Culture method for differentiating fibroblasts and keratinocytes into 3D skin models, vascular endothelial cells and hepatocytes

Abstract

The invention relates to the technical field of biological medicine application, in particular to a culture method for differentiating fibroblasts and keratinocytes into 3D skin models, vascular endothelial cells and hepatocytes. A 3D skin model, vascular endothelial cells and hepatocytes cultured by the chip can simultaneously simulate a complex three-dimensional tissue structure and a multifunctional humanized tissue of a cell microenvironment. In this way, preparation and evaluation research of in-vitro skin tissue can be achieved, and therefore clarification of occurrence mechanisms of skin-related diseases and selection of treatment strategies are further promoted. According to the method, the in-vivo exposure process that the exogenous chemicals enter blood after percutaneous exposure metabolism and then generate toxicity to the liver can be simulated, and it is shown that the chip has more advantages in the aspect of evaluating the toxicity of the exogenous chemicals, especially the exogenous chemicals with hepatotoxicity after percutaneous metabolism compared with an animal model in terms of species similarity, the 3R principle or the exposure level. Therefore, the invention can provide a new thought and method for the fields of biomedical engineering and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical applications, and particularly to a method for culturing fibroblasts and keratinocytes to differentiate into a 3D skin model, vascular endothelial cells, and hepatocytes. Background Art

[0002] Many exogenous chemicals are exposed through the skin. They have strong reactivity with the body and can clearly produce various toxic effects. However, after percutaneous exposure, they will first be metabolized by N-acetylase in the skin. Therefore, there will be a large toxic difference from the in vivo oral exposure test used to achieve the maximum exposure in traditional toxicological evaluation methods. And the differences in the skin and liver metabolic systems will also lead to misleading results in the in vitro toxicity test of 2D cultured cell lines using the liver S9 metabolic activation system. Therefore, considering the principles of reduction, replacement, and optimization and human relevance, skin chips have been developed. Skin chips, also known as skin microfluidic devices, are a type of dynamic perfusion in vitro 3D skin model. It can make the transport of nutrients more physiological, can simulate the 3D microenvironment of natural human skin, can observe the in vivo multiple 3D cell-cell interactions that cannot be reconstructed by traditional 2D models, and can perform high-throughput and low-cost rapid screening of the toxicity, efficacy, and delivery of chemicals. However, most of the existing skin microfluidic chips have complex structures, long pipelines, many auxiliary devices, are prone to contamination, require multiple people to operate when there are a large number of samples, and have poor usability. At the same time, none of them have toxic target cells for metabolites, so they cannot evaluate the metabolites that may have hepatotoxicity after percutaneous metabolism.

[0003] In view of this, the present invention provides a method for culturing fibroblasts and keratinocytes to differentiate into a 3D skin model, vascular endothelial cells, and hepatocytes. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for culturing fibroblasts and keratinocytes to differentiate into a 3D skin model, vascular endothelial cells, and hepatocytes in view of the deficiencies of the prior art.

[0005] To solve the above technical problems, the following technical solutions are adopted: A method for culturing fibroblasts and keratinocytes to differentiate into a 3D skin model, vascular endothelial cells, and hepatocytes, wherein the cultured fibroblasts and keratinocytes are placed in a chip for culturing, and the method includes the following steps: S1. First, prepare a fibroblast collagen mixture, then take 200 - 500 μL of the fibroblast collagen mixture and add it to the first culture chamber of the chip. Finally, gel at 37°C for 0.5 - 2 h to solidify the fibroblast collagen mixture. Then, fill the other chambers of the chip with 10% FBS DMEM culture medium and culture for 6 - 8 days.

[0006] S2. After culturing for 6 - 8 days as above, replace the 10% FBS DMEM culture medium with EpidermalizationⅠ, and drop 10 - 100 μL of keratinocyte suspension onto the surface of the fibroblast - collagen mixture, controlling the final density of the keratinocytes in the first culture chamber to be 500 - 5000 cells / mm 2 , after standing for 5 - 20 min, place it in an incubator and incubate at 37 °C for 0.5 - 2 h; finally, slowly drop EpidermalizationⅠ into the first culture chamber until it is full, and also fill the other chambers of the chip with EpidermalizationⅠ, and culture for 2 - 4 days.

[0007] S3. After culturing for 2 - 4 days as above, first aspirate EpidermalizationⅠ in the chip, and then fill it with EpidermalizationⅡ, and culture for 1 - 3 days.

[0008] S4. After culturing for 1 - 3 days as above, aspirate EpidermalizationⅡ in the chip, fill the second culture chamber, the third culture chamber, and the first reservoir with Cornification, perform air - liquid interface culture, and start flowing, and culture for 6 - 9 days to obtain a 3D skin model.

[0009] Based on the above - mentioned technical solution, a further improvement is that it further includes S5. After 4 - 6 days of the air - liquid interface culture in S4, continue the air - liquid interface culture, aspirate Cornification in the chip, rinse it once with PBS, fill the second culture chamber and the third culture chamber with ready - to - use matrix gel, and incubate at 37 °C for 0.5 - 2 h; S6. First aspirate the ready - to - use matrix gel in step S5, add vascular endothelial cell suspension to the second culture chamber, add hepatocyte suspension to the third culture chamber, control the final density of vascular endothelial cells and hepatocytes in the culture chamber to be 500 - 5000 cells / mm 2 , invert the chip, and incubate at 37 °C for 0.5 - 2 h; then aspirate the vascular endothelial cell suspension and hepatocyte suspension in the chip, rinse it once with PBS, add vascular endothelial cell suspension to the second culture chamber, add 10% FBS DMEM culture medium to the third culture chamber, and incubate at 37 °C for 0.5 - 2 h; then aspirate the vascular endothelial cell suspension in the chip, rinse it once with PBS, add 10% FBS DMEM culture medium to each chamber of the chip, and incubate at 37 °C overnight.

[0010] S7. On the second day after overnight incubation in step S6, aspirate the 10% FBS DMEM culture medium in the chip, replace it with Cornification, and start flowing, and maintain for 1 day.

[0011] S8. Cell growth is completed, and the chip can be used for exposure tests.

[0012] Based on the above technical solution, a further improvement is that the keratinocytes, fibroblasts, hepatocytes, and vascular endothelial cells include any one of human primary cells, cell lines, and cells obtained by stem cell differentiation.

[0013] Based on the above technical solution, a further improvement is that in step S1, the preparation method of the fibroblast collagen mixture is as follows: sequentially mix 10×DMEM, 1M NaOH, dH 2 O, high-concentration rat tail type I collagen, fetal bovine serum, and fibroblasts evenly and place them on ice to obtain the fibroblast collagen mixture.

[0014] Based on the above technical solution, a further improvement is that in step S5, during the immersion process of the 3D skin model, the liquid exchange generated by the flowing culture medium in the chip just immerses the edge of the 3D skin model, ensuring that the collagen is moist, the fibroblasts are immersed in culture, and the keratinocytes on the surface of the collagen come into contact with air and continue to differentiate.

[0015] Based on the above technical solution, a further improvement is that in step S5, the detection of the 3D skin model shows through paraffin section HE staining that the 3D skin can differentiate into a skin-like structure with an obvious stratum corneum.

[0016] Based on the above technical solution, a further improvement is that the chip includes a culture chamber, a liquid storage chamber, and a microfluidic channel. The liquid storage chamber includes a first liquid storage chamber and a second liquid storage chamber, and the microfluidic channel includes a first microfluidic channel and a second microfluidic channel.

[0017] The culture chamber is arranged in the middle of the chip. A first microfluidic channel for fluid inflow is provided on one side of the culture chamber, and the first microfluidic channel communicates with the first liquid storage chamber. A second microfluidic channel for fluid outflow is provided on the other side of the culture chamber, and the second microfluidic channel communicates with the second liquid storage chamber.

[0018] A height difference is formed between the first liquid storage chamber and the second liquid storage chamber in the vertical direction.

[0019] Based on the above technical solution, a further improvement is that the fluid flows out of the first liquid storage chamber under the action of gravity, first flows through the first microfluidic channel to the culture chamber, and then flows through the second microfluidic channel to the second liquid storage chamber; when the second liquid storage chamber is almost full, the fluid is sucked out and re-added to the first liquid storage chamber for flow circulation.

[0020] On the basis of the above-described technical solution, a further improvement is that the culture chambers are arranged in upper, middle and lower layers. The culture chambers include a first culture chamber, a second culture chamber and a third culture chamber. A porous filter membrane is provided in the culture chamber. The porous filter membrane includes a first porous filter membrane and a second porous filter membrane. A first porous filter membrane is provided between the first culture chamber and the second culture chamber, and a second porous filter membrane is provided between the second culture chamber and the third culture chamber. During the flow process, the fluid exchanges substances through the porous filter membrane.

[0021] On the basis of the above-described technical solution, a further improvement is that the material of the porous filter membrane includes a silicon-based membrane, a cellulose-based membrane, a synthetic polymer membrane, a chitosan-based membrane or a synthetic membrane.

[0022] Due to the adoption of the above technical solution, the following beneficial effects are achieved: The present invention provides a method for culturing fibroblasts and keratinocytes to differentiate into a 3D skin model, vascular endothelial cells and hepatocytes. The chip of the present invention has a three-layer culture chamber structure and is driven by gravity, with a simple structure and no auxiliary equipment. The method of the present invention includes culturing a 3D skin model differentiated from fibroblasts and keratinocytes in the chip, as well as vascular endothelial cells and hepatocytes in the chip.

[0023] The 3D skin model, vascular endothelial cells and hepatocytes cultured by the chip of the present invention can simultaneously simulate a multifunctional humanized tissue with a complex three-dimensional tissue structure and a cellular microenvironment. Thereby, the preparation and evaluation research of in vitro skin tissue can be realized, and further, the clarification of the pathogenesis of skin-related diseases and the selection of treatment strategies can be promoted.

[0024] The 3D skin model, vascular endothelial cells and hepatocytes cultured by the chip of the present invention. The method of the present invention can simulate the in vivo exposure process in which an exogenous chemical enters the blood after percutaneous exposure and metabolism and then reaches the liver to produce toxicity. Whether in terms of species similarity, the 3R principle or exposure level, it shows that the present chip has more advantages than animal models in evaluating the toxicity of exogenous chemicals, especially exogenous chemicals with hepatotoxicity after percutaneous metabolism. Therefore, the present invention can provide new ideas and methods for fields such as biomedical engineering.

[0025] The 3D skin model, vascular endothelial cells, and hepatocytes cultured on the chip of the present invention can be combined with other organs, integrating the tests of several different toxicological target organs to construct a skin chip for toxicological evaluation that can fully simulate the in-vivo environment and achieve satisfactory predictability. Based on the study of cell interactions, it enables high-throughput and low-cost rapid screening of various chemicals. Compared with a single keratinocyte model, the 3D skin model is more similar to human skin. Adding a collagen layer containing fibroblasts beneath the keratinocyte layer is equivalent to simulating the "dermis layer", which increases the resistance of keratinocytes to poisons. The 3D skin model is also applicable to the study of exogenous chemical metabolism because the three-dimensional tissue structure enhances the metabolic capacity of biotransformation enzymes in keratinocytes. In addition to metabolic research, due to the increased barrier function of the 3D skin model compared to the keratinocyte model, it is also used for skin penetration research of topically applied substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below with reference to the accompanying drawings: Figure 1 It is an exploded structural schematic diagram of the chip according to an embodiment of the present invention.

[0027] Figure 2 It is a top-view structural schematic diagram of the chip according to an embodiment of the present invention.

[0028] Figure 3 It is a three-dimensional structural schematic diagram of the chip according to an embodiment of the present invention.

[0029] Figure 4 It is an internal structural schematic diagram of the chip according to an embodiment of the present invention.

[0030] Figure 5 It is a structural schematic diagram of the culture chamber according to an embodiment of the present invention.

[0031] Figure 6 It is a schematic diagram of the flow pattern of fluid in the chip according to an embodiment of the present invention.

[0032] Figure 7 It is a reverse-side structural schematic diagram of the third chip substrate according to an embodiment of the present invention.

[0033] Figure 8 It is a culture diagram of the 3D skin model in the chip according to an embodiment of the present invention.

[0034] Figure 9 It is a HE staining diagram of paraffin sections of the differentiated 3D skin model in the chip, the differentiated keratinocytes in Transwell, and the differentiated 3D skin model in Transwell under the same culture conditions according to an embodiment of the present invention.

[0035] Figure 10Thickness diagrams of the differentiated 3D skin model in the chip, differentiated keratinocytes in Transwell, and the keratinocyte layer of the differentiated 3D skin model in Transwell under the same culture conditions in the embodiments of the present invention.

[0036] Figure 11 HE staining and immunohistochemistry diagrams of paraffin sections of the 3D skin model in the embodiments of the present invention.

[0037] Figure 12 Diagrams of the permeability of the vascular endothelial cell barrier to FITC-dextran in Transwell and in the chip in the embodiments of the present invention.

[0038] Figure 13 Diagram of the release of hepatocyte injury in the embodiments of the present invention.

[0039] Figure 14 Diagram of the release of hepatocyte injury after 2-AF exposure in the embodiments of the present invention.

[0040] Figure 15 Diagram of the analysis of related pathways of differentially expressed genes in hepatocytes after 2-AF exposure in the embodiments of the present invention.

[0041] Figure 16 Diagram of the release of liver injury in mice after 2-AF exposure in the embodiments of the present invention.

[0042] Figure 17 Diagram of the analysis of related pathways of differentially expressed genes in the liver of mice after 2-AF exposure in the embodiments of the present invention.

[0043] Reference numerals: 1 - culture chamber; 2 - first liquid storage chamber; 3 - second liquid storage chamber; 4 - first microfluidic pathway; 5 - second microfluidic pathway; 6 - first chip matrix; 7 - second chip matrix; 8 - third chip matrix; 9 - fourth chip matrix; 10 - fifth chip matrix.

[0044] 11 - first culture chamber; 12 - second culture chamber; 13 - third culture chamber; 14 - first porous filter membrane; 15 - second porous filter membrane. Detailed implementation manners

[0045] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention.

[0046] See Figure 1-17, a method for differentiating fibroblasts and keratinocytes into a 3D skin model and culturing vascular endothelial cells and hepatocytes. The fibroblasts and keratinocytes cultured by this method are placed in a chip for culturing, and the method includes the following steps: S1. First, prepare a fibroblast collagen mixture, then take 200 - 500 μL of the fibroblast collagen mixture and add it to the first culture chamber of the chip. Finally, gel it at 37°C for 0.5 - 2 h to solidify the fibroblast collagen mixture. Then fill the other chambers of the chip with 10% FBS DMEM culture medium and culture for 6 - 8 days. In this embodiment, the culture time is controlled to be 7 days. Among them, the 10% FBS DMEM culture medium refers to that the content of FBS in DMEM is 10%.

[0047] Among them, the preparation method of the fibroblast collagen mixture is as follows: Sequentially mix 10×DMEM, 1M NaOH, dH 2 O, high-concentration rat tail type I collagen, fetal bovine serum and fibroblasts evenly and place them on ice, control the final concentration of high-concentration rat tail type I collagen to be 3 - 8 mg / mL, and the final concentration of fibroblasts to be 0.5 - 2.0×10 5 cells / mL. In this embodiment, the final concentration of high-concentration rat tail type I collagen is 6 mg / mL, and the final concentration of fibroblasts is 0.9×10 5 cells / mL. Among them, 10×DMEM refers to DMEM culture medium with a 10-fold standard concentration.

[0048] In addition, it is necessary to prepare a keratinocyte differentiation culture medium according to Table 1 for use in the following steps. Table 1 Keratinocyte Differentiation Culture Medium Formula (500 mL) Reagent Name Stock Solution Concentration (×) Final Concentration Epidermalization Ⅰ (mL) Epidermalization Ⅱ (mL) Cornification (mL) <![CDATA[GlutaMAX TM > 50 4 mM 10 10 10 Adenine 500 40 μM 1 1 1 Hydrocortisone 500 1 μM 1 1 1 T3 500 20 nM 1 1 1 Recombinant Human Transferrin 500 10 μg / mL 1 1 1 Insulin 1000 10 μg / mL 0.5 0.5 0.5 Dual Antibody 100 / 5 5 5 DMEM (Calcium-Free) / / 363 363 237 Ham's F-12 / / 120 120 237 Progesterone 1000 2 nM 0.5 0.5 0 <![CDATA[CaCl 2 > 500 1.8 mM 0 1 1 FBS / / 0.5 0.5 10 Recombinant Human Epidermal Growth Factor 500 5 ng / mL 1 0 0 S2. After culturing for 6 - 8 days as above, replace the 10% FBS DMEM culture medium with EpidermalizationⅠ. Among them, do not add EpidermalizationⅠ to the first culture chamber, keep the original fibroblast collagen mixture, and then drop 10 - 100 μL of keratinocyte suspension on the surface of the fibroblast collagen mixture, control the final density of the keratinocytes in the first culture chamber to be 500 - 5000 cells / mm 2 , after standing for 5 - 20 min, put it into an incubator and incubate at 37°C for 0.5 - 2 h; finally, slowly drop EpidermalizationⅠ into the first culture chamber until it is full, and also fill the other chambers of the chip with EpidermalizationⅠ and culture for 2 - 4 days. In this embodiment, 50 μL of keratinocyte suspension is dropped, and the final density of the keratinocytes in the first culture chamber is 1500 cells / mm 2, The standing time is controlled at 15 min, the incubation time in the incubator is 1 h, and the final culture time is 3 days.

[0049] S3. After culturing for 2 - 4 days as above, first aspirate EpidermalizationⅠ in the chip, then fill it with EpidermalizationⅡ and culture for 1 - 3 days. In this embodiment, the culture is controlled for 2 days.

[0050] S4. After culturing for 1 - 3 days as above, aspirate EpidermalizationⅡ in the chip, fill the second culture chamber, the third culture chamber and the first liquid storage chamber with Cornification, perform air - liquid interface culture and start flowing, and culture for 6 - 9 days to obtain a 3D skin model. In this embodiment, the culture time is 7 days.

[0051] S5. After 4 - 6 days of the air - liquid interface culture in S4, continue the air - liquid interface culture, aspirate Cornification in the chip, rinse it once with PBS, fill the second culture chamber and the third culture chamber with ready - to - use matrix gel, and incubate at 37℃ for 0.5 - 2 h. In this embodiment, the air - liquid interface culture time in S4 is 5 days, the incubation temperature is controlled at 37℃, and the incubation time is 1 h.

[0052] S6. First aspirate the ready - to - use matrix gel in step S5, add vascular endothelial cell suspension to the second culture chamber, add hepatocyte suspension to the third culture chamber, control the final density of vascular endothelial cells and hepatocytes in the culture chamber to be 500 - 5000 cells / mm 2 , invert the chip, and incubate at 37℃ for 0.5 - 2 h; then aspirate the vascular endothelial cell suspension and hepatocyte suspension in the chip, rinse it once with PBS, add vascular endothelial cell suspension to the second culture chamber, add 10% FBS DMEM culture medium to the third culture chamber, and incubate at 37℃ for 0.5 - 2 h; then aspirate the vascular endothelial cell suspension in the chip, rinse it once with PBS, add 10% FBS DMEM culture medium to each chamber of the chip, and incubate at 37℃ overnight. In this embodiment, the final density of vascular endothelial cells and hepatocytes in the culture chamber is 1500 cells / mm 2 , the first incubation time when the chip is inverted is 1.5 h, and the second incubation time is 1.5 h.

[0053] S7. On the second day after overnight incubation in step S6, aspirate the 10% FBS DMEM culture medium in the chip, replace it with Cornification, and start flowing, and maintain for 1 day.

[0054] S8. When cell growth is completed, the chip can be used for exposure tests.

[0055] Based on the above technical solution, a further improvement is that the keratinocytes, fibroblasts, hepatocytes and vascular endothelial cells include any one of human primary cells, cell lines, and cells differentiated from stem cells.

[0056] As Figure 1-7 shown, the chip includes a culture chamber 1, a liquid storage chamber and a microfluidic channel. The liquid storage chamber includes a first liquid storage chamber 2 and a second liquid storage chamber 3. The microfluidic channel includes a first microfluidic channel 4 and a second microfluidic channel 5.

[0057] The culture chamber 1 is arranged in the middle of the chip. A first microfluidic channel 4 for fluid inflow is provided on one side of the culture chamber 1. The first microfluidic channel 4 communicates with the first liquid storage chamber 2. A second microfluidic channel 5 for fluid outflow is provided on the other side of the culture chamber 1. The second microfluidic channel 5 communicates with the second liquid storage chamber 3.

[0058] A height difference is formed between the first liquid storage chamber 2 and the second liquid storage chamber 3 in the vertical direction. In this embodiment, the height of the first liquid storage chamber 2 is greater than the height of the second liquid storage chamber 3.

[0059] Referring to Figure 6 , wherein, the fluid flows out from the first liquid storage chamber 2 under the action of gravity, first flows through the first microfluidic channel 4 to the culture chamber 1, and then flows through the second microfluidic channel 5 to the second liquid storage chamber 3; when the second liquid storage chamber 3 is almost full, the fluid is sucked out and re-added to the first liquid storage chamber 2 for flow circulation.

[0060] As a further description of this embodiment, the chip of this embodiment is a skin microfluidic chip, and at least three independent chambers are arranged inside it, namely the middle culture chamber 1, the left first liquid storage chamber 2 and the right second liquid storage chamber 3.

[0061] As a further description of this embodiment, the culture chamber 1 is connected to one or more microfluidic channels; the shape of the culture chamber 1 is rectangular or circular, and the area is not more than 200 mm 2 . In this embodiment, the culture chamber 1 is connected to an inflowing first microfluidic channel 4, the culture chamber 1 is connected to an outflowing second microfluidic channel 5, and the shape of the culture chamber 1 is circular.

[0062] Specifically, referring to Figure 5, the culture chambers 1 are arranged in upper, middle and lower positions. The culture chamber 1 includes a first culture chamber 11, a second culture chamber 12 and a third culture chamber 13. A porous filter membrane is provided in the culture chamber 1. The porous filter membrane includes a first porous filter membrane 14 and a second porous filter membrane. A first porous filter membrane 14 is provided between the first culture chamber 11 and the second culture chamber 12, and a second porous filter membrane 15 is provided between the second culture chamber 14 and the third culture chamber 15. During the flow process, the fluid exchanges substances through the porous filter membrane.

[0063] Among them, the first culture chamber 11 is located at the upper part of the entire culture chamber 1, the second culture chamber 12 is located at the middle part of the entire culture chamber 1, and the third culture chamber 13 is located at the lower part of the entire culture chamber 1.

[0064] Furthermore, the number of the first microfluidic channel 4 and the second microfluidic channel 5 can be set to one or more according to needs. In this embodiment, the first microfluidic channel 4 and the second microfluidic channel 5 are each one. Among them, the first microfluidic channel 4 and the second microfluidic channel 5 are arranged horizontally.

[0065] As a further description of this embodiment, the materials of the porous filter membrane include but are not limited to silicon-based membranes (such as PDMS), cellulose-based membranes (such as cellulose acetate), synthetic polymer membranes (such as nylon, PC, PET), chitosan-based membranes, and Strat-M® synthetic membranes, etc.

[0066] As a further description of this embodiment, the materials of the chip substrate include but are not limited to glass, stainless steel, and synthetic polymers (such as PMMA, PDMS, PET, PTFE, COC), etc.

[0067] As a further description of this embodiment, the chip includes a first chip substrate 6, a second chip substrate 7, a third chip substrate 8, a fourth chip substrate 9, and a fifth chip substrate 10 connected by bonding.

[0068] The skin microfluidic chip with multiple independent chambers prepared by this method can achieve relative isolation between different chambers, and various cells can be cultured independently; and the chip has a simple structure, low production cost, and a wide range of applications.

[0069] The microfluidic chip proposed in the present invention can simulate the in-vivo exposure process in which exogenous chemicals enter the blood after percutaneous exposure metabolism and then reach the liver to produce toxicity, and can evaluate the toxicity of exogenous chemicals, especially exogenous chemicals with hepatotoxicity after percutaneous metabolism. Therefore, the present invention can provide new ideas and methods for fields such as biomedical engineering.

[0070] As a further description of this embodiment, in step S5, this embodiment describes the 3D skin model culture and detection method in the chip: See Figure 8 It is a culture diagram of a 3D skin model in a chip, showing the submerged culture and air-liquid interface culture of the 3D skin model. During the submerged process of the 3D skin model, the liquid exchange generated by the flowing culture medium in the chip just allows the edge of the 3D skin model to be submerged, ensuring the wetness of collagen and the submerged culture of fibroblasts. However, the keratinocytes on the surface of the collagen can contact the air and continue to differentiate.

[0071] See Figure 9 It is a HE staining diagram of paraffin sections of the differentiated 3D skin model in the chip, the differentiated keratinocytes in Transwell, and the differentiated 3D skin model in Transwell under the same culture conditions in the embodiment of the present invention. Figure 10 It is a thickness diagram of the keratinocyte layer of the differentiated 3D skin model in the chip, the differentiated keratinocytes in Transwell, and the differentiated 3D skin model in Transwell under the same culture conditions in the embodiment of the present invention. Comparing the differentiated 3D skin model in the chip ( Figure 9 a), the differentiated keratinocytes in Transwell ( Figure 9 b), and the differentiated 3D skin model in Transwell ( Figure 9 c), it is found that when there are only keratinocytes in Transwell, the differentiated stratum corneum is thinner; when there is collagen in Transwell, the differentiated stratum corneum is slightly thicker; and the differentiated stratum corneum of keratinocytes in the chip is significantly thicker ( Figure 10 , ns indicates no significant difference, ** indicates p < 0.01), indicating that the collagen layer containing fibroblasts and fluid flow can promote keratinocyte differentiation to a certain extent.

[0072] See Figure 11 It is a hematoxylin-eosin staining and immunohistochemistry diagram of paraffin sections of the 3D skin model. Specifically, see Figure 11 (a). The detection of the 3D skin model by HE staining of paraffin sections shows that the 3D skin can differentiate into a skin-like structure with an obvious stratum corneum. As shown in Figure 11 (b) and Figure 11 (c), the expression of cytokeratin10 and filaggrin in immunohistochemistry indicates that keratinocytes have undergone terminal differentiation.

[0073] As a further illustration of this embodiment, the functional detection methods for vascular endothelial cells and hepatocytes in the chip in this embodiment are described: Barrier property of vascular endothelial cells. As shown in Figure 12The figure shows the permeability of the vascular endothelial cell barrier to FITC-dextran in Transwell and the chip (ns indicates no significant difference, ** indicates p < 0.01). The permeability (Papp value) of FITC-dextran in Transwell and the chip decreased by 91.66% and 75.99% respectively. Both the decreased proportion and the final value are similar to the research reports, indicating that a vascular endothelial cell barrier has been formed in the chip and is very close to the effect of the commercial chamber Transwell.

[0074] Hepatocyte injury release. Such as Figure 13 The figure shows the hepatocyte injury release (ns indicates no significant difference, ** indicates p < 0.01). The release of AST and UREA by hepatocytes with the same density in the 48-well cell culture plate and the chip showed no significant difference, indicating that there was no significant difference in the growth and metabolism of hepatocytes in the chip and the 48-well cell culture plate. Moreover, the flow of the culture medium in the chip significantly reduced the release of LDH, indicating that the growth state of hepatocytes in the chip was better.

[0075] As a further illustration of this embodiment, the toxic reaction of hepatocytes in the chip after exposure to the exogenous chemical 2-aminofluorene (2-AF) is described in this embodiment: Such as Figure 14 The figure shows the hepatocyte injury release in the chip after 2-AF exposure (ns indicates no significant difference, * indicates p < 0.05). Among them, Acetone is acetone, serving as the test solvent control group. Exposing 2-AF to the 3D skin model at a dose of 3000 μg / mL will significantly increase the release of AST by hepatocytes in the third culture chamber, indicating that it has a toxic effect on hepatocytes.

[0076] Such as Figure 15 The figure shows the analysis of the related pathways of differentially expressed genes in hepatocytes in the chip after 2-AF exposure. RNA sequencing of hepatocytes in the chip shows that the differentially expressed genes with biological significance are mainly concentrated in the pathways related to chemical metabolism, chemical carcinogens (DNA adducts), and cytochrome P450 enzymes, which is the same as the toxic mechanism of 2-AF and its metabolite 2-acetylaminofluorene (2-AAF).

[0077] As a further illustration of this embodiment, the toxic reaction of the liver in mice after exposure to the exogenous chemical 2-AF is described in this embodiment: Such as Figure 16Shown is the release diagram of liver injury in mice after 2-AF exposure. Among them, Acetone is acetone, serving as the test solvent control group. 2-AF was exposed to the dorsal skin of ICR male mice (body weight 18 ± 2 g) at doses of 200, 600, and 1800 mg / kg, which had no significant effect on the release of AST, UREA, and LDH in the mice, and there were no significant differences among the groups, indicating that the liver did not show damage in terms of blood biochemical indexes. As Figure 17 Shown is the analysis diagram of related pathways of differentially expressed genes in the liver of mice after 2-AF exposure. Among them Figure 17 (a) is the low-dose group, 17 (b) is the medium-dose group, and 17 (c) is the high-dose group.

[0078] RNA sequencing of mouse liver showed that the biologically significant pathways in the low- and medium-dose groups of 2-AF were histidine metabolism and glycerolipid metabolism pathways. The biologically significant pathways in the medium- and high-dose groups of 2-AF were related to chemical carcinogens (DNA adducts), chemical metabolism, and cytochrome P450 enzyme-related pathways, which were the same as the toxic action mechanisms of 2-AF and its metabolite 2-AAF.

[0079] As a further illustration of this embodiment, this embodiment describes the comparison results of the toxic reactions after exposure to the exogenous chemical 2-AF in in vitro tests (chips) and in vivo tests (mice): As shown in Table 2, in the in vitro test, the chip was exposed to 2-AF at 3000 μg / mL, and the percutaneous exposure amount was 54 μg; in the in vivo test, the mice were exposed to 2-AF at each dose, and the percutaneous exposure amounts were 3680, 10800, and 32760 μg. The percutaneous exposure amount of 2-AF in the in vivo test was approximately 68, 200, and 607 times that in the in vitro test. Calculated by the exposure amount per unit body surface area, the in vivo test was approximately 1.21, 3.62, and 10.89 times that in the in vitro test. In the in vitro test, the exposure dose extrapolated to humans for the chip was 12 mg / kg; in the in vivo test, the exposure dose extrapolated to humans for the mice was 15 - 134 mg / kg.

[0080] Table 2 In vitro and in vivo exposure amounts of 2-AF In Vitro Test (Chip) In Vivo Test (Mouse) In Vivo Test (Mouse) In Vivo Test (Mouse) Exposure Conditions 2-AF (Human) 2-AF (Low Dose Group) 2-AF (Medium Dose Group) 2-AF (High Dose Group) Exposure Dose (mg / mL) (mg / kg) 3 200 600 1800 Exposure Volume (μL) / Average Body Weight (g) 18 18.4 18 18.2 Transdermal Exposure Amount (μg) 54 3680 10800 32760 Transdermal Exposure Amount Multiple / 68 200 607 <![CDATA[Chip / mouse body surface area (cm 2 )]]> 1.13 63.42 62.5 62.96 <![CDATA[Exposure per unit body surface area (μg / cm 2 2)]] 48 58 173 520 Exposure Amount Multiple per Unit Body Surface Area / 1.21 3.62 10.89 Human Body Weight (kg) 70 / / / <![CDATA[Body surface area (cm 2 ).]]> 18004 / / / Extrapolated Exposure Dose to Human (mg / kg) 12 15 44 134 As shown in Table 3, among the same KEGG pathways in vitro and in vivo after 2-AF exposure, there were 5 and 4 KEGG functional enrichment pathways in the chip hepatocytes that were the same as those of the differentially expressed genes in the livers of mice in the medium- and high-dose groups of 2-AF, all mainly concentrated in chemical metabolism, chemical carcinogens (DNA adducts), and cytochrome P450 enzyme-related pathways, which were the same as the toxic action mechanisms of 2-AF and its metabolite 2-AAF, indicating that the toxic action mechanisms of 2-AF on hepatocytes / tissues after percutaneous exposure in in vitro and in vivo tests were basically the same.

[0081] Table 3 KEGG pathways that are the same in vivo and in vitro after 2-AF exposure Group Chip and Mouse Low Dose Group Chip and Mouse Medium Dose Group Chip and Mouse High Dose Group KEGG GnRH secretion Drug metabolism-other enzymes Drug metabolism-other enzymes Biosynthesis of cofactors Chemical carcinogenesis-DNA adducts Chemical carcinogenesis-DNA adducts Drug metabolism-cytochrome P450 Drug metabolism-cytochrome P450 Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome P450 The in vivo and in vitro test results show that the exposure of 2-AF in the chip is similar to the exposure in the medium and high-dose groups of mice, indicating that the chip can truly simulate the in vivo exposure process of 2-AF entering the blood after percutaneous exposure and metabolism, and then reaching the liver to produce toxicity. Calculated by extrapolating the exposure dose or percutaneous exposure amount to humans per unit body surface area, the chip can achieve the same toxicological effects as animal experiments at a lower exposure level (at least about 4-200 times lower).

[0082] Through the method for culturing fibroblasts and keratinocytes to differentiate into a 3D skin model in the chip and the method for culturing vascular endothelial cells and hepatocytes in the chip of the present invention, the in vivo exposure process of exogenous chemicals entering the blood after percutaneous exposure and metabolism and then reaching the liver to produce toxicity can be simulated. Whether in terms of species similarity, the 3R principle or exposure level, it shows that this chip has more advantages than animal models in evaluating the toxicity of exogenous chemicals, especially exogenous chemicals with hepatotoxicity after percutaneous metabolism.

[0083] The Chinese and English term explanations are shown in Table 4 below: Table 4 Chinese-English comparison table of term explanations English Chinese Epidermalization Ⅰ Epidermalization Culture Medium Ⅰ Epidermalization Ⅱ Epidermalization Culture Medium Ⅱ Cornification Cornification Culture Medium <![CDATA[GlutaMAX TM > Reagent Trade Name T3 Sodium 3,3',5-triiodo-L-thyronine DMEM Reagent Trade Name Ham's F-12 Reagent Trade Name FBS Fetal Bovine Serum KEGG Kyoto Encyclopedia of Genes and Genomes GnRH secretion Gonadotropin-Releasing Hormone Secretion Drug metabolism-other enzymes Drug Metabolism - Other Enzymes Biosynthesis of cofactors Biosynthesis of cofactors Chemical carcinogenesis-DNA adducts Chemical carcinogenesis-DNA adducts Drug metabolism-cytochrome P450 Drug metabolism - cytochrome P450 Metabolism of xenobiotics by cytochrome P450 Metabolism of xenobiotics by cytochrome P450 Transwell A consumable product name ICR male mice A commercial experimental mouse breed name The above are only specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent replacements or modifications made based on the present invention to solve basically the same technical problems and achieve basically the same technical effects are all covered by the protection scope of the present invention.

Claims

1. A method for culturing fibroblasts and keratinocytes into 3D skin models and vascular endothelial cells and hepatocytes, wherein the fibroblasts and keratinocytes cultured in the method are placed in a chip for culturing, and the method is characterized by comprising the following steps: S1. First, prepare a fibroblast-collagen mixture, then take 200-500 μL of the fibroblast-collagen mixture and add it to the first culture chamber of the chip, and finally gel at 37°C for 0.5-2 hours to solidify the fibroblast-collagen mixture, and then fill the other chambers of the chip with 10% FBS DMEM culture solution and culture for 6-8 days; S2. After 6-8 days of culture, the 10% FBS DMEM culture medium is replaced with Epidermalization I, and 10-100 μL of keratinocyte suspension is added to the surface of the fibroblast collagen mixture to control the final density of the keratinocytes in the first culture chamber to be 500-5000 / mm 2 After standing for 5-20 minutes, put it in an incubator and incubate it at 37°C for 0.5-2 hours; finally, slowly add Epidermalization I to the first culture chamber until it is full, and fill the other chambers of the chip with Epidermalization I, and culture for 2-4 days; S3. After 2-4 days of culture, remove Epidermalization I from the chip, then fill it with Epidermalization II and culture for 1-3 days; S4. After 1-3 days of the above culture, the Epidermalization II in the chip is aspirated, and the second culture chamber, the third culture chamber and the first liquid storage chamber are filled with Cornification to perform air-liquid interface culture and start flow. Culture for 6-9 days to obtain a 3D skin model.

2. The method for differentiating fibroblasts and keratinocytes into 3D skin models and vascular endothelial cells and hepatocytes according to claim 1, characterized in that: Also includes S5. After the air-liquid interface culture in S4 is carried out for 4-6 days, the air-liquid interface culture is continued, and the cornification in the chip is aspirated, rinsed once with PBS, and the second and third culture chambers are filled with ready-to-use matrix gel, and incubated at 37° C. for 0.5-2 h; S6. First, remove the ready-to-use matrix gel in step S5, add endothelial cell suspension to the second culture chamber, add hepatocyte suspension to the third culture chamber, and control the final density of endothelial cells and hepatocytes in the culture chamber to 500-5000 / mm 2 , invert the chip and incubate at 37°C for 0.5-2h; then remove the vascular endothelial cell suspension and hepatocyte suspension from the chip, rinse once with PBS, add the vascular endothelial cell suspension to the second culture chamber, add 10% FBSDMEM culture medium to the third culture chamber, and incubate at 37°C for 0.5-2h; then remove the vascular endothelial cell suspension from the chip, rinse once with PBS, add 10% FBS DMEM culture medium to each chamber of the chip, and incubate overnight at 37°C; S7. On the second day after the overnight stay in step S6, the 10% FBS DMEM culture medium in the chip was aspirated and replaced with Cornification, and the flow was started for 1 day; S8. Cell growth is complete and the chip can be used for exposure experiments.

3. The method for differentiating fibroblasts and keratinocytes into 3D skin models and vascular endothelial cells and hepatocytes according to claim 2, characterized in that: The keratinocytes, fibroblasts, hepatocytes and vascular endothelial cells include any one of human primary cells, cell lines and cells differentiated from stem cells.

4. The method for differentiating fibroblasts and keratinocytes into 3D skin models and vascular endothelial cells and hepatocytes according to claim 1, characterized in that: In step S1, the fibroblast collagen mixture is prepared as follows: 10×DMEM, 1M NaOH, dH2O, high concentration rat tail type I collagen, fetal bovine serum and fibroblasts are mixed in sequence and placed on ice to obtain a fibroblast collagen mixture.

5. The method for differentiating fibroblasts and keratinocytes into 3D skin models and culturing endothelial cells and hepatocytes according to claim 1, characterized in that: In step S5, during the immersion of the 3D skin model, the liquid exchange generated by the flowing culture medium in the chip just immerses the edge of the 3D skin model, ensuring that the collagen is moistened, the fibroblasts are immersed in the culture, and the keratinocytes on the surface of the collagen are exposed to the air to continue to differentiate.

6. The method for differentiating fibroblasts and keratinocytes into 3D skin models and culturing endothelial cells and hepatocytes according to claim 1, characterized in that: In step S5, the detection of the 3D skin model through HE staining of paraffin sections shows that the 3D skin can differentiate into a skin-like structure with a clear stratum corneum.

7. The method for differentiating fibroblasts and keratinocytes into 3D skin models and culturing endothelial cells and hepatocytes according to claim 1, characterized in that: The chip comprises a culture chamber, a liquid storage chamber and a microflow channel, wherein the liquid storage chamber comprises a first liquid storage chamber and a second liquid storage chamber, and the microflow channel comprises a first microflow channel and a second microflow channel. The culture chamber is arranged in the middle of the chip, a first micro-flow passage for fluid inflow is arranged on one side of the culture chamber, the first micro-flow passage is connected to the first liquid storage chamber, and a second micro-flow passage for fluid outflow is arranged on the other side of the culture chamber, the second micro-flow passage is connected to the second liquid storage chamber; The first liquid storage chamber and the second liquid storage chamber are formed with a height difference in a vertical direction.

8. The method for culturing fibroblasts and keratinocytes differentiated into 3D skin models and vascular endothelial cells and hepatocytes according to claim 7, characterized in that: The fluid flows out of the first liquid storage chamber under the action of gravity, first flows to the culture chamber through the first microflow path, and then flows to the second liquid storage chamber through the second microflow path; when the second liquid storage chamber is almost full, the fluid is sucked out and re-added to the first liquid storage chamber for flow circulation.

9. The method for differentiating fibroblasts and keratinocytes into 3D skin models and vascular endothelial cells and hepatocytes according to claim 7, characterized in that: The culture chambers are arranged in upper, middle and lower order, and include a first culture chamber, a second culture chamber and a third culture chamber. A porous filter membrane is provided in the culture chamber, and the porous filter membrane includes a first porous filter membrane and a second porous filter membrane. A first porous filter membrane is provided between the first culture chamber and the second culture chamber, and a second porous filter membrane is provided between the second culture chamber and the third culture chamber. During the flow process, the fluid exchanges substances through the porous filter membrane.

10. The method for culturing fibroblasts and keratinocytes into 3D skin models and vascular endothelial cells and hepatocytes according to claim 9, characterized in that: The material of the porous filter membrane includes silicon-based membrane, cellulose-based membrane, synthetic polymer membrane, chitosan-based membrane or synthetic membrane.

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