Skin model with controllable inflammation degree and construction method and application thereof

Abstract

The invention discloses a skin model with a controllable inflammation degree and a construction method and application thereof. The method comprises the following steps: inducing pluripotent stem cells to form keratinocytes, determining an induction condition range of an inflammation induction source in an optimal induction time range, and carrying out inflammation induction on the keratinocytes to obtain inflammatory keratinocytes under different induction conditions; according to the requirement of the inflammation degree, the inflammation keratinocytes and the normal keratinocytes are mixed in proportion, and a skin model corresponding to the inflammation degree is constructed. The model is constructed by inducing keratinocyte inflammation and then accurately regulating and controlling the mixing ratio of inflammation to normal keratinocytes. The method breaks through the traditional limitation that the normal skin model is firstly constructed and then inflammation is induced, the operation steps are simplified, the construction time is greatly shortened, and the construction efficiency is improved. Meanwhile, the proportion of inflammatory cells to normal cells can be changed, skin models with different inflammatory degrees can be precisely molded, and diversified research requirements are met.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering, and relates to an in vitro three-dimensional evaluation skin model, and in particular to a skin model with controllable inflammation degree, and a construction method and application thereof. Background Art

[0002] The skin is the largest organ in the human body. It not only serves as a protective barrier for the body, but also participates in various physiological processes, such as temperature regulation, sensory perception, and immune defense. However, the skin can also be affected by various internal and external factors and cause inflammatory reactions, such as ultraviolet radiation, chemical stimulation, bacterial infection, etc. Therefore, studying the occurrence and development mechanism of skin inflammation and finding effective treatment strategies are of great significance for maintaining skin health.

[0003] With the development of biological 3D printing technology, researchers have begun to use this technology to construct complex tissue and organ models to conduct research in a way that is closer to the real physiological environment. At the same time, the construction technology of inflammation models is also constantly improving in order to provide more accurate inflammation research models. The existing schemes for constructing inflammatory skin models are mainly in two ways: one is the cell inflammation model, that is, by culturing skin cells in vitro and adding specific inflammatory mediators to produce an inflammatory response; the other is to first construct a normal 3D skin model and then add pro-inflammatory agents to induce the normal skin model into an inflammatory skin model. Although the existing technology has solved the problem of constructing an inflammatory skin model to a certain extent, there are still at least the following limitations: First, although the cell inflammation model can simulate the inflammatory response, it uses a flat culture, which is quite different from the real in vivo culture environment, which affects the reliability of the experimental results. Secondly, the method of first constructing a normal 3D skin model and then inducing it into an inflammatory skin model is complicated and time-consuming. It is necessary to first construct and culture a normal 3D skin model, and then add pro-inflammatory factors to form inflammation after a period of time. Finally, the degree of inflammation is judged by tissue structure or inflammatory factors, and the process of inflammation and the degree of inflammation cannot be accurately controlled. Summary of the invention

[0004] Based on the deficiencies of the prior art, the present invention provides a skin model with controllable inflammation degree and a construction method and application thereof.

[0005] In a first aspect, the present invention provides a method for constructing a skin model with controllable inflammation degree, the method comprising the following steps: Step (1), inducing keratinocytes from pluripotent stem cells, and then performing cell passaging and amplification; Step (2), determining the induction condition range of the inflammation induction source within the optimal induction time range; Step (3), inducing inflammation on keratinocytes according to the induction condition range of the inflammatory induction source within the optimal induction time range, to obtain inflammatory keratinocytes under different induction conditions; according to the requirements of the degree of inflammation, the inflammatory keratinocytes and normal keratinocytes are mixed in proportion to construct a skin model corresponding to the degree of inflammation.

[0006] Preferably, the keratinocytes induced to differentiate in step (1) are polygonal, with clear cytoplasm, large nuclei containing 1-2 nucleoli, and have a typical cobblestone shape after the cells grow into a single layer.

[0007] Preferably, the inflammation inducing source in step (2) includes at least one of cytokines, pathogen-associated molecular patterns, chemical irritants, and ultraviolet radiation.

[0008] Preferably, if the inflammation inducing source is a chemical irritant or a cytokine, step (2) is specifically: Chemical irritants or cytokines were formulated into inflammation-inducing solutions at different concentrations; Different concentrations of inflammation induction solutions were used to induce inflammatory responses in the passaged and expanded keratinocytes, and the cell survival rates under treatment with different concentrations of inflammation induction solutions at different induction times were calculated; Determine whether the inflammatory keratinocytes after inflammation induction treatment have the conditions for inflammatory response, screen out inflammatory keratinocytes that have the conditions for inflammatory response and whose cell survival rate is ≥a at each induction time, and obtain the corresponding concentration range of inflammation induction solution; 0%<a<100%.

[0009] Preferably, if the inflammation inducing source is ultraviolet radiation, step (2) is specifically: The passaged and expanded keratinocytes were irradiated with ultraviolet radiation of different intensities to induce inflammatory response in the cells, and the cell survival rate under ultraviolet radiation treatment of different intensities at different induction times was calculated; Whether the inflammatory keratinocytes after inflammation induction treatment have the conditions for inflammatory response, the inflammatory keratinocytes with the conditions for inflammatory response and cell survival rate ≥ a at each induction time are screened out, and the corresponding ultraviolet irradiation intensity is obtained; 0%<a<100%.

[0010] Preferably, the conditions for the occurrence of inflammatory response include at least one condition: (1) Cell morphological changes; (2) Expression of inflammatory factors; (3) Changes in oxidative stress indicators; (4) Expression of genes or proteins related to inflammation.

[0011] Preferably, the mixing process of the inflammatory human keratinocytes and the normal human keratinocytes in step (3) is to add the inflammatory keratinocytes and the normal keratinocytes into the nozzle of the biological 3D droplet printer, and uniformly inoculate the mixed cells in an insert cell culture vessel containing a semipermeable membrane in the form of droplet printing through a preset spiral printing path, and then place the insert cell culture vessel in each culture well of a cell culture plate for culture, wherein the insert cell culture vessel and each culture well of the cell culture plate contain culture medium; after each culture period, the culture medium in the insert cell culture vessel is removed, and only fresh culture medium is added to the culture wells of the cell culture plate, and the culture is continued for a period of time, during which the fresh culture medium is replaced every day, thereby obtaining an inflammatory skin model.

[0012] Preferably, in step (3), if the skin model is a mild inflammation model, the ratio of the number of inflammatory keratinocytes to normal keratinocytes is: 1:500-1:100; If the skin model is a moderate inflammation model, the ratio of inflammatory keratinocytes to normal keratinocytes is: 1:99~1:50; If the skin model is a severe inflammation model, the ratio of inflammatory keratinocytes to normal keratinocytes is 1:49~1:10.

[0013] In a second aspect, the present invention provides a skin model with controllable inflammation level, which is constructed using the above method.

[0014] In a third aspect, the present invention provides the use of the above skin model in evaluating the soothing or barrier repair function of cosmetic raw materials or formulas, and the anti-inflammatory function of drugs.

[0015] Beneficial effects of the present invention: The present invention provides a method for constructing a skin model with controllable inflammation degree, which innovatively induces keratinocyte inflammation first, and then accurately regulates the mixing ratio of inflammation and normal keratinocytes to construct the model. This method breaks through the limitation of the traditional method of first constructing a normal skin model and then inducing inflammation, simplifies the operation steps, greatly shortens the construction time, improves the construction efficiency, and provides a more convenient way for skin inflammation research. At the same time, by changing the ratio of inflammatory cells to normal cells, skin models with different inflammation degrees can be accurately shaped to meet the diverse research needs. This precise control ability lays a solid foundation for in-depth exploration of the mechanism of inflammation occurrence and development, the mechanism of drug anti-inflammatory action, and the evaluation of cosmetic efficacy, making the research more in-depth and broad, and the results more accurate and reliable. In addition, the tissue morphology and inflammatory factor release characteristics of the inflammatory skin model obtained by the present invention are significant and stable. The tissue sections show typical inflammatory microstructures such as orderly lowering of the basal layer, nuclear pyknosis cells, and thinning of the cell layer; the skin model stably releases a large amount of inflammatory factors such as PGE2, IL-1α, IL-8, etc., highly simulates the real inflammatory skin state, and effectively improves the practical value and simulation accuracy of the model in skin inflammation related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 It is a flow chart of the method provided by an embodiment of the present invention.

[0018] Figure 2 This is an H&E staining image of a normal 3D skin model tissue section in Example 1.

[0019] Figure 3 This is an H&E staining image of the tissue section of the inflammatory skin model constructed in Example 1.

[0020] Figure 4 This is an H&E staining image of the tissue section of the inflammatory skin model constructed in Example 3.

[0021] Figure 5 This is an H&E staining image of the tissue section of the inflammatory skin model constructed in Example 5. DETAILED DESCRIPTION

[0022] As can be seen from the background technology, the traditional approach to constructing an inflammatory skin model has many limitations. Although the cell inflammation model can simulate the inflammatory response, its planar culture mode is significantly different from the real environment in the body, which greatly reduces the reliability of the experimental results. The method of first constructing a normal 3D skin model and then inducing inflammation is not only cumbersome and time-consuming, but also incapable of controlling the process and degree of inflammation, making it difficult to accurately control.

[0023] In this regard, the present invention proposes to induce keratinocytes into inflammatory cells before constructing a skin model with controllable inflammation degree, and to construct an inflammatory skin model with different inflammation degrees by controlling the mixing ratio of inflammatory cells and normal keratinocytes. Compared with first constructing a normal skin model and then inducing inflammation in the skin model, the operation is simpler, less time-consuming, and a more accurate inflammatory skin model with adjustable inflammation degree can be constructed. It effectively breaks through the shackles of the traditional construction paradigm, greatly simplifies the operation process, significantly shortens the construction time, greatly improves the construction efficiency, and opens up a new path for in-depth research on skin inflammation.

[0024] like Figure 1 As shown, the method comprises the following steps: Step (1), using pluripotent stem cells to induce keratinocytes, and then performing cell passaging and expansion.

[0025] Step (2), determining the induction condition range of the inflammatory induction source within the optimal induction time range.

[0026] Step (3), inducing inflammation on keratinocytes according to the induction condition range of the inflammatory induction source within the optimal induction time range, to obtain inflammatory keratinocytes under different induction conditions; according to the requirements of the degree of inflammation, the inflammatory keratinocytes and normal keratinocytes are mixed in proportion to construct a skin model corresponding to the degree of inflammation.

[0027] For example, the pluripotent stem cell in step (1) refers to a type of stem cell with self-renewal and multi-differentiation potential, which can be accurately transformed into a specific cell type under appropriate conditions. The pluripotent stem cells include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), human pluripotent stem cells, etc. In this example, commercial induced pluripotent stem cells (iPSCs) purchased by Zhongsheng Tracing Company were used to induce differentiation of keratinocytes.

[0028] For example, the keratinocytes induced to differentiate in step (1) are polygonal, with clear cytoplasm, large nuclei containing 1-2 nucleoli, and have a typical cobblestone shape after the cells grow into a single layer.

[0029] For example, the inflammation inducing source in step (2) includes at least one of cytokines, pathogen-associated molecular patterns (PAMPs), chemical irritants, and ultraviolet radiation; wherein: The cytokines are a class of protein molecules produced and secreted by cells, and their main function is to regulate signal transmission between cells. They are involved in many important biological processes, including immune response, cell growth, cell differentiation, inflammatory response and cell death. They mainly include interleukins, interferons, tumor necrosis factors, growth factors and chemical factors. In the process of inducing inflammatory response, one or more combinations of interleukins, tumor necrosis factors and chemical factors are commonly used.

[0030] The pathogen-associated molecular patterns (PAMPs) are molecular structures carried by pathogens such as bacteria, viruses, and fungi that can be recognized by the host immune system. These molecular patterns are commonly found in pathogens, but are usually absent in host cells, making them important targets for the immune system to attack pathogens. The presence of PAMPs can activate the host's immune response, including inflammatory responses.

[0031] The chemical irritant can be a single compound, such as sodium dodecyl sulfate (SDS), lipopolysaccharide (LPS), or a complex compound. Of course, the compound here can also be a drug.

[0032] If the inflammatory induction source is a chemical irritant or a cytokine, step (2) is specifically: Chemical irritants or cytokines were formulated into inflammation-inducing solutions at different concentrations; Different concentrations of inflammation induction solutions were used to induce inflammatory responses in the passaged and expanded keratinocytes, and the cell survival rates under treatment with different concentrations of inflammation induction solutions at different induction times were calculated; Determine whether the inflammatory keratinocytes after inflammation induction treatment have the conditions for inflammatory response, screen out the inflammatory keratinocytes that have the conditions for inflammatory response and whose cell survival rate is ≥ a at each induction time, and obtain the corresponding concentration range of inflammation induction solution; 0%<a<100%; If the inflammation inducing source is ultraviolet radiation, step (2) is specifically: The passaged and expanded keratinocytes were irradiated with ultraviolet radiation of different intensities to induce inflammatory response in the cells, and the cell survival rate under ultraviolet radiation treatment of different intensities at different induction times was calculated; After the inflammation induction treatment, the inflammatory keratinocytes were tested to determine whether they had the conditions for the occurrence of an inflammatory response. The inflammatory keratinocytes that had the conditions for the occurrence of an inflammatory response and whose cell survival rate was ≥ a at each induction time were screened out to obtain the corresponding ultraviolet irradiation intensity; 0%<a<100%; The conditions for the occurrence of the inflammatory response include at least one condition: (1) Cell morphological changes; (2) Expression of inflammatory factors; (3) Changes in oxidative stress indicators; (4) Expression of genes or proteins related to inflammation; For example, the inflammatory factors include interleukins, tumor necrosis factor, prostaglandins, etc., specifically IL-1α, IL-8, PGE2; If the inflammatory response conditions are expressed by inflammatory factors, the concentration of the inflammatory induction solution with a cell survival rate ≥ a and inflammatory factor statistical analysis P ≤ b at each induction time is selected as the concentration range for subsequent induction of keratinocyte inflammatory response, 0 < b < 1.

[0033] For example, the optimal induction time in step (2) is in the range of 24-72 hours.

[0034] For example, in step (3), according to the degree of inflammation, inflammatory keratinocytes and normal keratinocytes are mixed in proportion to construct a skin model corresponding to the degree of inflammation, specifically: In the model of mild inflammation, the ratio of inflammatory keratinocytes to normal keratinocytes is 1:500-1:100; In the moderate inflammation model, the ratio of inflammatory keratinocytes to normal keratinocytes is 1:99-1:50; In the model of severe inflammation, the ratio of inflammatory keratinocytes to normal keratinocytes is 1:49~1:10.

[0035] The main differences between skin models with different degrees of inflammation are: (1) Different thickness: The cell layer thickness of the skin model with high inflammation level is thinner.

[0036] (2) The arrangement of basal cells is different: the higher the degree of inflammation, the more chaotic the arrangement of basal cells.

[0037] (3) The number of cells with karyopyknosis is different: the higher the degree of inflammation, the greater the number of cells with karyopyknosis.

[0038] (4) Different levels of inflammatory factors: The higher the degree of inflammation, the higher the level of inflammatory factors secreted by the skin model.

[0039] For example, the mixing process of inflammatory human keratinocytes and normal human keratinocytes in step (3) is to add inflammatory keratinocytes and normal keratinocytes into the nozzle of a biological 3D droplet printer, and uniformly inoculate the mixed cells into an insert cell culture vessel containing a semipermeable membrane in the form of droplet printing through a preset spiral printing path, and then place the insert cell culture vessel in each culture well of a cell culture plate for initial immersion culture, wherein the insert cell culture vessel and each culture well of the cell culture plate contain culture medium; after each culture period, the culture medium in the insert cell culture vessel is removed, and only fresh culture medium is added to the culture wells of the cell culture plate, and the secondary culture is continued for a period of time, during which fresh culture medium is replaced every day, thereby obtaining an inflammatory skin model.

[0040] Specifically, the mixed cell seeding density was 3~9×10 5 Pieces / cm 2 The droplet printing parameters are: air pressure 0.05~0.15Mpa, speed 1~3mm / s, injection cycle 80~150ms, and valve opening time 100~400μs.

[0041] The initial immersion culture time is 2-3 days, and the immersion medium is KCs cell culture medium with filtered CaCl2 solution added to the final concentration of 0.08-0.8 mM. The secondary culture time is 5-10 days, and the culture medium is KCs cell culture medium with filtered CaCl2 solution added to the final concentration of 0.9-2.0 mM, and VC solution is added to the final concentration of 10-50 μg / mL.

[0042] The skin model prepared by the present invention can be characterized by barrier function. The higher the degree of inflammation, the worse the barrier function of the skin model, and the expression of various proteins related to the barrier function will decrease.

[0043] The barrier function of the skin refers to the ability of the skin to prevent harmful external substances (such as microorganisms, chemicals, ultraviolet rays, etc.) from entering the body, while preventing excessive loss of water and nutrients in the body. It is mainly caused by lipids and proteins secreted by the skin. Inflammatory reactions can lead to reduced lipid synthesis in the skin model and damage the junction proteins between keratinocytes, thereby weakening the barrier function. The embodiment of the present invention can reflect the barrier function of the skin model through the half effective time (ET50). The half effective time (ET50) refers to the time required for a certain concentration of a substance to reduce cell activity by 50%.

[0044] In addition, this embodiment also provides the application of the above-mentioned skin model with controllable inflammation level in evaluating the soothing or barrier repair function of cosmetic raw materials or formulas, as well as the anti-inflammatory function of drugs.

[0045] Based on this, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0046] Embodiment 1: like Figure 1 As shown, the embodiment of the present invention provides one of the methods for constructing a 3D skin model with controllable inflammation degree, taking iPSCs-induced differentiation of adult keratinocytes (KCs) as an example, specifically: Step (1) using iPSCs to induce differentiation of adult keratinocytes, the specific steps are as follows: Step 1-1, Matrigel culture plate coating: Thaw Matrigel overnight in a 4°C refrigerator and dilute Matrigel with refrigerated DMEM / F12 medium to a coating concentration of 0.01 mg / cm 2 The coated culture plate was placed at room temperature for at least 1 hour before use.

[0047] Matrigel is derived from the extracellular matrix protein extracts in mouse tumors. Its main components include key substances such as laminin, collagen IV, nestin, and heparin sulfate proteoglycans. While providing an ideal adherent growth environment for iPSCs, it accurately receives induced differentiation signals, strongly promotes cell differentiation into KCs and stabilizes cell functional status. Its components interact exquisitely with cell surface receptors, activate intracellular signaling pathways, accurately regulate cell physiological activities and gene expression, and make the behavior and phenotype of cells in in vitro culture highly close to the real state in vivo.

[0048] Step 1-2, recovery and seeding of iPSCs: The temperature of the thermostatic water bath was adjusted to 37°C, and the iPSCs complete culture medium was placed at room temperature 30 min in advance. The iPSCs complete culture medium contained: DMEM / F12, 15% knockout serum replacement, 1% glutamine, 1% NEAA, 1×10 -4 M2-ME, 4 ng / mL bFGF.

[0049] Take out the iPSCs cells to be revived from liquid nitrogen, place them in a 37°C water bath and shake gently until the ice crystals in the cell suspension completely disappear. Add the thawed cell suspension to a 15mL centrifuge tube, add 10mL DMEM / F12 culture medium drop by drop, and gently shake the centrifuge tube to mix. Place in a centrifuge, centrifuge at 1000rpm for 5min. After centrifugation, discard the supernatant, add 4mL of iPSCs complete culture medium containing Blebbistatin to resuspend, and centrifuge at 3×10 4 Cells / cm 2Inoculate into the wells of a culture plate.

[0050] Steps 1-3, induction of iPSCs into KCs: On the second day after inoculation, the iPSCs complete medium was replaced with differentiation medium, which contained: DMEM / F12, 1% NEAA, 0.4% BSA, 0.5 μg / mL hydrocortisone, 5 μg / mL NKH744, 10 ng / mL EGF, 8 μg / mL transferrin, and 20 μg / mL insulin.

[0051] Steps 1-4, Cultivation: Fresh differentiation medium was replaced every day, and cells were passaged when the confluence reached about 90%.

[0052] Steps 1-5: Continue to culture with differentiation medium after subculturing. After 4-5 subculturings, successfully differentiated KCs can be obtained.

[0053] The keratinocytes that have been successfully induced to differentiate are polygonal, with clear cytoplasm and large nuclei containing 1-2 nucleoli. After the cells have grown into a single layer, they have a typical paving stone shape. Cytokeratin-18 (CK18) is a positive marker for keratinocytes, and the cells that have been successfully induced to differentiate are positive for CK18 immunofluorescence staining.

[0054] Step (2), passage and expansion of successfully differentiated KCs, the specific steps are as follows: Step 2-1. Observe under a microscope. When the confluence of KCs reaches 60% to 80%, remove the culture medium in the culture bottle with a pipette tip, add phosphate buffer and shake gently to wash the cell surface twice.

[0055] Step 2-2, add an appropriate amount of 0.25% trypsin-0.02% EDTA solution, digest at 37°C for 3-6 minutes, and observe under a microscope that the cells begin to round up. Add an appropriate amount of DMEM high-glucose medium containing 10% serum to terminate the digestion.

[0056] Step 2-3: Use a pipette to gently blow the bottom of the culture flask to completely detach the cells, transfer the detached cells and culture medium into a centrifuge tube, and centrifuge at 1200 rpm for 5 minutes.

[0057] Step 2-4: resuspend the precipitate obtained by centrifugation in KCs medium and inoculate at a ratio of 1:2 for subculture. The KCs medium is commercially available Sciencell keratinocyte medium.

[0058] Step (3) determines the induction condition range of the inflammatory induction source under the optimal induction time range (24-72h), and the specific steps are as follows: Step 3-1, preparing SDS solutions of different concentrations; Weigh an appropriate amount of sodium dodecyl sulfate (SDS) powder and dissolve it in phosphate buffer (PBS) to prepare a solution with a concentration of 10 mg / mL.

[0059] Take an appropriate amount of the solution in step a) and dilute it twice with phosphate buffered saline (PBS) to prepare a solution with a concentration of 5 mg / mL.

[0060] According to the above method, gradient dilutions were performed in phosphate buffered saline (PBS) to prepare solutions with concentrations of 2.5 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.2 mg / mL, and 0.1 mg / mL, respectively, and placed in a 4°C refrigerator for later use.

[0061] Step 3-2: SDS-induced inflammatory KCs steps are as follows: S3-2-1 The successfully differentiated KCs were revived or subcultured and inoculated into a 48-well plate. The number of cells per well was about 5×10 4 indivual.

[0062] S3-2-2 was cultured to the second day, and fresh KCs culture medium was replaced, and prepared SDS solutions of different concentrations were added at a dilution ratio of 1:100, with 4 replicate wells set up for each concentration, recorded as the experimental group, and a group without SDS solution was set up as the control group.

[0063] S3-2-3 Culture to the third day, use a pipette to gently remove the culture medium in the well, and add prepared SDS solutions of different concentrations at a dilution ratio of 1:100.

[0064] S3-2-4 Culture to the 4th day, collect the culture base in the well into the cryopreservation tube and mark it for subsequent inflammatory factor detection. Add 200 μL of fresh KCs culture medium and 20 μL of CCK-8 solution to each well and incubate in a carbon dioxide incubator for 0.5-4 hours.

[0065] Step 3-3. Calculate cell survival rate Place the well plate incubated in step 3-2 in an ELISA reader and measure the absorbance at 450 nm, which is recorded as OD450.

[0066] The survival rate of keratinocytes under different SDS concentrations in the experimental group was calculated according to the following formula: Step 3-4: The culture medium collected in step S3-2-4 was used to detect inflammatory factors PGE2, IL-1α, and IL-8 according to the Elisa kit procedure. The data obtained from the experimental group and the control group were statistically analyzed.

[0067] Step 3-4, select the SDS concentration with cell survival rate ≥ 80% and statistical analysis P ≤ 0.05 of inflammatory factors PGE2, IL-1α, and IL-8 as the concentration range for subsequent induction of KCs inflammatory response. In this embodiment, the final concentration of SDS induction is 0.2 mg / mL.

[0068] Step (4), constructing a skin model with a mild inflammation degree, the specific steps are as follows: Step 4-1: Resuscitate or subculture the successfully differentiated KCs into T75 culture flasks.

[0069] Step 4-2: On the second day of culture, replace the KCs cell culture medium with fresh one, and add SDS solution at a dilution ratio of 1:100 to a final concentration of 0.2 mg / mL.

[0070] Step 4-3: On the third day of culture, use a pipette to gently remove the culture medium in the culture flask, replace it with fresh KCs cell culture medium, and add SDS solution at a dilution ratio of 1:100 to a final concentration of 0.2 mg / mL.

[0071] Step 4-4: On the fourth day of culture, remove the culture medium in the culture flask with a pipette, add phosphate buffer and shake gently to wash the cell surface twice.

[0072] Step 4-5: Add an appropriate amount of 0.25% trypsin-0.02% EDTA solution and digest at 37°C for 3-6 minutes. Under a microscope, cells begin to round up. Add an appropriate amount of DMEM high-glucose medium containing 10% serum to terminate digestion.

[0073] Step 4-6: Use a pipette to gently blow the bottom of the culture flask to completely detach the cells, transfer the detached cells and culture medium into a centrifuge tube, and centrifuge at 1200 rpm for 5 minutes.

[0074] Step 4-7: resuspend the cell pellet with immersion medium, mix it with normal KCs at a ratio of 1:200, add it to the nozzle of the biological 3D droplet printer, and evenly inoculate the cell suspension into the insert cell culture chamber containing a semipermeable membrane in the form of droplet printing through a preset spiral printing path, where the cell inoculation density is 6×10 5 Pieces / cm 2 The droplet printing parameters are: air pressure 0.07Mpa, speed 3mm / s, injection cycle 120ms, and valve opening time 400μs.

[0075] Step 4-8, placing the cell culture insert in each culture well of the cell culture plate for immersion culture, wherein the cell culture insert and each culture well of the cell culture plate are filled with culture medium; immersion culture for 2 to 3 days, wherein the immersion culture medium is a KCs cell culture medium with a filtered CaCl2 solution added thereto to a final concentration of 0.2 mM.

[0076] Step 4-9, after the immersion culture stage, enter the gas-liquid critical culture stage. In this stage, the culture medium in the cell culture device is removed, and only fresh culture medium is added to the culture wells of the cell culture plate. The culture is continued for 5 to 10 days, during which fresh gas-liquid critical culture medium is replaced every day to obtain an inflammatory skin model. The gas-liquid critical culture medium is a KCs cell culture medium with a filtered CaCl2 solution added to a final concentration of 1.5 mM, and a VC solution is added to a final concentration of 20 μg / mL.

[0077] Figure 2 This is the H&E staining image of a normal 3D skin model tissue section. Figure 3 This is an H&E staining image of the tissue section of the inflammatory skin model constructed by the present invention. Compared with the normal 3D skin model, the inflammatory skin model prepared by the present invention has the following characteristics:

[0078] Tissue morphology: From the results of H&E staining of tissue sections, the basal layer is arranged in a disordered manner and does not present a typical palisade shape; the cell layer has obvious nuclear pyknosis cells and the cell layer is thin.

[0079] Inflammatory factors: A large number of inflammatory factors, such as PGE2, IL-1α, and IL-8, are released in the culture medium of the 3D inflammatory skin model.

[0080] In addition, the barrier index ET50 of the inflammatory skin model prepared in Example 1 was 2.5h, which was a downward trend compared to the barrier index ET50 of the normal skin model of 3.0h.

[0081] Example 2: The inflammatory skin model constructed in Example 1 is used to evaluate the soothing effect of a cosmetic formula. The specific method is as follows:

[0082] a) preparing the cosmetic formula with pure water into working solutions with volume concentrations of 100%, 50%, 25% and 10%; adding the working solutions to the upper surface of the inflammatory skin model constructed in Example 1, 25 μL per well, and setting up a control group in which only pure water was added to the surface; b) The inflammatory skin model after adding the sample was cultured for 24 hours; c) Rinse the surface of the inflammatory skin model with PBS buffer along the side wall, and gently wipe the surface of the inflammatory skin model dry with a cotton swab. The same operation was performed on the control group; d) The inflammatory skin model was placed in a 1 mg / mL MTT solution and incubated in a carbon dioxide incubator for 3 hours; e) Transfer the inflammatory skin model to a new well plate, add 2 mL of isopropanol solution to each well, place at 4°C, and incubate for 18-24 hours; f) Use a pipette to evenly distribute the above solution, extract 200 μL from each well and add it to a 96-well plate, and measure the absorbance at 570 nm in an ELISA reader, which is recorded as OD570; g) Calculate the survival rate of the inflammatory skin model in the sample group using the following formula: h) Select the sample group with a survival rate of ≥80% in the inflammatory skin model for subsequent efficacy testing.

[0083] i) According to the results of i), 25 μL of cosmetic formula was added to the surface of the inflammatory skin model. After 24 hours, the inflammatory skin model culture medium was collected for the detection of inflammatory mediator PGE2 and inflammatory factors IL-1α and IL-8. Dexamethasone was used in the positive control group.

[0084] j) The stratum corneum thickness of the rinsed inflammatory skin model of the sample group was measured using an optical coherence scanner (OCT).

[0085] k) Add 1% Triton X-100 solution to perform ET50 test on the barrier function of the inflammatory skin model.

[0086] l) At the same time, the inflammatory skin model of the sample group was fixed for subsequent histological detection.

[0087] The experimental results showed that compared with the control group without the cosmetic formula, the H&E staining results of the inflammatory skin model tissue sections of the sample group showed that the basal cells were arranged more neatly, the thickness of the stratum corneum increased, and the expression of the inflammatory mediator PGE2 and the inflammatory factors IL-1α and IL-8 decreased significantly (P < 0.05), indicating that this cosmetic formula has the effect of soothing and repairing the skin barrier.

[0088] Embodiment 3: A method for constructing a 3D skin model with controllable inflammation degree is provided, specifically: Step (1), obtaining KCs that have been successfully differentiated, passaged and expanded from step (2) of Example 1; Step (2), determining the induction condition range of the inflammation induction source within the optimal induction time range, the specific steps are as follows: Step 2-1: Induce inflammatory KCs using ultraviolet irradiation of different intensities, as follows: S2-1-1 The successfully differentiated KCs were revived or subcultured and inoculated into 48-well plates. The number of cells per well was about 5×104 indivual.

[0089] S2-1-2 was cultured to the second day, the medium in the well plate was replaced with phosphate buffer, and then placed in a UV irradiator, the UV irradiation channel was set to UVA, and the irradiation dose was set to 1.0 J / cm 2 , 2.0J / cm 2 , 5.0J / cm 2 , 10.0J / cm 2 、15.0J / cm 2 , 20 J / cm 2 Each irradiation dose contains 4 replicates, recorded as the irradiation group. At the same time, a non-irradiation group was set up as the control group.

[0090] S2-1-3 After irradiation, replace the phosphate buffer in the well plate with fresh cell culture medium and continue culturing in a carbon dioxide incubator for 18 to 24 hours.

[0091] S2-1-4 Culture to the third day, collect the culture base in the wells into cryopreservation tubes and mark them for subsequent inflammatory factor detection. Add fresh 200 μL KCs culture medium and 20 μL CCK-8 solution to each well and incubate in a carbon dioxide incubator for 0.5-4 hours.

[0092] Step 2-2, calculate cell survival rate Place the well plate incubated in step 2-1 in an ELISA reader and measure the absorbance at 450 nm, which is recorded as OD450.

[0093] Calculate the survival rate of keratinocytes under different intensities of ultraviolet irradiation in the experimental group.

[0094] Step 2-3: The culture medium collected in step S2-1-4 was used to detect inflammatory factors PGE2, IL-1α, and IL-8 according to the Elisa kit procedure. The data obtained from the experimental group and the control group were statistically analyzed.

[0095] Step 2-4: Select the irradiation dose with a cell survival rate of ≥80% and a statistical analysis P of inflammatory factors PGE2, IL-1α, and IL-8 of ≤0.05 as the irradiation range for inducing KCs inflammatory response. In this embodiment, the ultraviolet irradiation intensity is selected to be 5.0 J / cm 2 .

[0096] Step (3), constructing a skin model with moderate inflammation, the specific steps are as follows: Step 3-1. Resuscitate or subculture the successfully differentiated KCs into a 100 mm culture dish.

[0097] Step 3-2: On the second day of culture, replace the culture medium with phosphate buffered saline, place the cells in a UV irradiator, adjust the channel to UVA, and adjust the irradiation dose to 5 J / cm 2 .

[0098] Step 3-3: After irradiation, replace the phosphate buffer in the culture dish with fresh cell culture medium and place it in a carbon dioxide incubator to continue culturing for 18 to 24 hours.

[0099] Step 3-4: On the third day of culture, remove the culture medium in the culture flask with a pipette, add phosphate buffer and shake gently to wash the cell surface twice.

[0100] Step 3-5: Add an appropriate amount of 0.25% trypsin-0.02% EDTA solution and digest at 37°C for 3-6 minutes. Under a microscope, cells begin to round up. Add an appropriate amount of DMEM high-glucose medium containing 10% serum to terminate digestion.

[0101] Step 3-6: Use a pipette to gently blow the bottom of the culture flask to completely detach the cells, transfer the detached cells and culture medium into a centrifuge tube, and centrifuge at 1200 rpm for 5 minutes.

[0102] Step 3-7: Resuspend the cell pellet with immersion medium, mix it with normal KCs at a ratio of 1:60, add it to the nozzle of the biological 3D droplet printer, and evenly inoculate the cell suspension into the insert cell culture chamber containing a semipermeable membrane in the form of droplet printing through a preset spiral printing path. The cell inoculation density is 4×10 5 Pieces / cm 2 The droplet printing parameters are: air pressure 0.05Mpa, speed 3mm / s, injection cycle 120ms, and valve opening time 400μs.

[0103] Step 3-8, placing the cell culture insert in each culture well of the cell culture plate for immersion culture, wherein the cell culture insert and each culture well of the cell culture plate contain culture medium; immersion culture for 2 to 3 days. The immersion culture medium is a KCs cell culture medium with a filtered CaCl2 solution added thereto to a final concentration of 0.5 mM.

[0104] Step 3-9, after the immersion culture stage, enter the gas-liquid critical culture stage. In this stage, the culture medium in the cell culture device is removed, and only fresh culture medium is added to the culture wells of the cell culture plate. The culture is continued for 5 to 10 days, during which fresh gas-liquid critical culture medium is replaced every day to obtain an inflammatory skin model. The gas-liquid critical culture medium is a KCs cell culture medium with a filtered CaCl2 solution added to a final concentration of 1.8 mM, and a VC solution is added to a final concentration of 30 μg / mL.

[0105] Figure 4 This is the tissue morphology of the inflammatory skin model constructed in this embodiment: from the results of H&E staining of tissue sections, the basal layer is arranged in a disordered manner and does not present a typical fence shape; the cell layer has obvious nuclear condensation cells and the cell layer thickness is thin. The culture medium of the 3D inflammatory skin model contains a large number of inflammatory factors, such as inflammatory factors PGE2, IL-1α, and IL-8. In addition, the barrier index ET50 of the inflammatory skin model prepared in this embodiment shows a downward trend.

[0106] Example 4: The inflammatory skin model constructed in Example 3 was used to evaluate the soothing effect of a cosmetic formula. The specific method is as follows:

[0107] a) Prepare the cosmetic formula with pure water into working solutions with volume concentrations of 100%, 50%, 25% and 10%; b) adding the working solution to the upper surface of the inflammatory skin model constructed in this example, 25 μL per well, and setting up a control group in which only pure water was added to the surface; c) The inflammatory skin model after adding the sample was cultured for 24 hours; d) Rinse the surface of the inflammatory skin model with PBS buffer along the side wall, and gently wipe the surface of the inflammatory skin model dry with a cotton swab. The same operation was performed on the control group; e) The inflammatory skin model was placed in a 1 mg / mL MTT solution and incubated in a carbon dioxide incubator for 3 hours; f) Transfer the inflammatory skin model to a new well plate, add 2 mL of isopropanol solution to each well, place at 4°C, and incubate for 18-24 hours; g) Use a pipette to evenly distribute the above solution, extract 200 μL from each well and add it to a 96-well plate. Detect the absorbance at 570 nm in an ELISA reader and record it as OD570. h) Calculate the survival rate of the inflammatory skin model in the sample addition group using the following formula: i) The sample group with a survival rate of ≥80% in the inflammatory skin model was selected for subsequent efficacy testing.

[0108] j) According to the results of i), 25 μL of cosmetic formula was added to the surface of the inflammatory skin model. After 24 hours, the inflammatory skin model culture medium was collected for the detection of inflammatory factors PGE2, IL-1α, and IL-8. Dexamethasone was used in the positive control group.

[0109] k) The washed and cleaned inflammatory skin model of the sample group was used to measure the thickness of the skin model using an optical coherence scanner (OCT).

[0110] l) Add 1% Triton X-100 solution to perform ET50 test on the barrier function of the inflammatory skin model.

[0111] m) At the same time, the inflammatory skin model of the sample group was fixed for subsequent histological detection.

[0112] The experimental results showed that compared with the control group without the cosmetic formula, the H&E staining results of the inflammatory skin model tissue sections of the sample group showed that the basal cells were arranged more neatly, the thickness of the stratum corneum increased, and the expression of inflammatory factors PGE2, IL-1α, and IL-8 decreased significantly (P < 0.05), indicating that this cosmetic formula has the effect of soothing and repairing the skin barrier.

[0113] Embodiment 5: A method for constructing a 3D skin model with controllable inflammation degree is provided, specifically: Step (1), obtaining KCs that have been successfully differentiated, passaged and expanded from step (2) of Example 1; Step (2), determining the induction condition range of the inflammation induction source within the optimal induction time range, the specific steps are as follows: Step 2-1: Induce inflammatory KCs using interleukin 13 (IL-13), as follows: S2-1-1 The successfully differentiated KCs were revived or subcultured and inoculated into 48-well plates. The number of cells per well was about 5×10 4 indivual.

[0114] S2-1-2 was cultured to the second day, and fresh KCs medium was replaced, and IL-13 solution was added to make the final concentration of IL-13 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL, respectively. Four replicate wells were set for each concentration, recorded as the experimental group, and a group without IL-13 was set as the control group.

[0115] S2-1-3 Culture until the third day, gently remove the culture medium in the well with a pipette, and repeat step S2-1-2.

[0116] S2-1-4 was cultured to the 4th day, and the culture base in the well was collected and placed in a cryopreservation tube and marked for subsequent inflammatory factor detection. Add 200 μL of fresh KCs culture medium to each well, and add 20 μL of CCK-8 solution, and incubate in a carbon dioxide incubator for 0.5-4 hours.

[0117] Step 2-2, calculate cell survival rate Place the well plate incubated in step 2-1 in an ELISA reader and measure the absorbance at 450 nm, which is recorded as OD450.

[0118] Calculate the survival rate of keratinocytes under different intensities of ultraviolet irradiation in the experimental group.

[0119] Step 2-3: The culture medium collected in step S2-1-4 was used to detect inflammatory factors PGE2, IL-1α, and IL-8 according to the Elisa kit procedure. The data obtained from the experimental group and the control group were statistically analyzed.

[0120] Step 2-4, select the irradiation dose with cell survival rate ≥ 80% and statistical analysis P ≤ 0.05 of inflammatory factors PGE2, IL-1α, and IL-8 as the irradiation range for subsequent induction of KCs inflammatory response. In this example, the final concentration of IL-13 in the culture medium is selected to be 10 ng / mL.

[0121] Step (3), constructing a skin model with severe inflammation, the specific steps are as follows: Step 3-1. Resuscitate or subculture the successfully differentiated KCs into a 100 mm culture dish.

[0122] Step 3-2: On the second day of culture, the culture medium was replaced with a culture medium containing 10 ng / mL IL-13.

[0123] Step 3-3: Culture until the third day and repeat step 3-2.

[0124] Step 3-4: On the fourth day of culture, remove the culture medium in the culture flask with a pipette, add phosphate buffer and shake gently to wash the cell surface twice.

[0125] Step 3-5: Add an appropriate amount of 0.25% trypsin-0.02% EDTA solution and digest at 37°C for 3-6 minutes. Under a microscope, cells begin to round up. Add an appropriate amount of DMEM high-glucose medium containing 10% serum to terminate digestion.

[0126] Step 3-6: Use a pipette to gently blow the bottom of the culture flask to completely detach the cells, transfer the detached cells and culture medium into a centrifuge tube, and centrifuge at 1200 rpm for 5 minutes.

[0127] Step 3-7: Resuspend the cell pellet with immersion medium, mix it with normal KCs at a ratio of 1:40, add it to the nozzle of the biological 3D droplet printer, and evenly inoculate the cell suspension into the insert cell culture chamber containing a semipermeable membrane in the form of droplet printing through a preset spiral printing path. The cell inoculation density is 6×10 5 Pieces / cm 2 The droplet printing parameters are: air pressure 0.05Mpa, speed 2mm / s, injection cycle 100ms, and valve opening time 200μs.

[0128] Step 3-8, placing the cell culture insert in each culture well of the cell culture plate for immersion culture, wherein the cell culture insert and each culture well of the cell culture plate contain culture medium; immersion culture for 2 days. The immersion culture medium is a KCs cell culture medium with a filtered CaCl2 solution added thereto to a final concentration of 0.1 mM.

[0129] Step 3-9, after the immersion culture stage, enter the gas-liquid critical culture stage. In this stage, the culture medium in the cell culture device is removed, and only fresh culture medium is added to the culture wells of the cell culture plate. The culture is continued for 5 to 10 days, during which fresh gas-liquid critical culture medium is replaced every day to obtain an inflammatory skin model. The gas-liquid critical culture medium is a KCs cell culture medium with a filtered CaCl2 solution added to a final concentration of 1.2 mM, and a VC solution is added to a final concentration of 30 μg / mL.

[0130] Figure 5 This is the tissue morphology of the severe inflammation skin model constructed in this example: from the results of H&E staining of the tissue section, the basal layer is arranged in a disordered manner and does not present a typical fence shape; there are a large number of pyknotic cells in the cell layer, and the thickness of the cell layer is significantly thinned. In addition, the barrier index ET50 of the inflammatory skin model prepared in this example shows a downward trend.

[0131] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

Claims

1. A method for constructing a skin model with controllable inflammation degree, characterized in that The method comprises the following steps: Step (1), inducing keratinocytes from pluripotent stem cells, and then performing cell passaging and amplification; Step (2), determining the induction condition range of the inflammation induction source within the optimal induction time range; Step (3), inducing inflammation on keratinocytes according to the induction condition range of the inflammatory induction source within the optimal induction time range, to obtain inflammatory keratinocytes under different induction conditions; according to the requirements of the degree of inflammation, the inflammatory keratinocytes and normal keratinocytes are mixed in proportion to construct a skin model corresponding to the degree of inflammation.

2. The method according to claim 1, characterized in that: The differentiated keratinocytes induced in step (1) are polygonal, with clear cytoplasm, large nuclei containing 1-2 nucleoli, and have a typical cobblestone shape after the cells have grown into a single layer.

3. The method according to claim 1, characterized in that: The inflammation inducing source in step (2) includes at least one of cytokines, pathogen-associated molecular patterns, chemical irritants, and ultraviolet radiation.

4. The method according to claim 3, characterized in that: If the inflammatory induction source is a chemical irritant or a cytokine, step (2) is specifically: Chemical irritants or cytokines were formulated into inflammation-inducing solutions at different concentrations; Different concentrations of inflammation induction solutions were used to induce inflammatory responses in the passaged and expanded keratinocytes, and the cell survival rates under treatment with different concentrations of inflammation induction solutions at different induction times were calculated; Determine whether the inflammatory keratinocytes after inflammation induction treatment have the conditions for inflammatory response, screen out inflammatory keratinocytes that have the conditions for inflammatory response and whose cell survival rate is ≥a at each induction time, and obtain the corresponding concentration range of inflammation induction solution; 0%<a<100%.

5. The method according to claim 3, characterized in that: If the inflammation inducing source is ultraviolet radiation, step (2) is specifically: The passaged and expanded keratinocytes were irradiated with ultraviolet radiation of different intensities to induce inflammatory response in the cells, and the cell survival rate under ultraviolet radiation treatment of different intensities at different induction times was calculated; Whether the inflammatory keratinocytes after inflammation induction treatment have the conditions for inflammatory response, the inflammatory keratinocytes with the conditions for inflammatory response and cell survival rate ≥ a at each induction time are screened out, and the corresponding ultraviolet irradiation intensity is obtained; 0%<a<100%.

6. The method according to claim 4 or 5, characterized in that: The conditions for the occurrence of the inflammatory response include at least one condition: (1) Cell morphological changes; (2) Expression of inflammatory factors; (3) Changes in oxidative stress indicators; (4) Expression of genes or proteins related to inflammation.

7. The method according to claim 1, characterized in that: The mixing process of the inflammatory human keratinocytes and the normal human keratinocytes in step (3) is to add the inflammatory keratinocytes and the normal keratinocytes into the nozzle of the biological 3D droplet printer, and uniformly inoculate the mixed cells in an insert cell culture vessel containing a semipermeable membrane in the form of droplet printing through a preset spiral printing path, and then place the insert cell culture vessel in each culture well of the cell culture plate for culture, wherein the insert cell culture vessel and each culture well of the cell culture plate contain culture medium; after each culture period, the culture medium in the insert cell culture vessel is removed, and only fresh culture medium is added to the culture wells of the cell culture plate, and the culture is continued for a period of time, during which the fresh culture medium is replaced every day, thereby obtaining an inflammatory skin model.

8. The method according to claim 1, characterized in that: In step (3), if the skin model is a mild inflammation model, the ratio of the number of inflammatory keratinocytes to normal keratinocytes is: 1:500~1:100; If the skin model is a moderate inflammation model, the ratio of inflammatory keratinocytes to normal keratinocytes is: 1:99~1:50; If the skin model is a severe inflammation model, the ratio of inflammatory keratinocytes to normal keratinocytes is 1:49~1:

10.

9. A skin model with controllable inflammation degree, constructed by the method according to any one of claims 1 to 8.

10. Use of the skin model as claimed in claim 9 in evaluating the soothing or barrier repairing function of cosmetic raw materials or formulas, and the anti-inflammatory function of drugs.

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