Microcolony-based 3D simulation human skin model, preparation method and application

Abstract

The invention belongs to the technical field of biology and medicine, and discloses a microcolony-based 3D simulated human skin model, a preparation method and application, and the preparation method comprises the following steps: preparing the 3D simulated human skin model; preparing to obtain the fungus-coated micro-capsule; and paving the microencapsulated microcapsules on the surface of the 3D simulated human skin model to obtain the microcolony-based 3D simulated human skin model. On the basis of a traditional 3D skin model, common skin symbiotic bacteria are wrapped in the microcapsules formed by cross-linking CS and TPP, direct contact between bacteria and between bacteria and the skin model is avoided due to existence of the microcapsules, bacterial secretions can be diffused out of the microcapsules through micropores in the surfaces of the microcapsules, material exchange with the outside is achieved, and the skin model has a good antibacterial effect. The survival time of bacteria is prolonged, meanwhile, skin proliferation is promoted to a certain extent, and the real skin environment of the human body is further restored.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biology and medicine, and in particular to a 3D simulated human skin model based on microcolonies, a preparation method and applications. BACKGROUND

[0002] Currently, various types of skin models have become an important cornerstone for skin disease research, laying a solid foundation for the development of this field. However, the incidence of skin diseases is rising year by year, highlighting the urgency of related research. At the same time, under the background of the EU's strategy to "gradually eliminate animal experiments" in 2024, the trend of using new 3D skin models to replace animal experiments has accelerated significantly. This trend undoubtedly puts higher demands on the construction of 3D skin models that can highly simulate real human environments and points out the direction for future development. Traditional three-dimensional (3D) skin models are obtained by isolating and culturing human keratinocytes and fibroblasts in vitro, and have been widely used and developed in the fields of skin diseases, tissue engineering, and beauty and cosmetics.

[0003] Healthy skin contains a variety of microbial flora and is a highly dynamic ecosystem composed of keratinocytes, immune cells, and hundreds of millions of skin symbiotic bacteria. Traditional skin models cannot truly simulate the physiological and immune homeostasis of the skin. For example, existing standard 3D skin models lack symbiotic microbial flora, which prevents them from reflecting the physiological and immune functions of the skin in real conditions. In addition, there are significant deviations in disease modeling and drug efficacy evaluation. Many skin diseases, such as atopic dermatitis, psoriasis, acne, and rosacea, are closely related to the imbalance of skin symbiotic flora. Existing models have significant deviations when simulating skin diseases due to their sterile state. Due to the lack of reduction in existing models, the data obtained has limited predictive value when translated to actual and clinical settings, leading to a high failure rate in clinical trials.

[0004] To address this deficiency, the present application is based on the preparation of traditional 3D skin models and focuses on restoring the symbiotic relationship between the skin and surface bacteria. Microcapsules are synthesized using the properties of chemical and biological materials to encapsulate Staphylococcus epidermidis, Staphylococcus aureus, lactic acid bacteria, or micrococci, and attach them to the surface of the skin model. The presence of microcapsules prevents direct contact between bacteria and bacteria, as well as between bacteria and the skin model. At the same time, bacteria can exchange substances with the skin model through the micro-pores on the surface of the microcapsules, and the presence of bacteria-encapsulating microcapsules can also promote skin proliferation. This preparation method is simple, does not require additional reagents or factors to maintain bacterial growth activity, and is suitable for large-scale production. SUMMARY

[0005] The present application aims to overcome the deficiencies in the prior art, and provides a 3D simulated human skin model based on microcolonies, a preparation method and application.

[0006] The technical scheme adopted by the present application to solve its technical problems is: A preparation method of a 3D simulated human skin model based on microcolonies, characterized by comprising the following steps: The 3D simulated human skin model is prepared; the bacteria-containing microcapsules are prepared; the bacteria-containing microcapsules are laid on the surface of the 3D simulated human skin model to obtain the 3D simulated human skin model based on microcolonies.

[0007] Further, the preparation method of the bacteria-containing microcapsules comprises the following steps: The bacteria slurry is obtained by centrifuging the skin symbiotic bacteria liquid; the organic solvent, the bacteria slurry and the chitosan powder are sequentially added into the conical flask to obtain a chitosan-bacteria solution; the sodium tripolyphosphate powder is sucked into the centrifuge tube and is added into the centrifuge sleeve device for centrifugation; finally, the CS-bacteria solution is uniformly dropped into the TPP solution to cross-link the two to obtain the microcapsules with uniform morphology and containing bacteria.

[0008] Further, the skin symbiotic bacteria comprises Staphylococcus epidermidis, Staphylococcus aureus, lactic acid bacteria or micrococcus; Alternatively, the preparation step of the bacteria liquid is that the skin symbiotic bacteria is inoculated in the super-clean bench, and the bacterial strain is cultured in the shaking bed to obtain the required bacterial strain.

[0009] Further, the specific preparation steps of the bacteria-containing microcapsules are as follows: The logarithmic growth period of the cultured Staphylococcus aureus liquid is added into a 10 mL centrifuge tube, and is placed in a centrifuge with a rotation speed of 4000-5000 rpm / min for centrifugation for 2-3 min to obtain the bacteria slurry; the bacteria slurry obtained by centrifugation is resuspended in 1 mL of 2% acetic acid solution; 0.4 g of chitosan powder is weighed, and 19 mL of 2% acetic acid solution, 1 mL of bacteria liquid and chitosan powder are sequentially added into a 100 mL conical flask, which is uniformly stirred with a glass rod to obtain a chitosan-bacteria solution, which is referred to as CS-bacteria solution; the CS-bacteria solution is added into a 1.5 mL second centrifuge tube in the self-made centrifuge sleeve device; 12.5 mL of 5% sodium tripolyphosphate solution is sucked into a 50 mL first centrifuge tube in the self-made centrifuge sleeve device, and the needle in the device is kept at a distance of 1-2 cm from the liquid level of the sodium tripolyphosphate solution; the centrifuge sleeve device is placed in the centrifuge, and is centrifuged at a rotation speed of 700 rpm / min for 10 min, so that the 2% CS-bacteria solution is uniformly dropped into the 5% TPP solution to obtain the microcapsules with uniform size and containing bacteria, i.e., the bacteria-containing microcapsules; The centrifugal sleeve device comprises a first centrifugal tube and a second centrifugal tube, the second centrifugal tube is coaxially and tightly detachably connected with the first centrifugal tube; the first centrifugal tube comprises a first centrifugal tube body and a first centrifugal tube cover, the first centrifugal tube body is arranged in a vertical direction and has an open top, the first centrifugal tube cover is tightly and coaxially detachably arranged on the upper opening of the first centrifugal tube body, and a second centrifugal tube mounting hole is integrally arranged on the first centrifugal tube cover; the first centrifugal tube body can contain CS-bacteria solution; The second centrifugal tube comprises a second centrifugal tube body, a second centrifugal tube cover and a needle, the second centrifugal tube body is arranged in a vertical direction and has an open top and bottom, the second centrifugal tube cover is movably and detachably arranged on the open top of the second centrifugal tube body, the second centrifugal tube body can contain 5% trisodium phosphate solution, the needle is tightly and detachably connected with the open bottom of the second centrifugal tube body, and the needle is arranged in a vertical direction; the upper surface of the second centrifugal tube body is arranged in a shape matching the inner surface of the second centrifugal tube mounting hole, the upper part of the second centrifugal tube body is coaxially and tightly detachably connected with the first centrifugal tube cover through the second centrifugal tube mounting hole, the upper surface of the second centrifugal tube body is flush with the upper surface of the first centrifugal tube cover, and the middle and lower parts of the second centrifugal tube body are arranged in the first centrifugal tube body.

[0010] Further, the preparation method of the 3D simulated human skin model comprises the following steps: (1) constructing a dermis layer: mixing fibroblasts with a mouse type I collagen solution, transferring the collagen gel solution containing fibroblasts to a Transwell insert chamber for culture, and obtaining a dermis layer after culture; (2) first epidermis inoculation: placing keratinocytes on the dermis layer in the Transwell chamber of step (1) and culturing in a culture box to allow the keratinocytes to adhere; adding keratinocyte culture medium into the Transwell chamber and fibroblast culture medium outside the Transwell chamber to immerse the culture medium, and obtaining an inner epidermis; (3) second epidermis inoculation: inoculating keratinocytes onto the inner epidermis of step (2), adding keratinocyte culture medium into the Transwell chamber and fibroblast complete culture medium outside the Transwell chamber for epidermis culture, and replacing the culture medium in time during the culture process; culturing until the keratinocytes are completely adsorbed on the inner epidermis layer and tightly connected to obtain an epidermis layer; after the epidermis layer is formed, the culture medium inside and outside the Transwell chamber is removed; (4) Co-culture: Add 1~2 mL of gas-liquid culture medium to the inside and outside of the chamber for gas-liquid culture to obtain a 3D simulated human skin model without microcapsules.

[0011] Further, the collagen gel in step (1) has a thickness of 1-3 mm; the dermis layer has a thickness of 1-2 mm; Alternatively, step (1) may also include the following operations: Fibroblast pretreatment: Fibroblasts were digested using trypsin; Prepare the collagen solution: The concentration of the collagen solution is 2~4 mg / mL, and the pH value of the collagen solution is adjusted to 7.1; Mixing collagen solution with fibroblasts: Under ice bath conditions, add collagen solution to culture medium containing fibroblasts, and then adjust the pH of the mixture to 7.1-7.3.

[0012] Alternatively, the density of fibroblasts in the collagen solution described in step (1) is 7 × 10⁻⁶. 4 ~12×10 4 / mL, with a collagen concentration of 1~3 mg / mL; Alternatively, the collagen solution containing fibroblasts described in step (1); and / or the keratinocyte density during the endodermal culture described in step (2) is 4 × 10⁻⁶. 5 -10×10 5 / mL; and / or the keratinocyte density during epidermal culture in step (3) is 2 × 10⁶. 5 -5×10 5 / mL; Alternatively, the ratio of the amount of keratinocytes used in the first epidermal inoculation in step (2) to the amount of keratinocytes used in the second epidermal inoculation in step (3) is 2-7.5:1.

[0013] Further, the keratinocyte inoculation method in step (2) is to inoculate the keratinocyte culture medium containing keratinocytes onto the surface of the dermis; or, the keratinocyte inoculation method in step (3) is to inoculate the keratinocyte culture medium containing keratinocytes onto the inner epidermis. Alternatively, the dermal cell culture medium formula is as follows: DMEM 8 g / L~15 g / L, F12 2 g / L~8 g / L, fetal bovine serum 0.1%~1%, L-glutamine 2 mol / L~5 mol / L, hydrocortisone 0.1%~0.5%, insulin 1 mg / L~5 mg / L, antibiotic i.e., penicillin-streptomycin mixture (100×) 0.1%~1.5%, with water as the solvent; where the percentages mentioned above are final mass concentrations. The keratinocyte culture medium formula is: DMEM 10 g / L~20 g / L, fetal bovine serum 5%~15%, L-glutamine 2 mol / L~5 mol / L, antibiotic, i.e. penicillin-streptomycin mixture (100x) 0.1%~1.5%, and the solvent is water; wherein the percentages are all final concentrations by mass; The gas-liquid culture medium formula is: DMEM 8 g / L~15 g / L, F12 2 g / L~8 g / L, fetal bovine serum 0.1%~1%, L-glutamine 2 mol / L~5 mol / L, hydrocortisone 0.1%~0.5%, insulin 1 mg / L~5 mg / L, palmitic acid 0.005 mM~0.008 mM, arachidonic acid 0.001 mM~0.002 mM, linoleic acid 0.005 mM~0.007 mM, BSA 0.005 mM~0.005 mM, and the solvent is water; wherein the percentages are all final concentrations by mass.

[0014] Further, the incubation method in step (1) is to place in a 37 o C incubator with 5% carbon dioxide for 0.2~0.5 h to form a collagen gel with a thickness of 1~3 mm; and then add dermal fibroblast complete culture medium into the Transwell chamber and outside the chamber respectively to make the liquid levels inside and outside the Transwell chamber even, and the dermal culture is performed for 2~5 d; Alternatively, the inner epidermis culture method in step (2) is to make the keratinocytes adhere after standing, and then add keratinocyte culture medium into the Transwell chamber and dermal fibroblast complete culture medium outside the chamber respectively to make the liquid levels inside and outside the Transwell chamber even, and immerse culture for 3~5 d; Alternatively, the epidermis layer culture method in step (3) is to make the keratinocytes adhere after standing, and then add keratinocyte culture medium 1~3 mL into the Transwell chamber and fibroblast culture medium 2~4 mL outside the chamber respectively to make the liquid levels inside and outside the Transwell chamber even, and immerse culture for 3~5 d; Alternatively, the gas-liquid culture method in step (4) is to add gas-liquid culture medium into the Transwell chamber and outside the chamber, and change the liquid every day for 10~12 d; Alternatively, the culture method of the strain in step (5) is to place the inoculated culture medium into a 37 o C shaker, and make the bacteria culture in the shaker for 12~24 h; Alternatively, the specific method of making the keratinocytes adhere in step (2) and step (3) is to place in a 5% carbon dioxide incubator at 37 o C for 1~3 h.

[0015] Alternatively, the fibroblast-containing collagen solution has mouse type I collagen or fetal bovine type I collagen.

[0016] The microcolony-based 3D simulated human skin model prepared by the preparation method.

[0017] Application of the microcolony-based 3D simulated human skin model in skin research and / or evaluation of samples.

[0018] The present application has the following advantages and positive effects: 1. The present application is based on the traditional 3D skin model construction method and constructs a microcolony-based 3D skin model. On the basis of the traditional 3D skin model, common skin symbiotic bacteria are coated in the microcapsules formed by the crosslinking of CS and TPP, the presence of the microcapsules avoids direct contact between bacteria and bacteria and between bacteria and the skin model, at the same time, bacterial secretions can diffuse to the outside of the microcapsules through the micropores on the surface of the microcapsules, realizing material exchange with the outside world, improving the survival time of the bacteria, and to some extent, promoting skin proliferation, and further restoring the real skin environment of the human body.

[0019] 2. The present application uses the paving technology, and the skin model and the bacteria-containing microcapsules are all paved layer by layer, so that the keratinocytes and fibroblasts in the dermis layer are arranged more closely, and the material exchange between the bacteria-containing microcapsules and the skin model is also strengthened, accelerating the formation of the 3D skin model. It is found through transdermal resistance test verification that the synthesized 3D skin model meets the requirements of the new 3D skin model, the resistance value increases and tends to be stable at about 200 Ω, the skin model develops well and has a complete skin barrier, providing a good 3D skin model for the paving of the bacteria-containing microcapsules.

[0020] 3. The skin model constructed by the present application optimizes the number of times of layer-by-layer paving, the inoculation density and proportion of keratinocytes at the time of twice paving, and the density of bacteria-containing microcapsules at the time of paving, avoiding the problem of insufficient nutrition caused by the accumulation of cells and bacteria-containing microcapsules, adhesion and competition for nutrition supply. Experiments prove that increasing the density of keratinocytes and bacteria-containing microcapsules at the time of the first epidermal inoculation, or using three or more paving methods, is easy to cause damage to the epidermal layer, apoptosis or death of the cells in the dermal layer and the bacteria in the microcapsules, and even damage to the dermal layer.

[0021] 4. On the basis of constructing the traditional 3D skin model, the present application introduces CS-bacteria-TPP microcapsules loaded with common human symbiotic bacteria (such as Staphylococcus epidermidis, Staphylococcus aureus, lactic acid bacteria or micrococcus) on the surface of the 3D skin model, so that the bacteria can secrete metabolites and other substances to the outside of the microcapsules through the micropores on the surface of the microcapsules, and the metabolites and other substances can also enter the microcapsules through the micropores on the surface of the microcapsules, realizing material exchange between the bacteria and the 3D skin model, and improving the survival time of the bacteria. Figure 6The H&E staining results showed that bacteria were encapsulated in microcapsules and spread onto the surface of the skin model, constructing an ecological balance system in which the skin model and microorganisms coexist. This system can highly simulate the normal human skin environment, meet most market demands, and has the potential for large-scale production.

[0022] 5. In this invention, collagen in the collagen solution interacts dynamically and bidirectionally with fibroblasts to form a skin model that closely resembles real skin, possessing good mechanical strength and morphology.

[0023] 6. This invention also utilizes a 3D simulated human skin model based on microcolonies, applicable to skin research / evaluation samples. This model can be used in skin research, including but not limited to dermatopathology studies and studies on burn or traumatic wound healing. The model can be used for sample efficacy evaluation, including but not limited to whitening and sun protection, skin barrier function, moisturizing and anti-aging, soothing and repair, and air pollution protection. The model can be applied to the skin biology evaluation of samples, including but not limited to drugs, cosmetics, and skincare products. The model can be used for sample safety evaluation, including but not limited to the detection of skin irritation, skin sensitization, and phototoxicity. Attached Figure Description

[0024] Figure 1 This is a histological diagram of the 3D simulated human skin model based on microcolonies prepared in Example 1 of this invention. Figure 2 This is a histological diagram of a recombinant human 3D skin model prepared in Example 2 of this invention; Figure 3 This is a microscopic morphological diagram of the microcapsules containing bacteria in this invention; Figure 4 The images show the test results of lactic acid bacteria secretions after microcapsules were encapsulated in the present invention; the first row of left images shows the phenol red solution before lactic acid secretion by the lactic acid bacteria, the first row of right images shows the phenol red solution after lactic acid secretion by the lactic acid bacteria, and the second row of left and right images are parallel experiments for the detection of lactic acid bacteria secretions; Figure 5 This is a plate coating result of the lactic acid bacteria supernatant in this invention; Figure 6 These are H&E staining images of a 3D skin model based on microcolonies used in this invention. Figure 7 These are electron microscope scans of the 3D skin model based on microcolonization used in this invention. Figure 8 This is a schematic diagram of a structural connection of the centrifugal sleeve device in this invention; Figure 9 for Figure 8 A schematic diagram of one structural connection of the second centrifuge tube. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0026] The various experimental operations involved in the specific embodiments are all conventional techniques in the art. For parts not specifically annotated herein, those skilled in the art can refer to various commonly used reference books, scientific and technological literature, or related instructions and manuals prior to the filing date of this invention for implementation. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0027] This invention constructs a 3D skin model based on microbial colonies, building upon traditional 3D skin model construction methods. Common skin symbiotic bacteria are encapsulated within microcapsules formed by cross-linking of CS and TPP, preventing direct contact between bacteria and between bacteria and the skin model. Simultaneously, bacterial secretions can diffuse through micropores on the microcapsule surface, facilitating material exchange with the external environment, increasing bacterial survival time, and promoting skin proliferation to some extent, further reproducing the real human skin environment.

[0028] In a first aspect, the present invention provides a method for preparing a 3D simulated human skin model based on microcolonies, the steps of which are as follows: (1) Construction of the dermis: Fibroblasts were mixed with mouse type I collagen solution. The collagen gel solution containing fibroblasts was transferred to a Transwell insert chamber for culture, and the dermis was obtained after culture.

[0029] (2) First epidermal inoculation: Place the keratinocytes on the dermis in the Transwell chamber of step (1) and culture them in an incubator to allow the keratinocytes to adhere to the wall. Add keratinocyte culture medium to the Transwell chamber and fibroblast culture medium to the outside of the Transwell chamber, so that the liquid levels are equal and the cells are submerged.

[0030] (3) Second epidermal inoculation: Keratinocytes are inoculated onto the inner epidermis described in step (2). Keratinocyte culture medium is added to the Transwell chamber, and fibroblast complete culture medium is added to the outside of the Transwell chamber for epidermal culture. The culture medium is changed in a timely manner during the culture process. The epidermal layer is obtained when the keratinocytes are completely adsorbed to the inner epidermal layer and tightly connected. After the epidermal layer is formed, the culture medium inside and outside the Transwell chamber is removed.

[0031] (4) Co-culture: Add 1~2 mL of gas-liquid culture medium to the inside and outside of the chamber for gas-liquid culture to obtain a 3D skin model without microcapsules.

[0032] (5) Preparation of bacterial strains: Inoculate the skin symbiotic bacteria in the clean bench and place them in a shaker to culture the bacterial strains to obtain the desired bacterial strains.

[0033] (6) Preparation of CS-bacteria-TPP microcapsules: The bacterial solution was centrifuged to obtain bacterial sludge. Organic solvent, bacterial sludge and chitosan powder were added to a conical flask in sequence to obtain chitosan-bacteria solution. Sodium tripolyphosphate powder was taken into a centrifuge tube and added to a self-made centrifuge module for centrifugation. Finally, the CS-bacteria solution was dripped into the TPP solution at a uniform rate to crosslink the two to obtain microcapsules with uniform morphology and containing bacteria.

[0034] (7) Spreading of microencapsulated bacteria: Spread microencapsulated bacteria onto the surface of the model using a pipette to construct a 3D simulated human skin model based on microcolonies.

[0035] The ratio of the amount of keratinocytes used in the first epidermal inoculation in step (2) to the amount of keratinocytes used in the second epidermal inoculation in step (3) is 2-7.5:1.

[0036] Optionally, the dermal cell culture medium is formulated as follows: DMEM 8 g / L~15 g / L, F12 2 g / L~8 g / L, fetal bovine serum 0.1%~1%, L-glutamine 2 mol / L~5 mol / L, hydrocortisone 0.1%~0.5%, insulin 1 mg / L~5 mg / L, and antibiotics 0.1%~1.5%.

[0037] Optionally, in step (6), when preparing the bacterial sludge, the Staphylococcus aureus bacterial solution needs to be added to a 10 mL centrifuge tube and centrifuged under the conditions of 4000~5000 rpm / min for 2~3 min.

[0038] Optionally, the keratinocyte culture medium formula is: DMEM 10 g / L~20 g / L, fetal bovine serum 5%~15%, L-glutamine 2 mol / L~5 mol / L, and antibiotics 0.1%~1.5%.

[0039] Alternatively, the microcapsules can encapsulate bacterial species that are common on the human skin surface, such as Staphylococcus epidermidis, lactic acid bacteria, or micrococci.

[0040] In an embodiment, a method for preparing a 3D simulated human skin model based on microcolonies, step (1) further includes the following operations: Fibroblast pretreatment: Fibroblasts were digested using trypsin; Prepare the collagen solution: The concentration of the collagen solution is 2~4 mg / mL, and the pH value of the collagen solution is adjusted to 7.1; Mixing collagen solution with fibroblasts: Under ice bath conditions, add collagen solution to culture medium containing fibroblasts, and then adjust the pH of the mixture to 7.1-7.3.

[0041] Alternatively, trypsin-EDTA can be used to digest fibroblasts.

[0042] Optionally, the collagen solution can be adjusted using 1 M NaOH.

[0043] Preferably, the collagen gel obtained in step (1) has a thickness of 1-3 mm; the dermis layer has a thickness of 1-2 mm.

[0044] Preferably, the density of fibroblasts in the collagen solution described in step (1) is 7 × 10⁻⁶. 4 ~12×10 4 / mL, with a collagen concentration of 1~3 mg / mL.

[0045] More preferably, the density of fibroblasts in the collagen solution described in step (1) is 10 × 10⁻⁶. 4 / mL.

[0046] Preferably, the density of keratinocytes during the endodermal layer culture in step (2) is 4 × 10⁻⁶. 5 ~10×10 5 / mL; More preferably, the density of keratinocytes during the endodermal layer culture in step (2) is 4 × 10⁻⁶. 5 / mL.

[0047] Preferably, the density of keratinocytes during epidermal culture in step (3) is 2 × 10⁻⁶. 5 ~5×10 5 / mL; More preferably, the density of keratinocytes during epidermal culture in step (3) is 5 × 10⁻⁶. 5 / mL.

[0048] Preferably, the method of inoculating keratinocytes in step (2) above is to inoculate the dermis with a culture medium containing keratinocytes; the method of inoculating keratinocytes in step (3) is to inoculate the inner epidermis with a culture medium containing keratinocytes.

[0049] Preferably, in some examples, the preparation method of the 3D simulated human skin model based on microcolonies, the preparation methods of steps (2) and (3) are as follows: Keratinocyte pretreatment: Keratinocytes are digested using trypsin; After digestion, the keratinocytes are added to the keratinocyte culture medium and mixed thoroughly.

[0050] Preferably, the incubation conditions for forming the collagen gel in step (1) above are as follows: the gel is added to a 24-well Transwell insert chamber and placed at 37°C. o C. In a 5% carbon dioxide incubator, gel the cells for 0.2-0.5 h. Add dermal cell culture medium to the inside and outside of the cell chamber to make the interface between the inside and outside of the cell chamber level. Culture for 2-5 days to allow fibroblasts and collagen to interact and form the dermis.

[0051] Preferably, in step (3), 1-3 mL of keratinocyte culture medium is added to the inner chamber and 2-4 mL of fibroblast culture medium is added to the outer chamber to make the inner and outer interfaces of the inner chamber level, and the culture is immersed for 3-5 days.

[0052] Preferably, in step (3), 1-3 mL of keratinocyte culture medium is added to the inner chamber and 2-4 mL of dermal fibroblast culture medium is added to the outer chamber, so that the liquid levels inside and outside the inner chamber are equal, and the keratinocytes are immersed and cultured for 1-3 days to allow them to adhere to the wall and grow.

[0053] Preferably, in step (4), the epidermis is cultured in a gas-liquid medium for 10-12 days, with the medium changed daily.

[0054] Preferably, the composition of the gas-liquid culture medium is as follows: DMEM 8 g / L~15 g / L, F12 2 g / L~8 g / L, fetal bovine serum 0.1%~1%, L-glutamine 2 mol / L~5 mol / L, hydrocortisone 0.1%~0.5%, insulin 1 mg / L~5 mg / L, palmitic acid 0.005 mM~0.008 mM, arachidonic acid 0.001 mM~0.002 mM, linoleic acid 0.005 mM~0.007 mM, and BSA 0.005 mM~0.005 mM.

[0055] More preferably, the specific method for allowing keratinocytes to adhere to the culture wall during the static incubation process described in steps (2) and (3) is to place them in a 5% carbon dioxide incubator at 37°C. o C, let stand for 1~3 hours.

[0056] Preferably, in step (5) during inoculation, 7 μL of Staphylococcus aureus cryopreservation solution and 5 mL of liquid culture medium are pipetted into a test tube in a clean bench and placed at 37°C. o In a shaker, Staphylococcus aureus is cultured for 12-24 hours.

[0057] Preferably, in step (6), the microbial sludge obtained by centrifugation is resuspended in 1 mL of 2% acetic acid solution.

[0058] The Transwell chamber described in this invention, also known as an insertable cell culture dish or a permeable chamber, resembles a transparent cup holder. Essentially, the upper chamber is the "cup" itself, with a polycarbonate membrane with 3 μm micropores at the bottom, and the lower chamber is the original pore of the well plate, which contains culture medium.

[0059] The Transwell chamber is placed in a culture plate. The chamber inside is called the upper chamber, and the chamber outside is called the lower chamber. The upper chamber contains the upper layer of culture medium, and the lower chamber contains the lower layer of culture medium. The upper and lower layers of culture medium are separated by a polycarbonate membrane.

[0060] The Transwell cell specifications used are selected according to the actual situation, including but not limited to 6-well, 12-well, and 24-well cells, as well as 75 mm diameter cells that are compatible with Transwell dishes.

[0061] Specifically, this invention provides a method for preparing a 3D simulated human skin model based on microcolonies. The Transwell chamber used is a 24-well plate Transwell chamber, prepared by nesting the 24-well plate Transwell chamber within a 24-well culture plate. The preparation method includes the following steps: (1) Construction of the dermis: Aspirate the culture medium containing fibroblasts into a 15 mL centrifuge tube, centrifuge the culture medium containing fibroblasts, collect the fibroblasts, then add 50-150 μL of culture medium and mix with mouse type I collagen solution to culture the dermis. Add 1-3 mL of complete dermal fibroblast culture medium to the upper chamber and 2-4 mL of complete dermal fibroblast culture medium to the lower chamber, so that the liquid levels in the upper and lower chambers are level, and culture the dermis to obtain the dermis. After the dermis culture is completed, remove the complete dermal fibroblast culture medium from the upper and lower chambers; (2) First epidermal inoculation: Take 80~120 μL of culture medium containing keratinocytes and inoculate it onto the dermis layer described in step (1) to carry out inner epidermal layer culture until the keratinocytes are completely attached to the surface of the dermal layer cells and are tightly connected to obtain the inner epidermal layer. (3) Second epidermal inoculation: Take 80~120 μL of culture medium containing keratinocytes and inoculate it onto the inner epidermal layer described in step (2) to carry out epidermal layer culture until the keratinocytes are completely attached to the inner epidermal layer and are tightly connected to obtain the epidermal layer; after the epidermal layer culture is completed, remove the culture medium from the upper and lower chambers.

[0062] (4) Co-culture: Add 1~2 mL of gas-liquid culture medium to the inside and outside of the chamber for gas-liquid culture to obtain a 3D skin model without microcapsules.

[0063] (5) Preparation of bacterial strains: Inoculate the skin symbiotic bacteria in the clean bench and place them in a shaker to culture the bacterial strains to obtain the desired bacterial strains.

[0064] (6) Preparation of CS-bacterial-TPP microcapsules: CS-bacterial solution was prepared in an Erlenmeyer flask. 12.5 mL of 5% sodium tripolyphosphate solution was taken and centrifuged. Then, 2% CS-bacterial solution was added dropwise to 5% sodium tripolyphosphate solution at a uniform rate to obtain uniformly sized microcapsules containing bacteria.

[0065] (7) Spreading of microcapsules: Use a pipette to spread microcapsules on the surface of the model to construct a 3D simulated human skin model of microcolon.

[0066] The ratio of keratinocytes used in step (2) to keratinocytes used in the second epidermal inoculation in step (3) is 2-7.5:1; the keratinocyte culture medium used in the inner and outer chambers of the Transwell is always kept equal.

[0067] Preferably, the collagen solution in step (1) contains mouse type I collagen or fetal bovine type I collagen.

[0068] More preferably, the collagen solution in step (1) is mouse type I collagen.

[0069] Collagen in the collagen solution interacts dynamically and bidirectionally with fibroblasts to form a skin model that closely resembles real skin, possessing good mechanical strength and morphology.

[0070] Secondly, the present invention also provides a 3D simulated human skin model based on microcolon prepared using the above-described preparation method.

[0071] Finally, this invention also provides the application of the above-mentioned microcolony-based 3D simulated human skin model in skin research / evaluation samples.

[0072] The 3D simulated human skin model based on microcolon provided by this invention can be used for skin research, including but not limited to skin pathology research and research on burn or trauma wound healing.

[0073] The microcolony-based 3D simulated human skin model described in this invention can be used for the efficacy evaluation of samples, including but not limited to whitening and sun protection, skin barrier, moisturizing and anti-aging, soothing and repair, and air pollution protection.

[0074] In detail, the invention described herein can be applied to the skin biology evaluation of samples, including but not limited to pharmaceuticals, cosmetics, and skin care products.

[0075] The microcolony-based 3D simulated human skin model of the present invention can be used for the safety evaluation of samples, including but not limited to the detection of skin irritation, skin sensitization and phototoxicity.

[0076] Specifically, the relevant preparation and testing methods are as follows: In the following embodiments of the present invention, a 3D simulated human skin model based on microcolonies is prepared using a culture plate nested with Transwell chambers. Specifically, Transwell chambers are placed within the culture plate, wherein the internal region of the Transwell chamber is referred to as the "upper chamber," and the external region (i.e., the remaining space within the culture plate) is referred to as the "lower chamber." During the preparation process, the upper and lower chambers are used for different cell cultures and nutrient supplies, respectively, forming a multi-layered environment that simulates the skin structure, ensuring that the novel 3D skin model exhibits good hierarchical differentiation and functional expression.

[0077] All test materials used in this invention are commercially available products and can be purchased through commercial channels.

[0078] Trypsin was purchased from Solarbio, trypsin-EDTA (0.25%), phenol red-free.

[0079] The dermal cell culture medium formula is as follows: DMEM 8 g / L~15 g / L, F12 (dichlorodifluoromethane, F12 is Ham F12 nutrient mixture) 2 g / L~8 g / L, fetal bovine serum 0.1%~1%, L-glutamine 2 mol / L~5 mol / L, hydrocortisone 0.1%~0.5%, insulin 1 mg / L~5 mg / L, antibiotic (penicillin-streptomycin mixture (100×)) 0.1%~1.5%, and water as solvent; wherein, the above percentages are all final mass concentrations.

[0080] The keratinocyte culture medium formula is as follows: DMEM 10 g / L~20 g / L, fetal bovine serum 5%~15%, L-glutamine 2mol / L~5 mol / L, antibiotic (penicillin-streptomycin mixture (100×)) 0.1%~1.5%, and water as the solvent; wherein, the above percentages are all final mass concentrations.

[0081] The aero-liquid culture medium formulation is as follows: DMEM 8 g / L~15 g / L, F12 (dichlorodifluoromethane, F12 is Ham F12 nutrient mixture) 2 g / L~8 g / L, fetal bovine serum 0.1%~1%, L-glutamine 2 mol / L~5 mol / L, hydrocortisone 0.1%~0.5%, insulin 1 mg / L~5 mg / L, palmitic acid 0.005 mM~0.008 mM, arachidonic acid 0.001 mM~0.002 mM, linoleic acid 0.005 mM~0.007 mM, BSA (bovine serum albumin) 0.005 mM~0.005 mM, and water as the solvent; where all percentages are final mass concentrations.

[0082] Example 1 A method for preparing a 3D simulated human skin model based on microcolonies includes the following steps: First, fibroblasts were digested using trypsin digestion solution. Then, DMEM medium containing fibroblasts was added to a 15 mL centrifuge tube. The DMEM medium containing fibroblasts was centrifuged, and the collected fibroblasts were added to 50-150 μL of DMEM medium. Finally, the DMEM medium containing fibroblasts was added to a mouse type I collagen solution with pH adjusted to 7.1 using 1 M NaOH, so that the density of the fibroblasts in the mouse type I collagen solution was 7 × 10⁻⁶. 4 ~12×10 4 / mL, the amount of mouse type I collagen is 80~120 μL, the concentration is 1~3 mg / mL, and a mouse type I collagen solution with fibroblasts is obtained; Add the mixed mouse type I collagen solution containing fibroblasts to a 24-well Transwell insert chamber and incubate at 37°C. o Gel was incubated in a 5% carbon dioxide incubator for 0.2–0.5 h to form a collagen gel with a thickness of 1–3 mm. Dermal cell culture medium was added separately to the chamber and outside of the cell, ensuring the interface between the chamber and the outside was level. The cells were cultured for 2–5 days at 37°C. o In a 5% carbon dioxide incubator, 400 μL of culture medium was added to the inner chamber and 1 mL of culture medium was added to the outer chamber, allowing dermal fibroblasts and collagen to interact and form the dermal layer.

[0083] First, keratinocytes were digested using trypsin digestion solution. Next, DMEM culture medium containing keratinocytes was transferred to a 15 mL centrifuge tube. The DMEM culture medium containing keratinocytes was then centrifuged, and the keratinocytes were collected and added to 50-150 μL of keratinocyte culture medium. This culture medium containing keratinocytes was then seeded onto the dermis and incubated at 37°C. o C. Place the cells in a 5% carbon dioxide incubator for 1-3 hours to allow the keratinocytes to adhere to the incubator wall. After standing for 1-3 hours to allow the keratinocytes to adhere to the wall, add 1-3 mL of keratinocyte culture medium to the inner chamber and 2-4 mL of fibroblast culture medium to the outer chamber to make the inner and outer interfaces level. Immerse and culture for 3-5 days to obtain the epidermal layer. Keratinocytes were digested with trypsin digestion solution. Then, DMEM culture medium containing keratinocytes was aspirated into a 15 mL centrifuge tube. The DMEM culture medium containing keratinocytes was then centrifuged, and 50-150 μL of keratinocyte culture medium was added to collect the keratinocytes. The culture medium containing keratinocytes was then inoculated onto the epidermal layer and placed in an incubator for 1-3 hours to allow the keratinocytes to adhere to the epidermal keratinocytes. Add 1-2 mL of gas-liquid culture medium to both the inner and outer chambers, and incubate at 5% CO2 and 37°C. o Culture in a C incubator for 10-12 days, changing the medium daily, to obtain a 3D skin model.

[0084] The 3D skin model was fixed in 4% paraformaldehyde, then dehydrated and embedded using an embedding machine. The embedded paraffin block was removed and sectioned using a microtome. The sections were then placed in a dewaxing machine and dewaxed using different volume concentrations of ethanol (anhydrous ethanol, 95%, 75%, 50%). Subsequently, the samples were stained with hematoxylin and eosin, followed by a gradient dehydration method with progressively increasing ethanol concentrations: each gradient of ethanol (50%, 75%, 95%, anhydrous ethanol, volume concentration) was left on the sample surface for 3–5 seconds, then immersed in anhydrous ethanol for 5 minutes, and finally dried in a ventilated area. The growth status of the 3D skin model was observed and photographed using an upright fluorescence microscope to analyze its growth. The 3D skin model prepared in this study was subjected to H&E staining, and the results are as follows: Figure 1As shown, the model exhibits a continuous and complete layered skin structure. The boundary between the epidermis and the underlying dermis is clear, and they are tightly bound together, forming a regular dermal-epidermal junction, indicating that cells established good interactions during culture. Observation revealed that the model not only met the overall thickness requirements but also demonstrated excellent uniformity. Both the epidermis and dermis maintained a high degree of consistency in thickness across different regions, without any localized areas of excessive thinning or abnormal thickening. This fully demonstrates the stability and reproducibility of the preparation process, ensuring the reliability and comparability of data in subsequent experiments.

[0085] Example 2 (In Example 1, keratinocytes were spread twice; in Example 2, keratinocytes were spread once). A method for preparing a recombinant human 3D skin model includes the following steps: First, fibroblasts were digested using trypsin digestion solution. Then, DMEM culture medium containing fibroblasts was transferred to a 15 mL centrifuge tube. The DMEM culture medium containing fibroblasts was centrifuged, and the fibroblasts were collected. 50–150 μL of DMEM culture medium was then added to the collected fibroblasts. Finally, the DMEM culture medium containing fibroblasts was added to a mouse type I collagen solution with pH adjusted to 7.1 using 1 M NaOH, so that the density of the fibroblasts in the mouse type I collagen solution was 7 × 10⁻⁶. 4 ~12×10 4 / mL, the amount of mouse type I collagen is 80~120 μL, and the concentration is 1~3 mg / mL; Add the mixed mouse type I collagen solution containing fibroblasts to a 24-well Transwell insert chamber and incubate at 37°C. o Gel was incubated in a 5% carbon dioxide incubator for 0.2–0.5 h to form a collagen gel with a thickness of 1–3 mm. Dermal cell culture medium was added separately to the chamber and outside of the cell, ensuring the interface between the chamber and the outside was level. The cells were cultured for 2–5 days at 37°C. o In a 5% carbon dioxide incubator, 400 μL of culture medium was added to the inner chamber and 1 mL of culture medium was added to the outer chamber, allowing dermal fibroblasts and collagen to interact and form the dermal layer.

[0086] First, keratinocytes were digested using trypsin digestion solution. Next, DMEM culture medium containing keratinocytes was transferred to a 15 mL centrifuge tube. The DMEM culture medium containing keratinocytes was then centrifuged, and the keratinocytes were collected and added to 50-150 μL of keratinocyte culture medium. This culture medium containing keratinocytes was then seeded onto the dermis and incubated at 37°C. o C. Place the cells in a 5% carbon dioxide incubator for 1-3 hours to allow them to adhere and grow. After standing for 1-3 hours to allow the keratinocytes to adhere and grow, add 1-3 mL of keratinocyte culture medium to the inner chamber and 2-4 mL of dermal fibroblast culture medium to the outer chamber, so that the liquid levels inside and outside the chamber are equal, and immerse and culture for 1-3 days until the keratinocytes adhere. After the previous step, gaseous and liquid culture medium gas was added to both the inner and outer chambers of the chamber, and the mixture was kept in 5% CO2 at 37°C. o Culture in a C incubator for 10-12 days, changing the medium daily, to obtain a 3D skin model.

[0087] The 3D skin model prepared in this study was subjected to H&E staining, as described in Example 1. The 3D skin model prepared in Example 2 is as follows. Figure 2 As shown, the model exhibits a continuous and complete layered skin structure. The boundary between the epidermis and the underlying dermis is clear, and they are tightly bound together, forming a regular dermal-epidermal junction. This indicates that the cells established good interactions during culture. However, observation revealed a shrinkage issue in the 3D skin model's volume; the overall diameter of the model decreased, and the thickness of the dermal collagen matrix significantly thinned, becoming denser, with slight curling or wrinkling at its edges. This demonstrates that the 3D skin model constructed using the method in Example 1 of this invention achieves improved skin quality, uniform thickness, and structural stability, effectively avoiding the volume shrinkage phenomenon caused by long culture times.

[0088] Example 3: Preparation of microcapsules containing bacteria The preparation method of CS-bacterial-TPP microcapsules based on chemical and biomass materials and the detection of secretions include the following steps: Add the cultured Staphylococcus aureus in the logarithmic growth phase to a 10 mL centrifuge tube and centrifuge at 5000 rpm for 2 min to obtain bacterial sludge. Resuspend the bacterial sludge in 1 mL of 2% acetic acid solution. Weigh 0.4 g of chitosan powder and add 19 mL of 2% acetic acid solution, 1 mL of bacterial culture, and chitosan powder to a 100 mL Erlenmeyer flask. Stir well with a glass rod to obtain chitosan-bacterial solution (i.e., chitosan-Staphylococcus aureus, abbreviated as CS-bacterial solution). Add the CS-bacterial solution to a 1.5 mL second centrifuge tube in a self-made centrifuge tube apparatus. Pipette 12.5 mL of 5% sodium tripolyphosphate (TPP) solution into a 50 mL first centrifuge tube in the self-made centrifuge tube apparatus, keeping the needle in the apparatus between 1 and 2 cm above the surface of the TPP solution. Place the centrifuge tube assembly into a centrifuge and centrifuge at 700 rpm for 10 min. Then, add 2% (w / w) CS-bacterial solution dropwise to a 5% TPP solution at a uniform rate to obtain the desired result. Figure 3Uniformly sized microcapsules containing bacteria are known as CS-bacterial-TPP microcapsules.

[0089] Among them, such as Figure 8 and Figure 9 As shown, the centrifuge tube device includes a first centrifuge tube 1 and a second centrifuge tube 2. The second centrifuge tube can be coaxially and detachably connected to the first centrifuge tube. The first centrifuge tube includes a first centrifuge tube body 11 and a first centrifuge tube cap 12. The first centrifuge tube body is arranged vertically and is shaped like a centrifuge tube with an open top. The first centrifuge tube cap is tightly and coaxially detachably disposed above the opening of the first centrifuge tube body, and a second centrifuge tube mounting through hole (not labeled in the figure) is integrally formed on the first centrifuge tube cap. The first centrifuge tube body can hold CS-bacterial solution. The second centrifuge tube includes a second centrifuge tube body 21, a second centrifuge tube cap 22, and a needle 3. The second centrifuge tube body is arranged vertically and is a centrifuge tube with openings at both the top and bottom. The second centrifuge tube cap is movably and detachably connected to the top opening of the second centrifuge tube body. The second centrifuge tube body can hold a 5% sodium tripolyphosphate solution. The needle is detachably and detachably connected to the bottom opening of the second centrifuge tube body and is arranged vertically. The shape of the upper outer surface of the second centrifuge tube body matches the shape of the inner surface of the second centrifuge tube mounting through hole. The upper part of the second centrifuge tube body can be coaxially and detachably connected to the first centrifuge tube cap through the second centrifuge tube mounting through hole. The upper surface of the second centrifuge tube body is flush with the upper surface of the first centrifuge tube cap. The lower middle part of the second centrifuge tube body extends downward into the first centrifuge tube body. Alternatively, the first centrifuge tube has a volume of 50 mL, the second centrifuge tube has a volume of 1.5 mL, and the bottom of the needle is positioned 1-2 cm above the 5% sodium tripolyphosphate solution contained in the first centrifuge tube.

[0090] The same method as described above was used to microencapsulate lactic acid bacteria, and the secretions were then analyzed. First, the lactic acid bacteria were activated and cultured to the logarithmic growth phase for CFU counting. Then, 1×10⁻⁶ CFU were used. 6 Lactic acid bacteria were activated by shaking tube culture. After reaching the logarithmic growth phase, they were microencapsulated and then placed in 5 mL LB medium for shaking tube culture for 24 h. The change in phenol red color indicated a change in pH from weakly alkaline to weakly acidic. The yellowing of phenol red confirmed that the lactic acid bacteria remained active and continued to release lactic acid. The supernatant was then aspirated and plate-spreading was performed to verify whether the color change was caused by the presence of lactic acid bacteria in the supernatant. The experiment proved that no lactic acid bacteria were present in the supernatant.

[0091] pass Figure 3It can be seen that this invention successfully prepared morphologically regular and structurally complete spherical microcapsules by encapsulating Staphylococcus aureus with chitosan and sodium tripolyphosphate. Observational results show that the synthesized microcapsules have good sphericity, smooth surfaces, and uniform sizes, confirming the significant advantages of this synthesis strategy in the controllable construction of microcapsule materials with good morphology. The color change range of phenol red is from pH 6.8 to 8.4; at pH 6.8, phenol red appears yellow, and when the pH increases to 8.4, phenol red turns red. Figure 4 The phenol red color changes from red to yellow, and in the plate coating experiment, the color changes on the plate (e.g., ...). Figure 5 As shown in the figure, there were no signs of lactic acid bacteria growth, proving that bacterial secretions can diffuse to the outside of the microcapsule through the micropores on the surface of the microcapsule, thereby achieving material exchange with the skin model.

[0092] Example 4 A method for constructing a 3D skin model loaded with microcapsules includes the following steps: Based on the 3D skin model without microcapsules constructed in Example 1, the CS-bacteria-TPP microcapsules prepared in Example 3 were applied to the surface of the 3D skin model using a pipette. First, the microcapsules prepared in Example 3 were resuspended in 1×PBS buffer solution (1 mL of PBS buffer solution, resulting in a bacterial concentration of 10⁻⁶ after resuspending the microcapsules). 4 In a solution containing microbial microcapsules (10 μL / 10 μL), 200 μL of the solution was pipetted onto the surface of the prepared 3D skin model to obtain a 3D simulated skin model based on microbial colonies.

[0093] The constructed 3D skin model containing microcapsules was subjected to H&E staining (steps same as in Example 1). Figure 6 As shown, the observation results indicate that the microcapsules were successfully and completely loaded onto the surface of the 3D skin model, and a stable host-microbe coexistence interface was initially formed. This provides a highly biomimetic and reliable in vitro research platform for in-depth research on the function of skin symbiotic bacteria, the interaction mechanism between the microbiota and the skin, and the efficacy evaluation of microecological regulators.

[0094] Example 5 Electron microscopy scanning of a microcolony-based 3D skin model includes the following steps: Electron microscopy (EM) was performed on a 3D skin model loaded with microcapsules constructed in Example 4. First, the 3D skin model loaded with microcapsules was freeze-dried in a vacuum freeze dryer for 24 hours, and then longitudinally sliced ​​to obtain longitudinal sections. The dried sample strips, with the longitudinal sections facing upwards, were adhered to the SEM sample stage using conductive double-sided carbon tape, ensuring good contact. Then, the sample stage was placed in an ion sputtering instrument, and a thin gold film was sputtered onto the sample surface. This made the non-conductive biological sample surface conductive, effectively dissipating charge during SEM observation and avoiding image distortion, drift, and overexposure caused by the "charge effect." The prepared sample stage was placed in the SEM sample chamber, and a vacuum was drawn. The accelerating voltage was set to 5–15 kV, and the working distance was set to approximately 8–12 mm to balance depth of field and signal intensity.

[0095] The morphology and structure of a 3D skin model loaded with microcapsules were observed using scanning electron microscopy (SEM), and a systematic evaluation was conducted. For example... Figure 7 As shown, a 3D skin model loaded with microcapsules was successfully constructed. The results showed that the cultured skin model and its surface microcapsules exhibited excellent morphological characteristics.

[0096] Example 6 Testing the transdermal resistance of a 3D simulated human skin model based on microcolonies includes the following steps: A Transwell culture chamber and a 24-well culture plate were combined, with the wells of the culture plate serving as the lower chambers, and a 3D skin model was cultured in the Transwell culture chamber. Add 1 mL of PBS receiving buffer to each well of the 24-well plate in the lower chamber (select an appropriate culture medium or other buffer according to the test substance; the selection of the receiving buffer needs to be verified to prove that it does not damage the test substance, does not affect the detection, and can ensure the normal survival of the skin model cells during the experiment). Then, calibrate the resistor meter after thoroughly cleaning the electrodes and place it in the culture medium to allow the resistor meter reading to stabilize. Slowly place the Transwell chamber containing the 3D simulated human skin model along one side of the well wall, ensuring that there are no air bubbles between the receiving buffer and the Transwell culture chamber. Choose an appropriate angle, with the electrode perpendicular to the Transwell chamber. Place the longer electrode outside the Transwell chamber and the shorter electrode inside, without contacting the cells. After the resistance meter reading stabilizes, record the resistance value. Measure the resistance in three directions for each Transwell chamber, and take the average as the actual TEER value for that well. Simultaneously measure the resistance of a cell-free Transwell chamber, designating it as a blank well. Then, the TEER value was measured daily using a resistance meter, and the data was recorded. When the TEER value increased and stabilized (140 Ω), the new 3D skin model was considered successfully constructed. The formula for calculating the resistance value (TEER value) is as follows:

[0097] From Table 1 and Figure 1 It can be seen that the thickness of the 3D simulated human skin model provided by the present invention is about 1~2 mm. The thickness, tissue layers and transcutaneous electrical resistance of the 3D simulated human skin model prepared by the present invention can meet the requirements of the new 3D skin model. That is, the skin structure is distinct, the resistance value increases and tends to stabilize at about 200 Ω, the skin model is well developed and has a complete skin barrier, and can be used as a reliable permeability and irritation test model, indicating that the 3D skin model has been successfully constructed.

[0098] Table 1 Performance test results of the 3D simulated human skin model based on microbial colonies

[0099] The control group was the model prepared in Example 2, and the experimental group was the model prepared in Example 4. D1, D2, D3, and D4 refer to the first day, the second day, the third day, and the fourth day, respectively.

[0100] Comparative Example 1 Its preparation method is exactly the same as that in Example 4, except that: The CS-bacterial-TPP microcapsules were prepared using the method described in Example 3, but sodium tripolyphosphate was replaced with citrate. However, citrate has two fewer negative charges per molecule than sodium tripolyphosphate, resulting in a weaker degree of cross-linking. Furthermore, the binding of citrate to chitosan is significantly weakened in environments with a pH greater than 6.5, thus failing to form microcapsules with good mechanical strength.

[0101] Comparative Example 2 Its preparation method is exactly the same as that in Example 4, except that: In the preparation of CS-bacterial-TPP microcapsules, the preparation method described in Example 3 was used, but chitosan was replaced with polyethyleneimine (PEI). However, PEI has limitations compared to chitosan, including poor biocompatibility, high cytotoxicity, difficult biodegradation, and higher cost. When bacteria were encapsulated using both systems, the bacterial survival rate after encapsulation with the chitosan-sodium tripolyphosphate system was 95%, significantly higher than the 35% survival rate after encapsulation with the polyethyleneimine-sodium tripolyphosphate system. This is detrimental to the survival of both bacteria and cells.

[0102] By comparing Example 4, Comparative Example 1 and Comparative Example 2, it can be seen that sodium tripolyphosphate and chitosan have a synergistic effect in the method of the present invention, which can synergistically improve the relevant performance of the 3D skin model loaded with microcapsules.

[0103] In summary, the index value of the skin model of this invention is 200 Ω, and it tends to stabilize after four consecutive days of transdermal resistance testing, which means it meets the standard.

[0104] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing a 3D simulated human skin model based on microcolonies, characterized in that: Includes the following steps: A 3D simulated human skin model was prepared; microcapsules containing bacteria were prepared; the microcapsules were spread onto the surface of the 3D simulated human skin model to obtain a 3D simulated human skin model based on microcolonies.

2. The preparation method according to claim 1, characterized in that: The method for preparing the bacterial microcapsules includes the following steps: The bacterial symbiotic solution of the skin was centrifuged to obtain bacterial sludge. Organic solvent, bacterial sludge and chitosan powder were added to an Erlenmeyer flask in sequence to obtain chitosan-bacteria solution. Sodium tripolyphosphate powder was taken into a centrifuge tube and added to a centrifuge sleeve device for centrifugation. Finally, the CS-bacteria solution was added dropwise into the TPP solution at a uniform rate to cross-link the two to obtain microcapsules with uniform morphology and containing bacteria.

3. The preparation method according to claim 2, characterized in that: The skin symbiotic bacteria include Staphylococcus epidermidis, Staphylococcus aureus, lactic acid bacteria, or micrococci; Alternatively, the preparation steps of the bacterial solution are as follows: inoculating the skin symbiotic bacteria in a clean bench, culturing the bacterial strains in a shaker, and obtaining the desired bacterial strains.

4. The preparation method according to claim 2, characterized in that: The specific preparation steps of the bacterial microcapsules are as follows: Each culture of *Staphylococcus aureus* in the logarithmic growth phase is added to a 10 mL centrifuge tube and centrifuged at 4000-5000 rpm for 2-3 minutes to obtain bacterial sludge. The bacterial sludge is resuspended in 1 mL of 2% acetic acid solution. 0.4 g of chitosan powder is weighed and added to a 100 mL Erlenmeyer flask in sequence with 19 mL of 2% acetic acid solution, 1 mL of bacterial culture, and chitosan powder. The mixture is stirred evenly with a glass rod to obtain chitosan-bacterial solution, abbreviated as CS-bacterial solution. The CS-bacterial solution is added to a 1.5 mL second centrifuge tube in a self-made centrifuge sleeve device. 12.5 mL of 5% sodium tripolyphosphate solution is pipetted into a 50 mL first centrifuge tube in the self-made centrifuge sleeve device, with the needle in the device maintaining a distance of 1-2 cm from the sodium tripolyphosphate solution level. The centrifuge sleeve device is placed in a centrifuge and centrifuged at 700 rpm for 10 minutes. min, and add 2% CS-bacterial solution at a constant rate to 5% TPP solution to obtain uniformly sized microcapsules containing bacteria, i.e., bacterial microcapsules. The centrifuge tube device includes a first centrifuge tube and a second centrifuge tube. The second centrifuge tube is coaxially and detachably connected to the first centrifuge tube. The first centrifuge tube includes a first centrifuge tube body and a first centrifuge tube cap. The first centrifuge tube body is arranged vertically and is shaped like a centrifuge tube with an open top. The first centrifuge tube cap is coaxially and detachably disposed above the opening of the first centrifuge tube body, and a second centrifuge tube mounting through hole is integrally formed on the first centrifuge tube cap. The first centrifuge tube body can hold CS-bacterial solution. The second centrifuge tube includes a second centrifuge tube body, a second centrifuge tube cap, and a needle. The second centrifuge tube body is arranged vertically and is open at both the top and bottom. The second centrifuge tube cap is movably and detachably connected to the top opening of the second centrifuge tube body. The second centrifuge tube body can hold a 5% sodium tripolyphosphate solution. The needle is detachably and detachably connected to the bottom opening of the second centrifuge tube body and is arranged vertically. The shape of the upper outer surface of the second centrifuge tube body matches the shape of the inner surface of the second centrifuge tube mounting through hole. The upper part of the second centrifuge tube body can be coaxially and detachably connected to the first centrifuge tube cap through the second centrifuge tube mounting through hole, and the upper surface of the second centrifuge tube body is flush with the upper surface of the first centrifuge tube cap. The lower middle part of the second centrifuge tube body extends downward into the first centrifuge tube body.

5. The preparation method according to claim 1, characterized in that: The method for preparing the 3D simulated human skin model includes the following steps: (1) Construction of the dermis: Fibroblasts were mixed with mouse type I collagen solution, and the collagen gel solution containing fibroblasts was transferred to a Transwell insert chamber for culture. The dermis was obtained after culture. (2) First epidermal inoculation: Place the keratinocytes on the dermis in the Transwell chamber of step (1) and culture them in an incubator to allow the keratinocytes to adhere to the wall; add keratinocyte culture medium to the Transwell chamber and fibroblast culture medium to the outside of the Transwell chamber, and immerse them in the culture until the liquid levels are equal to obtain the inner epidermis. (3) Second epidermal inoculation: Keratinocytes are inoculated onto the inner epidermis described in step (2). Keratinocyte culture medium is added to the Transwell chamber and fibroblast complete culture medium is added to the outside of the Transwell chamber for epidermal culture. The culture medium is changed in time during the culture process. The cells are cultured until the keratinocytes are completely adsorbed to the inner epidermis and tightly connected to it, thus obtaining the epidermis; after the epidermis is formed, the culture medium inside and outside the Transwell chamber is removed; (4) Co-culture: Add 1~2 mL of gas-liquid culture medium to the inside and outside of the chamber for gas-liquid culture to obtain a 3D simulated human skin model without microcapsules.

6. The preparation method according to claim 5, characterized in that: The collagen gel in step (1) has a thickness of 1-3 mm; the dermis layer has a thickness of 1-2 mm; Alternatively, step (1) may also include the following operations: Fibroblast pretreatment: Fibroblasts were digested using trypsin; Prepare the collagen solution: The concentration of the collagen solution is 2~4 mg / mL, and the pH value of the collagen solution is adjusted to 7.1; Mixing collagen solution with fibroblasts: Under ice bath conditions, add collagen solution to culture medium containing fibroblasts, and then adjust the pH of the mixture to 7.1-7.3; Alternatively, the density of fibroblasts in the collagen solution described in step (1) is 7 × 10⁻⁶. 4 ~12×10 4 / mL, collagen concentration is 1~3 mg / mL; Alternatively, the collagen solution containing fibroblasts described in step (1); and / or the keratinocyte density during the endodermal culture described in step (2) is 4 × 10⁻⁶. 5 -10×10 5 / mL; and / or the keratinocyte density during epidermal culture in step (3) is 2 × 10⁶. 5 -5×10 5 / mL; Alternatively, the ratio of the amount of keratinocytes used in the first epidermal inoculation in step (2) to the amount of keratinocytes used in the second epidermal inoculation in step (3) is 2-7.5:

1.

7. The preparation method according to claim 5, characterized in that: The method of inoculating keratinocytes in step (2) is to inoculate the keratinocyte culture medium containing keratinocytes onto the surface of the dermis; or, the method of inoculating keratinocytes in step (3) is to inoculate the keratinocyte culture medium containing keratinocytes onto the inner epidermis. Alternatively, the dermal cell culture medium formula is as follows: DMEM 8 g / L~15 g / L, F12 2 g / L~8 g / L, fetal bovine serum 0.1%~1%, L-glutamine 2 mol / L~5 mol / L, hydrocortisone 0.1%~0.5%, insulin 1 mg / L~5 mg / L, antibiotic i.e., penicillin-streptomycin mixture (100×) 0.1%~1.5%, with water as the solvent; where the percentages mentioned above are final mass concentrations. The keratinocyte culture medium formula is as follows: DMEM 10 g / L~20 g / L, fetal bovine serum 5%~15%, L-glutamine 2 mol / L~5 mol / L, antibiotic i.e. penicillin-streptomycin mixture (100×) 0.1%~1.5%, and water as solvent; wherein, the above percentages are all final mass concentrations; The aero-liquid culture medium formulation is as follows: DMEM 8 g / L~15 g / L, F12 2 g / L~8 g / L, fetal bovine serum 0.1%~1%, L-glutamine 2 mol / L~5 mol / L, hydrocortisone 0.1%~0.5%, insulin 1 mg / L~5 mg / L, palmitic acid 0.005 mM~0.008 mM, arachidonic acid 0.001 mM~0.002 mM, linoleic acid 0.005 mM~0.007 mM, BSA 0.005 mM~0.005 mM, and water as the solvent; all percentages mentioned above are final mass concentrations.

8. The preparation method according to claim 5, characterized in that: The cultivation method described in step (1) is to place the plant at 37°C. o In a 5% carbon dioxide incubator, gels were formed for 0.2–0.5 h to create a collagen gel with a thickness of 1–3 mm. Dermal fibroblast complete culture medium was added to both the transwell chamber and the transwell chamber to ensure that the liquid levels of the culture medium inside and outside the transwell chamber were equal. The dermal culture was carried out for 2–5 days. Alternatively, the method for endodermal culture described in step (2) is to allow the keratinocytes to adhere to the wall after static incubation, then add keratinocyte culture medium to the Transwell chamber and dermal fibroblast complete culture medium to the outside of the Transwell chamber to make the liquid levels inside and outside the chamber equal, and immerse and culture for 3-5 days. Alternatively, the method for culturing the epidermal layer in step (3) is to allow the keratinocytes to adhere to the wall after static incubation, add 1-3 mL of keratinocyte culture medium to the Transwell chamber and 2-4 mL of fibroblast culture medium to the outside of the Transwell chamber to make the liquid levels inside and outside the chamber equal, and immerse and culture for 3-5 days. Alternatively, the gas-liquid culture method described in step (4) involves adding gas-liquid culture medium both inside and outside the Transwell chamber, and changing the medium daily for 10-12 days. Alternatively, in step (5), the culture method for the bacterial strain is to place the inoculated culture medium into a 37°C container. o In a shaker, bacteria are cultured for 12-24 hours. Alternatively, the specific method for allowing keratinocytes to adhere to the culture vessel as described in steps (2) and (3) is to place them in a 5% carbon dioxide incubator at 37°C. o C, let stand for 1~3 hours; Alternatively, the collagen solution containing fibroblasts may contain mouse type I collagen or fetal bovine type I collagen.

9. A 3D simulated human skin model based on microcolonies prepared by any one of the preparation methods described in 1 to 8.

10. The application of a microcolony-based 3D simulated human skin model as described in claim 9 in skin research and / or evaluation samples.

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