internal contamination and prevent evaporation of water in the culture medium.

[0044] The dermal culture connection layer 2, dermal culture layer 3, epidermal culture layer 4, upper chip 5 and culture cavity plug are assembled in sequence on the upper surface of the carrier layer 1; during installation, the first culture cavity, the second culture cavity and the third culture cavity need to correspond to the culture cavity holes up and down; UV sterilization is required for 1 hour before use.

[0045] According to the second aspect of the present application, a vascularized skin model is proposed. The vascularized skin model is formed by inserting a microneedle into the microvascular channel 7 of the vascularized skin chip, perfusing a skin equivalent in the culture chamber, removing the microneedle after the skin equivalent solidifies, perfusing endothelial cells in the microvascular channel 7, and using Luer connectors at both ends of the microvascular channel 7 to form a closed pipeline to form a microvascular channel 7 in the culture chamber; using a syringe pump to perfuse epidermal cell culture medium at the culture medium inlet 82, and perfusing endothelial culture medium in the microvascular channel 7 to form a vascularized skin model; the vascularized skin chip is the above-mentioned vascularized skin chip. The above-mentioned skin equivalent is a mixture of human fibroblast matrix gel and human epidermal keratinocytes; the above-mentioned endothelial cells can be human umbilical vein endothelial cells. The above-mentioned human fibroblasts, epidermal keratinocytes and human umbilical vein endothelial cells can all be obtained through commercial channels, and can also be self-screened according to ethical requirements. In this application, the human epidermal keratinocytes used are HaCaT cells, the human fibroblasts are HFF cells, and the human umbilical vein cells are HUVEC cells.

[0046] The above-mentioned injection pumps can be of appropriate number, for example, there can be two, one injection pump is connected to the inlet of the culture medium channel, and the other injection pump is connected to the inlet of the microvascular channel 7. The outlet of the microvascular channel 7 and the outlet of the culture medium channel are connected to the waste liquid container through a pipeline for collecting waste liquid. The above-mentioned waste liquid container can be a waste liquid bottle, and the cover of the waste liquid bottle is seamlessly connected to the pipeline to avoid contamination inside the waste liquid bottle, thereby causing contamination inside the chip. In the present application, the injection rate of the epidermal culture medium is 1μL / min-2μL / min, and the injection rate of the endothelial culture medium is 1μL / min-2μL / min.

[0047] In the vascularized skin model prepared in the present application, after the endothelial cells form microvessels and the upper epidermis is formed, the supply of endothelial cell culture medium and epidermal cell culture medium can be stopped, and the liquid in the culture medium channel and microvessel channel 7 can be replaced for the next step of research.

[0048] In order to provide enough space for endothelial cells to form blood vessels inside the dermis, the pore diameter of the microneedle can be 0.2mm-0.4mm, preferably 0.25mm. It can promote cell migration and distribution, support nutrient and gas exchange, optimize microfluidics, enhance physiological relevance, and facilitate operation and observation, providing important support for the establishment of an efficient and reliable vascularized skin model.

[0049] According to the third aspect of the present application, an application of a vascularized skin model is proposed, which is the application of the vascularized skin model in in vitro non-therapeutic scientific research, drug testing, drug screening, drug metabolism, toxicity research or disease model.

[0050] The vascularized skin chip, vascularized skin model and their applications proposed in this application are described below with reference to embodiments.

[0051] 1. Example Example 1 (1) Place the sterilized vascularized skin chip in a clean bench, open the culture chamber plug, and insert a microneedle into the microvascular channel; (2) A human fibroblast matrix gel mixture was perfused into the culture chamber holes and the first culture chamber through the third culture chamber on the upper chip to prepare the dermis layer; the human fibroblast matrix gel mixture included matrigel matrix gel (8 mg / mL-13 mg / mL), bovine fibrinogen (5 mg / mL-10 mg / mL), bovine thrombin (100 U / mL-200 U / mL) and human dermal fibroblasts (5×10 6 / mL -6×10 6 pieces / mL); (3) After the matrix gel solidifies, a human epidermal keratinocyte suspension is perfused into the second culture chamber through the third culture chamber. The concentration of keratinocytes in the human epidermal keratinocyte suspension is 1×10 7 / mL -2×10 7 Pieces / mL; (4) After the matrix gel solidifies, remove the microneedle and perfuse the endothelial cell suspension into the microvascular channel to form an endothelial layer. The concentration of endothelial cells in the endothelial cell suspension is 1×10 6 / mL -2×10 6 Pieces / mL; (5) After the endothelial layer and human epidermal keratinocytes have attached, turn on the syringe pump to infuse the endothelial culture medium and epidermal cell culture medium respectively; (6) Observe the cell status inside the vascularized skin chip. After the vascularized skin model is constructed, stop supplying endothelial culture medium and epidermal cell culture medium and proceed to the next step.

[0052] Note: There is no order of precedence for steps (3) and (4) above.

[0053] 2. Experimental Results 2.1 Morphology of the vascularized skin model Figure 4 Schematic diagram of the structure of the vascularized skin model prepared by the vascularized skin chip in this application, Figure 4 It can be seen that the epidermis of the vascularized skin model constructed in the present application is in direct contact with the dermis, the endothelial cells form microvessels inside the dermis, and the endothelial cells are also in direct contact with the dermis, which fully simulates the connection between the dermis and the epidermis and the blood vessels, indicating that the vascularized skin chip prepared in the present application can be used to restore the real state and microenvironment of the skin.

[0054] Figure 5 a in the figure is a top view of the vascularized skin model prepared by the vascularized skin chip in this application under a microscope, Figure 5 b in the figure is a local enlarged view. Figure 5 As can be seen from b, the microvessels pass through the dermis, and fibroblasts are evenly distributed on both sides of the microvessels with good morphology and high cell activity. Therefore, the vascularized skin chip in this application is used to construct a vascularized skin model.

[0055] 2.2 Immunofluorescence validation of vascularized skin model In this application, CD31 antibody was used to verify endothelial cells, vimentin antibody was used to verify fibroblasts, and DAPI was used to stain cell nuclei. Figure 6 a in the figure is an immunofluorescence image of a top view of the vascularized skin model in this application, Figure 6b is a side view of the vascularized skin model in this application, Figure 6 As can be seen from a and b, the endothelial cells are evenly distributed to form a tubular structure, indicating that the vascularized skin chip in the present application can successfully construct microvessels, and accumulation of fibroblasts can be seen around the microvessels.

[0056] The preferred embodiments of the present application are described in detail above; however, the present application is not limited to the specific details in the above embodiments. Within the technical concept of the present application, a variety of simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the protection scope of the present application.

[0057] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

[0058] In addition, the various implementation modes of the present application may be arbitrarily combined, and as long as they do not violate the concept of the present application, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A vascularized skin chip, characterized in that: The material of the vascularized skin chip does not include polydimethylsiloxane; the vascularized skin chip includes, from bottom to top: a dermis culture chip layer and an epidermis culture chip layer; Wherein, a culture cavity which is connected vertically is arranged between the dermis culture chip layer and the epidermis culture chip layer; The dermis culture chip layer is provided with a microvascular channel (7), and the microvascular channel (7) is used to construct microvessels in the dermis layer.

2. The vascularized skin chip according to claim 1, characterized in that: The dermis culture chip layer comprises: a carrier layer (1), the carrier layer (1) is arranged at the bottom of the vascularized skin chip, and the carrier layer (1) is made of a transparent material; A dermis culture layer (3), wherein a first culture cavity (31) penetrating the dermis culture layer (3) is provided in the middle of the dermis culture layer (3) and is used for culturing the dermis layer; A dermis culture connection layer (2), the dermis culture connection layer (2) being used to connect the carrier layer (1) and the dermis culture layer (3), and a culture cavity (21) is provided on the dermis culture connection layer (2).

3. The vascularized skin chip according to claim 1, characterized in that: The microvascular channel (7) is arranged in the dermis culture layer (3) of the dermis culture chip layer, and the microvascular channel (7) is connected to the culture cavity and is parallel to the radial direction of the culture cavity.

4. The vascularized skin chip according to claim 2, characterized in that: The thickness of the carrier layer (1) is 1.0mm-1.3mm; The thickness of the dermal culture connection layer (2) is 0.2mm-0.3mm; The thickness of the dermal culture layer (3) is 3.3 mm-3.6 mm; The diameters of the first culture chamber (31) and the culture chamber hole (21) are 4.5 mm to 4.9 mm.

5. The vascularized skin chip according to claim 3, characterized in that: The outer diameter of the microvascular channel (7) is 1.5 mm-1.8 mm, and the inner diameter is 0.8 mm-0.9 mm.

6. The vascularized skin chip according to claim 1, characterized in that: The epidermal culture chip layer comprises an epidermal culture layer (4) and an upper chip (5); The epidermal culture layer (4) is arranged below the upper chip (5), and a second culture chamber (41) is arranged in the middle, and A culture medium channel, the culture medium channel being connected to the second culture chamber (41) in the radial direction, and being used for providing nutrients to the epidermis.

7. The vascularized skin chip according to claim 6, characterized in that: A third culture chamber (51) is provided in the middle of the upper chip (5), and A culture medium inlet (82) and a culture medium outlet (81), wherein the culture medium inlet (82) and the culture medium outlet (81) penetrate the upper chip (5) and are respectively connected to two ends of the culture medium channel to provide culture medium for the culture medium channel.

8. The vascularized skin chip according to claim 6, characterized in that: The thickness of the epidermal culture layer (4) is 0.2 mm-0.3 mm, and the thickness of the upper chip (5) is 5.2 mm-5.5 mm; the length of the culture medium channel is 21.0 mm-22.0 mm, the width of the culture medium channel is 1.4 mm-1.6 mm, and the diameter of the second culture cavity (41) is 4.5 mm-4.9 mm.

9. The vascularized skin chip according to claim 7, characterized in that: The diameter of the third culture chamber (51) is 4.5 mm-4.9 mm.

10. The vascularized skin chip according to claim 1, characterized in that: A culture cavity plug (6) is arranged on the top of the vascularized skin chip. The culture cavity plug (6) is made of a transparent material and has a diameter of 4.9 mm to 5.2 mm and a thickness of 5.0 mm to 5.3 mm.

11. A vascularized skin model, characterized in that The vascularized skin model is formed by inserting a microneedle into the microvascular channel of the vascularized skin chip, perfusing the culture chamber with skin equivalents, removing the microneedle after the skin equivalents solidify, and perfusing endothelial cells into the microvascular channel to form a microvascular channel in the culture chamber; the pore diameter of the microneedle is 0.2 mm-0.4 mm; The vascularized skin chip is the vascularized skin chip described in any one of claims 1-10.

12. The vascularized skin model according to claim 11, characterized in that: Epidermal cell culture medium was perfused at the culture medium inlet using a syringe pump, and endothelial culture medium was perfused at the microvascular channel to form a vascularized skin model.

13. Use of a vascularized skin model, characterized in that: The application is the use of a vascularized skin model in in vitro non-therapeutic scientific research, drug testing, drug screening, drug metabolism, toxicity research or disease models, and the vascularized skin model is the vascularized skin model described in any one of claims 11-12.

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