3D skin model based on photocuring bio-printing and construction method thereof

Abstract

The invention discloses a 3D skin model based on photocuring bio-printing and a construction method thereof, and the construction method comprises the following steps: A, printing bio-ink by adopting a 3D printing technology to obtain a corium layer; b, inoculating keratinocytes or epidermal stem cells to the surface of a corium layer, standing for a certain time, and then carrying out immersion culture; c, the dermis layer obtained after immersion culture is completed is added into a gas-liquid culture medium for gas-liquid culture, and the 3D skin model is obtained; wherein laminin is added into the gas-liquid culture medium in the step C. By adopting the construction scheme of combining 3D printing of the corium layer with artificial culture of the epidermal layer, the batch stability can be improved, the artificial culture period can be shortened, the cost can be reduced, and the quality of the model can be improved; the epidermal layer and the corium layer of the prepared 3D skin model are tightly connected, the epidermal layer is completely differentiated, and the 3D skin model can be widely applied to safety evaluation and efficacy evaluation of cosmetics or drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biological tissue engineering, and in particular to a 3D skin model based on photocuring bioprinting and a construction method thereof. Background Art

[0002] The skin is the largest organ in the human body and serves as a natural protective barrier, protecting against external stimuli and maintaining a stable internal environment. The skin is primarily composed of the dermis and epidermis, connected by a basement membrane. The dermis primarily consists of dermal fibroblasts and collagen, while the epidermis comprises four layers: the basal layer, the spinous layer, the granular layer, and the stratum corneum, formed by the continuous differentiation of epidermal stem cells located in the basal layer.

[0003] Previous cosmetic irritation and corrosion tests were conducted on live animals. Due to species differences between animals and humans, animal experiments cannot fully and truly reflect the safety and effectiveness of cosmetics, and experiments can cause great pain or even death to animals. With the establishment of animal welfare and the 3R principle, the European Union has enacted relevant laws and regulations to prohibit the sale of cosmetics and raw materials that have been tested on animals. Based on this, 3D in vitro skin testing models have become a new trend to replace animal testing. They use human cells obtained through in vitro culture. The European Union has already used three-dimensional (3D) skin models for cosmetic safety evaluation (EU new cosmetics regulation EC1223 / 2009), confirming that 3D skin models can be used for skin irritation testing of chemicals and cosmetic raw materials. The human nature of the material enables 3D skin models to truly reflect the efficacy of cosmetics.

[0004] A 3D full-thickness skin model is a three-dimensional skin model that contains both the epidermis and dermis. The model is usually constructed by reconstructing the epidermis on the de-epidermated dermis. That is, after constructing the dermis, the constructed dermis is used as the basis for the growth of keratinocytes. Human keratinocytes are cultured three-dimensionally on the constructed dermis, and a 3D full-thickness skin model is obtained by performing gas-liquid phase culture on them.

[0005] The representative artificial culture scheme (CN104726396A) specifically includes:

[0006] (1) Fibroblasts and epidermal cells (human keratinocytes) were cultured separately; (2) Vitamin C was used to induce the culture of fibroblasts to construct a dermis with good cell density and thickness; (3) Human keratinocytes were inoculated on the surface of the dermis to construct a double-layer skin including the epidermis; (4) Full-thickness skin was obtained through air-liquid phase culture.

[0007] While traditional manual construction methods can successfully create full-thickness skin models with clear structures and comprehensive properties, they are time-consuming and subject to significant variability between batches, such as differences in appearance and epidermal differentiation. This results in poor reproducibility in cosmetic efficacy research. Therefore, reliable, high-quality, and reproducible skin model construction technology is urgently needed for testing.

[0008] The booming development of bioprinting technology in the biomanufacturing field has provided new insights into tissue-engineered skin construction. 3D printed skin can effectively address batch-to-batch variability. The key technical challenge lies in leveraging the raw materials used in 3D bioprinting, namely bio-ink, to create a dense, stable skin model that mimics skin function.

[0009] Lee et al. used extrusion bioprinting technology based on a layer-by-layer printing method, that is, printing skin tissue in a collagen-fibroblast-collagen-keratinocyte-collagen layer (Lee, W., et al., Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication. Biomaterials, 2009. 30(8): p. 1587-95), which solved the problem of poor repeatability of artificially constructed models. However, when bioprinting full-layer skin models, it is difficult to achieve ideal resolution and simulate the fine structure of each layer of skin. At the same time, there is the problem of incomplete differentiation of the epidermis, especially the structure of the epidermis and the dermal epidermal junction (DEJ) in the skin model. In addition, mechanical factors during the printing process can easily cause the proteins and structures in the DEJ to denature, making the full-layer printed skin model unable to reproduce the material response ability or regeneration potential of real skin.

[0010] While 3D skin bioprinting has made some progress, it's still not widely adopted. This is primarily due to the low biomimetic properties of the printed skin samples, which limits their applicability. Printing at the junction of the epidermis and dermis requires high resolution to achieve high-precision structures. Otherwise, the properties of the entire skin layer cannot be reflected, hindering its application in cosmetic skin irritation testing and efficacy evaluation.

[0011] Based on this, a model construction method that takes into account both batch stability and high structural precision is needed at this stage. Summary of the Invention

[0012] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a 3D skin model based on photocuring bioprinting and a construction method thereof.

[0013] The purpose of the present invention is achieved through the following technical solutions:

[0014] In a first aspect, the present invention provides a method for constructing a 3D skin model, comprising the following steps:

[0015] A. Use 3D printing technology to print the bio-ink to obtain the dermis layer;

[0016] B. Inoculate keratinocytes or epidermal stem cells onto the surface of the dermis, let them stand for a certain period of time, and then perform immersion culture for 3 to 7 days;

[0017] C. Add the skin model after immersion culture to the air-liquid culture medium for air-liquid culture for 7 to 14 days to obtain a 3D skin model;

[0018] Wherein, laminin is added to the gas-liquid culture medium in step C.

[0019] As a preferred solution, in step A, the bio-ink comprises the following components: 0.5% to 5% by weight of methylated gelatin, 0.15% to 0.5% by weight of collagen, and 0.2×10 6 ~1×10 6 The dermis layer prepared by the bio-ink of the present invention is more tightly connected to the epidermis layer.

[0020] As a preferred embodiment, in step B, the inoculation amount of the keratinocytes or epidermal stem cells is 0.4×10 6 ~1×10 6 indivual.

[0021] As a preferred embodiment, in step B, the standing time is 4 to 6 hours.

[0022] As a preferred embodiment, in step B, the immersion culture medium used in the immersion culture comprises the following composition: 40% to 80% DMEM, 10% to 50% F12, 1% to 20% fetal bovine serum, 0.1 to 0.7 μg / ml of hydrocortisone, 10 to 50 μg / ml of adenine hydrochloride, 0.1 to 10 μM of isoproterenol, and 2×10 -13 ~2×10 -9 M, insulin 1-10 μg / ml and epidermal growth factor 1-20 ng / ml.

[0023] As a preferred embodiment, in step C, the gas-liquid culture medium comprises the following components: 40% to 80% DMEM, 10% to 50% F12, 4 to 12 mg / ml bovine serum albumin, 0.1 to 0.7 μg / ml hydrocortisone, 1 to 10 μg / ml insulin, 10 to 100 μg / ml ascorbic acid, 5 to 30 μg / ml laminin, and 1 to 20 ng / ml KGF growth factor.

[0024] As a preferred embodiment, in step C, the specific steps of the air-liquid culture are: placing a three-dimensional steel wire mesh in the culture dish, padding a piece of sterile filter paper on the steel wire mesh, adding air-liquid culture medium until the liquid level of the culture medium is level with the filter paper, and then placing the skin model after the immersion culture on the sterile filter paper for air-liquid culture for 7 to 14 days.

[0025] As a preferred solution, in step B, the immersion culture time is 7 days; in step C, the air-liquid culture time is 7 days. The 3D skin model obtained under this culture time has better effect.

[0026] As a preferred solution, in step A, the specific steps of printing are:

[0027] A1. Import the model, set printing parameters and slice it, and start printing;

[0028] A2. The deposition platform is lowered to a height one floor above the bottom of the trough;

[0029] A3. The light source emits light, the DMD displays the slice pattern, and the light is reflected to the bottom of the trough;

[0030] A4. After exposure, the bio-ink is solidified and the light machine stops emitting light;

[0031] A5. The deposition platform is raised to a layer height, and the printing is completed to obtain the dermis layer.

[0032] As a preferred solution, in steps A2 and A5, the layer height is 10 μm to 200 μm.

[0033] As a preferred solution, in step A3, the intensity of the light source is 10 mW / cm 2 ~15mW / cm 2 .

[0034] As a preferred solution, in step A4, the exposure time is 10s to 20s.

[0035] In a second aspect, the present invention provides a 3D skin model constructed according to the aforementioned method.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1) This invention utilizes a 3D-printed dermis combined with artificially cultured epidermis. The dermis is first printed using 3D photocuring technology. Keratinocytes are then manually inoculated and cultured in specific culture media, both submerged and in air-liquid environments, ultimately completing the construction of a full-thickness skin model. This method combines the advantages of 3D printing and artificial culture, improving batch stability, reducing the artificial culture cycle, lowering costs, and improving model quality.

[0038] 2) The present invention uses specific immersion culture medium and air-liquid culture medium for immersion culture and air-liquid culture, which can enable the model epidermis to grow and differentiate well, thereby forming a multi-layered and densely differentiated epidermis with clear visibility of the basal layer, spinous layer, granular layer and stratum corneum; and further, by adding laminin to the air-liquid culture medium, the inoculated keratinocytes can be well attached to the dermis obtained by 3D printing, thereby achieving a tight connection between the epidermis and dermis.

[0039] 3) The 3D skin model constructed by the present invention can be widely used in the safety evaluation and efficacy evaluation of cosmetics or medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0041] Figure 1 This is a photo of the appearance of the 3D skin model prepared in Example 1;

[0042] Figure 2 HE staining results of the 3D skin model prepared in Example 1. The subscript size is 500 μm.

[0043] Figure 3 HE staining results of the 3D skin model prepared in Example 2. The subscript size is 500 μm.

[0044] Figure 4 HE staining results of the 3D skin model prepared in Example 3. The subscript size is 500 μm.

[0045] Figure 5 HE staining results of the 3D skin model prepared in Comparative Example 1;

[0046] Figure 6 This is a photo of the appearance of the skin model prepared in Comparative Example 2;

[0047] Figure 7 HE staining results of the skin model prepared in Comparative Example 2;

[0048] Figure 8 HE staining results of the skin model prepared in Comparative Example 3, the subscript size is 500 μm;

[0049] Figure 9 This is the HE staining result of the skin model prepared in Comparative Example 4. The subscript size is 500 μm. DETAILED DESCRIPTION

[0050] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0051] The terms "preferred," "preferably," "more preferred," and the like, used herein, refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0052] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers indicating, for example, the amounts of ingredients used in the description and claims should be understood to be modified in all cases by the term "about". Therefore, unless otherwise indicated, the numerical parameters set forth in the following description and the appended claims are approximate values ​​that vary according to the desired properties to be obtained by the present invention. At least without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant figures and by applying ordinary rounding techniques.

[0053] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0054] In a specific embodiment of the present invention, a method for constructing a 3D skin model is provided, comprising the following steps:

[0055] A. Using 3D printing technology, the bio-ink is printed to obtain a dermis layer. The specific steps of the printing are as follows:

[0056] A1. Import the model, set printing parameters and slice it, and start printing;

[0057] A2. The deposition platform is lowered to a height of 10 μm to 200 μm from the bottom of the trough;

[0058] A3, light source emits light with an intensity of 10mW / cm 2 ~15mW / cm 2 ,DMD displays the slice pattern, and the light is reflected to the bottom of the trough;

[0059] A4, after exposure for 10s to 20s, the bio-ink (including the following components: 0.5% to 5% by weight of methylated gelatin, 0.15% to 0.5% by weight of collagen and 0.2×10 6 ~1×10 6 pcs / ml) are solidified and the light machine stops emitting light;

[0060] A5. The deposition platform is raised to a layer height of 10μm to 200μm, and the dermis layer is obtained after printing.

[0061] B. 0.4×10 6 ~1×10 6 The keratinocytes or epidermal stem cells were inoculated onto the surface of the dermis, allowed to stand for 4 to 6 hours, and then submerged in an immersion culture medium for 3 to 7 days; the immersion culture medium used included the following composition: 40% to 80% DMEM, 10% to 50% F12, 1% to 20% fetal bovine serum, 0.1 to 0.7 μg / ml hydrocortisone, 10 to 50 μg / ml adenine hydrochloride, 0.1 to 10 μM isoproterenol, and 2×10 -13 ~2×10 -9 M, insulin 1-10 μg / ml and epidermal growth factor 1-20 ng / ml;

[0062] C. Place a three-dimensional steel mesh in a culture dish, place a piece of sterile filter paper on the steel mesh, add air-liquid culture medium until the liquid level of the culture medium is level with the filter paper, and then place the skin model after immersion culture on the sterile filter paper for air-liquid culture for 7-14 days to obtain a 3D skin model; the air-liquid culture medium used includes the following composition: 40%-80% DMEM, 10%-50% F12, 4-12 mg / ml bovine serum albumin, 0.1-0.7 μg / ml hydrocortisone, 1-10 μg / ml insulin, 10-100 μg / ml ascorbic acid, 5-30 μg / ml laminin, and 1-20 ng / ml KGF growth factor.

[0063] All of the above conditions can be used to prepare a 3D skin model with a relatively tight connection between the epidermis and the dermis and complete differentiation of the epidermis.

[0064] In the following examples, methylated gelatin was purchased from EFL; collagen was purchased from Sichuan Mingrang Biotechnology Co., Ltd.

[0065] The fibroblasts, keratinocytes and epidermal stem cells used in the present invention are all conventional cells, which can be obtained by purchase, donation, etc., or separated by conventional methods, and can all be used in the present invention; specifically, the fibroblasts, keratinocytes and epidermal stem cells used in the following examples were all independently separated and identified by our company using conventional methods, and the ethics number is JIEC-S70(2023).

[0066] Example 1

[0067] This embodiment provides a method for constructing a 3D skin model, and the specific steps are as follows:

[0068] (1) Using a 3D light-curing printer, bio-ink was printed to obtain dermal tissue; the bio-ink included: 4.5% by weight of methylated gelatin, 0.15% by weight of collagen, and 1×10 fibroblasts. 6 / ml, the printing steps are as follows:

[0069] a) Import the model, set printing parameters and slice it, and start printing;

[0070] b) The deposition platform is lowered to a height of 100 μm from the bottom of the trough;

[0071] c) Light source output (15mW / cm 2 ), DMD displays the slice pattern, and the light is reflected to the bottom of the trough;

[0072] d) After a certain exposure time (15s), the bio-ink is solidified and the light machine stops emitting light;

[0073] e) The sedimentation platform is raised by one layer height;

[0074] f) Printing is completed to obtain the dermis layer.

[0075] (2) Construct a full-thickness skin model. The steps are as follows:

[0076] a) Keratinocyte seeding: 1×10 6 Keratinocytes were inoculated onto the surface of the dermis and placed in an incubator for 6 hours. The steel ring was then removed and immersion culture medium was added to cover the top of the model.

[0077] b) Submerged culture: The model prepared in the above steps was submerged for a total of 7 days. The medium was changed every other day during the submerged culture. The submerged culture medium used contained the following components: 60% DMEM, 30% F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 24 μg / ml adenine hydrochloride, 1 μM isoproterenol, 2×10 -11 M, insulin 5 μg / ml and epidermal growth factor 10 ng / ml.

[0078] c) Air-liquid culture: Place a three-dimensional steel mesh in a 10 cm culture dish, place a piece of sterile filter paper on the mesh, and add air-liquid culture medium until the liquid level is level with the filter paper. After the immersion culture is completed, the medium is aspirated and the dermis after immersion culture is placed on the filter paper for air-liquid culture. Culture for a total of 7 days. No further treatment is performed during the entire air-liquid culture period, and constant temperature and humidity are maintained. The air-liquid culture medium contains the following components: 60% DMEM, 30% F12, 8 mg / ml bovine serum albumin, 0.4 μg / ml hydrocortisone, 5 μg / ml insulin, 50 μg / ml ascorbic acid, 10 μg / ml laminin (laminin 521), and 10 ng / ml KGF growth factor.

[0079] d) Harvesting: After the air-liquid culture is completed, a full-thickness skin model, i.e., a 3D skin model, is obtained.

[0080] Example 2

[0081] This embodiment provides a method for constructing a 3D skin model, and the specific steps are as follows:

[0082] (1) A 3D light-curing printer was used to print the bio-ink to obtain dermal tissue; the bio-ink included: 0.35% by weight of methylated gelatin, 0.45% by weight of collagen, and 1×10 fibroblasts. 6 / ml; the printing steps are as follows:

[0083] a) Import the model, set printing parameters and slice it, and start printing;

[0084] b) The deposition platform is lowered to a height of 100 μm from the bottom of the trough;

[0085] c) Light source output (10mW / cm 2 ), DMD displays the slice pattern, and the light is reflected to the bottom of the trough;

[0086] d) After a certain exposure time (15s), the bio-ink is solidified and the light machine stops emitting light;

[0087] e) The sedimentation platform is raised by one layer height;

[0088] f) Printing is completed to obtain the dermis layer.

[0089] (2) Construction of a full-thickness skin model, including:

[0090] a) Keratinocyte seeding: 1×10 6 Keratinocytes were inoculated onto the surface of the dermis and placed in an incubator for 5 hours. The steel ring was then removed and immersion culture medium was added to cover the top of the model.

[0091] b) Submerged culture: The model prepared in the above steps was submerged for a total of 7 days. The medium was changed every other day during the submerged culture. The submerged culture medium used contained the following components: 60% DMEM, 30% F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 24 μg / ml adenine hydrochloride, 1 μM isoproterenol, 2×10 -11 M, insulin 5 μg / ml and epidermal growth factor 10 ng / ml.

[0092] c) Air-liquid culture: Place a three-dimensional steel mesh in a 10 cm culture dish, place a piece of sterile filter paper on the mesh, and add culture medium until the liquid level is level with the filter paper. After the immersion culture is completed, the culture medium is aspirated, and the dermis after immersion culture is placed on the filter paper for air-liquid culture. Culture for a total of 7 days. No further treatment is performed during the entire air-liquid culture period, and constant temperature and humidity are maintained. The air-liquid culture medium contains the following components: 60% DMEM, 30% F12, 8 mg / ml bovine serum albumin, 0.4 μg / ml hydrocortisone, 5 μg / ml insulin, 50 μg / ml ascorbic acid, 5 μg / ml laminin (laminin 521), and 10 ng / ml KGF growth factor.

[0093] d) Harvesting: After the air-liquid culture is completed, a full-thickness skin model, i.e., a 3D skin model, is obtained.

[0094] The full-thickness skin models (i.e., 3D skin models) from Examples 1 and 2 were harvested and fixed with 4% paraformaldehyde. The next day, the skin was washed twice with PBS, wiped with filter paper, embedded in OCT, and stored in a -80°C freezer overnight. The next day, frozen sections were cut and stained according to the HE staining kit instructions. After staining, photos were taken under a microscope.

[0095] Figure 1 Shown are photos of the appearance of the 3D skin model prepared in Example 1. From the appearance photos, it can be seen that the skin model prepared in Example 1 has a regular appearance and uniform skin size, and the appearance difference between batches is small.

[0096] Figure 2 The HE staining results of the skin model prepared in Example 1 are shown. Figure 2As can be seen from the microphotographs, the skin model prepared in Example 1 has a regular appearance and uniform size. HE staining shows that the dermis and epidermis are tightly connected, the basal layer, spinous layer, granular layer and stratum corneum are clearly visible, and the epidermis is fully differentiated.

[0097] Figure 3 The HE staining results of the 3D skin model prepared in Example 2 are shown. Figure 3 It can be seen from the microphotographs that the dermis and epidermis of the skin model prepared in Example 2 are tightly connected, the basal layer, spinous layer, granular layer and stratum corneum are clearly visible, and the epidermis is fully differentiated.

[0098] Example 3

[0099] This embodiment provides a method for constructing a 3D skin model, and the specific steps are as follows:

[0100] (1) A 3D light-curing printer was used to print the bio-ink to obtain dermal tissue; the bio-ink included: 4.5% by weight of methylated gelatin, 0.15% by weight of collagen, and 1×10 fibroblasts. 6 / ml; the printing steps are as follows:

[0101] a) Import the model, set printing parameters and slice it, and start printing;

[0102] b) The deposition platform is lowered to a height of 100 μm from the bottom of the trough;

[0103] c) Light source output (10mW / cm 2 ), DMD displays the slice pattern, and the light is reflected to the bottom of the trough;

[0104] d) After a certain exposure time (20s), the bio-ink is solidified and the light machine stops emitting light;

[0105] e) The sedimentation platform is raised by one layer height;

[0106] f) Printing is completed to obtain the dermis layer.

[0107] (2) Construction of a full-thickness skin model, including:

[0108] a) Epidermal stem cell seeding: 1×10 6 Epidermal stem cells were seeded onto the surface of the dermis and placed in an incubator for 4 hours. The steel ring was then removed and immersion culture medium was added to cover the top of the model.

[0109] b) Submerged culture: The model prepared in the above steps was submerged for a total of 7 days. The medium was changed every other day during the submerged culture. The submerged culture medium used contained the following components: 60% DMEM, 30% F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 24 μg / ml adenine hydrochloride, 1 μM isoproterenol, 2×10 -11 M, insulin 5 μg / ml and epidermal growth factor 10 ng / ml.

[0110] c) Air-liquid culture: Place a three-dimensional steel mesh in a 10 cm culture dish, place a piece of sterile filter paper on the mesh, and add culture medium until the liquid level is level with the filter paper. After the immersion culture is completed, the culture medium is aspirated, and the dermis after immersion culture is placed on the filter paper for air-liquid culture. Culture for a total of 7 days. No further treatment is performed during the entire air-liquid culture period, and constant temperature and humidity are maintained. The air-liquid culture medium contains the following components: 60% DMEM, 30% F12, 8 mg / ml bovine serum albumin, 0.4 μg / ml hydrocortisone, 5 μg / ml insulin, 50 μg / ml ascorbic acid, 20 μg / ml laminin, and 10 ng / ml KGF growth factor.

[0111] d) Harvest: After the air-liquid culture is completed, a full-thickness skin model, i.e., a 3D skin model, is obtained. In the 3D skin model prepared in this example, the dermis and epidermis are tightly connected, and the epidermis is fully differentiated.

[0112] Figure 4 The HE staining results of the skin model prepared in Example 3 are shown. Figure 4 As can be seen from the microphotographs, the skin model prepared in Example 3 has a regular appearance and uniform size. HE staining shows that the dermis and epidermis are tightly connected, the epidermis is fully differentiated, the basal layer, spinous layer, granular layer and stratum corneum are clearly visible, and the epidermis is fully differentiated.

[0113] Comparative Example 1

[0114] This comparative example provides a method for constructing a 3D skin model, and the specific steps are as follows:

[0115] (1) A 3D light-curing printer was used to print the bio-ink (compared to Example 1, the bio-ink did not add collagen), and the bio-ink included: methylated gelatin with a mass percentage of 4.5% and fibroblasts 1×10 6 / ml, the printing steps are as follows:

[0116] a) Import the model, set printing parameters and slice it, and start printing;

[0117] b) The deposition platform is lowered to a height of 100 μm from the bottom of the trough;

[0118] c) Light source output (15mW / cm 2 ), DMD displays the slice pattern, and the light is reflected to the bottom of the trough;

[0119] d) After a certain exposure time (15s), the bio-ink is solidified and the light machine stops emitting light;

[0120] e) The sedimentation platform is raised by one layer height;

[0121] f) Printing is completed, and the dermis tissue is obtained.

[0122] (2) Construction of a full-thickness skin model, including:

[0123] a) Keratinocyte seeding: 1×10 6 Keratinocytes were inoculated onto the surface of the dermis and placed in an incubator for 6 hours. The steel ring was then removed and immersion culture medium was added to cover the top of the model.

[0124] b) Submerged culture: The model prepared in the above steps was submerged for a total of 7 days. The medium was changed every other day during the submerged culture. The submerged culture medium used contained the following components: 60% DMEM, 30% F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 24 μg / ml adenine hydrochloride, 1 μM isoproterenol, 2×10 -11 M, insulin 5 μg / ml and epidermal growth factor 10 ng / ml.

[0125] c) Air-liquid culture (compared to Example 1, the air-liquid culture medium lacks laminin): A three-dimensional steel mesh was placed in a 10 cm culture dish, and a piece of sterile filter paper was placed on the mesh. Air-liquid culture medium was added until the liquid level was level with the filter paper. After the immersion culture was completed, the culture medium was aspirated, and the dermis after immersion culture was placed on the filter paper for air-liquid culture. The culture was carried out for a total of 7 days. No further treatment was performed during the entire air-liquid culture period, and constant temperature and humidity were maintained. The air-liquid culture medium comprised the following components: 60% DMEM, 30% F12, 8 mg / ml bovine serum albumin, 0.4 μg / ml hydrocortisone, 5 μg / ml insulin, 50 μg / ml ascorbic acid, and 10 ng / ml KGF growth factor.

[0126] d) Harvesting: After the air-liquid culture is completed, a full-thickness skin model, i.e., a 3D skin model, is obtained.

[0127] The 3D skin model prepared in this comparative example was harvested and fixed with 4% paraformaldehyde. The next day, it was washed twice with PBS, wiped with filter paper, embedded in OCT, and placed in a -80°C refrigerator overnight. The next day, frozen sections were sectioned and stained according to the instructions of the HE staining kit. After staining, photos were taken under a microscope.

[0128] Figure 5 Shown are the HE staining results of the above-mentioned 3D skin model. The network structure of the dermis is loose, a large number of hollow structures are formed at the junction of the true epidermis, and the epidermis is incompletely differentiated.

[0129] Comparative Example 2

[0130] This comparative example provides a method for constructing a skin model (manually constructed throughout the process), and the specific steps are as follows:

[0131] (1) Construction of the dermis, including:

[0132] Take 1.5% collagen solution, adjust the pH to 7.0-7.5 with NaHCO3, dilute the collagen concentration to 0.5% with sterile water, and then add 0.5×10 6 Fibroblasts were added to 6 cm culture dishes at a volume of 10 ml per dish and placed in an incubator for 4 hours to allow the collagen to solidify. Culture medium was added to cover the surface of the collagen and the collagen was allowed to shrink. After the diameter of the collagen gel shrank to 2 cm, keratinocytes were inoculated.

[0133] (2) Construction of a full-thickness skin model, including:

[0134] a) Keratinocyte seeding: 1×10 6 Keratinocytes were inoculated onto the surface of the dermis and placed in an incubator for 6 hours. The steel ring was then removed and immersion culture medium was added to cover the top of the model.

[0135] b) Submerged culture: The model prepared in the above steps was submerged for a total of 7 days. The medium was changed every other day during the submerged culture. The submerged culture medium used contained the following components: 60% DMEM, 30% F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 24 μg / ml adenine hydrochloride, 1 μM isoproterenol, 2×10 -11 M, insulin 5 μg / ml and epidermal growth factor 10 ng / ml.

[0136] c) Air-liquid culture: Place a three-dimensional steel mesh in a 10 cm culture dish, place a piece of sterile filter paper on the mesh, and add air-liquid culture medium until the liquid level is level with the filter paper. After the immersion culture is completed, the medium is aspirated and the dermis after immersion culture is placed on the filter paper for air-liquid culture. Culture for a total of 7 days. No further treatment is performed during the entire air-liquid culture period, and constant temperature and humidity are maintained. The air-liquid culture medium contains the following components: 60% DMEM, 30% F12, 8 mg / ml bovine serum albumin, 0.4 μg / ml hydrocortisone, 5 μg / ml insulin, 50 μg / ml ascorbic acid, 10 μg / ml laminin (laminin 521), and 10 ng / ml KGF growth factor.

[0137] d) Harvest: After the completion of the air-liquid culture, a full-thickness skin model was obtained.

[0138] The full-thickness skin model prepared in this comparative example was harvested and fixed with 4% paraformaldehyde. The next day, the skin was washed twice with PBS, wiped with filter paper, embedded in OCT, and placed in a -80°C refrigerator overnight. The next day, the skin was frozen and sectioned, and stained according to the instructions of the HE staining kit. After staining, the skin was photographed under a microscope.

[0139] Figure 6 and Figure 7 The appearance photos and HE staining results of the skin model are shown respectively. Figure 6 It can be seen from the figure that the manually constructed skin model has irregular shapes and uneven sizes. Figure 7 HE staining showed that the true epidermis was not dense enough and the epidermis was not completely differentiated.

[0140] Comparative Example 3

[0141] This comparative example provides a method for constructing a 3D skin model, and the specific steps are as follows:

[0142] (1) A 3D light-curing printer was used to print the bio-ink, which included 4.5% by weight of methylated gelatin, 0.15% by weight of collagen, and 1×10 fibroblasts. 6 / ml, the printing steps are as follows:

[0143] a) Import the model, set printing parameters and slice it, and start printing;

[0144] b) The deposition platform is lowered to a height of 100 μm from the bottom of the trough;

[0145] c) Light source output (15mW / cm 2 ), DMD displays the slice pattern, and the light is reflected to the bottom of the trough;

[0146] d) After a certain exposure time (15s), the bio-ink is solidified and the light machine stops emitting light;

[0147] e) The sedimentation platform is raised by one layer height;

[0148] f) Printing is completed, and the dermis tissue is obtained.

[0149] (2) Construction of a full-thickness skin model, including:

[0150] a) Keratinocyte seeding: 1×10 6 Keratinocytes were inoculated onto the surface of the dermis and placed in an incubator for 6 hours. The steel ring was then removed and immersion culture medium was added to cover the top of the model.

[0151] b) Submerged culture: The model prepared in the above steps was submerged for a total of 7 days. The medium was changed every other day during the submerged culture. The submerged culture medium used contained the following components: 60% DMEM, 30% F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 24 μg / ml adenine hydrochloride, 1 μM isoproterenol, 2×10 -11 M, insulin 5 μg / ml and epidermal growth factor 10 ng / ml.

[0152] c) Air-liquid culture (compared to Example 1, the air-liquid culture medium lacks laminin): A three-dimensional steel mesh was placed in a 10 cm culture dish, and a piece of sterile filter paper was placed on the mesh. Air-liquid culture medium was added until the liquid level was level with the filter paper. After the immersion culture was completed, the culture medium was aspirated, and the dermis after immersion culture was placed on the filter paper for air-liquid culture. The culture was carried out for a total of 7 days. No further treatment was performed during the entire air-liquid culture period, and constant temperature and humidity were maintained. The air-liquid culture medium comprised the following components: 60% DMEM, 30% F12, 8 mg / ml bovine serum albumin, 0.4 μg / ml hydrocortisone, 5 μg / ml insulin, 50 μg / ml ascorbic acid, and 10 ng / ml KGF growth factor.

[0153] d) Harvesting: After the air-liquid culture is completed, a full-thickness skin model, i.e., a 3D skin model, is obtained.

[0154] The full-thickness skin model used in this comparative example was harvested and fixed with 4% paraformaldehyde. The next day, the skin was washed twice with PBS, wiped with filter paper, embedded in OCT, and stored in a -80°C refrigerator overnight. The next day, the skin was frozen and sectioned, and stained according to the instructions of the HE staining kit. After staining, the skin was photographed under a microscope.

[0155] Figure 8 Shown are the HE staining results of the above-mentioned full-thickness skin model. The dermis structure is relatively dense, and the dermis-epidermal junction is relatively tight.

[0156] The results of Example 1 and Comparative Examples 1-3 show that the skin model constructed entirely manually in Comparative Example 2 exhibits significant batch-to-batch appearance differences and low stability compared to the skin model obtained in Example 1 (3D printing the dermis layer + manually constructing the epidermis layer). Furthermore, the density of the epidermal junction in Comparative Example 2 is significantly worse than that in Example 1. Only the basal layer and stratum corneum are visible in the epidermis layer of Comparative Example 2, and the degree of differentiation is not as complete as in Example 1. Furthermore, due to the different compositions of the bio-ink and the air-liquid culture medium, the skin model obtained in Comparative Example 1 forms a large number of hollow structures at the epidermal junction, meaning that it cannot function as a full-thickness skin model. Epidermal differentiation is incomplete, with the basal layer and stratum corneum visible. Although the skin model obtained in Comparative Example 3 has a denser dermal structure and a tighter epidermal junction than the skin model obtained in Example 1, the dermal structure and skin junction tightness of the skin model obtained in Comparative Example 3 are both inferior to those in Example 1. In Comparative Example 3, epidermal differentiation is incomplete, with the basal layer and stratum corneum visible. Therefore, the skin model construction method protected by the present invention includes the advantages of batch stability and controllability, as well as the advantages of stable model morphology, comprehensive differentiation and complete barrier function.

[0157] Comparative Example 4

[0158] This comparative example provides a method for constructing a 3D skin model, and the specific steps are as follows:

[0159] (1) A 3D light-curing printer was used to print the bio-ink, which included 4.5% by weight of methylated gelatin, 0.15% by weight of collagen, and 1×10 fibroblasts. 6 / ml, the printing steps are as follows:

[0160] a) Import the model, set printing parameters and slice it, and start printing;

[0161] b) The deposition platform is lowered to a height of 100 μm from the bottom of the trough;

[0162] c) Light source output (15mW / cm 2 ), DMD displays the slice pattern, and the light is reflected to the bottom of the trough;

[0163] d) After a certain exposure time (15s), the bio-ink is solidified and the light machine stops emitting light;

[0164] e) The sedimentation platform is raised by one layer height;

[0165] f) Printing is completed, and the dermis tissue is obtained.

[0166] (2) Construction of a full-thickness skin model, including:

[0167] a) Laminin coating: Discard the dermal culture medium, open the lid and air dry for 2 minutes, coat the surface of the dermis with 100 μl of 10 μg / ml laminin (laminin 521), evenly disperse it on the surface, cover the lid and place in an incubator for 30 minutes to obtain the laminin-coated dermis.

[0168] b) Keratinocyte seeding: The laminin-coated dermis was removed and 1×10 6 Keratinocytes were inoculated onto the surface of the dermis and placed in an incubator for 6 hours. The steel ring was then removed and immersion culture medium was added to cover the top of the model.

[0169] c) Submerged culture: The model prepared in the above steps was submerged for a total of 7 days. The medium was changed every other day during the submerged culture. The submerged culture medium used contained the following components: 60% DMEM, 30% F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 24 μg / ml adenine hydrochloride, 1 μM isoproterenol, and 2×10 -11 M, insulin 5 μg / ml and epidermal growth factor 10 ng / ml.

[0170] d) Air-liquid culture: Place a three-dimensional steel mesh in a 10 cm culture dish, place a piece of sterile filter paper on the mesh, and add air-liquid culture medium until the liquid level is level with the filter paper. After the submerged culture is completed, the medium is aspirated, and the submerged dermis is placed on the filter paper for air-liquid culture. Culture for a total of 7 days. No further treatment is performed during the entire air-liquid culture period, and constant temperature and humidity are maintained. The air-liquid culture medium contains the following components: 60% DMEM, 30% F12, 8 mg / ml bovine serum albumin, 0.4 μg / ml hydrocortisone, 5 μg / ml insulin, 50 μg / ml ascorbic acid, and 10 ng / ml KGF growth factor.

[0171] e) Harvesting: After the air-liquid culture is completed, a full-thickness skin model, i.e., a 3D skin model, is obtained.

[0172] The 3D skin model prepared in this comparative example was harvested and fixed with 4% paraformaldehyde. The next day, it was washed twice with PBS, wiped with filter paper, embedded in OCT, and placed in a -80°C refrigerator overnight. The next day, frozen sections were sectioned and stained according to the instructions of the HE staining kit. After staining, photos were taken under a microscope.

[0173] Figure 9The HE staining results of the 3D skin model are shown. Compared with Comparative Example 3, there was no significant improvement in the dermal-epidermal junction. Compared with Example 1, the structural density of the dermis and the tightness of the skin junction were both worse than those in Example 1, indicating that a skin model with a tighter connection between the dermis and epidermis can only be achieved after adding laminin to the air-liquid culture medium.

[0174] Application Examples

[0175] The skin models prepared in Example 1 and Comparative Examples 1-4 were subjected to skin irritation tests using stimulants and compared to human skin tests. The results showed that the test results of the 3D skin model prepared in Example 1 were comparable to those of the human skin tests. However, the test results of the skin models prepared in Comparative Examples 1-4 differed somewhat from those of the human skin tests. This demonstrates that the skin models prepared in the present invention can better simulate human skin.

[0176] The present invention has many specific application paths, and the above is only a preferred embodiment of the present invention. It should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, several improvements can be made without departing from the principles of the present invention, and these improvements should also be considered as the scope of protection of the present invention.

Claims

1. A method for constructing a 3D skin model, characterized in that: The following steps are involved: A. Use 3D printing technology to print the bio-ink to obtain the dermis layer; B. Inoculate keratinocytes or epidermal stem cells onto the surface of the dermis, let them stand for a certain period of time, and then perform immersion culture for 3 to 7 days; C. Add the skin model after immersion culture to the air-liquid culture medium for air-liquid culture for 7 to 14 days to obtain a 3D skin model; Wherein, laminin is added to the gas-liquid culture medium in step C.

2. The method for constructing a 3D skin model according to claim 1, wherein: In step A, the bio-ink comprises the following components: 0.5% to 5% by weight of methylated gelatin, 0.15% to 0.5% by weight of collagen, and 0.2×10 6 ~1×10 6 pcs / ml.

3. The method for constructing a 3D skin model according to claim 1, wherein: In step B, the inoculation amount of the keratinocytes or epidermal stem cells is 0.4×10 6 ~1×10 6 indivual.

4. The method for constructing a 3D skin model according to claim 1, wherein: In step B, the standing time is 4 to 6 hours.

5. The method for constructing a 3D skin model according to claim 1, wherein: In step B, the immersion culture medium used in the immersion culture comprises the following composition: 40% to 80% DMEM, 10% to 50% F12, 1% to 20% fetal bovine serum, 0.1 to 0.7 μg / ml of hydrocortisone, 10 to 50 μg / ml of adenine hydrochloride, 0.1 to 10 μM of isoproterenol, 2×10 -13 ~2×10 -9 M, insulin 1-10 μg / ml and epidermal growth factor 1-20 ng / ml.

6. The method for constructing a 3D skin model according to claim 1, wherein: In step C, the gas-liquid culture medium includes the following components: 40% to 80% DMEM, 10% to 50% F12, 4 to 12 mg / ml bovine serum albumin, 0.1 to 0.7 μg / ml hydrocortisone, 1 to 10 μg / ml insulin, 10 to 100 μg / ml ascorbic acid, 5 to 30 μg / ml laminin, and 1 to 20 ng / ml KGF growth factor.

7. The method for constructing a 3D skin model according to claim 1, wherein: In step C, the specific steps of the air-liquid culture are: placing a three-dimensional steel wire mesh in a culture dish, padding a piece of sterile filter paper on the steel wire mesh, adding air-liquid culture medium until the liquid level of the culture medium is level with the filter paper, and then placing the skin model after the immersion culture on the sterile filter paper for air-liquid culture for 7 to 14 days.

8. The method for constructing a 3D skin model according to claim 1, wherein: In step A, the specific steps of printing are: A1. Import the model, set printing parameters and slice it, and start printing; A2. The deposition platform is lowered to a height one floor above the bottom of the trough; A3. The light source emits light, the DMD displays the slice pattern, and the light is reflected to the bottom of the trough; A4. After exposure, the bio-ink is solidified and the light machine stops emitting light; A5. The deposition platform is raised to a layer height, and the printing is completed to obtain the dermis layer.

9. The method for constructing a 3D skin model according to claim 8, wherein: In steps A2 and A5, the layer height is 10 μm to 200 μm; In step A3, the intensity of the light source is 10 mW / cm 2 ~15mW / cm 2 In step A4, the exposure time is 10s to 20s.

10. A 3D skin model constructed according to the method according to any one of claims 1 to 9.

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