Culture kit for in-vitro recombination of 3D skin epidermis and use method

Abstract

The invention relates to the technical field of biology, and discloses a culture kit for in-vitro recombination of a 3D skin epidermis and a use method, the culture kit comprises: a kit body, the top end of which is provided with a top cover; the protection assembly is arranged in the box body, the protection assembly comprises a base, the outer wall of the base is arranged in the box body, a connecting rod is fixed to the top end of the base, and a plurality of sponge plates are fixed to the outer wall of the connecting rod; the reagent bottle is arranged on the inner wall of the sponge plate, and three top ends of the reagent bottle are in threaded connection with sealing covers on the inner wall of the sponge plate; the three reagent bottles are respectively filled with a reagent A, a reagent B and a reagent C; by adopting the technical scheme that a reagent A skin printing ink, a reagent B skin fixing liquid and a reagent C3D skin induction liquid which are required by 3D skin epidermal layer printing are prepared in advance and packaged into the kit, the technical effects of remarkably improving the operation convenience and repeatability are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture kit for in vitro reconstructing 3D skin epidermis and a method for using the kit. Background Art

[0002] Skin is the largest organ in the human body and has important functions in protection, regulation and perception. However, large-area skin damage (such as burns and trauma) is one of the major problems in clinical medicine. In the existing technology, autologous skin transplantation or skin substitutes are often used for repair, but these technologies have obvious shortcomings: autologous skin transplantation relies on healthy skin donors from patients, which is often difficult to implement in patients with large-area injuries due to insufficient donors; although skin substitutes have certain replacement functions, their structure and function are far from simulating real skin, and are prone to problems such as poor biocompatibility and insufficient regeneration ability.

[0003] 3D bioprinting technology can accurately construct three-dimensional structures by stacking printing materials layer by layer, and is gradually being applied to the construction of artificial skin. However, existing 3D bioprinting technology still faces the following challenges in the field of skin construction: the preparation of printing consumables is complex and has poor stability, and users need to configure printing inks by themselves, which can easily lead to insufficient repeatability and stability of experimental results, insufficient functionality and consistency of printed tissues, and lack of optimized ink composition and growth factor ratios, resulting in incomplete printed skin tissue structures and low biological activity.

[0004] In the efficacy and safety evaluation of cosmetics and drugs, traditional experimental models mainly include animal models and monolayer cell culture models. However, due to ethical restrictions and species differences, the results of animal models are quite different from the reactions of human skin, and the reference value of experimental data is limited; although the monolayer cell model is simple to operate, it cannot truly reflect the three-dimensional structure and barrier function of the skin, and it is difficult to meet the needs of high-precision experiments. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a culture kit and method for using the in vitro reconstructed 3D skin epidermis, which solves the problem that some seriously injured patients do not even have enough intact skin tissue for medical personnel to collect.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: a culture kit for in vitro reconstructing 3D skin epidermis, comprising:

[0007] The box body has a top cover disposed on the top thereof;

[0008] A protection component is arranged inside the box body, the protection component comprises a base, the outer wall of the base is arranged on the box body, a connecting rod is fixed on the top of the base, and a plurality of sponge plates are fixed on the outer wall of the connecting rod;

[0009] A reagent bottle is arranged on the inner wall of the sponge board, and the reagent bottle is provided with three top ends threadedly connected with sealing caps on the inner wall of the sponge board;

[0010] The three reagent bottles are respectively filled with reagent A, reagent B and reagent C.

[0011] Furthermore, a protective component for fixing and protecting reagent bottles is provided inside the box body, and the protective component includes a base fixed to the bottom of the box body, the outer wall of the base is fixedly connected to the inner wall of the box body, a plurality of vertically arranged connecting rods are fixed to the top of the base, a plurality of sponge plates for buffering and protecting the reagent bottles are fixed around the outer walls of the connecting rods, the sponge plates are fixedly connected to the connecting rods to form an annular structure, a accommodating cavity is provided on the inner wall of each sponge plate, and a reagent bottle is placed in the accommodating cavity, and there are three reagent bottles in total, which are used to contain reagent A, reagent B and reagent C respectively, and sealing caps are threadedly connected to the tops of the reagent bottles, the outer walls of the reagent bottles are tightly fitted to the inner walls of the accommodating cavity of the sponge plate to prevent the reagent bottles from shaking, colliding or leaking during transportation and use, and the box body implements fixation and buffering protection of the reagent bottles through the protective component, thereby ensuring the stability and safety of the test kit during transportation and use.

[0012] Preferably, reagent A: skin printing ink, made of the following ingredients: 3% sodium alginate, 1% gelatin, 10 ng / ml epidermal growth factor (EGF) and 10 ng / ml fibroblast growth factor (FGF);

[0013] Reagent B: Skin fixative, consisting of 2% calcium chloride solution;

[0014] Reagent C: 3D skin induction solution, which is a mixture of basal culture medium and epidermal cell-specific culture medium at a ratio of 7:3, and supplemented with 20 ng / ml EGF, 20 ng / ml FGF, 0.4 μg / ml hydrocortisone, 10 ng / ml insulin, 8 μg / ml vitamin C, 10 ng / ml IBMX and 100XHKGS.

[0015] Furthermore, reagent A is a skin printing ink, which is made of the following ingredients: 3% sodium alginate, 1% gelatin, 10ng / ml epidermal growth factor (EGF) and 10ng / ml fibroblast growth factor (FGF), wherein sodium alginate, as the matrix material of the printing ink, can provide a suitable microenvironment for cells and has excellent moisture retention and stability, gelatin, as a supporting material, provides the necessary viscoelasticity and morphological stability for the printed skin structure, EGF and FGF, as important growth factors, can promote the proliferation and differentiation of epidermal cells and the repair and regeneration of skin tissue respectively; reagent B is a skin fixative, composed of a 2% calcium chloride solution, and calcium chloride reacts with collagen or similar components in the printing material to quickly fix the printed skin tissue, thereby ensuring the integrity and stability of the printed skin morphology; reagent C is a 3D skin induction solution, which is a mixture of a basal culture medium and a special culture medium for epidermal cells in a ratio of 7:3, and a variety of induction factors are added, including 20ng / ml EGF and 20ng / ml FGF promotes cell proliferation and differentiation, 0.4μg / ml hydrocortisone regulates the balance of cell proliferation and differentiation, 10ng / ml insulin enhances cell metabolic activity, 8μg / ml vitamin C acts as an antioxidant to enhance the cell's antioxidant capacity and collagen synthesis capacity, 10ng / ml IBMX promotes the cell's secretion of important structural proteins, and 100XHKGS provides essential growth factors and nutrients. The composition design of reagent C can fully meet the needs of cell proliferation, differentiation and functionalization during 3D skin culture.

[0016] Preferably, the preparation method of the reagent A comprises the following steps:

[0017] Dissolve sodium alginate with saline, vortex for 20 minutes, and filter with a 0.22 μm filter membrane to sterilize after complete dissolution;

[0018] The gelatin was sterilized by high pressure at 121°C, added into the sodium alginate solution according to proportion, dissolved in a water bath at 80°C, and added with EGF and FGF after cooling, and mixed well to obtain the product.

[0019] Further, firstly, the required amount of sodium alginate is weighed, and physiological saline is used as a solvent to dissolve the sodium alginate. The solution is placed on a vortex shaker for 20 minutes to ensure that the sodium alginate is completely dissolved. The dissolved sodium alginate solution is filtered and sterilized through a 0.22 μm filter membrane to ensure the sterility and purity of the solution. Then, the required amount of gelatin is weighed, divided and placed in a high-pressure steam sterilizer at 121° C. for sterilization. When used, the sterilized gelatin is gradually added to the aforementioned sterilized sodium alginate solution according to a predetermined ratio, and the mixed solution is placed at 80° C. The mixture is heated in a water bath to accelerate the dissolution of gelatin. During the process, continuous stirring or vortex oscillation is required to ensure that the gelatin is completely dissolved and the solution is uniform. After the gelatin is completely dissolved, the solution is cooled to room temperature. Subsequently, 10 ng / ml of epidermal growth factor (EGF) and 10 ng / ml of fibroblast growth factor (FGF) are added according to the formula ratio, and the mixture is fully stirred or oscillated until the components are evenly distributed. Finally, ink reagent A for skin printing is obtained. The entire preparation process needs to be operated in a sterile environment to ensure the stability and safety of the product.

[0020] Preferably, the preparation method of reagent B comprises the following steps:

[0021] Weigh calcium chloride and dissolve it in pure water;

[0022] After dissolution, the solution was sterilized by filtration through a 0.22 μm filter membrane to prepare a 2% calcium chloride solution.

[0023] Further, weigh the required amount of calcium chloride, accurately calculate its mass according to the formula ratio, select high-purity calcium chloride powder without impurities, gradually add it to an appropriate amount of pure water, and use a magnetic stirrer or a manual stirring rod to fully stir the solution while adding to ensure that the calcium chloride is completely dissolved in the water to avoid local supersaturation and cause the formation of precipitation. If necessary, it can be heated appropriately to accelerate the dissolution process; after the calcium chloride is completely dissolved, the resulting solution is placed at room temperature and allowed to stand, and the clarity and uniformity of the solution are checked to ensure that there are no obvious particles or impurities, and then the solution is filtered through a filter membrane with a pore size of 0.22 μm. The membrane filtration step should be completed under a sterile operating environment to ensure that no impurities or contaminants are introduced during the filtration process. Finally, a calcium chloride solution with a concentration of 2% is obtained, and the prepared solution is placed in a sterilized reagent bottle and sealed for storage to ensure the sterility and stability of the reagent. The environmental conditions must be strictly controlled during the entire preparation process and the operation must be performed in accordance with the specifications to ensure that the quality of reagent B meets the use requirements.

[0024] Preferably, the preparation method of reagent C comprises the following steps:

[0025] Mix the basal medium and epidermal cell-specific medium in a ratio of 7:3;

[0026] The preparation is prepared by sequentially adding EGF, FGF, hydrocortisone, insulin, vitamin C, I BMX and HKGS and mixing them evenly.

[0027] Furthermore, according to the formula requirements, appropriate amounts of basal culture medium and epidermal cell-specific culture medium were taken and accurately mixed in a volume ratio of 7:3, and the mixed solution was placed in a stirring device for low-speed stirring to ensure that the two culture media were fully and evenly mixed to avoid differences in subsequent culture effects due to uneven distribution of components; after the culture media were evenly mixed, 20 ng / ml of epidermal growth factor (EGF) and 20 ng / ml of fibroblast growth factor (FGF) were added in sequence according to the set concentrations, and gently stirred to dissolve them evenly, followed by adding 0.4 μg / ml of hydrocortisone and 10 ng / ml of insulin, and continuing to stir to ensure complete mixing, and at the same time adding 8 μg / ml of vitamin C. C is used as an antioxidant to enhance the antioxidant capacity of cells, and 10ng / ml IBMX is added to promote the secretion of structural proteins by cells. Finally, a certain concentration of 100XHKGS solution is added as a key component for supplementing growth factors and nutrients. The entire mixing process must be carried out under sterile conditions, and appropriate stirring or oscillation must be performed after adding each component to ensure the uniformity of the solution and the effectiveness of the components. Finally, the induction solution for 3D skin culture is obtained, and the prepared reagent C is packaged in sterilized reagent bottles and sealed for storage to maintain the stability and functionality of the solution during subsequent use. The entire operation must be carried out in strict accordance with sterile operation specifications to ensure that the quality and safety of the product meet the experimental and application requirements.

[0028] A method for culturing an in vitro reconstructed 3D skin epidermis layer using a kit, comprising the following steps:

[0029] Reagent A was placed in a 37°C water bath and heated for 20 minutes, and the skin cells and reagent A were mixed and 3D printed;

[0030] After printing, fix the skin tissue on a cold bench for 1 min using reagent B and wash it twice with saline;

[0031] The cleaned skin tissue is placed in reagent C and cultured for 21 to 32 days.

[0032] Preferably, the method further comprises: observing the integrity of the skin tissue by HE staining after culturing for 21 to 32 days.

[0033] Preferably, the method further comprises the following steps:

[0034] The cultured 3D skin tissue was fixed with 4% formaldehyde, dehydrated with alcohol in stages, and transparentized with xylene;

[0035] The dehydrated and transparent tissue is embedded, sliced ​​and baked for HE staining or immunohistochemistry experiments.

[0036] Furthermore, reagent A is heated in a 37°C water bath for 20 minutes to ensure that the components in reagent A are completely dissolved and reach the optimal state of use, the required skin cells are fully mixed with the heated reagent A, and the mixture is printed using 3D printing technology. During the printing process, the parameters of the printer are ensured to be reasonably set to ensure the integrity and accuracy of the printed skin epidermal structure; after printing, the printed skin tissue is transferred to a cold stage, and the printed skin tissue is immediately fixed with reagent B for 1 minute. The morphology and structure of the skin tissue are kept stable through the chemical fixation of reagent B, and then the fixed skin tissue is washed with sterile saline for 2 times to remove impurities or unreacted components that may remain during the printing process; after washing, the treated skin tissue is transferred to reagent C for culture for 21 to 32 days. During the culture process, reagent C provides a suitable environment for epidermal cell growth. The method further comprises the following steps: placing the cultured 3D skin tissue in a 4% formaldehyde solution for fixation to stabilize the tissue morphology, followed by gradient dehydration using 50%, 70%, 80%, 90%, 95%, and 100% alcohol in sequence, placing the dehydrated tissue in a xylene solution for transparency treatment to improve the optical visibility and embedding effect of the tissue, then embedding and slicing the transparent tissue, and baking the slices on a 60°C baking machine for 4 hours for use in HE staining or immunohistochemistry experiments. The entire operation must be performed under sterile conditions to ensure that the quality and functionality of the tissue during the culture process meet the experimental requirements.

[0037] Preferably, the expression levels of fibronectin and keratin 10 in skin tissue are detected by immunohistochemistry.

[0038] Further, the cultured 3D skin tissue is fixed with 4% formaldehyde to ensure the stability of the tissue structure and the retention of the antigen, and then the fixed tissue is subjected to gradient alcohol dehydration treatment, using 50%, 70%, 80%, 90%, 95%, and 100% alcohol dehydration from low concentration to high concentration to remove moisture from the tissue. After dehydration, the tissue is placed in xylene for transparent treatment to further increase the optical visibility of the tissue; the transparent tissue needs to be paraffin embedded and sliced ​​to a thickness of 4-6 microns, and the sliced ​​tissue is fixed on a glass slide and dried on a 60°C slide oven for subsequent experimental operations; after slicing, antigen retrieval is performed by microwave heating using an appropriate buffer such as sodium citrate or EDTA solution to expose the antigen site, followed by incubation in 3% hydrogen peroxide solution to block endogenous peroxidase activity, and then adding a blocking solution such as 5% normal goat serum or bovine serum to incubate at room temperature for 30 minutes to reduce nonspecific antibody binding; after blocking, add The tissue slides were incubated overnight in a 4°C refrigerator with specific primary antibody solutions for fibronectin and keratin 10 (CK10) to ensure that the antibodies were fully bound to the target antigens. After incubation, the slides were washed 3 times with PBS buffer for 5 minutes each time to remove unbound antibodies, and then the secondary antibody solution containing the labeled enzyme was added and incubated at room temperature for 1 hour, and then washed again with PBS 3 times; after washing, SABC (streptavidin-biotin complex) reagent was added for incubation for 30 minutes, and then DAB (3,3'-diaminobenzidine) color development solution was added to control the color development time to obtain the appropriate signal intensity, and finally hematoxylin was used to lightly stain the cell nucleus for observation; after staining, the slides were sequentially dehydrated with gradient alcohol, transparentized with xylene, and sealed with neutral gum. After the seal was dried, the expression distribution and level of fibronectin and keratin 10 were observed under a microscope, and the signal intensity was quantitatively analyzed using microscopic image analysis software to evaluate the biological function and integrity of skin tissue.

[0039] Preferably, the method can significantly improve the operational efficiency of 3D skin epidermal layer printing and culturing, and ensure the biological functional integrity and consistency of cultured skin tissue.

[0040] The present invention provides a culture kit and a method for using the in vitro reconstructed 3D skin epidermis layer, which has the following beneficial effects:

[0041] 1. The present invention adopts a technical solution that reagent A (skin printing ink), reagent B (skin fixative) and reagent C (3D skin induction solution) required for 3D skin epidermal printing are pre-prepared and packaged into a kit, achieving a technical effect of significantly improving the ease of operation and repeatability. Compared with the technical solution in the prior art that requires users to configure each reagent component by themselves, it solves the shortcomings of complex reagent preparation process, high time cost and difficulty for beginners to accurately grasp the ratio.

[0042] 2. The present invention achieves the technical effect of significantly improving the integrity and biological activity of printed skin tissue by optimizing the formula of reagent A (containing 3% sodium alginate, 1% gelatin, 10ng / ml EGF, 10ng / ml FGF) and the temperature control treatment technical solution during the printing process. Compared with the printing instability caused by the single printing ink component and poor solubility in the prior art, the problem of incomplete morphology and low cell activity of printed skin tissue is solved, and the functionality and consistency of the printed tissue are improved.

[0043] 3. The present invention achieves the technical effect of supporting multifunctional 3D skin epidermal layer culture through the technical solution of scientifically matching basal culture medium, epidermal cell culture medium and multiple induction factors in reagent C. Compared with the technical solution in the prior art that lacks a dedicated kit and limits the culture process to specific experiments (such as clinical repair experiments), it solves the shortcomings of the prior art in the application of cosmetic efficacy evaluation (such as moisturizing, anti-inflammatory, barrier repair) and safety testing (such as skin toxicity and irritation experiments), making it have a wider range of practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 A perspective view of the present invention;

[0045] Figure 2 It is a partial structural schematic diagram of the protection component of the present invention;

[0046] Figure 3 It is a schematic diagram of the internal structure of the box body of the present invention;

[0047] Figure 4 It is a partial structural schematic diagram of a reagent bottle of the present invention;

[0048] Figure 5 is a flow chart of the method of the present invention;

[0049] Figure 6 This is the 3D skin HE staining image of the present invention;

[0050] Figure 7 This is a 3D printed epidermal protein inspection diagram of the present invention.

[0051] Among them, 1. box body; 2. top cover; 3. protection component; 301. base; 302. connecting rod; 303. sponge board; 4. reagent bottle; 5. sealing cover. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] Please see attached Figure 1 - Attachment Figure 7 :

[0054] Example 1: Example with innovative ingredients and process parameters within the range

[0055] step:

[0056] Preparation of Reagent A

[0057] 3% sodium alginate was taken, dissolved with physiological saline, vortexed for 20 minutes, and sterilized by filtration using a 0.22 μm filter membrane.

[0058] After high-pressure sterilization, gelatin (1%) was added to the above solution, dissolved in a water bath at 80°C, and 10 ng / ml of epidermal growth factor (EGF) and 10 ng / ml of fibroblast growth factor (FGF) were added and mixed to obtain the solution.

[0059] Preparation of Reagent B

[0060] Take 2% calcium chloride, dissolve it in pure water, and filter it through a 0.22 μm filter membrane for sterilization.

[0061] Preparation of Reagent C

[0062] The basal culture medium and the epidermal cell-specific culture medium were mixed in a ratio of 7:3, and 20 ng / ml of EGF, 20 ng / ml of FGF, 0.4 μg / ml of hydrocortisone, 10 ng / ml of insulin, 8 μg / ml of vitamin C, 10 ng / ml of IBMX and 100XHKGS were added and mixed thoroughly.

[0063] Cultivation steps

[0064] Heat reagent A in a 37°C water bath for 20 minutes, mix with skin cells and perform 3D printing.

[0065] After printing, fix it with reagent B on a cold bench for 1 minute and then wash it twice with saline.

[0066] The washed tissue was cultured in Reagent C for 21 to 32 days.

[0067] result:

[0068] The printed skin tissue has complete morphology, clear layers, high cell activity, normal expression levels of fibronectin and keratin 10 in the tissue, and the cultured skin has good barrier function and biological activity.

[0069] Summarize:

[0070] This embodiment shows that when the innovative ingredients and process parameters are within the recommended range, high-quality skin tissue can be obtained, reflecting the superiority of the technology of the present invention.

[0071] Example 2: Example with innovative ingredients below the range value

[0072] step:

[0073] The sodium alginate content in reagent A was reduced to 1.5%, and the EGF and FGF contents in reagent C were reduced to 10 ng / ml. Other operations were the same as in Example 1.

[0074] result:

[0075] The printed skin tissue morphology was defective, some areas were incomplete, cell activity was significantly reduced, the expression levels of fibronectin and keratin 10 in the tissue were low, and the barrier function was poor.

[0076] Summarize:

[0077] When the innovative ingredient is lower than the recommended range, the reagent's ability to support skin cells is weakened, resulting in poor tissue formation and biological activity, indicating the necessity of the innovative ingredient being within the recommended range.

[0078] Example 3: Example with innovative ingredients above the range value

[0079] step:

[0080] The sodium alginate content in reagent A was increased to 5%, and the EGF and FGF contents in reagent C were increased to 30 ng / ml. Other operations were the same as in Example 1.

[0081] result:

[0082] The printed skin tissue had a complete morphology, but due to the high sodium alginate content, the viscosity increased during printing, making operation difficult. In addition, some cells proliferated too quickly during the culture process, resulting in uneven tissue thickness and an abnormally increased expression level of keratin 10.

[0083] Summarize:

[0084] When the innovative ingredients are higher than the recommended range, although the formation is complete, the operation complexity increases and the cells grow excessively, affecting the functionality of the skin tissue, which shows the importance of ingredient optimization.

[0085] Comparative Experimental Design

[0086] Comparative Example 1: Comparison between the prior art solution lacking EGF and the technical solution of the present invention

[0087] Existing technical solutions:

[0088] The reagents were prepared according to Example 1, but EGF was not added to reagent A and reagent C.

[0089] result:

[0090] The printed skin tissue morphology is poor, the layers are unclear, the expression level of fibronectin is reduced, and the skin barrier function is significantly weakened.

[0091] analyze:

[0092] EGF is a key component that promotes the proliferation and differentiation of epidermal cells. The lack of EGF leads to insufficient cell activity and incomplete tissue structure, indicating that the addition of EGF is an important factor in the successful printing of skin tissue.

[0093] Comparative Example 2: Comparison between the prior art solution lacking FGF and the technical solution of the present invention

[0094] Existing technical solutions:

[0095] The reagents were prepared according to Example 1, but FGF was not added to reagent A and reagent C.

[0096] result:

[0097] The morphology of the printed tissue was relatively complete, but the cell proliferation rate was slow after culture, the expression level of keratin 10 in the tissue was lower than the normal range, and the overall biological activity was poor.

[0098] analyze:

[0099] FGF plays an important role in promoting cell proliferation and collagen synthesis. The lack of FGF will weaken the growth ability of tissues and lead to insufficient biological functionality.

[0100] Comparative Example 3: Comparison between the prior art and the process of the present invention

[0101] Existing technical solutions:

[0102] Use traditional 2D culture methods or non-optimized 3D printing inks (without sodium alginate and gelatin).

[0103] result:

[0104] The morphology of the printed tissue was severely damaged, the cell activity was extremely low, fibronectin and keratin 10 were almost not expressed, and the cultured skin tissue lacked barrier function.

[0105] analyze:

[0106] Traditional 2D culture methods and non-optimized printing inks were unable to provide sufficient support, demonstrating the key role of the optimized formula and 3D printing technology of the present invention in skin tissue construction.

[0107] Experimental data table

[0108] Table 1: Comparison of skin tissue formation integrity and biological activity

[0109]

[0110] Table 2: Safety test results

[0111]

[0112]

[0113] Table 3: Barrier function test results

[0114] Experimental groups Water loss rate reduction (%) Improved antioxidant capacity (%) Embodiment 1 (present invention) 75 80 Example 2 55 60 Example 3 70 75 Comparative Example 1 40 50 Comparative Example 2 45 55 Comparative Example 3 20 30

[0115] Comparative experimental results analysis

[0116] The necessity of innovative ingredients EGF and FGF

[0117] Experimental data show that in the absence of EGF or FGF, cell proliferation and differentiation are significantly affected, resulting in a decrease in skin tissue molding quality and biological activity, indicating that these two components are the key to promoting cell function recovery.

[0118] Advantages of optimized formula

[0119] In Example 1, the ratio of sodium alginate and gelatin within the recommended range effectively improves the viscoelasticity and solubility of the printing ink, ensuring the integrity of skin tissue and cell biological activity.

[0120] Effect of process parameters on performance

[0121] The present invention optimizes the preparation of printing ink and culture conditions, making the operations of tissue molding and culture more efficient, and the cultured skin tissue is significantly superior to the existing technical solutions in terms of functionality and safety.

[0122] A kit-based in vitro reconstructed 3D skin epidermis culture method, the core of which is to evaluate the function and tissue integrity of epidermal cells by using optimized printing materials (reagent A), fixative (reagent B) and induction culture medium (reagent C) and specific culture procedures, combined with immunohistochemistry experiments to detect the cultured skin tissue.

[0123] Tissue integrity testing after culture

[0124] In this embodiment, the integrity of the cultured 3D skin tissue was detected by HE staining, and the specific operation was as follows:

[0125] Tissue fixation: The skin tissue cultured for 21 to 32 days is taken out and immediately immersed in a 4% formaldehyde solution for 4-6 hours. The fixed tissue is used for subsequent dehydration and staining steps. The role of formaldehyde fixation is to cross-link the protein molecules in the tissue and maintain the stability of cell morphology and structure.

[0126] Gradient dehydration: The fixed tissue is placed in 50%, 70%, 80%, 90%, 95%, and 100% alcohol solutions in sequence. The dehydration time for each step is 1 hour to ensure that the water in the tissue is gradually replaced by alcohol to prevent structural deformation of the tissue due to rapid dehydration.

[0127] Transparency: The dehydrated tissue is immersed in xylene for 40 minutes to replace the alcohol inside the tissue, thereby improving its optical transparency.

[0128] Staining: Place the sliced ​​tissue in a hematoxylin solution for staining for 2 to 5 minutes, then rinse with tap water to remove background staining, then immerse the slice in ammonia water for anti-blueing, and finally add eosin staining solution for staining for 1 minute. Hematoxylin is mainly used to stain the nucleus, while eosin is used to stain the cytoplasm and matrix.

[0129] Dehydration, clearing, and sectioning

[0130] In this embodiment, the fixed skin tissue needs to be further processed to facilitate subsequent experimental operations. The specific steps are as follows:

[0131] Dehydration treatment:

[0132] The fixed skin tissue was placed in alcohol solutions of different concentrations (50%, 60%, 70%, 80%, 90%, 95%, 100%) step by step, and dehydrated for 30 minutes in each step.

[0133] Transparency processing:

[0134] Soak the dehydrated tissue in xylene solution, using xylene I and xylene II successively, each for 40 minutes.

[0135] The effect of clearing is to replace the alcohol in the tissue with xylene, making the tissue easier to embed and improving the optical properties.

[0136] Slicing and toasting:

[0137] The transparent tissue is embedded in paraffin. After the paraffin is solidified, it is cut into thin slices of 4-6 microns using a microtome. The slices must be kept flat and free of cracks.

[0138] After slicing, place the slices in a 60°C slice oven for 4 hours to remove residual moisture and ensure that the slices are firmly attached.

[0139] Immunohistochemistry

[0140] In this example, the expression levels of fibronectin and keratin 10 (CK10) in skin tissue were detected by immunohistochemistry, and the specific operation was as follows:

[0141] Antigen retrieval: Immerse the sections in sodium citrate buffer and microwave for 10 minutes to expose antigenic sites, thereby improving antibody binding efficiency.

[0142] Endogenous enzyme blocking: Incubate the sections in 3% hydrogen peroxide solution for 30 minutes to block the interference of endogenous peroxidase.

[0143] Antibody incubation:

[0144] Blocking solution (5% normal goat serum) was added and incubated at room temperature for 30 minutes to reduce nonspecific binding.

[0145] Primary antibodies (anti-CK10 and anti-Fibronectin) were added and incubated overnight at 4°C.

[0146] After washing, enzyme-labeled secondary antibody was added and incubated at room temperature for 1 hour.

[0147] Chromogenicity and staining:

[0148] Use DAB (3,3'-diaminobenzidine) colorimetric solution for color development, and control the color development time to not exceed 5 minutes.

[0149] Hematoxylin stains nuclei to further enhance tissue contrast.

[0150] Sealing observation:

[0151] After dehydration with graded alcohol and clearing with xylene, the slides were mounted with neutral gum.

[0152] The cells were observed and photographed under a microscope, and the expression levels of CK10 and fibronectin were quantitatively evaluated using image analysis software.

[0153] Principles and beneficial effects

[0154] Tissue integrity testing

[0155] HE staining can be used to visually observe the structural integrity and cell arrangement of the skin epidermis. Transparency and gradient dehydration ensure that the slices are smooth and avoid tissue deformation, which helps to evaluate whether cell differentiation is normal during the culture process.

[0156] Dehydration and sectioning

[0157] The gradient treatment of dehydration and transparency can effectively avoid tissue collapse or shrinkage, and the sectioning step further ensures the clear presentation of tissue structure, providing high-quality samples for subsequent immunohistochemical detection.

[0158] Immunohistochemistry experiment Immunohistochemistry uses specific antibodies to detect the expression of target proteins and verify the differentiation and functional characteristics of cells. CK10 is used as a keratinocyte marker to reflect the differentiation of the epidermis, and Fibronectin is used as a matrix marker to verify the integrity of the tissue structure and matrix function, ensuring the functionality and application value of the printed skin tissue.

[0159] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A culture kit for reconstructing 3D skin epidermis in vitro, characterized in that: include: A box body (1) having a top cover (2) disposed on its top; A protection component (3) is arranged inside the box body (1), the protection component (3) comprises a base (301), the outer wall of the base (301) is arranged on the box body (1), a connecting rod (302) is fixed to the top of the base (301), and a plurality of sponge plates (303) are fixed to the outer wall of the connecting rod (302); A reagent bottle (4) is arranged on the inner wall of the sponge board (303), and the reagent bottle (4) is provided with three top ends threadedly connected with sealing caps (5) on the inner wall of the sponge board (303); The three reagent bottles (4) are respectively filled with reagent A, reagent B and reagent C.

2. The in vitro 3D skin epidermis reconstructing culture kit according to claim 1, characterized in that: Reagent A: Skin printing ink, made of the following ingredients: 3% sodium alginate, 1% gelatin, 10 ng / ml epidermal growth factor (EGF), and 10 ng / ml fibroblast growth factor (FGF); Reagent B: Skin fixative, consisting of 2% calcium chloride solution; Reagent C: 3D skin induction solution, which is a mixture of basal culture medium and epidermal cell-specific culture medium in a ratio of 7:3, and supplemented with 20 ng / ml EGF, 20 ng / ml FGF, 0.4 μg / ml hydrocortisone, 10 ng / ml insulin, 8 μg / ml vitamin C, 10 ng / ml IBMX and 100X HKGS.

3. The in vitro 3D skin epidermis reconstructing culture kit according to claim 1, characterized in that: The preparation method of the reagent A comprises the following steps: Dissolve sodium alginate with saline, vortex for 20 minutes, and filter with a 0.22 μm filter membrane to sterilize after complete dissolution; The gelatin was sterilized by high pressure at 121°C, added into the sodium alginate solution according to proportion, dissolved in a water bath at 80°C, and added with EGF and FGF after cooling, and mixed well to obtain the product.

4. The in vitro 3D skin epidermis reconstructing culture kit according to claim 1, characterized in that: The preparation method of reagent B comprises the following steps: Weigh calcium chloride and dissolve it in pure water; After dissolution, the solution was sterilized by filtration through a 0.22 μm filter membrane to prepare a 2% calcium chloride solution.

5. The in vitro 3D skin epidermis reconstructing culture kit according to claim 1, characterized in that: The preparation method of reagent C comprises the following steps: Mix the basal medium and epidermal cell-specific medium in a ratio of 7:3; The preparation is prepared by sequentially adding EGF, FGF, hydrocortisone, insulin, vitamin C, IBMX and HKGS and mixing them evenly.

6. A method for culturing an in vitro reconstructed 3D skin epidermis layer using a kit, comprising the in vitro reconstructed 3D skin epidermis layer culturing kit according to claims 1-5, characterized in that: The following steps are involved: Reagent A was placed in a 37°C water bath and heated for 20 minutes, and the skin cells and reagent A were mixed and 3D printed; After printing, fix the skin tissue on a cold bench for 1 min using reagent B and wash it twice with saline; The cleaned skin tissue is placed in reagent C and cultured for 21 to 32 days.

7. The method for culturing in vitro reconstructed 3D skin epidermis using a kit according to claim 6, characterized in that: The method further comprises: observing the integrity of the skin tissue by HE staining after culturing for 21 to 32 days.

8. The method for culturing in vitro reconstructed 3D skin epidermis using a kit according to claim 6, characterized in that: The following steps are also included: The cultured 3D skin tissue was fixed with 4% formaldehyde, dehydrated with alcohol in stages, and transparentized with xylene; The dehydrated and transparent tissue is embedded, sliced ​​and baked for HE staining or immunohistochemistry experiments.

9. The method for culturing in vitro reconstructed 3D skin epidermis using a kit according to claim 6, characterized in that: Immunohistochemistry was used to detect the expression levels of fibronectin and keratin 10 in skin tissue.

10. The method for culturing in vitro reconstructed 3D skin epidermis using a kit according to claim 6, characterized in that: The method can significantly improve the operational efficiency of 3D skin epidermal layer printing and cultivation, and ensure the biological functional integrity and consistency of cultured skin tissue.

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