Vascularized skin chip and method of constructing the same

Abstract

The application discloses a kind of vascularized skin chip and its construction method, it is related to biomedical engineering technical field, the chip includes hardware structure and software system, hardware adopts polyimide material and realizes detachable connection of each layer, from top to bottom in order to second chip, epidermis culture chip layer, dermis culture chip layer and first chip, integrated electrode is used for monitoring;Software system contains data acquisition, analysis and control module, built-in vascularization evaluation, impedance analysis unit and culture medium flow regulation unit, its construction method includes chip assembly and information acquisition, dermis layer vascularization induction and evaluation, epidermis layer construction and differentiation monitoring, combined culture and dynamic regulation.The application is innovated by hardware material, multidimensional dynamic monitoring and the synergistic design of intelligent control, fully solves the deficiency of existing skin chip in physiological simulation degree, detection dimension and environmental controllability, significantly improves the functional authenticity and experimental reliability of skin model.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical engineering, and particularly relates to a vascularized skin chip and a construction method thereof. BACKGROUND

[0002] Skin, as the largest organ of the human body, its barrier function, mechanical strength and response characteristics to external stimuli are the core research objects of life science research, drug development and cosmetic safety evaluation. In vitro skin model simulates the structure and function of human skin, providing an important experimental platform for the above research, and has become one of the key technologies to replace animal experiments. The ordinary skin model is usually composed of dermis and epidermis, which mainly simulates the barrier function, mechanical strength and response characteristics to external stimuli of the skin.

[0003] At present, the current skin chip technology still has certain limitations: on the one hand, it is difficult to accurately restore and replicate the three-dimensional environment of human skin, which leads to the inability to truly simulate the actual barrier function, mechanical strength and response to external stimuli of the skin; on the other hand, in the detection of skin barrier function, although various methods such as dye penetration test, skin permeability test, skin electrical impedance test can be used for detection from different angles, but the existing skin chip can only support one of the test methods, and cannot realize the synchronous experiment of multiple skin chips in multiple detection modes, which is not conducive to ensuring the singleness of experimental conditions. In addition, the skin chip adopts one-piece design and cannot be disassembled, which brings great difficulty to the end-point analysis of in vitro tissues; furthermore, the culture environment regulation of the existing model mainly depends on static or semi-static mode, and lacks dynamic feedback mechanism based on real-time monitoring data. At the same time, the existing technology does not fully consider the possible adverse effects of skin chip materials (such as polydimethylsiloxane) on skin model construction. Therefore, it is necessary to propose a vascularized skin chip and a construction method thereof to solve the above problems. SUMMARY

[0004] The present application provides a vascularized skin chip and a construction method thereof, which solves the deficiencies of the prior art in physiological simulation, detection dimension, structural flexibility and dynamic regulation.

[0005] To achieve the above purpose, according to the embodiment of the present application in one aspect, a vascularized skin chip is provided, which comprises a hardware structure and a software system;

[0006] The hardware structure comprises a second chip, an epidermis culture chip layer, a dermis culture chip layer and a first chip arranged in sequence from top to bottom, and each layer is detachably connected by polyimide material; wherein the first chip is used to provide a bottom accommodating space for the dermis culture medium, the second chip is arranged above the first chip and is used to supply and store the culture medium or supply the skin stimulant;

[0007] The first chip is provided with a first electrode, and the second chip is provided with a second electrode;

[0008] The dermis culture chip layer is sequentially provided with a dermis culture connecting layer, a carrier layer and a first chip connecting layer between the first chip;

[0009] The software system communicates with the first electrode, the second electrode and an external monitoring device, and comprises a data acquisition module, a data analysis module and a control module, the data analysis module is internally provided with a blood vesselization evaluation unit and an electrical impedance analysis unit, and the control module is internally provided with a culture medium flow adjusting unit.

[0010] Further improvement lies in that the dermis culture chip layer comprises a dermis culture layer, the dermis culture layer is provided with a first culture cavity in the middle, and the dermis culture layer is used for constructing a dermis layer;

[0011] The first chip connecting layer and the dermis culture connecting layer are respectively provided with a first culture cavity hole and a second culture cavity hole corresponding to the first culture cavity in the middle.

[0012] Further improvement lies in that the carrier layer is a microporous membrane, the carrier layer covers the first culture cavity hole and is used for separating the first culture cavity hole from the first culture cavity, and the first chip connecting layer is further provided with a pair of dermis culture medium channels symmetrically relative to the first culture cavity hole, and each dermis culture medium channel is communicated with the first culture cavity hole.

[0013] Further improvement lies in that the dermis culture connecting layer is further provided with a pair of dermis culture medium inlet hole channels symmetrically relative to the second culture cavity hole;

[0014] The dermis culture layer is further provided with a pair of dermis culture medium outlet hole channels symmetrically relative to the first culture cavity, and each dermis culture medium inlet hole channel and dermis culture medium outlet hole channel is communicated with the dermis culture medium channel on the first chip connecting layer.

[0015] Further improvement lies in that the epidermis culture chip layer comprises a second culture cavity arranged through and four epidermis culture medium channels arranged in an array outside the second culture cavity, the epidermis culture medium channels are communicated with the second culture cavity and are used for providing epidermis culture medium.

[0016] Further improvement lies in that the second chip comprises a third culture cavity arranged through and an epidermis culture medium inlet (a), an epidermis culture medium outlet (b), a dermis culture medium inlet (c) and a dermis culture medium outlet (d) arranged in an array outside the third culture cavity;

[0017] The third culture cavity is communicated with the second culture cavity on the epidermis culture chip layer;

[0018] The dermis culture medium inlet (c) and the dermis culture medium outlet (d) are communicated with the dermis culture medium hole passage and the dermis culture medium hole passage respectively, and are communicated with the epidermis culture medium passage on the epidermis culture chip layer.

[0019] The application further provides a construction method of the vascularized skin chip.

[0020] S1: chip assembly and information acquisition, the first chip, the first chip connecting layer, the carrier layer, the dermis culture connecting layer, the dermis culture chip layer, the epidermis culture chip layer and the second chip are sequentially sealed and connected by polyimide material to form a detachable chip whole; the software system is initialized, the data acquisition module is communicated with the first electrode, the second electrode and the external monitoring equipment, and the signal acquisition accuracy and the equipment parameters are calibrated;

[0021] S2: dermis layer construction and vascularization induction, the dermis cell suspension is inoculated into the first culture cavity, the vascular endothelial cell suspension is inoculated after the dense cell layer is formed, the culture medium containing the vascular induction factor is continuously supplied through the dermis culture medium passage, and the vascular network characteristics are analyzed in real time through the vascularization evaluation unit;

[0022] S3: epidermis layer construction, the epidermis cell suspension is inoculated into the second culture cavity, the culture medium containing the differentiation induction factor is continuously supplied through the epidermis culture medium passage, and the epidermis differentiation state is monitored through the electric resistance and impedance analysis unit;

[0023] S4: combined culture and dynamic regulation, the fresh culture medium or the skin stimulant is regularly supplied through the second chip, and the culture medium supply is optimized based on the detection data through the culture medium flow regulation unit.

[0024] Further improvement lies in that in step S2, the vascular network characteristics are analyzed in real time through the vascularization evaluation unit, and the specific steps are as follows:

[0025] S21, information acquisition, the vascular fluorescence image of the vascular endothelial cell specific fluorescent label in the first culture cavity is acquired through the external monitoring equipment, and the vascular fluorescence image covers the complete growth area of the dermis layer vascular network;

[0026] S22, image preprocessing, the acquired vascular fluorescence image is sequentially executed:

[0027] Gaussian filter denoising, the vascular fluorescence image is smoothed by using a Gaussian kernel function;

[0028] Adaptive histogram equalization enhances the contrast of the vascular region and the background;

[0029] Threshold segmentation based on the OTSU algorithm to obtain a binary vascular network region image;

[0030] S23, feature extraction, performing on the binary blood vessel network region image:

[0031] The morphological thinning algorithm processes the binary blood vessel network region to obtain a single-pixel width blood vessel skeleton and retains the blood vessel topological structure;

[0032] Identify the blood vessel branch point, count the total number of branch points, and the branch point is a pixel point with more than or equal to three connected pixels in the skeleton;

[0033] Segment the blood vessel segment, count the total number of blood vessel segments and the number of connected blood vessel segments, and the connected blood vessel segment is a blood vessel segment participating in forming a continuous path; combine the image resolution to calculate the total length of the blood vessel and the area of the image analysis region;

[0034] S24, quantifying blood vessel network features, including branch density, length proportion, and connectivity index;

[0035] S25, blood vessel network feature judgment, if any of the blood vessel network features reaches the corresponding preset threshold, it is determined that the blood vessel network is mature, a blood vesselization completion signal is fed back, and an epidermis layer construction step is triggered.

[0036] Further improvement lies in that in step S3, when the electrical impedance analysis unit monitors the epidermis differentiation state, the following steps are included:

[0037] S31, information acquisition, applying a multi-frequency alternating excitation signal to the epidermis layer in the second culture cavity of the epidermis culture chip layer through the first electrode and the second electrode, the excitation signal frequency range is 10Hz-1MHz; synchronously collecting the voltage signal and the corresponding excitation current signal output by the electrode;

[0038] S32, signal preprocessing, performing filtering processing on the collected voltage signal and current signal, removing environmental noise and interference signal, and retaining effective electrophysiological signal;

[0039] S33, impedance parameter calculation, calculating the epidermis evaluation features including impedance modulus and phase angle based on the preprocessed signal;

[0040] S34, differentiation state analysis, evaluating the epidermis differentiation degree through the epidermis evaluation features, including:

[0041] Monitoring the stability of the low-frequency impedance modulus, when the impedance modulus fluctuation amplitude is less than or equal to the preset fluctuation threshold value within a continuous preset time length, it indicates that the epidermis barrier function tends to be mature;

[0042] Monitoring the change trend of the phase angle with the frequency, when the phase angle change curve slope is less than or equal to the preset slope threshold value within the full frequency range, it indicates that the epidermis cell differentiation state is stable;

[0043] S35, skin differentiation evaluation and judgment, when the stability of the low-frequency impedance modulus value and the change trend of the phase angle both meet the above conditions, it is determined that the epidermis is maturely differentiated, a signal of epidermis construction completion is fed back, and a combined culture step is triggered.

[0044] Further improvement lies in that the medium supply is optimized based on the detection data by the medium flow regulation unit, including the following steps:

[0045] S41, information acquisition, the concentration of nutrient components Md and Me in the dermis medium and epidermis medium supplied by the second chip is collected in real time by an external monitoring device 、 and the current medium supply flow is collected by a flow sensor 、 ; wherein is the current concentration of the dermis medium, is the current concentration of the epidermis medium, Md is the metabolite concentration of the dermis medium, and Me is the metabolite concentration of the epidermis medium;

[0046] S42, signal preprocessing, the collected concentration data and flow data are subjected to sliding average filtering processing to remove transient fluctuation noise and obtain smooth effective concentration values 、 and flow values 、 ;

[0047] S43, deviation calculation, based on the preset target concentration of the dermis medium and the target concentration of the epidermis medium , the concentration deviation is calculated, including:

[0048] the deviation of the dermis medium concentration from the target value ;

[0049] the deviation of the epidermis medium concentration from the target value ;

[0050] S44, flow regulation calculation, the adjusted flow is calculated based on the deviation value by using an adaptive PID control algorithm;

[0051] dermis medium adjusted flow , the formula is , wherein, 、 、 are the proportional, integral, and differential coefficients of the dermis medium, respectively;

[0052] epidermis medium adjusted flow, the formula is , wherein, 、 , respectively are the epidermis culture medium ratio, integral, differential coefficient;

[0053] S45, dynamic adjustment, according to the calculated and , through the dermis culture medium inlet and outlet of the second chip, and the epidermis culture medium inlet and outlet, respectively adjust the culture medium supply rate of the dermis culture medium channel and the epidermis culture medium channel;

[0054] S46, stability judgment and optimization, when and Both are maintained within the preset deviation allowable range, it is judged that the culture environment is stable; if and Any of the deviations exceeds the preset deviation allowable range, trigger alarm and optimize the ratio, integral, differential coefficient of the dermis culture medium or the epidermis culture medium, until the concentration returns to the target range, ensure that the epidermis layer and the dermis layer nutrition supply balance.

[0055] Compared with the prior art, the beneficial effects of the present application are:

[0056] (1) The skin chip of the present application adopts polyimide material to realize the detachable connection of each chip layer, avoiding the potential toxicity and drug adsorption problem of traditional PDMS material; the non-contact design of the electrode reduces the physical and chemical interference to the tissue, and the detachable characteristic facilitates the end-point tissue analysis and repeated use of the chip, solving the problems of difficult disassembly, high cost and poor material compatibility of the existing one-piece chip.

[0057] (2) The skin chip of the present application adopts the design of up-and-down through culture cavity, simulating the physiological environment of real skin tissue, on the one hand, the first chip and the second chip with or without electrode can be used according to the need, improving the flexibility of the skin chip; on the other hand, the electrode does not directly contact with the dermis layer or the epidermis layer, reducing the physical and chemical interference to the skin tissue.

[0058] (3) The present application quantifies the vascular network features in real time through the vascularization evaluation unit, dynamically monitors the vascular maturity combined with the fluorescence image preprocessing and feature extraction algorithm; the resistance impedance analysis unit monitors the epidermis differentiation state at multiple frequencies, realizes the synchronous correlation analysis of vascularization and epidermis function, solving the limitations of single detection mode and lack of dynamic correlation of the existing technology.

[0059] (4) The culture medium flow regulation unit of the present application based on adaptive PID algorithm dynamically optimizes the supply rate by real-time acquisition of nutrient and metabolite concentration, combined with the stability judgment mechanism to accurately maintain the balance of the culture environment, solving the problem of nutrient imbalance caused by traditional static control, and ensuring long-term stable culture of the model.

[0060] In summary, this invention, through innovative hardware materials, multi-dimensional dynamic monitoring, and intelligent control, comprehensively addresses the shortcomings of existing skin chips in terms of physiological simulation, detection dimensions, and environmental controllability, significantly improving the functional realism and experimental reliability of skin models. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the overall structure of Embodiment 1 of the vascularized skin chip proposed in this invention;

[0062] Figure 2 for Figure 1 The diagram shows the exploded structure.

[0063] Figure 3 This is a flowchart of the method for constructing a vascularized skin chip according to Embodiment 2 of the present invention.

[0064] Marked in the image:

[0065] 1. First chip; 11. First electrode;

[0066] 2. First chip connection layer; 21. First culture chamber pore; 22. Dermal culture medium channel;

[0067] 3. Carrier layer;

[0068] 4. Dermal culture connecting layer; 41. Second culture chamber pore; 42. Dermal culture medium inlet channel;

[0069] 5. Dermal culture chip layer; 51. First culture chamber; 52. Dermal culture layer; 53. Dermal culture medium outlet channel;

[0070] 6. Epidermal culture chip layer; 61. Second culture chamber; 62. Epidermal culture medium channel;

[0071] 7. Second chip; 71. Second electrode; 7a. Epidermal culture medium inlet; 7b. Epidermal culture medium outlet; 7c. Dermal culture medium inlet; 7d. Dermal culture medium outlet. Detailed Implementation

[0072] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0073] Example 1

[0074] like Figure 1 and Figure 2As shown, a vascularized skin chip includes a hardware structure and a software system;

[0075] The hardware structure includes, from top to bottom, a second chip (7), an epidermal culture chip layer (6), a dermal culture chip layer (5), and a first chip (1). Each layer is detachably connected by polyimide material. The first chip (1) provides a bottom space for the dermal culture medium, and the second chip (7) is located above the first chip (1) and is used to supply and store the culture medium or supply skin irritants.

[0076] The first chip 1) is provided with a first electrode 11, and the second chip 7) is provided with a second electrode 71).

[0077] A dermal culture connecting layer 4, a carrier layer 3, and a first chip connecting layer 2 are sequentially provided between the dermal culture chip layer 5) and the first chip 1).

[0078] The software system communicates with the first electrode 11), the second electrode 71) and external monitoring equipment, including a data acquisition module, a data analysis module and a control module. The data analysis module has a built-in vascularization assessment unit and an electrical impedance analysis unit, and the control module has a built-in culture medium flow rate adjustment unit.

[0079] It should be noted that the first chip 1 and the second chip 7 mentioned above are not directly used for culturing skin models;

[0080] Specifically, the dermal culture chip layer 5 includes a dermal culture layer 52, in which a first culture chamber 51 is provided, and the dermal culture layer 52 is used to construct the dermal layer;

[0081] The first chip connection layer (2) and the dermal culture connection layer (4) are respectively provided with a first culture cavity hole (21) and a second culture cavity hole (41) corresponding to the first culture cavity (51).

[0082] It should be noted that in the chip where electrodes are present, the first electrode 11 and the second electrode 71 disposed on the first chip 1 and the second chip 7 correspond to each other in the vertical direction with each culture chamber.

[0083] To avoid the chip material affecting the experiment, the skin chip material may not contain polydimethylsiloxane (PDMS), thus avoiding the absorption of drugs by PDMS and improving the authenticity of the experiment.

[0084] Each layer of the aforementioned skin chip is independently designed and can be disassembled. Installation is simple, requiring only reassembly. The chip layers are connected by polyimide material, or are made entirely of polyimide, making assembly easier and providing relatively high sealing without the need for additional seals.

[0085] The skin chip proposed in this application features a detachable design that facilitates sample collection and analysis, and allows for further detection directly on the chip. Secondly, the detachable design allows for the reuse of the skin chip, thereby reducing experimental costs. It also facilitates direct observation of samples under a microscope and even imaging and analysis of separated tissues.

[0086] Gravity perfusion eliminates the need for complex external culture medium pumping systems, reducing operating costs. It allows for the simultaneous culture of multiple skin chips, making standardized operation of skin chips more convenient and facilitating high-throughput testing. Furthermore, the absence of external contact between the culture medium and the environment avoids the possibility of skin chip contamination.

[0087] The interconnected culture chamber better simulates the layered structure and barrier function of real skin, providing bidirectional fluid flow to facilitate the effective exchange of nutrients and metabolic waste, promoting cell growth and metabolism. Simultaneously, this design allows for more realistic studies of drug and compound penetration processes, making it suitable for transdermal drug delivery and skincare product testing. Furthermore, the interconnected design allows for the creation of different microenvironmental conditions at both ends of the chamber, effectively simulating the differences between the internal and external environments of the skin, further enhancing the physiological relevance of the experiments.

[0088] For skin models constructed in the same batch, different tests can be performed simultaneously using skin chips with and without electrodes. Since the other structures of the chips are completely identical, the consistency of experimental conditions can be guaranteed. Secondly, when using chemical irritants or other experimental procedures that may cause electrode corrosion, skin chips without electrodes should be selected. Electrode-equipped chips can be used in skin electrophysiology, drug penetration, and barrier function detection (current and voltage are set according to actual test requirements). In electrode-equipped chips, the first electrode 11 and the second electrode 71 do not directly contact the tissue, avoiding interference and potential damage to the tissue and maintaining a stable culture environment. Furthermore, this helps to obtain clearer and more stable signals, improving test accuracy. In addition, this design reduces electrode contamination and wear, extends electrode lifespan, and reduces maintenance frequency. It facilitates standardized and automated operation, supports high-throughput testing, and improves experimental efficiency and consistency.

[0089] To construct the dermal layer, specifically, the dermal culture chip layer 5 may include a dermal culture layer 52 with a first culture cavity 51 in the middle. The dermal culture layer 52 is used to construct the dermal layer. The first chip connecting layer 2 and the dermal culture connecting layer 4 are both disposed below the dermal culture layer 52 and are used to connect the dermal culture layer 52 and the first chip 1. The first chip connecting layer 2 and the dermal culture connecting layer 4 are respectively provided with a first culture cavity hole 21 and a second culture cavity hole 41 corresponding to the first culture cavity 51. A carrier layer 3 is also disposed between the dermal culture connecting layer 4 and the first chip connecting layer 2. In the skin chip without electrodes, the material of the first chip 1 is glass; in the skin chip with electrodes, the material of the first chip 1 is ITO glass (indium tin oxide glass), which is obtained by etching an ITO (indium tin oxide) conductive coating on the glass. The materials of the first chip connecting layer 2 and the dermal culture connecting layer 4 can be suitable materials, such as polyethylene terephthalate (PET), polyvinyl chloride (PVC), and preferably polyimide (PI). Double-sided adhesive tape is applied to both the upper and lower surfaces of the first chip connecting layer 2 and the dermal culture connecting layer 4 to connect and fix the dermal culture layer 52 and the first chip 1, sealing them to prevent culture medium leakage and effectively avoiding the possibility of skin model contamination. The material of the double-sided adhesive tape is the same as that of the first chip connecting layer 2 and the dermal culture connecting layer 4. The dermal culture layer 52 is made of polymethyl methacrylate (PMMA), which has high optical transparency, UV resistance, and high hardness and compressive strength. The carrier layer 3 is a microporous membrane that covers the first culture chamber pore 21, separating the first culture chamber pore 21 from the first culture chamber 51. The diameter of the carrier layer 3 is larger than the diameter of the first culture chamber pore 21. The carrier layer 3 is fixed between the first chip connecting layer 2 and the dermal culture connecting layer 4 by the double-sided adhesive tape. The carrier layer 3 can be made of a suitable material, such as polycarbonate. The aforementioned carrier layer 3 is used for direct inoculation of skin equivalents to construct the dermis. The lower layer of carrier layer 3 is in direct contact with the dermal culture medium. The pore size of carrier layer 3 is 0.4μm-10μm. The aforementioned carrier layer 3 allows the culture medium to pass through, but the skin equivalent cannot pass through the carrier layer 3. The culture medium does not directly contact the skin equivalent, providing culture conditions that are closer to the physiological environment for the construction of the dermis. Secondly, it also facilitates the removal of the skin model after culture for further analysis.

[0090] To provide nutrients to the culture medium, a dermal culture medium channel 22 can be specifically provided on the first chip connection layer 2. The space defined by the sidewall of the first culture chamber 21, the first chip 1, and the carrier layer 3 is filled with culture medium to provide nutrients to the dermal layer on the carrier layer 3. The dermal culture medium can serve as a conductive solution for the first electrode 11 on the first chip 1. The culture medium channel is used to infuse the first culture chamber 21 with culture medium, ensuring uniform distribution of nutrients and helping to maintain the stability of the culture environment. Avoiding direct contact between the culture medium and the skin equivalent helps to enhance the skin barrier function and support diverse experimental designs.

[0091] In order to ensure that the bottom of the dermis layer is in full contact with the culture medium through the carrier layer 3, the dermis culture connecting layer 4 and the dermis culture layer 52 can be provided with dermis culture medium inlet channel 42 and dermis culture medium outlet channel 53, which are connected to the dermis culture medium channel 22 on the first chip connecting layer 2. By using gravity, the culture medium is more evenly distributed in the first culture chamber pore 21 and can be in full contact with the carrier layer 3.

[0092] To construct a skin model similar to that in vivo, specifically, the thickness of the dermal culture layer 52 can be 3mm-4mm, preferably 3.5mm; the thickness of the aforementioned dermal culture layer 52 is close to the thickness of the dermal layer of human skin, which helps to better reproduce the physiological structure of the skin. The thickness of the first chip connecting layer 2 and the dermal culture connecting layer 4 can be 0.2mm-0.4mm.

[0093] The thickness of the carrier layer 3 is preferably 0.3 mm, and the thickness of the carrier layer 3 can be 0.01 mm to 0.02 mm, preferably 0.017 mm. The above-mentioned dermal culture connecting layer 4 and carrier layer 3 provide appropriate support and separation for the dermal culture layer 52, ensuring effective isolation between the culture medium and the skin equivalent. At the same time, it allows sufficient culture medium to provide nutrition to the cells through the carrier layer 3, maintaining healthy cell growth. The diameter of the first culture chamber 51, the first culture chamber pore 21 and the second culture chamber pore 41 can be 5.0 mm to 6.0 mm, preferably 5.5 mm, which can provide sufficient growth space for the skin equivalent, which is conducive to the construction of the dermal layer. It can also ensure that the culture medium can effectively penetrate, ensuring the stability of the cell culture environment and enhancing the reliability and reproducibility of experimental results.

[0094] To construct a skin model that accurately reflects the structure of real skin, specifically, an epidermal culture chip layer 6 can be disposed on top of the dermal culture chip layer. The epidermal culture chip layer 6 may include: a second culture chamber 61, which is disposed throughout the epidermal culture chip layer 6; and an epidermal culture medium channel 62, symmetrically located on both sides of the second culture chamber 61 and connected to it, for providing epidermal culture medium. The epidermal culture chip layer 6 can be made of a suitable material, such as polyethylene terephthalate (PET), polyvinyl chloride (PVC), and preferably polyimide (PI). By disposing the epidermal culture chip layer 6 on top of the dermal culture chip layer 5 and designing a structure including the second culture chamber 61 and the epidermal culture medium channel 62, the layered structure of skin can be realistically simulated, providing an independent and controllable culture environment for the epidermis and dermis. The connection between the epidermal culture medium channel and the second culture chamber 61 ensures that the epidermal layer receives a uniform nutrient supply while avoiding cross-contact between the epidermal culture medium and the dermal culture medium, better simulating the physiological separation between the two tissue layers. This design optimizes the interaction between the epidermis and dermis, promoting cell growth, differentiation, and functional expression. It enables more precise high-throughput experiments such as skin substitute construction, drug penetration studies, and skin toxicity testing, providing a comprehensive and near-physiological skin simulation platform with wide applications in dermatological research, cosmetic testing, and drug development.

[0095] To correspond to the diameter of the dermis and construct a near-human epidermis, specifically, the thickness of the epidermal culture chip layer 6 can be 0.2mm-0.4mm, preferably 0.3mm, and the diameter of the second culture chamber 61 can be 5mm-6mm, preferably 5.5mm. Double-sided adhesive tape is provided on the upper and lower sides of the epidermal culture chip layer 6 to connect the upper and lower dermal culture layers 52 and the second chip 7. The double-sided adhesive tape can be of a suitable material; in this application, it is made of the same material as the epidermal culture chip layer 6. The epidermal culture chip layer 6 is fixed above the dermal culture layer 52, realistically simulating the structure and function of the skin epidermis, providing suitable space and uniform culture medium supply, supporting the growth and differentiation of epidermal cells. The reasonable size design ensures effective penetration of the culture medium, avoids uneven distribution, optimizes culture conditions, and is suitable for long-term culture and high-throughput experiments.

[0096] Specifically, for storing the culture medium, a second chip 7 can be disposed above the epidermal culture chip layer 6. The second chip 7 includes: a third culture chamber 72, which communicates with the second culture chamber 61 on the epidermal culture chip layer 6; a dermal culture medium inlet 7c and a dermal culture medium outlet 7d, which communicate with the dermal culture medium inlet channel 42 and the dermal culture medium outlet channel 53 on the dermal culture layer, respectively; and an epidermal culture medium inlet 7a and an epidermal culture medium outlet 7b, which communicate with the epidermal culture medium channel 62 on the epidermal culture chip layer 6. The material of the second chip 7 is polymethyl methacrylate (PMMA), which has good light transmittance. In the chip with electrodes, the second electrode 71 on the second chip 7 is a wire disposed in the third culture chamber 72. The length of the wire in the third culture chamber 72 is 0.5mm-1mm. The length of the wire is not limited to the above length, as long as it can contact the epidermal culture medium. The wire passes through the wire hole on the second chip 7. The wire hole can be disposed in an appropriate position, such as on the top of the second chip 7 or on the side of the second chip 7. The diameter of the aforementioned wire hole is 0.8 mm, facilitating wire passage. To maintain the airtightness of the skin chip, AB glue is used to seal the wire hole. The AB glue can be any suitable material, such as silicone sealant or polyurethane sealant. The aforementioned dermal culture medium inlet 7c and dermal culture medium outlet 7d can be positioned appropriately, for example, on the second chip 7. This prevents the dermal culture medium from overflowing during the skin chip's cultivation process. Positioning them on the upper surface of the second chip 7 provides a uniform pressure distribution for the culture medium within the first culture chamber pore 21, ensuring independent nutrient supply and waste removal between the epidermal culture chip layer 6 and the dermal culture layer 52, thus more realistically simulating the physiological functions of the skin. The placement of the epidermal and dermal culture medium inlets and outlets on the upper surface of the second chip 7 allows for the storage of a larger amount of culture medium, providing an appropriate amount for long-term skin chip cultivation. By setting different inlets and outlets for the dermal and epidermal culture media, mutual interference between the two is avoided, allowing each layer to grow under optimal conditions.

[0097] To facilitate the perfusion of the skin equivalent and the perfusion and storage of the culture medium, specifically, the thickness of the second chip 7 can be 4mm-5mm, preferably 4.6mm, and the diameter of the third culture chamber 72 can be 5mm-6mm, preferably 5.5mm. This design allows the chip to accommodate the culture medium and complex fluid systems, and also provides stable structural support for the skin chip.

[0098] To prevent contamination of the skin model within the skin chip, the skin chip may further include a culture chamber plug 8 and a sealing cap 9. The culture chamber plug 8 has a diameter of 5mm-6mm, preferably 5mm. The culture chamber plug 8 is made of a transparent material to facilitate observation of the skin model's growth during the culture process. The culture chamber plug can be made of a suitable, elastic material; in this application, it is made of silicone. The culture chamber plug 8 is used to block the third culture chamber 72, and its diameter is slightly smaller than that of the third culture chamber 72. The thickness of the culture chamber plug 8 is 5.3mm.

[0099] In this embodiment, it should be noted that this application only addresses the shortcomings of existing skin chips, such as difficulty in accurately reproducing skin function and the reliance on a single detection method, and does not involve improvements in other aspects. The working principle of this vascularized skin chip is described below:

[0100] In the specific assembly and application of the skin chip of the present invention, the chips are assembled from bottom to top as follows: a first chip 1, a first chip connecting layer 2, a carrier layer 3, a dermal culture connecting layer 4, a dermal culture layer 52, an epidermal culture chip layer 6, a second chip 7, a culture chamber plug 8, and a sealing cap 9. Double-sided adhesive is provided on the upper and lower surfaces of the first chip connecting layer 2, the dermal culture connecting layer 4, and the epidermal culture chip layer 6. During culture, the chip layers are sealed together, ensuring the integrity and airtightness of the skin chip. After culture, when the skin model needs to be removed entirely or partially for histological analysis, each layer can be disassembled sequentially. For subsequent culture, the cleaned chip layers are reassembled, sterilized, and ready for use. Compared to a one-piece molded chip, the detachable chip in this application is reusable, reducing experimental costs and facilitating the removal of the skin model for further operations.

[0101] Example 2

[0102] Please refer to the following: Figure 3 As shown, based on the vascularized skin chip provided in Embodiment 1 of this application, Embodiment 2 of this application proposes another method for constructing a vascularized skin chip. Embodiment 2 is merely a preferred embodiment of Embodiment 1, and the implementation of Embodiment 2 will not affect the individual implementation of Embodiment 1.

[0103] Specifically, Embodiment 2 of this application provides a method for constructing a vascularized skin chip, based on the vascularized skin chip provided in Embodiment 1, including the following steps:

[0104] S1: Chip assembly and information acquisition. To ensure the sealed connection of each layer of the chip structure and the alignment of functional channels, avoid culture medium leakage, and ensure accurate signal acquisition, specifically, the first chip 1, the first chip connecting layer 2, the carrier layer 3, the dermal culture connecting layer 4, the dermal culture chip layer 5, the epidermal culture chip layer 6, and the second chip 7 are sequentially sealed and connected using polyimide material to form a detachable chip assembly. This allows the first culture chamber 51, the second culture chamber 61, and the third culture chamber 72 to be vertically connected, with each culture medium channel correspondingly connected. To establish a collaborative working mechanism between the software, hardware, and external devices, and to ensure the accuracy of data acquisition, during software system initialization, the data acquisition module establishes an electrical signal transmission link with the first electrode 11 and the second electrode 71 through an interface protocol. Simultaneously, it connects to external monitoring devices (such as a fluorescence microscope and a concentration sensor) to calibrate the signal acquisition accuracy and device parameters, laying the foundation for subsequent culture monitoring.

[0105] S2: Dermal Layer Construction and Angiogenesis Induction. To construct a dermal layer with a functional vascular network in vitro and simulate the nutrient supply structure of dermal tissue in vivo, specifically, a dermal cell suspension is inoculated into the first culture chamber 51 and cultured in a constant temperature environment until the cells adhere and fuse to form a dense dermal cell layer, providing structural support for vascularization. Subsequently, a vascular endothelial cell suspension is inoculated onto the surface of the dermal cell layer, and dermal culture medium containing angiogenesis-inducing factors is continuously supplied through the dermal culture medium inlet 7c and the dermal culture medium channel 22. Angiogenesis-inducing factors include, but are not limited to, vascular endothelial growth factor and fibroblast growth factor. To monitor the formation status of the vascular network in real time and ensure that the maturity of vascularization meets the experimental requirements, the vascular network characteristics are analyzed in real time through the vascularization assessment unit, quantifying branch density, length ratio, and connectivity index. When the vascular network characteristics reach a preset threshold, the vascular network is judged to be mature, ensuring the functional integrity of the dermal layer.

[0106] S3: Epidermal layer construction. In order to induce epidermal cell differentiation to form an epidermal layer with barrier function and simulate the outer protective structure of the skin, specifically, after the dermal vascular network is stabilized, epidermal cell suspension is inoculated into the second culture chamber 61, and culture medium containing differentiation inducing factors is continuously supplied through the epidermal culture medium channel 62 to promote the stratified differentiation of epidermal cells; and the epidermal differentiation status is monitored through the electrical impedance analysis unit to ensure that the epidermal barrier function meets the target.

[0107] S4: Co-culture and dynamic regulation. To maintain the long-term stable culture of the skin model and support research on its response to external stimuli, specifically, the second chip 7 is used to periodically replace the dermal and epidermal culture media with fresh ones, remove metabolic waste, and replenish nutrients. To achieve precise regulation of the culture environment and ensure that the nutrient supply matches the metabolic needs of the cells, the culture medium flow rate adjustment unit optimizes the culture supply based on detection data and dynamically adjusts the supply rate of the dermal culture medium channel 22 and the epidermal culture medium channel 62 based on concentration deviations, so that the concentration of nutrients is maintained within the target range, avoiding the impact of nutrient excess or deficiency on the function of the skin model.

[0108] To accurately monitor the formation and maturity of the dermal vascular network and provide a reliable initiation signal for epidermal construction, specifically, in step S2, the vascular network characteristics are analyzed in real time through a vascularization assessment unit, as follows:

[0109] S21, Information Acquisition: In order to obtain complete morphological data of the vascular network and ensure that the analysis covers the entire growth area, specifically, the fluorescence images of blood vessels in the first culture chamber 51 that are specifically fluorescently labeled with vascular endothelial cells (such as CD31 fluorescence staining) are acquired by external monitoring equipment (such as a fluorescence microscope). The vascular fluorescence images completely cover the growth area of ​​the dermal vascular network, providing raw data support for subsequent analysis.

[0110] S22, Image preprocessing: To eliminate image noise interference, enhance the distinction between blood vessels and background, and improve the accuracy of blood vessel region recognition, specifically, the acquired blood vessel fluorescence images are processed sequentially as follows:

[0111] Gaussian filtering noise reduction, using a Gaussian kernel function. A smoothed vascular fluorescence image, where (x,y) are the image pixel coordinates, x and y represent the horizontal and vertical coordinates in the vascular fluorescence image, respectively, and σ is a preset filtering coefficient;

[0112] Adaptive histogram equalization enhances the contrast between the vascular region and the background;

[0113] Threshold segmentation based on the OTSU algorithm is used to obtain a binary image of the vascular network region, which clearly separates the vascular structure from the background.

[0114] S23, Feature Extraction: To accurately extract the topological parameters of the vascular network and quantify vascular morphological features, specifically, the following steps are performed on the binarized vascular network region image:

[0115] The morphological thinning algorithm processes the binary vascular network region to obtain a single-pixel-width vascular skeleton while preserving the vascular topology.

[0116] Identify blood vessel branch points and count the total number of branch points Nb. A branch point is a pixel in the skeleton that has ≥3 connected pixels.

[0117] The blood vessel segments are segmented, and the total number of blood vessel segments Nt and the number of connected blood vessel segments Nc are counted. Connected blood vessel segments are those that participate in the formation of continuous pathways. The total length of blood vessels Lv and the area of ​​the image analysis region A are calculated based on the image resolution, providing basic parameters for quantitative analysis.

[0118] S24. In order to objectively characterize the richness, coverage and integrity of vascular networks through quantitative parameters, specifically, quantify vascular network characteristics, including branch density, length ratio and connectivity index, and convert vascular morphology into comparable numerical indicators.

[0119] The logic for obtaining vascular network features is as follows:

[0120] The branch density Bd is calculated by the total number of branch points and the area of ​​the image analysis region. The formula is: Bd=Nb / A. The branch density represents the abundance of vascular branches.

[0121] The length ratio is calculated by comparing the total length of the blood vessel with the area of ​​the image analysis region. The formula is Lr=Lv / A. The length ratio represents the spatial coverage of the blood vessel.

[0122] The connectivity index is calculated by combining the total number of vessel segments with the number of connected vessel segments. The formula is Cz=Nc / Nt. The connectivity index characterizes the integrity of the vascular network.

[0123] S25, Vascular network feature judgment: In order to accurately determine whether the vascular network is mature and ensure that the timing of epidermal layer construction is reasonable, specifically, if any feature of the vascular network reaches its corresponding preset threshold, the vascular network is determined to be mature, a vascularization completion signal is fed back, and the epidermal layer construction step is triggered to ensure the temporality and reliability of skin model construction.

[0124] To objectively assess the epidermal differentiation status and barrier function maturity through electrophysiological signals, and to provide a precise basis for initiating co-culture, specifically, in step S3, when the electrical impedance analysis unit monitors the epidermal differentiation status, the following steps are included:

[0125] S31, Information Acquisition: To comprehensively capture the response signals of the epidermal layer under different electrophysiological characteristics and ensure the integrity of the analysis, specifically, multi-frequency AC excitation signals are applied to the epidermal layer in the second culture chamber 61 of the epidermal culture chip layer 6 through the first electrode 11 and the second electrode 71. The frequency range of the excitation signals is 10Hz-1MHz, which can cover the response range of the resistance and capacitance characteristics of the epidermal layer; the voltage signal U and the corresponding excitation current signal I output by the electrodes are acquired simultaneously to provide raw data for impedance calculation.

[0126] S32, Signal preprocessing: In order to eliminate the influence of environmental electromagnetic interference, baseline drift and other noise on the signal and ensure the accuracy of subsequent parameter calculations, specifically, the acquired voltage signal U and current signal I are filtered (such as low-pass filtering and notch filtering) to remove environmental noise and interference signals, retain the effective signal that can reflect the electrophysiological characteristics of the epidermis, and improve the signal-to-noise ratio.

[0127] S33, Impedance parameter calculation: In order to characterize the electrophysiological properties and barrier function of the epidermis through quantitative parameters, specifically, epidermal assessment characteristics are calculated based on the preprocessed signal, including impedance magnitude and phase angle, as follows:

[0128] The impedance value Z of the epidermis is calculated based on the voltage signal U and the corresponding excitation current signal I. The formula is: Z=U / I. The impedance value is used to reflect the electrophysiological characteristics of the epidermis.

[0129] Identify the real part Re(Z) and the imaginary part Im(Z) of the impedance, where Re(Z) characterizes the skin layer resistance and Im(Z) characterizes the skin layer capacitance.

[0130] Calculate the impedance magnitude and phase angle based on the real and imaginary parts of the impedance, respectively; the formula for calculating the impedance magnitude is as follows: The impedance modulus is used to reflect the overall barrier function strength of the epidermis; the formula for calculating the phase angle is... The phase angle is used to reflect the proportion of the capacitive component in the impedance of the epidermis.

[0131] S34, Differentiation Status Analysis: To determine the maturity of epidermal differentiation through parameter variation patterns, specifically, the degree of epidermal differentiation is assessed using epidermal evaluation characteristics, including:

[0132] Monitor the stability of the impedance modulus in the low-frequency band. When the fluctuation amplitude of the impedance modulus is less than or equal to the preset fluctuation threshold within a preset duration, it indicates that the epidermal barrier function is maturing.

[0133] Monitor the trend of phase angle change with frequency. When the slope of the phase angle change curve in the whole frequency range is less than or equal to the preset slope threshold, it indicates that the epidermal cell differentiation state is stable and the cell membrane and cell layer structure tends to be perfect.

[0134] S35, Epidermal Differentiation Assessment: To accurately determine whether the epidermal layer has matured and to ensure that the timing of co-culture initiation is appropriate, specifically, when the stability of the low-frequency impedance modulus and the trend of phase angle change both meet the above conditions, the epidermal layer is determined to be mature, a signal indicating completion of epidermal construction is fed back, and the co-culture step is triggered, ensuring the functional integrity of the skin model and the accuracy of the experimental timing.

[0135] To achieve dynamic and precise control of the culture medium supply, ensure nutritional balance and metabolic stability in the epidermis and dermis, and provide a suitable environment for the long-term culture of the skin model, the culture medium supply is optimized based on detection data through a culture medium flow regulation unit, including the following steps:

[0136] S41, Information Acquisition: To comprehensively understand the real-time status of the culture medium and provide data support for regulatory decisions, specifically, the concentrations of nutrients in the dermal and epidermal culture media supplied by the second chip 7 are collected in real time through external monitoring devices (such as concentration sensors). , (Reflecting nutrient supply adequacy) and metabolite concentrations Md and Me (reflecting cell metabolic state), while simultaneously collecting the current culture medium supply flow rate via a flow sensor. , (as an adjustment benchmark); where This is the current concentration of the dermal culture medium. Md represents the current concentration of the epidermal culture medium, Md represents the concentration of metabolites in the dermal culture medium, and Me represents the concentration of metabolites in the epidermal culture medium, ensuring that the data covers key indicators of nutrient supply and metabolic feedback.

[0137] S42, Signal preprocessing: To eliminate the interference of transient fluctuation noise on the data and ensure the stability and reliability of the analysis data, specifically, moving average filtering is performed on the collected concentration and flow data to remove transient fluctuation noise and obtain smoothed effective concentration values. , and flow value , To avoid misjudgment of adjustment due to sudden noise;

[0138] S43, Deviation Calculation: To quantify the difference between the current culture medium concentration and the target concentration, and to clarify the direction and magnitude of adjustment, specifically, based on the preset target concentration of the dermal culture medium. and the target concentration of epidermal culture medium Calculate the concentration deviation, including:

[0139] Deviation between dermal culture medium concentration and target value ;

[0140] Deviation between epidermal culture medium concentration and target value ;

[0141] Through deviation value , It directly reflects the state of excess or deficiency in nutrient supply;

[0142] S44, Flow regulation calculation: In order to achieve precise dynamic flow regulation based on deviation and adapt to changes in cell metabolic needs, specifically, an adaptive PID control algorithm is used to calculate the regulated flow based on the deviation value.

[0143] Flow rate adjustment of dermal culture medium The formula is ,in, , , These are the proportion of dermal culture medium, integral, and differential coefficient, respectively;

[0144] The flow rate of the epidermal culture medium is adjusted using the following formula: ,in, , , These are the proportion of the epidermal culture medium, the integral, and the differential coefficient, respectively.

[0145] S45, dynamic adjustment, in order to translate the calculated optimized flow rate into the actual supply rate and ensure that the adjustment effect is implemented, specifically, based on the calculation... and The dermal culture medium inlet 7c, outlet 7d and epidermal culture medium inlet 7a, outlet 7b of the second chip 7 are used to adjust the culture medium supply rate of dermal culture medium channel 22 and epidermal culture medium channel 62 respectively, so that the nutrient supply matches the cell demand in real time.

[0146] S46, Stability Assessment and Optimization: To continuously monitor the stability of the culture environment, correct deviations in a timely manner, and optimize adjustment parameters, specifically, when... and When all deviations remain within the preset allowable range, the culture environment is considered stable; if and If any deviation exceeds the preset allowable deviation range, an alarm will be triggered and the proportion, integral, and differential coefficients of the dermal or epidermal culture medium will be optimized until the concentration returns to the target range, ensuring a balanced nutrient supply to the epidermis and dermis, and improving the functional stability and experimental reliability of the skin model.

[0147] Following step S4, step S5 is also included: endpoint analysis and data summarization, which includes the following steps:

[0148] S51, Skin Model Acquisition and Histological Characterization:

[0149] To visually verify the structural integrity and functional cell distribution of the skin model, specifically:

[0150] After the culture cycle is completed, taking advantage of the detachable characteristics of the polyimide sealing connection between the chip layers, the first chip 1, the epidermal culture chip layer 6, the dermal culture chip layer 5 and other components are separated in sequence, and the skin model in the second culture chamber 61 (epidermal-dermal composite structure) and the first culture chamber 51 (vascularized dermis) is completely extracted.

[0151] HE staining was used to observe the tissue layering and vascular morphology of the epidermis (stratum corneum, stratum granulosum, stratum spinosum, and stratum basale) and dermis; immunofluorescence staining (such as keratin K10 labeling of epidermal differentiated cells and CD31 labeling of vascular endothelial cells) was used to locate the spatial distribution of functional cell populations, providing a visual basis for structural effectiveness.

[0152] S52, the entire process data is aggregated into dynamic monitoring data of the integrated cultivation process, and a multi-dimensional analysis dataset is constructed, specifically:

[0153] The time-varying curves of vascular network features in step S2 are summarized; the dynamic data of epidermal assessment features in step S3, and the concentration deviations of dermal and epidermal culture media, PID adjustment parameters, and flow control records in step S4 are stored in a preset data storage module; the data of each module are linked by timestamps to form a full-process data matrix of angiogenesis → epidermal differentiation → environmental regulation, covering the core functional dimensions of model construction.

[0154] S53, Data statistics and correlation analysis are used to uncover patterns in parameter relationships and verify the stability of the model's physiological function. Specifically:

[0155] The data acquisition module calls statistical analysis algorithms (such as linear regression and Pearson correlation analysis): Single parameter trend fitting: For parameters such as Bd (vascular branch density) and |Z| (epidermal impedance), it fits the curve of their change with culture time, generates parameter trend graphs, and intuitively displays the dynamic evolution law of functional indicators.

[0156] Cross-module correlation analysis: Calculation of vascularization parameters Bd and Lr and epidermal function parameter |Z|. The correlation coefficient was used to clarify the functional synergistic relationship between the vascular network and the epidermal layer, providing quantitative support for the verification of the physiological correlation of the model.

[0157] S54, functional evaluation and chip reuse, to comprehensively verify the effectiveness of the model and reduce experimental costs, specifically:

[0158] (1) Functional assessment

[0159] By integrating histological staining images, parametric trend plots, and correlation conclusions, a functional assessment report of the vascularized skin model was generated, validating the model's physiological relevance from the following dimensions:

[0160] Vascular network integrity: Vascular function is assessed through branch density, length ratio, and connectivity indicators;

[0161] Epidermal barrier function: Epidermal differentiation and barrier maturity are assessed by impedance modulus and phase angle;

[0162] (2) Chip reuse

[0163] The disassembled chip layers (such as the first chip 1, the epidermal culture chip layer 6, and the dermal culture connecting layer 4) are subjected to a cleaning and sterilization process:

[0164] Ultrasonic cleaning removes residual culture medium and cell debris;

[0165] Ultraviolet sterilization inactivates residual microorganisms and restores the biocompatibility of the chip surface;

[0166] The processed chips can be reassembled for experiments, reducing material waste costs.

[0167] The above embodiments are only used to illustrate the technical methods of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical methods of the present invention without departing from the spirit and scope of the technical methods of the present invention.

Claims

1. A vascularized skin chip, characterized in that, This includes both hardware architecture and software systems; The hardware structure includes a second chip (7), an epidermal culture chip layer (6), a dermal culture chip layer (5), and a first chip (1) arranged sequentially from top to bottom. Each layer is detachably connected by polyimide material. The first chip (1) is used to provide a bottom space for the dermal culture medium, and the second chip (7) is arranged above the first chip (1) to supply and store the culture medium or supply skin irritants. The first chip (1) is provided with a first electrode (11), and the second chip (7) is provided with a second electrode (71). Between the dermal culture chip layer (5) and the first chip (1), there is a dermal culture connecting layer (4), a carrier layer (3), and a first chip connecting layer (2) in sequence; the dermal culture chip layer (5) includes a dermal culture layer (52), and a first culture cavity (51) is provided in the middle of the dermal culture layer (52), which is used to construct the dermal layer; the first chip connecting layer (2) and the dermal culture connecting layer (4) are respectively provided with a first culture cavity hole (21) and a second culture cavity hole (41) corresponding to the first culture cavity (51); The software system communicates with the first electrode (11), the second electrode (71) and external monitoring equipment, and includes a data acquisition module, a data analysis module and a control module. The data analysis module has a built-in vascularization assessment unit and an electrical impedance analysis unit, and the control module has a built-in culture medium flow rate adjustment unit. Specifically, the vascular network characteristics are analyzed in real time through a vascularization assessment unit, as follows: S21, Information Acquisition: Acquire vascular fluorescence images labeled with vascular endothelial cell-specific fluorescence in the first culture chamber (51) through external monitoring equipment, wherein the vascular fluorescence images cover the complete growth area of ​​the dermal vascular network; S22, Image preprocessing, performing the following steps sequentially on the acquired vascular fluorescence images: Gaussian filtering was used for noise reduction, and the blood vessel fluorescence image was smoothed using a Gaussian kernel function. Adaptive histogram equalization enhances the contrast between the vascular region and the background; Threshold segmentation based on the OTSU algorithm is used to obtain a binary image of the vascular network region; S23, Feature extraction, performed on the binarized vascular network region image: The morphological thinning algorithm processes the binary vascular network region to obtain a single-pixel-width vascular skeleton while preserving the vascular topology. Identify blood vessel branch points and count the total number of branch points. The branch points are pixels in the skeleton that have three or more connected pixels. The blood vessel segments are segmented, and the total number of blood vessel segments and the number of connected blood vessel segments are counted. The connected blood vessel segments are those that participate in the formation of a continuous pathway. The total length of the blood vessels and the area of ​​the image analysis region are calculated based on the image resolution. S24 quantifies vascular network characteristics, including branch density, length ratio, and connectivity index; S25, vascular network feature judgment: if any feature in the vascular network reaches its corresponding preset threshold, the vascular network is judged to be mature, a vascularization completion signal is fed back, and the epidermal layer construction step is triggered.

2. The vascularized skin chip according to claim 1, characterized in that, The carrier layer (3) is a microporous membrane, which covers the first culture chamber pore (21) and is used to separate the first culture chamber pore (21) from the first culture chamber (51). The first chip connection layer (2) is also provided with a pair of dermal culture medium channels (22) symmetrical about the first culture chamber pore (21), and each dermal culture medium channel (22) is connected to the first culture chamber pore (21).

3. The vascularized skin chip according to claim 1, characterized in that, The dermal culture connecting layer (4) is also provided with a pair of dermal culture medium inlet channels (42) symmetrical about the second culture chamber pore (41). The dermal culture layer (52) is also provided with a pair of dermal culture medium outlet channels (53) symmetrical about the first culture chamber (51), and each of the dermal culture medium inlet channel (42) and dermal culture medium outlet channel (53) is connected to the dermal culture medium channel (22) on the first chip connection layer (2).

4. The vascularized skin chip according to claim 1, characterized in that, The epidermal culture chip layer (6) includes a second culture chamber (61) that runs through it, and four epidermal culture medium channels (62) arrayed outside the second culture chamber (61). The epidermal culture medium channels (62) are connected to the second culture chamber (61) and are used to provide epidermal culture medium.

5. A vascularized skin chip according to claim 3, characterized in that, The second chip (7) includes a third culture chamber (72) that is disposed through the third culture chamber (72) and an array of epidermal culture medium inlet (7a), epidermal culture medium outlet (7b), dermal culture medium inlet (7c) and dermal culture medium outlet (7d) located outside the third culture chamber (72). The third culture chamber (72) is connected to the second culture chamber (61) on the epidermal culture chip layer (6); The dermal culture medium inlet (7c) and dermal culture medium outlet (7d) are respectively connected to the dermal culture medium outlet channel (53) and the dermal culture medium inlet channel (42); and are connected to the epidermal culture medium channel (62) on the epidermal culture chip layer (6).

6. A method for constructing a vascularized skin chip, based on the vascularized skin chip according to any one of claims 1-5, characterized in that, Includes the following steps: S1: Chip assembly and information acquisition: The first chip (1), the first chip connection layer (2), the carrier layer (3), the dermal culture connection layer (4), the dermal culture chip layer (5), the epidermal culture chip layer (6), and the second chip (7) are sequentially sealed and connected with polyimide material to form a detachable chip assembly; the software system is initialized, the data acquisition module establishes communication with the first electrode (11), the second electrode (71), and the external monitoring equipment, and calibrates the signal acquisition accuracy and equipment parameters; S2: Dermal layer construction and vascularization induction. Dermal cell suspension is inoculated into the first culture chamber (51). After the dense cell layer is formed, vascular endothelial cell suspension is inoculated. Culture medium containing vascularization induction factor is continuously supplied through the dermal culture medium channel (22). The vascular network characteristics are analyzed in real time through the vascularization assessment unit. S3: Epidermal layer construction, epidermal cell suspension is inoculated into the second culture chamber (61), culture medium containing differentiation inducing factors is continuously supplied through the epidermal culture medium channel (62), and the epidermal differentiation status is monitored through the electrical impedance analysis unit; S4: Co-culture and dynamic regulation, fresh culture medium or skin irritants are supplied periodically through the second chip (7), and the culture medium supply is optimized based on the detection data through the culture medium flow regulation unit.

7. The method for constructing a vascularized skin chip according to claim 6, characterized in that, In step S3, when the electrical impedance analysis unit monitors the epidermal differentiation state, it includes the following steps: S31, Information acquisition: Apply a multi-frequency AC excitation signal to the epidermal layer in the second culture chamber (61) of the epidermal culture chip layer (6) through the first electrode (11) and the second electrode (71), wherein the frequency range of the excitation signal is 10Hz-1MHz; and synchronously acquire the voltage signal and the corresponding excitation current signal output by the electrode. S32, Signal preprocessing, performs filtering on the acquired voltage and current signals to remove environmental noise and interference signals, and retains effective electrophysiological signals; S33, Impedance parameter calculation, calculates skin evaluation characteristics based on the preprocessed signal, including impedance magnitude and phase angle; S34, Differentiation status analysis, assesses the degree of epidermal differentiation through epidermal assessment characteristics, including: Monitor the stability of the impedance modulus in the low-frequency band. When the fluctuation amplitude of the impedance modulus is less than or equal to the preset fluctuation threshold within a preset duration, it indicates that the epidermal barrier function is maturing. Monitor the trend of phase angle change with frequency. When the slope of the phase angle change curve in the whole frequency range is less than or equal to the preset slope threshold, it indicates that the epidermal cell differentiation state is stable. S35, Epidermal Differentiation Assessment: When the stability of the low-frequency impedance modulus and the trend of phase angle change both meet the above conditions, the epidermal layer is determined to be mature, a signal indicating completion of epidermal construction is fed back, and the co-culture step is triggered.

8. The method for constructing a vascularized skin chip according to claim 7, characterized in that, Optimizing the culture medium supply based on detection data using a culture medium flow control unit includes the following steps: S41, Information Acquisition: Real-time acquisition of nutrient concentrations in the dermal and epidermal culture media supplied by the second chip (7) via external monitoring equipment. , The concentrations of metabolites Md and Me are also collected simultaneously via a flow sensor, which monitors the current culture medium supply flow rate. , ;in This is the current concentration of the dermal culture medium. Md represents the current concentration of the epidermal culture medium, Md represents the concentration of metabolites in the dermal culture medium, and Me represents the concentration of metabolites in the epidermal culture medium. S42, Signal preprocessing: Perform moving average filtering on the acquired concentration and flow data to remove instantaneous fluctuation noise and obtain smoothed effective concentration values. , and flow value , ; S43, Deviation calculation, based on the preset target concentration of dermal culture medium. and the target concentration of epidermal culture medium Calculate the concentration deviation, including: Deviation between dermal culture medium concentration and target value ; Deviation between epidermal culture medium concentration and target value ; S44, Flow regulation calculation: The adjusted flow rate is calculated based on the deviation value using an adaptive PID control algorithm. Flow rate adjustment of dermal culture medium The formula is ,in, , , These are the proportion of dermal culture medium, integral, and differential coefficient, respectively. The flow rate of the epidermal culture medium is adjusted using the following formula: ,in, , , These are the proportion of the epidermal culture medium, the integral, and the differential coefficient, respectively. S45, dynamic adjustment, based on calculations. and The culture medium supply rates of the dermal culture medium channel (22) and the epidermal culture medium channel (62) are adjusted respectively through the dermal culture medium inlet (7c), outlet (7d) and epidermal culture medium inlet (7a) and outlet (7b) of the second chip (7); S46, Stability assessment and optimization, when and When all deviations remain within the preset allowable range, the culture environment is considered stable; if and If any deviation exceeds the preset allowable deviation range, an alarm will be triggered and the proportion, integral, and derivative coefficients of the dermal culture medium or epidermal culture medium will be optimized until the concentration returns to the target range.

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