In-vitro model of keloid disease as well as construction method and application of in-vitro model

Abstract

The invention provides a keloid disease in-vitro model as well as a construction method and application thereof, and relates to the technical field of cell biology. The construction method comprises the following steps: constructing a full-thickness skin model with an epidermal layer and a corium layer by utilizing a cell culture chamber or a cell culture micro-fluidic chip, and then performing induced culture by adopting TGF-beta to obtain the keloid disease in-vitro model. According to the construction method, by selecting the concentration and culture time of TGF-beta in the induction process, keloid-like change of the full-thickness skin model can be effectively promoted, and the pathological state of keloid is truly simulated; meanwhile, the human fibroblasts and the human keratinocytes are adopted to prepare the in-vitro model with the full-thickness skin structure, the in-vitro model has the human attribute, the individual difference of species difference bases is reduced, and the real effectiveness of the model is greatly improved; the defect that keloid which is a special human disease cannot be effectively simulated by an animal model is overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular, to an in vitro model of keloid disease, a construction method thereof, and an application thereof. Background Art

[0002] Keloid is a pathological scar formed by abnormal proliferation of fibroblasts and excessive deposition of collagen during the healing process of human skin injury. Keloid is a pathological state, characterized by out-of-control collagen synthesis and metabolism, manifested as invasive growth beyond the original wound boundary, continuous and progressive, into the surrounding normal skin tissue, accompanied by destruction of the surrounding normal skin appendages. Keloids are more common in areas with greater skin tension such as the presternal area, the shoulder and back area, the double submandibular area, and the earlobe. There is currently no radical and effective treatment for keloids, with a high recurrence rate, seriously affecting the appearance and causing serious impacts on the quality of life and psychology of patients.

[0003] Since keloid is a human-specific disease that is difficult to replicate in animal models, reconstructing an in vitro biological model with the physiological structure of human skin and the characteristics of keloid disease is an important tool for studying the pathological mechanism of keloid and testing potential treatment methods.

[0004] Existing in vitro models of keloid include monolayer cell culture models, three-dimensional collagen gel models, co-culture models, organoid models, artificial skin models, and in vitro transplantation models of keloid. Among them, monolayer cell culture models, three-dimensional collagen gel models, and co-culture models can only simulate the characteristics of a few skin cells such as fibroblasts, lacking the simulation of complex skin physiological structures; organoid models and in vitro transplantation models of keloid are constructed relying on patient-derived keloid tissues. Although they can simulate the complex cell composition and microenvironment of keloid, it is difficult to maintain the in vivo disease characteristics under ex vivo conditions and cannot reflect the pathological process and final outcome of keloid over several years. Therefore, the simulation of the pathological characteristics of keloid is limited.

[0005] Therefore, it is very necessary and urgent to research and develop an in vitro model of keloid disease that can simulate the main pathological phenotypes such as thickening of the epidermis and dermis of keloid, excessive proliferation of fibroblasts, changes in extracellular matrix components, and secretion of inflammatory factors such as IL6, as well as a construction method thereof, for application in the mechanism research and drug development research of keloid.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The object of the present invention is to provide an in vitro model of keloid disease and a construction method thereof. The in vitro model of keloid disease can simulate the main pathological features of keloid disease, such as thickening of the epidermis and dermis, increased skin hardness, over-proliferation of fibroblasts, changes in extracellular matrix components, and secretion of inflammatory factors such as IL6.

[0008] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: A construction method of an in vitro model of keloid disease provided by the present invention, the construction method includes: constructing a full-thickness skin model with an epidermis layer and a dermis layer by using a cell culture chamber or a cell culture microfluidic chip, wherein the dermis layer is formed by mixing a keloid fibroblast suspension with a rigid substrate hydrogel precursor solution and culturing into a gel; the Young's modulus of the rigid substrate hydrogel is 30 KPa to 200 kPa; the epidermis layer is formed by culturing keratinocytes on the dermis layer; The induction culture is air-liquid culture. On the epidermis layer side of the full-thickness skin model in the cell culture chamber or the cell culture microfluidic chip is gas culture, and on the dermis layer side of the full-thickness skin model is liquid culture; During the air-liquid culture process, the culture medium for liquid culture on the dermis layer side is an induction culture medium containing TGF-β, and the concentration of TGF-β in the induction culture medium is 10 to 25 ng / mL; The induction culture is carried out in a cell culture incubator, and the induction culture time is 1 to 5 days to obtain an in vitro model of keloid disease.

[0009] Further, the gas environment in the cell culture incubator is 95% air and 5% CO2; the temperature in the cell culture incubator is 37 °C.

[0010] Further, the construction method of the full-thickness skin model includes: (A) Dermis layer construction: Mix a keloid fibroblast suspension with a rigid substrate hydrogel precursor solution to obtain a cell hydrogel mixture; then add the cell hydrogel mixture into a cell culture chamber or a microfluidic chip to form a gel, and after gel formation, use a fibroblast culture medium to culture the dermis layer to form a dermal matrix layer; (B) Epidermis layer construction: Add a keratinocyte suspension on the dermal matrix layer, and add a keratinocyte liquid medium in the cell culture chamber or the microfluidic chip to culture the epidermis layer, so that keratinocytes form proliferating keratinocytes on the dermal matrix layer; (C) Full-thickness skin model construction: Aspirate the culture medium on the surface of keratinocytes in the cell culture chamber or the microfluidic chip, and add an air-liquid culture medium to culture the full-thickness skin to form a full-thickness skin model with an epidermis layer and a dermis layer.

[0011] Further, the keloid fibroblast suspension is a human fibroblast suspension, and the cell concentration of fibroblasts in the human fibroblast suspension is 0.5×10 6 cells / mL to 2×10 7 cells / mL; Preferably, the human fibroblasts are primary human fibroblasts of passages 2 to 6.

[0012] Further, the density of keratinocytes in the keratinocyte suspension in step (B) is 1.5×10 5 cells / cm 2 to 8×10 5 cells / cm 2 ; Preferably, the keratinocytes are primary human keratinocytes of passages 2 to 6.

[0013] Further, step (A) of culturing the dermis layer is carried out in a cell culture incubator, and the culture time is 3 to 7 days; Further, step (B) of culturing the epidermis layer is carried out in a cell culture incubator, and the culture time is 1 to 2 days; Further, step (C) of culturing the full-thickness skin is carried out in a cell culture incubator, and the culture time is 10 to 14 days; Preferably, the gas environment in the cell culture incubator is 95% air and 5% CO2; the temperature in the cell culture incubator is 37°C.

[0014] An in vitro model of keloid disease provided by the present invention is mainly prepared by the construction method of the above in vitro model of keloid disease.

[0015] Further, the in vitro model of keloid disease includes: an epidermis layer and a dermis layer; The epidermis layer includes keratinocytes and has the disease characteristics of a typical stratum corneum morphology and thickening of the epidermis layer; the dermis layer includes skin fibroblasts and has the disease characteristics of fibroblast hyperplasia, transformation into myofibroblasts, collagen deposition in the extracellular matrix, and secretion of cytokine IL6.

[0016] Use of the above in vitro model of keloid disease provided by the present invention in the research on the pathogenic mechanism of keloid, drug development, and evaluation of treatment effects.

[0017] Compared with the prior art, the beneficial effects of the present invention are: The construction method of the keloid disease in vitro model provided by the present invention uses a cell culture chamber or a cell culture microfluidic chip to construct a full-thickness skin model with an epidermis layer and a dermis layer, and then uses TGF-β to induce and culture the full-thickness skin model to obtain a keloid disease in vitro model. The construction method of this application can effectively promote the keloid-like changes of the full-thickness skin model by selecting the concentration of TGF-β and the culture time during the induction process. The main characteristics of the keloid in vitro disease model obtained according to the construction method of the present invention are as follows: the epidermis layer thickens, the fibroblasts in the dermis layer proliferate, the collagen in the extracellular matrix of the dermis layer is excessively deposited, the mechanical strength increases, the expressions of inflammatory factors TNF-α, IL6, and MCP-1 increase, and the expression of fibrosis-related genes changes. This model can truly simulate the pathological state of keloids, reduce species differences and individual differences, greatly improve the authenticity and effectiveness of the model, and make up for the defect that the unique human disease of keloids cannot be effectively simulated by animal models.

[0018] At the same time, the keloid disease model reconstructed in vitro in this application has a more realistic skin structure, can simultaneously reflect the pathological changes of keloids in the dermis layer and the epidermis layer; and through TGF-β induction, it overcomes the problem that other in vitro models can only reflect the characteristics of early keloids and cannot effectively simulate the final characteristics of keloids formed in the case of a long disease course, making the model more authentic. The keloid disease in vitro model of the present invention can be widely used in the research on the pathogenesis of keloids, drug development, and evaluation of treatment effects.

[0019] In addition, this application uses human fibroblasts and human keratinocytes to prepare an in vitro model with a full-thickness skin structure. Therefore, the keloid disease in vitro model of this application has a human origin, reduces species differences and individual differences, greatly improves the authenticity and effectiveness of the model, and makes up for the defect that the unique human disease of keloids cannot be effectively simulated by animal models. Brief Description of the Drawings

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is a schematic structural diagram of the cell culture chamber and the microfluidic chip of the present invention; Figure 2 It is a tissue morphology comparison diagram between the keloid disease in vitro model prepared in Example 1 provided in Experimental Example 1 of this application and the control group; Figure 3a The comparative graph of Vimentin immunofluorescence staining for characterizing fibroblast proliferation between the keloid disease in vitro model prepared in Example 1 provided in Experimental Example 1 of this application and the control group; Figure 3b The comparative graph of immunofluorescence staining of myofibroblasts (α-SMA) for evaluating fibroblast fibrosis between the keloid disease in vitro model prepared in Example 1 provided in Experimental Example 1 of this application and the control group; Figure 4 The comparative graph of immunohistochemical detection between the keloid disease in vitro model prepared in Example 1 provided in Experimental Example 1 of this application and the control group; Figure 5 The comparative graph of ELISA determination of the content of IL-6 in the culture supernatant between the keloid disease in vitro model prepared in Example 1 provided in Experimental Example 1 of this application and the control group. Detailed implementation manners

[0022] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] According to one aspect of the present invention, a method for constructing a keloid disease in vitro model, the construction method includes: Using a cell culture insert or a cell culture microfluidic chip to construct a full-thickness skin model with an epidermis layer and a dermis layer, wherein the dermis layer is formed by mixing a keloid fibroblast suspension with a rigid substrate hydrogel precursor solution and culturing to form a gel; The Young's modulus of the rigid substrate hydrogel is 30 KPa to 200 kPa; The epidermis layer is formed by culturing keratinocytes on the dermis layer; The induction culture is air-liquid culture. On the epidermis layer side of the full-thickness skin model in the cell culture insert or the cell culture microfluidic chip is gas culture, and on the dermis layer side of the full-thickness skin model is liquid culture; During the air-liquid culture process, the culture medium for liquid culture on the dermis layer side is an induction medium containing TGF-β, and the concentration of TGF-β in the induction medium is 10 to 25 ng / mL; The induction culture is carried out in a cell culture incubator, and the induction culture time is 1 to 5 days to obtain a keloid disease in vitro model.

[0024] The construction method of the in vitro model of keloid disease provided by the present invention uses a cell culture insert or a cell culture microfluidic chip to construct a full-thickness skin model with an epidermis layer and a dermis layer, and then uses TGF-β to induce the culture of the full-thickness skin model to obtain an in vitro model of keloid disease, wherein: the induction culture is air-liquid culture, the epidermis layer side of the full-thickness skin model in the cell culture insert or the cell culture microfluidic chip is for gas culture, and the dermis layer side of the full-thickness skin model is for liquid culture; during the air-liquid culture process, the culture medium for liquid culture on the dermis layer side is an induction medium containing TGF-β, and the concentration of TGF-β in the induction medium is 10-25 ng / mL; the induction culture is carried out in a cell culture incubator, and the induction culture time is 1-5 days.

[0025] Through long-term research, the above construction method of the in vitro model of keloid disease obtained by selecting the concentration of TGF-β and the culture time during the induction process overcomes the problem that other in vitro models can only reflect the characteristics of early keloids and cannot effectively simulate the final characteristics of keloids formed under the condition of a long disease course, making the model more authentic. It has been verified that the in vitro model of keloid disease prepared by the above method can simulate the main pathological characteristics of keloid disease such as thickening of the epidermis and dermis layers of keloids, excessive proliferation of fibroblasts, changes in the components of the extracellular matrix, and secretion of inflammatory factors such as IL6.

[0026] It should be noted that when preparing the keloid dermal gel in this application, the Young's modulus of the selected rigid substrate hydrogel is 30 KPa - 200 kPa. The rigid substrate hydrogel with the above Young's modulus of 30 KPa - 200 kPa can significantly improve the mechanical strength and hardness after gelation and is suitable for the treatment scenario of keloids that require high-strength support.

[0027] That is, this application selects materials with relatively high hardness (Young's modulus between 30 KPa - 200 kPa) such as crosslinked gelatin (GelMA), PVA hydrogel, PLGA hydrogel, or polyurethane hydrogel. These hydrogel materials can better simulate the hardness and mechanical strength of keloids through different design strategies (such as double-network structure, nanocomposite, biomimetic structure, etc.).

[0028] It should be noted that the concentration of TGF-β in the above induction medium can be, but is not limited to, 10 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL, 20 ng / mL, 23 ng / mL, or 25 ng / mL, and can also be any value between 10 ng / m - 25 ng / mL. The induction culture time can be, but is not limited to, 1 day, 2 days, 3 days, 4 days, or 5 days, and can also be any time between 1 day - 5 days.

[0029] Specifically, the method for constructing the in vitro model of keloid disease includes: (1) Dermis construction: Mix the keloid fibroblast suspension with the hard substrate hydrogel precursor solution to obtain a cell hydrogel mixture; then add the cell hydrogel mixture into a cell culture chamber or a microfluidic chip to form a gel. After gel formation, use fibroblast medium for dermal layer culture to form a dermal matrix layer. Figure 1 FIG. is a schematic structural diagram of the cell culture chamber and microfluidic chip of the present invention. Among them: Figure 1 In (A) is the cell culture chamber; Figure 1 In (B) is the cell culture microfluidic chip; In a preferred embodiment of the present invention, the keloid fibroblast suspension in step (A) is a human fibroblast suspension, and the cell concentration of fibroblasts in the human fibroblast suspension is 1×10 6 cells / mL to 1×10 7 cells / mL; In a preferred embodiment of the present invention, the dermal layer culture in step (A) is carried out in a cell culture incubator, and the culture time is 3 to 7 days; The conditions of the cell culture incubator are: culture at 37°C and 5% CO2 for 3 to 7 days, and change the medium with fibroblast medium every other day for the growth of the dermal matrix layer.

[0030] It should be noted that the present invention has no special restrictions on the selection of fibroblast medium, and a basal medium supplemented with specific growth factors well-known to those skilled in the art can be used.

[0031] Preferably, the method for constructing the dermal layer includes: taking the 2nd to 6th generation primary human fibroblasts, mixing the cell suspension with the hard substrate hydrogel precursor solution, adding it to the upper chamber of a Transwell culture dish with a 24-well plate specification, and adding 50 - 150 μL of the cell hydrogel mixture to each well to form a gel. After gel formation, add fibroblast medium to the lower chamber of the Transwell culture dish, and also add 100 - 200 μL of fibroblast medium to the upper chamber, and culture at 37°C and 5% CO2 for 3 - 7 days, and change the medium every other day for the growth of the dermal matrix layer.

[0032] In a preferred embodiment of the present invention, the Young's modulus of the hard substrate hydrogel is 30 KPa to 200 kPa (referring to the strength of the hydrogel after gel formation without adding fibroblasts). The hard substrate hydrogel with the above Young's modulus of 30 KPa to 200 kPa can significantly improve the mechanical strength and hardness after gel formation, and is suitable for the treatment scenario of keloid that requires high-strength support.

[0033] (B) Epidermis layer construction: Add keratinocyte suspension onto the dermal matrix layer, and add keratinocyte subculture medium into the cell culture chamber or microfluidic chip for epidermis layer culture, so that keratinocytes form proliferating keratinocytes on the dermal matrix layer.

[0034] In a preferred embodiment of the present invention, the keratinocytes in step (B) are the 2nd to 6th passage primary human keratinocytes, and the concentration of keratinocytes in the keratinocyte suspension is 1.5×10 5 cells / cm 2 ~8×10 5 cells / cm 2 ; In a preferred embodiment of the present invention, the epidermis layer culture in step (B) is carried out in a cell culture incubator, and the culture time is 1 to 2 days; The conditions of the cell culture incubator are: culture at 37°C and 5% CO2 for 1 to 2 days for keratinocyte proliferation.

[0035] In a preferred embodiment of the present invention, the composition of the keratinocyte subculture medium includes: the medium is DMEM / F12 medium, supplemented with 10% bovine pituitary extract, 10 ng / mL epidermal growth factor (EGF), 0.4 μg / mL hydrocortisone, 1.8×10 −4 M (mol / L) adenine, 5 μg / mL insulin, 2×10 −9 M triiodothyronine, 5 μg / mL transferrin and 1% penicillin-streptomycin.

[0036] Preferably, the method for constructing the epidermis layer includes: aspirate the fibroblast medium in the hollow plate after the culture in step (A) above, add 0.8 - 1.2 mL of keratinocyte subculture medium in the lower layer, take the 2nd to 6th passage primary human keratinocytes, make a cell suspension with the subculture medium, add it above the dermal layer of the Transwell or microfluidic chip in step one above, add 100 - 200 uL of cell suspension to each well, add 0.8~1.2 mL of subculture medium in the lower layer, and place it in an incubator at 37°C and 5% CO2 for 1 to 2 days for keratinocyte proliferation.

[0037] (C) Full-thickness skin model construction: Aspirate the medium on the surface of keratinocytes in the cell culture chamber or microfluidic chip, and add air-liquid medium for full-thickness skin culture to form a full-thickness skin model with an epidermis layer and a dermal layer.

[0038] In a preferred embodiment of the present invention, the full-thickness skin culture in step (C) is carried out in a cell culture incubator, and the culture time is 10 to 14 days; The cell incubator conditions are as follows: Cultivate for 10 - 14 days under the conditions of 37°C and 5% CO₂, and change the medium with air-liquid medium every other day for keratinocyte differentiation.

[0039] In a preferred embodiment of the present invention, the air-liquid medium is a medium prepared by adding 10% fetal bovine serum, 10% bovine pituitary extract, 1 mM calcium chloride, 10 ng / mL epidermal growth factor (EGF), 0.4 μg / mL hydrocortisone, 1.8×10 −4 M adenine, 5 μg / mL insulin, 2×10 −9 M triiodothyronine, 5 μg / mL transferrin, and 1% penicillin-streptomycin to DMEM / F12 medium or DMEM medium.

[0040] Preferably, the method for constructing the full-thickness skin model includes: aspirating the upper-layer medium in the culture chamber in step (B), changing to gas culture, changing the lower-layer medium to 700 μL air-liquid medium, and continuously culturing under the conditions of 37°C and 5% CO₂ for 10 - 14 days, changing the medium every other day for keratinocyte differentiation, and in vitro recombining to form a full-thickness skin model with an epidermal layer and a dermal layer.

[0041] (D) TGF-β induced culture: The induced culture is air-liquid culture. The epidermal layer side of the full-thickness skin model in the cell culture chamber or the cell culture microfluidic chip is gas culture, and the dermal layer side of the full-thickness skin model is liquid culture; During the air-liquid culture process, the medium for liquid culture on the dermal layer side is an induction medium containing TGF-β, and the concentration of TGF-β in the induction medium is 10 - 25 ng / mL; The induced culture is carried out in a cell incubator, and the time of the induced culture is 1 - 5 days.

[0042] In a preferred embodiment of the present invention, the step (D) induced culture is carried out in a cell incubator; the cell incubator conditions are: cultivate for 1 - 5 days under the conditions of 37°C and 5% CO₂ to obtain an in vitro skin model with the phenotype of keloid disease.

[0043] In a preferred embodiment of the present invention, the TGF-β induction medium is prepared by adding a TGF-β inducer with a final concentration of 10 - 25 ng / mL on the basis of the above air-liquid medium.

[0044] Preferably, the TGF-β induction medium is: adding 10% fetal bovine serum, 10% bovine pituitary extract, 1 mM calcium chloride, 10 ng / mL epidermal growth factor (EGF), 0.4 μg / mL hydrocortisone, 1.8×10 −4M adenine, 5 μg / mL insulin, 2×10 −9 M triiodothyronine, 5 μg / mL transferrin, 1% penicillin-streptomycin, and an inducer of 10 - 25 ng / mL TGF-β.

[0045] According to one aspect of the present invention, an in vitro model of keloid disease, which is mainly prepared by the method for constructing the in vitro model of keloid disease as described above.

[0046] The in vitro model of keloid disease provided by the present invention is mainly prepared by the method for constructing the in vitro model of keloid disease as described above. The in vitro model of keloid disease has a human origin and has the physiological structure of the full-thickness skin with an epidermis layer and a dermis layer, where: the epidermis layer has the typical morphology of the stratum corneum and the disease characteristics of epidermal thickening; the dermis layer has the disease characteristics of fibroblast hyperplasia, transformation into myofibroblasts, collagen deposition in the extracellular matrix, and secretion of cytokine IL6.

[0047] Therefore, the in vitro model of keloid disease in this application can simulate the main pathological phenotypes such as thickening of the epidermis and dermis layers of keloid, excessive proliferation of fibroblasts, changes in the composition of the extracellular matrix, and secretion of inflammatory factors such as IL6, and can be used for the mechanism research and drug development research of keloid.

[0048] According to one aspect of the present invention, the application of the above in vitro model of keloid disease in the research of the pathogenic mechanism of keloid, drug development, and evaluation of treatment effects.

[0049] The in vitro model of keloid disease provided by the present invention can be widely applied in the research of the pathogenic mechanism of keloid, drug development, and evaluation of treatment effects.

[0050] Next, the technical solutions of the present invention will be further described in conjunction with the embodiments.

[0051] Taking the Transwell cell culture insert (Corning 3470) as an example, the following embodiments illustrate the method for constructing the in vitro model of keloid disease in this application.

[0052] The list of abbreviations, English, and definitions of key terms in the following embodiments is as follows: 1. Fibroblasts (Fb): Fibroblasts.

[0053] 2. Keratinocytes (Kc): Keratinocytes.

[0054] 3. ECM: Extracellular matrix.

[0055] 4. TGF-β: Transforming growth factor β.

[0056] 5, HE: Hematoxylin and Eosin.

[0057] Example 1 An in vitro model of keloid disease, and the construction method of the in vitro model of keloid disease includes: (I). In vitro construction of the dermis layer: 1. Take human fibroblasts cultured to passages P2 - P6, wash twice with buffer, digest with 2 mL of Digestive Solution I for 2 min, terminate digestion with 2 - fold volume of buffer, count simultaneously, centrifuge at 1000 rpm for 5 min, remove the supernatant, and obtain a cell suspension.

[0058] 2. Mix the matrix hydrogel (type I collagen hydrogel) with fibroblasts (operate on ice). In a 120 μL dermal gel system, each well contains 80 μL of gel + 20 mL of fibroblast medium + 20 μL of cell suspension.

[0059] 3. Add 120 μL of the cell hydrogel solution to the upper chamber of a Transwell cell culture insert with a 24 - well plate specification. After pre - cooling the plate on an ice box, add the liquid, and transfer it to a 37 °C incubator for 30 min.

[0060] 4. Add 1 mL of PBS buffer to the lower chamber. After adding 200 μL to the upper chamber for rinsing once, add 200 μL to the upper chamber and 1 mL of fibroblast medium to the lower chamber for dermal layer culture.

[0061] The dermal layer culture is carried out in a cell incubator, and the gas environment in the cell incubator is 95% air and 5% CO2, and the temperature is 37 °C; The culture time of the dermal layer culture is 5 days, and the medium is changed every other day.

[0062] The cell concentration of fibroblasts in the human fibroblast suspension is 2×10 6 cells / ml; The fibroblast medium is DMEM medium supplemented with 10% FBS.

[0063] The matrix hydrogel is a rigid substrate hydrogel with a Young's modulus of 30 KPa - 200 kPa.

[0064] (II). In vitro construction of the epidermis layer: 1. Aspirate the fibroblast medium in the well plate, and add 1 mL of sub - liquid medium to the lower layer.

[0065] 2. Digest keratinocytes, resuspend keratinocytes with sub - liquid medium, and dilute to a certain concentration of cell suspension after counting.

[0066] 3. Add 200 μL of keratinocyte suspension to the upper layer of each well, and add 1 mL of keratinocyte subculture medium to the lower layer for epidermal layer culture.

[0067] The epidermal layer culture is carried out in a cell incubator. The gas environment in the cell incubator is 95% air and 5% CO2, and the temperature is 37 °C; the culture time of the epidermal layer culture is 1 day, and the medium is changed every other day.

[0068] The concentration of keratinocytes in the keratinocyte suspension is 5×10 5 cells / cm 2 ; The keratinocyte subculture medium is DMEM / F12 medium supplemented with 10% bovine pituitary extract, 10 ng / mL epidermal growth factor (EGF), 0.4 μg / mL hydrocortisone, 1.8×10 −4 M adenine, 5 μg / mL insulin, 2×10 −9 M triiodothyronine, 5 μg / mL transferrin and 1% penicillin-streptomycin.

[0069] (III). In vitro construction of the full-thickness skin model: Aspirate the upper-layer medium, replace the lower-layer medium with 600 μL of air-liquid medium, keep the upper layer dry, and carry out full-thickness skin culture to form a full-thickness skin model with an epidermal layer and a dermal layer.

[0070] The full-thickness skin culture is carried out in a cell incubator, and the culture time is 14 days; the gas environment in the cell incubator is 95% air and 5% CO2; the temperature in the cell incubator is 37 °C.

[0071] The air-liquid medium is: DMEM / F12 medium supplemented with 10% fetal bovine serum, 10% bovine pituitary extract, 1 mM calcium chloride, 10 ng / mL epidermal growth factor (EGF), 0.4 μg / mL hydrocortisone, 1.8×10 −4 M adenine, 5 μg / mL insulin, 2×10 −9 M triiodothyronine, 5 μg / mL transferrin and 1% penicillin-streptomycin.

[0072] (IV). Induction of the keloid disease model: On the basis of the full-thickness skin model in step (III), replace the lower-layer air-liquid medium with TGF-β induction medium, maintain air-liquid culture, and induce culture in a cell incubator for 5 days to harvest the in vitro model of the keloid disease.

[0073] The gas environment in the cell incubator is 95% air and 5% CO2; the temperature in the cell incubator is 37 °C.

[0074] The TGF-β induction medium is: DMEM / F12 medium supplemented with 10% fetal bovine serum, 10% bovine pituitary extract, 1 mM calcium chloride, 10 ng / mL epidermal growth factor (EGF), 0.4 μg / mL hydrocortisone, 1.8×10 −4 M adenine, 5 μg / mL insulin, 2×10 −9 M triiodothyronine, 5 μg / mL transferrin, 1% penicillin-streptomycin, and 15 ng / mL TGF-β inducer.

[0075] The TGF-β inducer is MCE HY-P70543.

[0076] Example 2 The in vitro model of keloid disease in this example is the same as that in Example 1, except that in step (iv) of the construction method, the concentration of the TGF-β inducer in the TGF-β induction medium is 10 ng / mL TGF-β inducer.

[0077] Example 3 The in vitro model of keloid disease in this example is the same as that in Example 1, except that in step (iv) of the construction method, the concentration of the TGF-β inducer in the TGF-β induction medium is 25 ng / mL TGF-β inducer.

[0078] Comparative Example 1 The in vitro model of keloid disease in this example is the same as that in Example 1, except that in step (iv) of the construction method, the concentration of the TGF-β inducer in the TGF-β induction medium is 5 ng / mL TGF-β inducer.

[0079] Comparative Example 2 The in vitro model of keloid disease in this example is the same as that in Example 1, except that in step (iv) of the construction method, the concentration of the TGF-β inducer in the TGF-β induction medium is 30 ng / mL TGF-β inducer.

[0080] Comparative Example 3 The in vitro model of keloid disease in this example is the same as that in Example 1, except that in step (iv) of the construction method, the induction culture time in the cell culture incubator is 10 days.

[0081] Experimental Example 1 To demonstrate that the in vitro model of keloid disease prepared in this application can simulate the main pathological phenotypic characteristics of keloid diseases, such as thickening of the epidermis and dermis, over-proliferation of fibroblasts, changes in extracellular matrix components, and secretion of inflammatory factors such as IL6.

[0082] Now, phenotypic identification is performed on the in vitro model of keloid disease prepared in Example 1, as follows: (1) Tissue morphology: The epidermal layer thickening was evaluated by HE staining of pathological sections ( Figure 2 ) Figure 2 This is a comparison diagram of the tissue morphology between the keloid disease in vitro model prepared in Example 1 of this application and the control group.

[0083] Note: The control group in this experimental example and each attached figure is the full-thickness skin model constructed in step (iii) of Example 1, that is, the control group is the full-thickness skin model without undergoing the TGF-β induction treatment in step (iv). The scar group is the keloid disease in vitro model prepared in Example 1.

[0084] (2) Fibroblast proliferation and phenotypic conversion: Fibroblast proliferation was characterized by Vimentin immunofluorescence staining, and the presence of fibroblast fibrosis was evaluated by myofibroblast (α-SMA) immunofluorescence staining; Figure 3a This is a comparison diagram of fibroblast proliferation characterized by Vimentin immunofluorescence staining between the keloid disease in vitro model prepared in Example 1 of this application and the control group.

[0085] Figure 3b This is a comparison diagram of the evaluation of fibroblast fibrosis by myofibroblast (α-SMA) immunofluorescence staining between the keloid disease in vitro model prepared in Example 1 of this application and the control group.

[0086] (3) ECM components: Immunohistochemical detection of procollagen type I (ProCOL-1) and immunofluorescent detection of collagen type III (Col III) showed increased expression, and the ratio of type I / III collagen changed ( Figure 4 ) Figure 4 This is a comparison diagram of immunohistochemical detection between the keloid disease in vitro model prepared in Example 1 of this application and the control group.

[0087] (4) Inflammatory factors: ELISA determination showed an upregulation of IL-6 in the culture supernatant ( Figure 5 ) Figure 5 This is a comparison diagram of the content of IL-6 in the culture supernatant determined by ELISA between the keloid disease in vitro model prepared in Example 1 of this application and the control group.

[0088] Experimental Example 2 In this experimental example, the keloid disease in vitro models prepared in Examples 2 and 3 of this application and Comparative Examples 1 to 3 were subjected to phenotype identification (the specific characterization method was the same as that in Experimental Example 1), and the results were as follows: (1) Tissue morphology: The in vitro models of keloid diseases prepared in Examples 2 and 3 of the present application have tissue morphology similar to that in Example 1. HE staining of pathological sections shows that the epidermal layer thickens in a dose-dependent manner; while in Comparative Example 1, there is no obvious thickening; in Comparative Example 2, there is excessive thickening; and in Comparative Example 3, the stratum corneum shows a loose state.

[0089] (2) Proliferation and phenotypic transformation of fibroblasts: The in vitro models of keloid diseases prepared in Examples 2 and 3 of the present application have the same tissue morphology as that in Example 1, and the proliferation of fibroblasts is obvious. While in Comparative Example 1, the fibroblasts do not proliferate significantly; in Comparative Example 2, the fibroblasts have excessive proliferation with poor state; and in Comparative Example 3, the fibroblasts show death.

[0090] (3) ECM components: The in vitro models of keloid diseases prepared in Examples 2 and 3 of the present application have the same tissue morphology as that in Example 1. Immunohistochemical detection of procollagen type I (ProCOL-1) and immunofluorescence detection of collagen type III (Col III) both show increased expression; while in Comparative Example 1, there is no obvious increase in expression; in Comparative Example 2, the expression increases significantly; and in Comparative Example 3, there is no obvious increase in expression.

[0091] (4) Inflammatory factors: The in vitro models of keloid diseases prepared in Examples 2 and 3 of the present application have the same tissue morphology as that in Example 1, and ELISA determination shows an up-regulation of IL-6 in the culture supernatant; while in Comparative Example 1, there is no obvious up-regulation; in Comparative Example 2, there is obvious up-regulation; and in Comparative Example 3, there is obvious up-regulation.

[0092] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an in vitro model of keloid disease, characterized in that, The construction method includes: Using a cell culture chamber or a cell culture microfluidic chip to construct a full-thickness skin model with an epidermis and a dermis. Among them, the dermis is formed by mixing a keloid fibroblast suspension with a rigid substrate hydrogel precursor solution and culturing to form a gel. The Young's modulus of the rigid substrate hydrogel is 30 KPa to 200 kPa. The epidermis is formed by culturing keratinocytes on the dermis. Subsequently, TGF-β is used to induce the culture of the full-thickness skin model to obtain an in vitro model of keloid disease. The induction culture is air-liquid culture. On the epidermis side of the full-thickness skin model in the cell culture chamber or the cell culture microfluidic chip is gas culture, and on the dermis side of the full-thickness skin model is liquid culture. During the air-liquid culture process, the culture medium for liquid culture on the dermis side is an induction medium containing TGF-β, and the concentration of TGF-β in the induction medium is 10 to 25 ng / mL. The induction culture is carried out in a cell culture incubator, and the induction culture time is 1 to 5 days to obtain an in vitro model of keloid disease.

2. The method for constructing an in vitro model of keloid disease according to claim 1, wherein The gas environment in the cell culture incubator is 95% air and 5% CO2; the temperature in the cell culture incubator is 37°C.

3. The method for constructing an in vitro model of keloid disease according to claim 1, wherein The construction method of the full-thickness skin model includes: (A) Dermis construction: Mix a keloid fibroblast suspension with a rigid substrate hydrogel precursor solution to obtain a cell hydrogel mixture. Subsequently, add the cell hydrogel mixture into a cell culture chamber or a microfluidic chip to form a gel. After gel formation, use a fibroblast culture medium to culture the dermis to form a dermal matrix layer. (B) Epidermis construction: Add a keratinocyte suspension onto the dermal matrix layer, and add a keratinocyte liquid medium into the cell culture chamber or the microfluidic chip to culture the epidermis, so that keratinocytes form proliferating keratinocytes on the dermal matrix layer. (C) Full-thickness skin model construction: Aspirate the culture medium on the surface of the keratinocytes in the cell culture chamber or the microfluidic chip, and add an air-liquid culture medium to culture the full-thickness skin to form a full-thickness skin model with an epidermis and a dermis.

4. The method for constructing an in vitro model of keloid disease according to claim 3, wherein The keloid fibroblast suspension in step (A) is a human fibroblast suspension, and the cell concentration of fibroblasts in the human fibroblast suspension is 0.5×10 6 cells / mL to 2×10 7 cells / mL; Preferably, the human fibroblasts are the 2nd to 6th generation primary human fibroblasts.

5. The method for constructing an in vitro model of keloid disease according to claim 3, wherein, The density of keratinocytes in the keratinocyte suspension in step (B) is 1.5×10 5 cells / cm 2 ~8×10 5 cells / cm 2 ; Preferably, the keratinocytes are the 2nd to 6th generation primary human keratinocytes.

6. The method for constructing an in vitro model of keloid disease according to claim 3, wherein, The dermis culture in step (A) is carried out in a cell culture incubator, and the culture time is 3 to 7 days. And / or, the epidermis culture in step (B) is carried out in a cell culture incubator, and the culture time is 1 to 2 days. And / or, the full-thickness skin culture in step (C) is carried out in a cell culture incubator, and the culture time is 10 to 14 days. Preferably, the gas environment in the cell culture incubator is 95% air and 5% CO2; the temperature in the cell culture incubator is 37°C.

7. An in vitro model of keloid disease, characterized in that, The in vitro model of keloid disease is mainly prepared by the construction method of the in vitro model of keloid disease according to any one of claims 1 to 6.

8. The in vitro model of keloid disease according to claim 7, wherein The in vitro model of keloid disease includes: an epidermis and a dermis. The epidermis layer includes keratinocytes and has the disease characteristics of the typical morphology of the stratum corneum and thickening of the epidermis layer; the dermis layer includes skin fibroblasts and has the disease characteristics of fibroblast hyperplasia, transformation into myofibroblasts, collagen deposition in the extracellular matrix, and secretion of cytokine IL6.

9. Use of the keloid disease in vitro model according to claim 7 or 8 in the research on the pathogenic mechanism of keloid, drug development, and evaluation of treatment effects.

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