Skin chip, skin model and application thereof

Abstract

The invention discloses a skin chip, a skin model and application of the skin chip and the skin model. The material of the skin chip does not contain polydimethylsiloxane; the skin chip comprises a first chip and a second chip, wherein the first chip is used for providing a bottom accommodating space for a dermal culture medium; the dermal culture chip layer and the epidermal culture chip layer are arranged at the top end of the first chip, and the second chip is used for supplying and storing a culture medium or supplying skin irritants; wherein the skin chip adopts a gravity perfusion mode; the skin chip is detachable, and all layers of the skin chip are connected through a polyimide material or are made of the polyimide material; a culture cavity between the dermal culture chip layer and the epidermal culture chip layer is through up and down; the first chip and the second chip are not provided with electrodes, or the first chip and the second chip are respectively provided with a first electrode and a second electrode. According to the skin chip provided by the invention, the experiment flexibility is enhanced, and the cost is reduced; and a real culture environment can be simulated.

Description

Technical Field

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

[0002] Ordinary skin models are composed of the dermis and epidermis. The skin model mainly simulates the skin's barrier function, mechanical strength and response to external stimuli.

[0003] At present, skin chips cannot restore and replicate the three-dimensional environment of human skin very well, and cannot truly restore the skin's real barrier function, mechanical strength and response to external stimuli. Multiple methods can be used to detect skin barrier function from multiple aspects, such as dye penetration test, skin permeability test, skin electrical impedance test, etc. At present, skin chips can only meet one test method, and it is impossible to realize multiple skin chips and multiple detection methods together to ensure the uniformity of experimental conditions. Moreover, the skin chip is integrally formed and cannot be disassembled, making the endpoint analysis of in vitro tissue more difficult. In addition, the adverse effects of the material of the skin chip (for example: polydimethylsiloxane) on the construction of the skin model are not taken into account.

[0004] Therefore, how to provide a skin chip with high biocompatibility and flexibility and convenient for endpoint analysis of skin models has become a technical problem that needs to be solved in the field. Summary of the invention

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

[0006] According to the first aspect of the present application, a skin chip is proposed, the material of which does not contain polydimethylsiloxane; the skin chip comprises: a first chip: the first chip is used to provide a bottom accommodation space for a dermal culture medium; and a dermal culture chip layer and an epidermal culture chip layer are arranged on the top of the first chip, and a second chip, the second chip is used to supply and store culture medium or supply skin irritants; wherein the skin chip adopts gravity perfusion to provide culture medium for the skin model; the skin chip is detachable, and the first chip, the dermal culture chip layer, the epidermal culture chip layer and the second chip are connected by polyimide material or are made of polyimide material; the culture cavity between the dermal culture chip layer and the epidermal culture chip layer is through-connected from top to bottom; no electrodes are arranged on the first chip and the second chip, or a first electrode and a second electrode are arranged on the first chip and the second chip respectively, and the first electrode and the second electrode are not in direct contact with the skin model cultured in the dermal culture chip layer and the epidermal culture chip layer.

[0007] Specifically, the dermis culture chip layer includes a dermis culture layer with a first culture cavity in the middle, and the dermis culture layer is used to construct the dermis layer; the first chip connection layer and the dermis culture connection layer are both arranged below the dermis culture layer, for connecting the dermis culture layer and the first chip, and the first culture cavity hole and the second culture cavity hole corresponding to the first culture cavity are respectively arranged in the middle of the first chip connection layer and the dermis culture connection layer; wherein a carrier layer is also arranged between the dermis culture connection layer and the first chip connection layer.

[0008] Specifically, the carrier layer is a microporous membrane, and the carrier layer covers the first culture cavity hole and is used to separate the first culture cavity hole from the first culture cavity.

[0009] Specifically, a dermal culture medium channel is further provided on the first chip connection layer. The dermal culture medium channel is symmetrically arranged on both sides of the first culture cavity hole and is communicated with the first culture cavity hole.

[0010] Specifically, a dermal culture medium inlet channel and a dermal culture medium outlet channel are further provided on the dermal culture connection layer and the dermal culture layer, and are communicated with the dermal culture medium channel on the first chip connection layer.

[0011] Specifically, the thickness of the dermis culture layer is 3mm-4mm; the thickness of the first chip connection layer and the dermis culture connection layer is 0.2mm-0.4mm, the thickness of the carrier layer is 0.01mm-0.02mm, and the diameters of the first culture cavity, the first culture cavity hole and the second culture cavity hole are 5.0mm-6.0mm.

[0012] Specifically, the epidermal culture chip layer is arranged on the top of the dermal culture chip layer, and the epidermal culture chip layer includes: a second culture cavity, which is arranged to penetrate the epidermal culture chip layer; and an epidermal culture medium channel, which is symmetrically arranged on both sides of the second culture cavity and connected to the second culture cavity for providing epidermal culture medium.

[0013] Specifically, the thickness of the epidermal culture chip layer is 0.2 mm-0.4 mm, and the diameter of the second culture cavity is 5 mm-6 mm.

[0014] Specifically, the second chip is arranged above the epidermal culture chip layer, and the second chip includes: a third culture cavity, which is communicated with the second culture cavity on the epidermal culture chip layer; a dermal culture medium inlet and a dermal culture medium outlet, which are respectively communicated with the dermal culture medium inlet channel and the dermal culture medium outlet channel on the dermal culture layer; an epidermal culture medium inlet and an epidermal culture medium outlet, which are communicated with the epidermal culture medium channel on the epidermal culture chip layer.

[0015] Specifically, the thickness of the second chip is 4 mm-5 mm, and the diameter of the third culture chamber is 5 mm-6 mm.

[0016] Specifically, the skin chip further comprises a culture cavity plug and a sealing cover, and the diameter of the culture cavity plug is 5mm-6mm.

[0017] According to a second aspect of the present application, a skin model is proposed. The skin model is obtained by culturing a skin equivalent in a skin chip. The skin chip is the skin chip described above.

[0018] According to the third aspect of the present application, an application of a skin model is proposed, which is the application of the skin model in vitro for non-therapeutic scientific research, drug testing, drug screening, cosmetic testing, toxicity assessment, and disease model, and the skin model is the above-mentioned skin model.

[0019] The skin chip proposed in the present application does not use polydimethylsiloxane, thus avoiding the problem of low biocompatibility of the skin chip and effectively avoiding the potential toxic effects of polydimethylsiloxane on cells; the detachable skin chip design makes the skin chip convenient for endpoint analysis, making the skin chip reusable and reducing experimental costs; the culture chamber design that runs through from top to bottom simulates the physiological environment of real skin tissue; in addition, the first chip and the second chip with or without electrodes can be used as needed, thereby improving the flexibility of the use of the skin chip; on the other hand, the electrodes do not directly contact the dermis or epidermis, thereby reducing physical and chemical interference with the skin tissue.

[0020] Other features and advantages of the present application will be described in detail in the subsequent detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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 skin chip according to a preferred embodiment of the present application.

[0022] Figure 2 Schematic top view of a skin chip according to a preferred embodiment of the present application.

[0023] Figure 3 It is a three-dimensional schematic diagram of a skin chip according to a preferred embodiment of the present application.

[0024] Figure 4 This is a schematic diagram of a skin chip culture device according to a preferred embodiment of the present application.

[0025] Figure 5This is a schematic diagram of drug delivery on a skin chip according to a preferred embodiment of the present application.

[0026] Figure 6 This is a flow chart of culturing a skin model on a skin chip according to a preferred embodiment of the present application.

[0027] Figure 7 This is a morphological diagram of the skin model cultured in a skin chip according to a preferred embodiment of the present application.

[0028] Figure 8 A diagram of cell activity for cosmetic testing on a skin model cultured in a skin chip according to a preferred embodiment of the present application. 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] A skin chip is a miniaturized, integrated skin model system suitable for rapid screening and testing of large numbers of samples. The most important purpose of constructing a skin model in vitro is to be consistent with the morphology and microenvironment of the skin in situ, so as to truly restore representative skin functions in vitro. Therefore, the chip design needs to meet the construction requirements of a multi-layer skin structure, especially the differentiation of the dermis and epidermis, the growth status and interaction of fibroblasts and keratinocytes. Secondly, in actual operation, after the construction is completed, the skin model must be taken out to facilitate other analyses. At present, chips are usually made in one piece by plasma bonding and cannot be disassembled.

[0031] like Figure 1 and Figure 3 As shown, according to the first aspect of the present application, a skin chip is proposed, the material of which does not contain polydimethylsiloxane; the skin chip comprises: a first chip 1: the first chip 1 is used to provide a bottom accommodation space for the dermal culture medium; and a dermal culture chip layer and an epidermal culture chip layer 6 arranged on the top of the first chip 1, and a second chip 7, the second chip is used to supply and store culture medium or supply skin irritants; wherein the skin chip adopts gravity perfusion to provide culture medium for the skin model; the skin chip is detachable, and the first chip 1, the dermal culture chip layer, the epidermal culture chip layer 6 and the second chip 7 are connected by polyimide material or are made of polyimide material; the culture cavity between the dermal culture chip layer and the epidermal culture chip layer 6 is through-connected from top to bottom; no electrodes are arranged on the first chip 1 and the second chip 7, or a first electrode i and a second electrode ii are respectively arranged on the first chip 1 and the second chip 7, and the first electrode i and the second electrode ii are not in direct contact with the skin model cultured in the dermal culture chip layer and the epidermal culture chip layer 6.

[0032] The skin chip comprises a first chip 1, a dermis culture chip layer, an epidermis culture chip layer 6 and a second chip 7 from bottom to top, and the first chip 1 and the second chip 7 are not directly used for culturing a skin model.

[0033] In the chip where the electrodes exist, the first electrode i and the second electrode ii provided on the first chip 1 and the second chip 7 correspond to the culture chamber in the vertical direction.

[0034] In order to avoid the influence of the chip material on the experiment, the material of the above-mentioned skin chip may not contain polydimethylsiloxane (PDMS), so as to avoid the absorption of drugs by polydimethylsiloxane and improve the authenticity of the experiment.

[0035] Each layer of the skin chip is independently arranged and can be disassembled, and can be installed when in use. The chip layers of the skin chip are connected by polyimide material or are made of polyimide material, making the skin chip assembly more convenient and relatively high in sealing, without the need for additional sealing parts.

[0036] The skin chip proposed in this application has a detachable design that facilitates sample collection and analysis, and can be further tested directly on the chip; secondly, the detachable design allows the skin chip to be reused, thereby reducing experimental costs. It is convenient to directly observe samples under a microscope and even image and analyze separated tissues.

[0037] The gravity perfusion method does not require an external complex culture medium pumping system, which can reduce the cost of use. It can realize the simultaneous culture of multiple skin chips, making the standardized operation of skin chips more convenient and easier to achieve high-throughput testing. The culture medium is not connected to the outside world, which also avoids the possibility of contamination of the skin chip.

[0038] The vertical connection of the culture chamber can better simulate the layered structure and barrier function of real skin, provide two-way fluid exchange, facilitate the effective exchange of nutrients and metabolic waste, and promote cell growth and metabolism; at the same time, this design can also more realistically study the penetration process of drugs and compounds, and is suitable for transdermal drug delivery and skin care product testing. In addition, the vertical connection design facilitates the setting of different microenvironmental conditions at both ends of the chamber, which is suitable for simulating the difference between the internal and external environments of the skin, further enhancing the physiological relevance of the experiment.

[0039] For skin models constructed in the same batch, skin chips with and without electrodes can be used for different tests. Since the other structures of the chips are completely consistent, the singleness of the experimental conditions can be guaranteed; secondly, when using chemical stimulation or other experimental operations that may cause electrode corrosion, skin chips without electrodes should be selected. Chips with electrodes can be used in skin electrophysiology, drug penetration, barrier function detection, etc. (current and voltage are set according to actual test needs); in chips with electrodes, the first electrode i and the second electrode ii are not in direct contact with the tissue, avoiding interference and potential damage to the tissue by the electrodes and maintaining the stability of the culture environment; secondly, it helps to obtain clearer and more stable signals and improve the accuracy of the test; in addition, this design reduces electrode contamination and loss, extends the service life of the electrode, and reduces the maintenance frequency; it is easy to standardize and automate operations, and can also support high-throughput testing to improve experimental efficiency and consistency.

[0040] In order to construct the dermis layer, specifically, the dermis culture chip layer may include a dermis culture layer 5, with a first culture cavity 51 disposed in the middle, and the dermis culture layer 5 is used to construct the dermis layer; the first chip connection layer 2 and the dermis culture connection layer 4 are both disposed below the dermis culture layer 5, and are used to connect the dermis culture layer 5 with the first chip, and the first chip connection layer 2 and the dermis 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; wherein a carrier layer is also disposed between the dermis culture connection layer 4 and the first chip connection layer 2. In a skin chip without electrodes, the material of the first chip 1 is glass; in a 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 glass, and the material of the first chip connection layer 2 and the dermis culture connection layer 4 can be a suitable material, for example, polyethylene terephthalate (PET), polyvinyl chloride (PVC), preferably, polyimide (PI). Double-sided tape is provided on the upper and lower surfaces of the first chip connection layer 2 and the dermis culture connection layer 4, which is used to connect and fix the dermis culture layer 5 and the first chip 1, seal, prevent leakage of the culture medium, and effectively avoid the possibility of contamination of the skin model. The material of the double-sided tape is the same as that of the first chip connection layer 2 and the dermis culture connection layer 4. The material of the dermis culture layer 5 is polymethyl methacrylate (PMMA), which has high optical transparency, resistance to ultraviolet radiation, and high hardness and compressive strength. The carrier layer is a microporous membrane, which covers the first culture cavity hole 21 and is used to separate the first culture cavity hole 21 from the first culture cavity 51. The diameter of the carrier layer is larger than the diameter of the first culture cavity hole 21, and the carrier layer is fixed between the first chip connection layer 2 and the dermis culture connection layer 4 by double-sided tape; the carrier layer can be made of an appropriate material, such as polycarbonate. The above-mentioned carrier layer is used for directly inoculating skin equivalents to construct the dermis layer. The lower layer of the carrier layer is in direct contact with the dermis culture medium. The pore size of the carrier layer is 0.4μm-10μm. The above-mentioned carrier layer allows the culture medium to pass through, but the skin equivalent cannot pass through the carrier layer. The culture medium is not in direct contact with the skin equivalent, which provides culture conditions that are closer to the physiological environment for the construction of the dermis layer. Secondly, it is also convenient to take out the skin model after the culture is completed for the next step of analysis.

[0041] In order to provide nutrients to the culture medium, specifically, the first chip connection layer 2 may also be provided with a dermal culture medium channel, which is symmetrically arranged on both sides of the first culture cavity 21 and is connected to the first culture cavity 21. The space defined by the side wall of the first culture cavity 21 and the first chip 1 and the carrier layer is filled with culture medium, which is used to provide nutrients to the dermis layer on the carrier layer. The dermal culture medium can be used as a conductive solution for the first electrode i on the first chip 1. The culture medium channel is used to perfuse the culture medium into the first culture cavity 21, which can ensure uniform distribution of nutrients and help 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 diversified experimental designs.

[0042] In order to make the bottom of the dermis layer fully contact with the culture medium through the carrier layer, specifically, the dermis culture connection layer 4 and the dermis culture layer 5 can also be provided with a dermis culture medium inlet channel and a dermis culture medium outlet channel, which are connected to the dermis culture medium channel on the first chip connection layer 2, and use gravity to make the culture medium more evenly distributed in the first culture cavity 21 and can fully contact with the carrier layer.

[0043] In order to construct a skin model similar to that in vivo, specifically, the thickness of the dermis culture layer 5 can be 3mm-4mm, preferably 3.5mm; the thickness of the dermis culture layer 5 is close to the thickness of the dermis layer of human skin, which helps to better reproduce the physiological structure of the skin. The thickness of the first chip connection layer 2 and the dermis culture connection layer 4 can be 0.2mm-0.4mm, preferably 0.3mm, and the thickness of the carrier layer can be 0.01mm-0.02mm, preferably 0.017mm. The dermis culture connection layer 4 and the carrier layer provide appropriate support and separation for the dermis culture layer 5, ensuring the effective isolation of the culture medium and the skin equivalent. At the same time, sufficient culture medium is allowed to provide nutrition to the cells through the carrier layer to maintain healthy cell growth. The diameter of the first culture cavity 51, the first culture cavity hole 21 and the second culture cavity hole 41 can be 5.0mm-6.0mm, preferably 5.5mm, which can provide sufficient growth space for the skin equivalent, facilitate the construction of the dermis layer, and ensure that the culture medium can effectively penetrate, thereby ensuring the stability of the cell culture environment and enhancing the reliability and repeatability of the experimental results.

[0044] In order to construct a skin model that can restore the real skin structure, specifically, the epidermal culture chip layer 6 can be arranged on the top of the dermal culture chip layer, and the epidermal culture chip layer 6 can include: a second culture cavity 61, which is arranged to penetrate the epidermal culture chip layer 6; and an epidermal culture medium channel, which is symmetrically arranged on both sides of the second culture cavity 61 and communicated with the second culture cavity 61 to provide epidermal culture medium. The above-mentioned epidermal culture chip layer 6 can be made of appropriate materials, such as polyethylene terephthalate (PET), polyvinyl chloride (PVC), preferably polyimide (PI). By arranging the epidermal culture chip layer 6 on the top of the dermal culture chip layer and designing a structure including the second culture cavity 61 and the epidermal culture medium channel, the hierarchical structure of the skin can be truly simulated, and an independent and controllable epidermal and dermal culture environment can be provided. The connection design of the epidermal culture medium channel and the second culture cavity 61 ensures that the epidermal layer can obtain uniform nutrient supply, while avoiding cross-contact between the epidermal culture medium and the dermal culture medium, and better simulating the physiological separation between the two layers of tissue. This design optimizes the interaction between the epidermis and the dermis, which is beneficial to promoting cell growth, differentiation and functional performance. It can achieve more accurate skin substitute construction, drug penetration research, skin toxicity testing and other high-throughput experiments, providing a comprehensive and physiological skin simulation platform, which is widely used in skin science research, cosmetic testing and drug development.

[0045] In order to correspond to the diameter of the dermis layer, an epidermis layer close to the real human body is constructed. Specifically, the thickness of the epidermis culture chip layer 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. The upper and lower sides of the above-mentioned epidermis culture chip layer 6 are respectively provided with double-sided tape, which is used to connect the dermis culture layer 5 and the second chip 7 on the upper and lower sides. The above-mentioned double-sided tape can be a suitable material. In this application, it is the same material as the epidermis culture chip layer 6. The epidermis culture chip layer 6 is fixed on the top of the dermis culture layer 5, which can truly simulate the structure and function of the skin epidermis layer, provide suitable space and uniform culture medium supply, and support the growth and differentiation of epidermal cells. Reasonable size design ensures the effective penetration of the culture medium, avoids uneven distribution, optimizes the culture conditions, and is suitable for long-term culture and high-throughput experiments.

[0046] like Figure 1 and Figure 3As shown, in order to store the culture medium, specifically, the second chip 7 can be arranged above the epidermal culture chip layer 6, and the second chip 7 includes: a third culture cavity 71, the third culture cavity 71 is connected to the second culture cavity 61 on the epidermal culture chip layer 6; a dermal culture medium inlet 7c and a dermal culture medium outlet 7d, respectively connected to the dermal culture medium inlet channel and the dermal culture medium outlet channel on the dermal culture layer; an epidermal culture medium inlet 7a and an epidermal culture medium outlet 7b, which are connected to the epidermal culture medium channel on the epidermal culture chip layer. The material of the second chip 7 is polymethyl methacrylate (PMMA) with good light transmittance. In the chip with electrodes, the second electrode ii on the second chip 7 is a wire arranged in the third culture cavity 71, and the length of the wire in the third culture cavity 71 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, and the wire hole can be arranged at an appropriate position, for example, it can be arranged on the top of the second chip 7, or it can be arranged on the side of the second chip 7. The diameter of the wire hole is 0.8 mm, which is convenient for the wire to pass through. In order to maintain the sealing state of the skin chip, the wire hole is sealed with AB glue. The AB glue can be made of appropriate materials, such as silicone sealant or polyurethane sealant. The dermal culture medium inlet 7c and the dermal culture medium outlet 7d can be set at appropriate positions. For example, setting them on the second chip 7 can prevent the dermal culture medium from overflowing during the culture process of the skin chip. Setting them on the upper surface of the second chip 7 can provide uniform pressure distribution for the culture medium in the first culture cavity 21, and can ensure independent nutrient supply and waste removal between the epidermal culture chip layer 6 and the dermal culture layer 5, so as to simulate the physiological function of the skin more realistically. The epidermal culture medium and dermal culture medium inlet and outlet are set on the upper surface of the second chip 7, so that more culture medium can be stored inside, providing an appropriate amount of culture medium for long-term culture of the skin chip. By setting different inlets and outlets for the culture medium of the dermis and epidermis, mutual interference between the two is avoided, so that each layer can grow under the most suitable conditions.

[0047] In order 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 4 mm-5 mm, preferably 4.6 mm, and the diameter of the third culture chamber 71 can be 5 mm-6 mm, preferably 5.5 mm. This design allows the chip to accommodate the culture medium and a complex fluid system, and also provides a stable structural support for the skin chip.

[0048] In order to prevent the skin model in the skin chip from being contaminated, specifically, the skin chip may further include a culture cavity plug 8 and a sealing cover 9. The diameter of the culture cavity plug 8 is 5mm-6mm, preferably 5mm. The culture cavity plug 8 is made of a transparent material, which is convenient for observing the growth state of the skin model during the culture process. The material of the culture cavity plug may be an appropriate elastic material. In the present application, the material of the culture cavity plug is a silicone plug. The culture cavity plug 8 is used to plug the third culture cavity 71, and its diameter is slightly smaller than the third culture cavity 71. The thickness of the culture cavity plug 8 is 5.3mm, as shown in FIG. Figure 5 As shown, when the drug is administered to treat the skin model, the drug addition process can be completed by directly opening the culture chamber plug 8. In the present application, the sealing cover can be made of a suitable material, such as polymethyl methacrylate with good light transmittance. The sealing cover 9 covers the second chip 7, and a certain gap is left between the inner surface of the sealing cover 9 and the second chip 7 to ensure that the culture medium inside the chip flows smoothly and avoid uneven flow rate or blockage. In addition, it can also prevent the evaporation of water in the culture medium during the culture process, resulting in an increase in the concentration of nutrients or metabolic waste in the culture medium, which will have an adverse effect on the skin model cultured therein.

[0049] The chip is assembled from bottom to top in sequence, including the first chip 1, the first chip connection layer 2, the carrier layer 3, the dermis culture connection layer 4, the dermis culture layer 5, the epidermis culture chip layer 6, the second chip 7, the culture cavity plug 8 and the sealing cover 9. The upper and lower surfaces of the first chip connection layer 2, the dermis culture connection layer 4 and the epidermis culture chip layer 6 are provided with double-sided tape. During the culture, the layers of chips are sealed and connected to ensure the integrity and sealing of the skin chip. After the culture is completed, when the skin model needs to be removed in whole or in part for histological analysis, it is only necessary to disassemble the layers in sequence. When cultured again, the washed layers of chips are assembled again and can be used after sterilization. Compared with the one-piece chip, the detachable chip in this application can be reused, which reduces the experimental cost and is also convenient for removing the skin model for the next step.

[0050] According to a second aspect of the present application, a skin model is proposed. The skin model is obtained by culturing a skin equivalent in a skin chip. The skin chip is the skin chip described above.

[0051] The skin equivalent is a mixture of human fibroblast matrix gel and human epidermal keratinocytes. The human fibroblasts and epidermal keratinocytes can be obtained through commercial channels or can be screened according to ethical requirements. In this application, human epidermal keratinocytes and human dermal fibroblasts are separated according to ethical requirements. The isolation method of the above cells is separated according to conventional methods, and the skin model is constructed after the separated cells are stable.

[0052] like Figure 6 As shown, the skin model is as follows: after the human fibroblast matrix gel mixture is perfused into the culture chamber, after the matrix gel is solidified, a suspension containing human epidermal keratinocytes is perfused into the culture chamber; the epidermal culture medium and the dermal culture medium are respectively perfused through the epidermal culture medium inlet and the dermal culture medium inlet; after the culture chamber plug 8 plugs the third culture chamber 71, the sealing cover 9 is covered on the second chip 7, and the skin chip is placed on a pumpless rocker shaker (such as Figure 4 As shown), the swing angle can be set as needed. In this application, the swing angle is 15°, so that the epidermal culture medium and the dermal culture medium flow back and forth, and the dynamic perfusion of the culture medium is achieved by gravity perfusion, which is used to simulate the human microenvironment. Secondly, there is no complex fluid perfusion system, and it is easier to prepare high-throughput skin chips, which provides higher stability and reproducibility for high-throughput drug screening. When it is necessary to use a skin chip with electrodes to detect skin barrier function or other tests, the first electrode i and the second electrode ii on the first chip 1 and the second chip 7 are respectively connected to the power supply.

[0053] After the skin equivalent is cultured for about 2 days, the epidermal culture medium is no longer added, so that the epidermal layer is directly in contact with the air to form an air-liquid interface, further stimulating keratinocytes to differentiate into stratum corneum to form a skin model. The dermal culture medium is a complete fibroblast culture medium, and the epidermal culture medium is a complete keratinocyte culture medium. The above culture media can be obtained through commercial channels.

[0054] The skin chip can be placed on a pumpless rocker and a chip holder can be used to fix the skin chip on the pumpless rocker. Secondly, the chip holder is also provided with a chip fixing device. The chip fixing device can be in an appropriate form, for example, it can be a groove suitable for placing the skin chip. In the present application, a positioning column is used as the chip fixing device, which corresponds to the positioning hole on the skin chip to prevent the skin chip from moving. In addition, a plurality of positioning columns can be provided on the chip holder to fix a plurality of skin chips, so as to ensure that a plurality of skin chips are cultured at the same time and maintain the consistency of culture conditions.

[0055] According to the third aspect of the present application, an application of a skin model is proposed, which is the application of the skin model in vitro for non-therapeutic scientific research, drug testing, drug screening, cosmetic testing, toxicity assessment, and disease model, and the skin model is the above-mentioned skin model.

[0056] exist Figure 1-Figure 3 The skin chip with electrodes in the present application is shown in FIG. 1 , and the skin model is constructed using a skin chip without electrodes in the embodiment.

[0057] 1. Example Example 1 A1. Place the sterilized skin chip in a clean bench and open the sealing cover and culture chamber plug; A2. A human fibroblast matrix gel mixture is perfused into the first culture chamber through the third culture chamber on the second chip 7 to prepare the dermis layer. The human fibroblast matrix gel mixture includes matrigel matrix gel (8-13 mg / mL), bovine fibrinogen (5-10 mg / mL), bovine thrombin (100-200 U / mL) and human dermal fibroblasts (5×10 6 -6×10 6 / mL); after the matrix gel is solidified, the dermal culture medium is perfused into the dermal culture medium inlet on the third chip, so that the dermal culture medium evenly fills the first culture cavity hole on the dermal culture connection layer; A3. After the dermis layer is constructed, human epidermal keratinocyte suspension is perfused into the second culture chamber through the third culture chamber to make it adhere to the surface of the matrix gel. The concentration of keratinocytes in the human epidermal keratinocyte suspension is 1×10 6 -5×10 6 Pieces / mL; A4. Epidermal cell culture medium is perfused into the interior through the epidermal culture medium inlet on the second chip 7; A5. Cover the culture chamber with a plug and a sealing cover, and culture in a 37°C, 5% CO2 incubator on a pumpless shaker. After 2 days of culture, remove the epidermal culture medium. After 14 days of culture, the epidermis and dermis are formed, and the next step can be performed.

[0058] 2. Experimental Methods 2.1 Morphological analysis of the skin model After the skin model is constructed, the skin chip is opened, the skin model is taken out, the skin model is dehydrated, embedded, sliced, and HE-stained (hematoxylin-eosin staining) before the morphology of the skin model is observed under an ordinary optical microscope at 100-200 times.

[0059] 2.2 Cosmetic irritation testing on skin models The skin model was constructed and stimulated with isopropyl alcohol (analytical grade), hexyl salicylate (analytical grade), cyclamen aldehyde (analytical grade), and 5% potassium hydroxide at the piston. The amount of the above chemicals added was based on covering the epidermis. After the stimulation period (15min-60min), the chemicals were cleaned with PBS buffer or 0.9% saline. The positive control was 5% SDS aqueous solution, and the negative control was water or phosphate buffer (PBS).

[0060] After culturing the skin model for 36-48 h, cell viability was identified according to the instructions of CCK8 (Cell Counting Kit-8).

[0061] 3. Experimental Results 3.1 Morphology of the skin model Figure 7 This is the picture of the skin model after HE staining in this application, Figure 7 It can be seen that in the skin model, the upper dense epidermis layer and the lower dermis layer are tightly combined, indicating that the skin chip in the present application can be used to construct a skin model.

[0062] 3.2 Response of skin models to cosmetic stimuli Figure 8 A, B, C and D in the test are the cell viability of the skin model after the skin model is stimulated by isopropyl alcohol, hexyl salicylate, cyclamen aldehyde and 5% potassium hydroxide. According to the OECD439 (Guidelines for in vitro skin irritation tests issued by the Organization for Economic Cooperation and Development), the cell viability is above 50%, which is considered non-irritating. Figure 8 From A and B in the figure, it can be seen that the cell viability of the skin model treated with isopropyl alcohol and hexyl salicylate is above 50%, which is a non-irritating chemical. Figure 8 From C and D in the figure, it can be seen that the cell viability of the skin model treated with cyclamen aldehyde and 5% potassium hydroxide is below 50%, which is an irritant chemical. The experimental results are in line with the standard requirements. This shows that the skin model constructed in this application has a certain sensitivity to different chemical stimuli, indicating that the skin model constructed in this application has a complete structure and has a complete skin barrier function.

[0063] 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.

[0064] 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.

[0065] 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. Skin chip, characterized in that: The material of the skin chip does not contain polydimethylsiloxane; the skin chip includes: First chip (1): the first chip (1) is used to provide a bottom accommodation space for the dermal culture medium; and a dermis culture chip layer and an epidermis culture chip layer (6) arranged on the top of the first chip (1), and A second chip (7), the second chip being used for supplying and storing a culture medium or supplying a skin irritant; Wherein, the skin chip uses gravity perfusion to provide culture medium for the skin model; The skin chip is detachable; the first chip (1), the dermis culture chip layer, the epidermis culture chip layer (6) and the second chip (7) are connected by a polyimide material or are made of a polyimide material; the culture cavity between the dermis culture chip layer and the epidermis culture chip layer (6) is connected vertically; The first chip (1) and the second chip (7) are not provided with electrodes, or the first chip (1) and the second chip (7) are provided with a first electrode (i) and a second electrode (ii) respectively, and the first electrode (i) and the second electrode (ii) are not in direct contact with the skin model cultured in the dermis culture chip layer and the epidermis culture chip layer (6).

2. The skin chip according to claim 1, characterized in that: The dermis culture chip layer comprises a dermis culture layer (5) with a first culture cavity (51) arranged in the middle, and the dermis culture layer (5) is used to construct a dermis layer; The first chip connection layer (2) and the dermis culture connection layer (4) are both arranged below the dermis culture layer (5) and are used to connect the dermis culture layer (5) and the first chip (1); a first culture cavity hole (21) and a second culture cavity hole (41) corresponding to the first culture cavity (51) are respectively arranged between the first chip connection layer (2) and the dermis culture connection layer (4); A carrier layer (3) is further provided between the dermis culture connection layer (4) and the first chip connection layer (2).

3. The skin chip according to claim 2, characterized in that: The carrier layer (3) is a microporous membrane, and the carrier layer (3) covers the first culture cavity hole (21) and is used to separate the first culture cavity hole (21) from the first culture cavity (51).

4. The skin chip according to claim 2, characterized in that: The first chip connection layer (2) is also provided with a dermal culture medium channel, which is symmetrically arranged on both sides of the first culture cavity hole (21) and is connected to the first culture cavity hole (21).

5. The skin chip according to claim 2, characterized in that: The dermis culture connection layer (4) and the dermis culture layer (5) are also provided with a dermis culture medium inlet channel and a dermis culture medium outlet channel, which are connected to the dermis culture medium channel on the first chip connection layer (2).

6. The skin chip according to claim 2, characterized in that: The thickness of the dermal culture layer (5) is 3 mm to 4 mm; The thickness of the first chip connection layer (2) and the dermis culture connection layer (4) is 0.2 mm-0.4 mm; The thickness of the carrier layer (3) is 0.01 mm-0.02 mm; The diameters of the first culture chamber (51), the first culture chamber hole (21) and the second culture chamber hole (41) are 5.0 mm to 6.0 mm.

7. The skin chip according to claim 1, characterized in that: The epidermis culture chip layer (6) is arranged on top of the dermis culture chip layer. The epidermis culture chip layer (6) comprises: a second culture cavity (61), the second culture cavity (61) being arranged throughout the epidermis culture chip layer (6); and An epidermal culture medium channel is symmetrically arranged on both sides of the second culture chamber (61) and is connected to the second culture chamber (61) for providing an epidermal culture medium.

8. The skin chip according to claim 7, characterized in that: The thickness of the epidermal culture chip layer (6) is 0.2 mm to 0.4 mm. The diameter of the second culture chamber (61) is 5 mm-6 mm.

9. The skin chip according to claim 1, characterized in that: The second chip (7) is arranged above the epidermis culture chip layer (6). The second chip (7) comprises: a third culture cavity (71), the third culture cavity (71) being in communication with the second culture cavity (61) on the epidermis culture chip layer (6); The dermal culture medium inlet (7c) and the dermal culture medium outlet (7d) are respectively connected to the dermal culture medium inlet channel and the dermal culture medium outlet channel on the dermal culture layer (5); The epidermal culture medium inlet (7a) and the epidermal culture medium outlet (7b) are in communication with the epidermal culture medium channel on the epidermal culture chip layer (6).

10. The skin chip according to claim 9, characterized in that: The thickness of the second chip (7) is 4 mm to 5 mm. The diameter of the third culture chamber (71) is 5 mm-6 mm.

11. The skin chip according to claim 1, characterized in that: The skin chip further comprises a culture cavity plug (8) and a sealing cover (9), and the diameter of the culture cavity plug (8) is 5 mm to 6 mm.

12. A skin model, characterized in that The skin model is obtained by culturing a skin equivalent in a skin chip, and the skin chip is the skin chip according to any one of claims 1-11.

13. Application of a skin model, characterized in that The application is the use of the skin model for scientific research, drug testing, drug screening, cosmetic testing, toxicity assessment, and disease model in vitro for non-therapeutic purposes, and the skin model is the skin model described in claim 12.

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