A cross-scale vascularized skin model and a method of preparing the same

Abstract

The application relates to a cross-scale vascularized skin model and a preparation method thereof, and belongs to the technical field of tissue / organ bionic manufacturing. The method disclosed by the application constructs submillimeter blood vessel trunks by taking micro-scale vascularized cell balls as micro-blood-vessel construction units, combining axis printing and a sacrificial material technology, and inducing cell ball budding and trunk blood vessel connection in post-culture, and for the first time realizes integrated and cross-scale integrated construction from micron-scale capillary networks to submillimeter blood vessels. Through the cooperation of a bionic multi-component biological ink system and a cross-scale printing process, a hierarchical blood vessel network with physiological similarity is constructed, so that the skin model is significantly improved in structural integrity and functional bionics. The introduction of the cross-scale vascular network effectively solves the core bottlenecks of limited nutrient transport, long-term survival difficulty of cells and large deviation of drug evaluation results from real in-vivo conditions in traditional skin models.

Description

Technical Field

[0001] This invention belongs to the field of tissue / organ biomimetic manufacturing technology, specifically relating to a multi-scale vascularized skin model and its preparation method. Background Technology

[0002] As the largest organ in the human body, the skin performs many vital physiological functions, including protecting against external stimuli, regulating body temperature, and maintaining water balance, serving as the body's first line of defense for health. However, with increasing environmental pollution, frequent traffic accidents, and the spread of diseases, skin lesions are on the rise. These lesions not only severely impact patients' quality of life but may also trigger systemic health problems. Currently, the research and evaluation of drugs for treating skin lesions mainly rely on two-dimensional cell culture and animal experimental models. While two-dimensional models are beneficial for high-throughput screening, their overly simplified planar environment makes it difficult to replicate the anisotropic structure, intercellular interactions, and three-dimensional conformation of the extracellular matrix of human skin, thus limiting the accuracy of drug efficacy assessment. Animal models, on the other hand, suffer from species differences, long cycles, and poor reproducibility, and contradict the strategic goals of improving R&D efficiency and reducing reliance on animal experiments outlined in the Ministry of Industry and Information Technology's pharmaceutical industry development plan. Therefore, developing highly biomimetic in vitro models that can simulate the structure and function of human skin has become an urgent need in the field of drug development.

[0003] To overcome the limitations of two-dimensional cell and animal experiments, researchers have developed three-dimensional skin models based on biocompatible materials. These models primarily consist of an epidermis containing keratinocytes, a dermis containing fibroblasts, or a dermis-epidermis composed of both types of cells, and have achieved initial commercialization. However, existing models still have significant shortcomings in terms of structural integrity and biological function regulation, with the core bottleneck being the absence of a vascular network. The lack of a vascular system not only blocks nutrient transport and metabolic waste removal, making it difficult to maintain cell survival within the model in the long term, thus affecting the model's biological function, but also leads to a significant deviation between drug evaluation results and the actual situation in vivo. The human epidermis, as the only avascular tissue, contrasts sharply with the underlying dermis, which is rich in a complex vascular system. Skin anatomy studies have shown that the vascular network within the dermis has typical trans-scale characteristics, with vessel diameters ranging from 5 µm at the capillary level to 500 µm in the deep vascular plexus. This structural transition from sub-millimeter to micrometer scale, coupled with the coexistence of an avascular epidermis and a vascular dermis, constitutes the core scientific challenge in preparing cross-scale vascularized skin models, which also severely restricts their value as a drug development tool. Summary of the Invention

[0004] The purpose of this invention is to provide a cross-scale vascularized skin model and its preparation method, in order to solve the technical problem that existing skin models are difficult to integrate and construct cross-scale vascular networks in the same model, resulting in simple structure and lack of anisotropic features in the printed skin models, which cannot meet the technical needs of high-biomimetic skin models for nutrient transport networks and drug development.

[0005] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a method for preparing a multi-scale vascularized skin model, comprising the following steps: S1: Constructing microscale angiogenesis units; S2: Formulate various bio-inks for constructing the vascular layer, dermis, and epidermis, respectively; S3: Using coaxial printing and extrusion printing processes, bio-inks of the vascular layer, dermis layer and epidermis layer are deposited according to a predetermined spatial configuration. The coaxial printing process is used to construct sub-millimeter-scale vascular trunks with endothelialized cavities. The bio-ink of the vascular layer contains the microscale angiogenesis units. S4: A hollow submillimeter-scale vascular channel is formed inside the vascular layer through a sacrificial material removal process; through a post-culture process, the microscale angiogenesis unit is induced to sprout and connect with the channel of its adjacent submillimeter-scale vascular trunk, thereby forming a cross-scale vascular network from the submillimeter-scale trunk to the micrometer-scale capillary, resulting in a cross-scale vascularized skin model.

[0006] Furthermore, in S1, the step of constructing the microscale angiogenesis unit includes: Endothelial cells and stromal cells were co-cultured to allow them to self-assemble into microscale vascularized cell spheres with budding potential; The endothelial cells are human umbilical vein endothelial cells, and the matrix cells are fibroblasts.

[0007] Furthermore, in S2, the vascular layer bio-ink comprises a first structural material, a first cellular component, and the microscale angiogenesis unit; The dermal bio-ink contains a second structural material and fibroblasts; The epidermal bio-ink contains a third structural material and keratinocytes.

[0008] Furthermore, in S2, the first structural material comprises gelatin, methacrylamide gelatin, decellularized extracellular matrix, and a photoinitiator; the first cell component comprises human umbilical vein endothelial cells; In the dermal bio-ink, the concentration of gelatin is 7%-10% (w / v), the concentration of methacrylamide gelatin is 8%-15% (w / v), the concentration of decellularized extracellular matrix is ​​1%-3% (w / v), the concentration of photoinitiator is 0.25% (w / v), and the concentration of microscale vascularized cell spheroids is 1×10⁻⁶. 4 -5×10 4 The concentration of cells / mL and human umbilical vein endothelial cells was 5 × 10⁶. 6 -10×10 6 per mL.

[0009] Furthermore, in the vascular layer bio-ink, the density of microscale angiogenesis units is configured such that the capillary network formed after budding can functionally match the submillimeter-scale vascular channels.

[0010] Further, the second structural material is GelMA, dECM, and LAP; the concentration of dECM in the dermal bio-ink is 1%-3% (w / v), the concentration of LAP is 0.25% (w / v), and the concentration of GelMA is 8%-15% (w / v); the concentration of fibroblasts is 0.5 × 10⁻⁶. 6 -2×10 6 cells / mL; The third structural material is gelalin; in the epidermal bio-ink, the concentration of gelalin is 7%-10% (w / v), and the concentration of keratinocytes is 1×10⁻⁶. 6 -6×10 6 per mL.

[0011] Furthermore, in S3, in the coaxial printing process, the structural material containing the microscale angiogenesis unit is used as the outer phase fluid, and the mixture containing endothelial cells and sacrificial material is used as the inner phase fluid.

[0012] Furthermore, in S4, the specific operation of forming a hollow sub-millimeter-level vascular channel inside the vascular layer through the sacrificial material removal process is as follows: the obtained skin model is placed in an incubator at 37°C, and the inner phase of the skin model dissolves to form a sub-millimeter-level hollow channel.

[0013] Furthermore, in S6, the post-culture process includes sequential immersion culture for promoting the growth and proliferation of skin cells and gas-liquid interface culture for promoting epidermal differentiation, skin barrier function, and the formation of cross-scale vascular networks. In S6, the immersion culture lasts for 3-5 days; the gas-liquid interface culture lasts for 14-21 days.

[0014] The present invention also discloses a cross-scale vascularized skin model, which is prepared using the above-described preparation method.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for preparing a multi-scale vascularized skin model. By using microscale vascularized cell spheres as microvascular building blocks, and combining coaxial printing and sacrificial material technology to construct sub-millimeter-scale vascular trunks, and inducing cell sphere budding and connection with the trunk vessels during post-culture, it achieves for the first time an integrated, multi-scale construction from a micrometer-scale capillary network to sub-millimeter-scale vessels. Through the synergy of a biomimetic multi-component bio-ink system and the multi-scale printing process, a hierarchical vascular network with physiological similarity is constructed, significantly improving the skin model's structural integrity and functional biomimicry. The introduction of the multi-scale vascular network effectively solves the core bottlenecks of traditional skin models, such as limited nutrient transport, difficulty in long-term cell survival, and large deviations between drug evaluation results and in vivo realities. The vascularized skin model prepared by this invention more closely resembles the human physiological state and can be used for research on skin disease mechanisms, evaluation of transdermal drug absorption, toxicity screening, and efficacy verification, potentially reducing reliance on animal experiments and meeting the strategic needs of the pharmaceutical industry's development plan.

[0016] Furthermore, by designing and preparing a biomimetic multi-component bio-ink system adapted to the epidermis, dermis, and blood vessels, using a composite ink containing vascularized cell spheres as a microscale building block and a gelatin-based sacrificial ink as a sub-millimeter-scale vascular backbone material, and relying on a cross-scale bioprinting method that integrates coaxial printing, extrusion printing, and sacrificial material technology, the integrated construction from micron-scale capillary networks to sub-millimeter-scale blood vessels is finally achieved. This provides a new method for manufacturing highly biomimetic and functional skin models, and also provides a more reliable tool for drug development.

[0017] Furthermore, by using a sacrificial material removal process, the model is placed in a 37°C culture environment. The gelatin in the inner phase dissolves to form sub-millimeter-scale hollow channels, and endothelial cells adhere to the channel wall to rapidly form the vascular endothelial layer. Coaxial printing allows the dispersed cell spheres in the outer phase to be deposited around the sub-millimeter-scale vascular channels with the ink during the printing process. Under the induction of the post-culture process, they sprout and connect with the main blood vessels, thereby simultaneously realizing the cross-scale integration of sub-millimeter-scale blood vessels and micron-scale capillary networks.

[0018] Furthermore, the post-culture process employs immersion culture and gas-liquid interface culture to promote the growth and differentiation of skin cells, thereby enabling them to possess excellent biological functions. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the process for preparing the cross-scale vascularized skin model of the present invention; Figure 2This is a schematic diagram of the bioprinting of the in vitro biomimetic skin model prepared according to the present invention. Detailed Implementation

[0020] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0021] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0022] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0023] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0024] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0025] Existing skin models generally lack highly biomimetic vascular networks. This invention provides a method for preparing a cross-scale vascularized skin model, such as... Figure 1 and Figure 2 As shown, it includes the following steps: S1: Microscale vascularized cell spheroids were prepared using the low-adhesion well plate method; S2: Prepare bio-ink for the vascular layer, bio-ink for the dermis layer, and bio-ink for the epidermis layer respectively; S3: Load the vascular layer bio-ink into the coaxial nozzle of the bioprinting system, and load the dermal layer bio-ink and the epidermal layer bio-ink into two different extrusion nozzles respectively; S4: Bioprinting of cross-scale vascularized skin models in the order of vascular layer, dermis, and epidermis; S5: Place the printed skin model in an incubator to sacrifice the gelatin and obtain submillimeter-scale vascular channels; S6: The model obtained in S5 is subjected to immersion culture and gas-liquid interface culture in sequence to obtain a cross-scale vascularized skin model.

[0026] Preferably, the microscale vascularized cell spheres in S1 are obtained by culturing a mixture of human umbilical vein endothelial cells and fibroblasts in a low-adhesion well plate.

[0027] Preferably, the vascular layer bio-ink component in step S2 includes gelatin, methacrylated gelatin (GelMA), decellularized extracellular matrix (dECM), photoinitiator (Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), microscale cell spheroids, and human umbilical vein endothelial cells. The dermal layer bio-ink component in step S2 includes GelMA, dECM, LAP, and fibroblasts. The epidermal layer bio-ink component in step S2 includes gelatin and keratinocytes. The concentration of gelatin is 7-10% (w / v), the concentration of GelMA is 8-15% (w / v), the concentration of dECM is 1-3% (w / v), and the concentration of LAP is 0.25% (w / v).

[0028] Preferably, the coaxial printhead described in S3 uses GelMA / dECM / LAP composite ink fused with microscale vascularized cell spheres as the outer phase for coaxial printing, and gelatin mixed with human umbilical vein endothelial cells as the inner phase.

[0029] Preferably, the submillimeter-scale vascular channel described in S5 is formed by placing the model in a 37°C culture environment after printing. The inner phase gelatin dissolves to form a submillimeter-scale hollow channel, and endothelial cells adhere to the tube wall to quickly form a vascular endothelial layer. Coaxial printing allows the dispersed cell spheres in the outer phase to be deposited around the submillimeter-scale vascular channel with the ink during the printing process. Under the induction of the post-culture process, they sprout and connect with the main blood vessel, thereby simultaneously realizing the cross-scale integration of submillimeter-scale blood vessels and micron-scale capillary networks.

[0030] Preferably, the immersion culture and gas-liquid interface culture described in S6 are for the purpose of promoting the growth and differentiation of skin cells, thereby enabling them to have good biological functions.

[0031] Existing bioprinted skin models suffer from structural uniformity, particularly lacking biomimetic vascular networks. Most studies rely on post-printing static cell self-assembly to form micron-scale capillary networks or sacrificial material post-processing to obtain sub-millimeter-scale vessels. This makes it difficult to integrate cross-scale vascular networks within a single model, resulting in simple structures and a lack of anisotropic characteristics, failing to meet the needs of highly biomimetic skin models for nutrient transport networks and drug development. This invention takes a novel approach, designing and preparing a biomimetic multi-component bio-ink system adapted to the epidermis, dermis, and blood vessels. It uses a composite ink containing vascularized cell spheres as the microscale building block and gelatin-based sacrificial ink as the sub-millimeter-scale vascular backbone material. Utilizing a cross-scale bioprinting method integrating coaxial printing, extrusion printing, and sacrificial material technology, it ultimately achieves integrated construction from micron-scale capillary networks to sub-millimeter-scale vessels, providing a new method for manufacturing highly biomimetic, high-functional skin models and a more reliable tool for drug development.

[0032] In other words, this invention discloses a method for preparing a cross-scale vascularized skin model. Its core lies in the first realization of an integrated, cross-scale construction from a micron-level capillary network to a sub-millimeter-level blood vessel, systematically solving the core bottlenecks that have long existed in traditional skin models, such as limited nutrient transport, difficulty in cell survival, and large deviations between drug evaluation results and the actual situation in vivo.

[0033] This invention uses microscale vascularized cell spheres as microvascular construction units, combining coaxial printing and sacrificial material technology to construct sub-millimeter-scale vascular trunks. During post-culture, cell spheres are induced to sprout and connect with the trunk vessels, thus overcoming the technical obstacle in existing technologies where vascular networks cannot simultaneously maintain the fine structure of microcirculation and the patency of trunk vessels. Through this cross-scale vascular network design, the skin model achieves significant improvements in both structural integrity and functional biomimicry: sub-millimeter-scale vascular channels ensure efficient transport of culture medium and nutrients within the model, while the micron-scale capillary network simulates the microcirculatory environment of nutrient exchange and metabolic waste removal in real skin. The synergistic effect of these two technologies enables the model to maintain high cell viability and tissue function over a long period, overcoming the limitations of traditional skin models such as limited thickness and susceptibility to necrosis in the central region.

[0034] This invention also constructs a biomimetic multi-component bio-ink system adapted to the epidermis, dermis, and vascular structures. Specifically, the vascular layer bio-ink contains gelatin, methacrylamide gelatin (GelMA), decellularized extracellular matrix (dECM), photoinitiator (LAP), microscale cell spheroids, and human umbilical vein endothelial cells; the dermis layer bio-ink contains GelMA, dECM, LAP, and fibroblasts; and the epidermis layer bio-ink contains gelatin and keratinocytes. By precisely controlling the concentration of each component (e.g., GelMA 8-15% w / v, dECM 1-3% w / v, etc.), this ink system simulates the extracellular matrix microenvironment of different skin layers, providing suitable physical and biochemical signals for cell adhesion, proliferation, and differentiation. In particular, the simultaneous introduction of microscale cell spheroids and free endothelial cells into the vascular layer bio-ink provides both seed units for microvascular construction and ensures rapid spreading of the endothelial layer on the vessel wall.

[0035] In terms of printing technology, this invention relies on a cross-scale bioprinting method that integrates coaxial printing, extrusion printing, and sacrificial material technology. In coaxial printing, a GelMA / dECM / LAP composite ink containing microscale vascularized cell spheres serves as the outer phase, while a gelatin-mixed human umbilical vein endothelial cell layer serves as the inner phase. After printing, the model is placed in a 37°C incubation environment. The gelatin in the inner phase dissolves to form sub-millimeter-scale hollow channels, while the endothelial cells rapidly adhere to the channel walls, forming a complete endothelial layer. Meanwhile, the dispersed cell spheres in the outer phase are deposited around the channels with the ink and induced to sprout during subsequent culture, actively connecting with the main blood vessels. This process automatically integrates the micrometer-scale capillary network and sub-millimeter-scale blood vessels without additional operations, significantly reducing process complexity and improving reproducibility.

[0036] Finally, the present invention employs a post-culture process that combines immersion culture with gas-liquid interface culture, which not only ensures the full growth and differentiation of skin cells in a three-dimensional environment, but also simulates the physiological conditions of the epidermis exposed to air, making the skin model highly similar to real human skin in terms of tissue structure (such as epidermal layering and dermal density) and barrier function (such as transdermal absorption and metabolic activity).

[0037] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0038] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0039] Example 1 A method for preparing a multi-scale vascularized skin model includes the following steps: S1: Microscale vascularized cell spheres were prepared by mixing human umbilical vein endothelial cells and fibroblasts using a low-adhesion well plate method; S2: Prepare vascular layer bio-inks (7% Gelalin, 8% GelMA, 3% dECM, 0.25% LAP, (w / v)) and microscale cell spheroids 3×10⁻⁶. 4 6 × 10⁶ cells / mL and human umbilical vein endothelial cells 6 (cells / mL), dermal bio-ink (8% GelMA, 3% dECM, 0.25% LAP, (w / v) and human fibroblasts 1×10 6 (cells / mL) and epidermal bio-ink (7% Gelalin and 3 × 10 human keratinocytes) 6 (pcs / mL) S3: Vascular layer bio-ink (8% GelMA, 3% dECM, 0.25% LAP, (w / v) and microscale cell spheres) is loaded into the outer phase of the coaxial nozzle of the bioprinting system, vascular layer bio-ink (7% Gelalin and human umbilical vein endothelial cells) is loaded into the inner phase of the coaxial nozzle of the bioprinting system, and dermal layer bio-ink (8% GelMA, 3% dECM, 0.25% LAP and human fibroblasts) and epidermal layer bio-ink (7% Gelalin and human keratinocytes) are loaded into two different extrusion nozzles respectively. S4: Bioprinting of cross-scale vascularized skin models in the order of vascular layer, dermis, and epidermis; S5: The printed skin model is placed in a 37°C culture environment. The inner phase gelatin dissolves to form sub-millimeter hollow channels. Endothelial cells adhere to the channel wall and quickly form the vascular endothelial layer. Coaxial printing allows the dispersed cell spheres in the outer phase to be deposited around the sub-millimeter vascular channels with the ink during the printing process. They also sprout under the induction of the post-culture process and connect with the main blood vessels, thereby simultaneously realizing the cross-scale integration of sub-millimeter blood vessels and micron-level capillary networks. S6: The model obtained in S5 was subjected to immersion culture for 4 days and gas-liquid interface culture for 14 days in sequence to obtain a cross-scale vascularized skin model.

[0040] Example 2 A method for preparing a multi-scale vascularized skin model includes the following steps: S1: Microscale vascularized cell spheres were prepared by mixing human umbilical vein endothelial cells and fibroblasts using a low-adhesion well plate method; S2: Prepare vascular layer bio-inks (10% Gelalin, 15% GelMA, 1% dECM, 0.25% LAP, (w / v)) and microscale cell spheroids 5×10⁻⁶. 4 5 × 10⁶ cells / mL and human umbilical vein endothelial cells 6 (cells / mL), dermal bio-ink (15% GelMA, 1% dECM, 0.25% LAP, (w / v) and human fibroblasts 0.5×10) 6 (cells / mL) and epidermal bio-ink (10% Gelalin and 1×10 human keratinocytes) 6 (pcs / mL) S3: The vascular layer bio-ink (15% GelMA, 1% dECM, 0.25% LAP and microscale cell spheres) is loaded into the outer phase of the coaxial nozzle of the bioprinting system, the vascular layer bio-ink (10% Gelalin and human umbilical vein endothelial cells) is loaded into the inner phase of the coaxial nozzle of the bioprinting system, and the dermal layer bio-ink (15% GelMA, 1% dECM, 0.25% LAP and human fibroblasts) and the epidermal layer bio-ink (10% Gelalin and human keratinocytes) are loaded into two different extrusion nozzles respectively. S4: Bioprinting of cross-scale vascularized skin models in the order of vascular layer, dermis, and epidermis; S5: The printed skin model is placed in a 37°C culture environment. The inner phase gelatin dissolves to form sub-millimeter hollow channels. Endothelial cells adhere to the channel wall and quickly form the vascular endothelial layer. Coaxial printing allows the dispersed cell spheres in the outer phase to be deposited around the sub-millimeter vascular channels with the ink during the printing process. They also sprout under the induction of the post-culture process and connect with the main blood vessels, thereby simultaneously realizing the cross-scale integration of sub-millimeter blood vessels and micron-level capillary networks. S6: The model obtained in S5 was subjected to immersion culture for 3 days and gas-liquid interface culture for 21 days in sequence to obtain a cross-scale vascularized skin model.

[0041] Example 3 A method for preparing a multi-scale vascularized skin model includes the following steps: S1: Microscale vascularized cell spheres were prepared by mixing human umbilical vein endothelial cells and fibroblasts using a low-adhesion well plate method; S2: Prepare vascular layer bio-inks (8% Gelalin, 12% GelMA, 2% dECM, 0.25% LAP, (w / v)) and microscale cell spheroids 5×10⁻⁶. 4 10 × 10⁶ cells / mL and human umbilical vein endothelial cells 6 (cells / mL), dermal bio-ink (12% GelMA, 2% dECM, 0.25% LAP, (w / v) and human fibroblasts 2×10 6 (cells / mL) and epidermal bio-ink (8% Gelalin and 6×10 human keratinocytes) 6 (pcs / mL) S3: Vascular layer bio-ink (12% GelMA, 2% dECM, 0.25% LAP, (w / v) and microscale cell spheres) is loaded into the outer phase of the coaxial nozzle of the bioprinting system, vascular layer bio-ink (8% Gelalin and human umbilical vein endothelial cells) is loaded into the inner phase of the coaxial nozzle of the bioprinting system, and dermal layer bio-ink (12% GelMA, 2% dECM, 0.25% LAP, (w / v) and human fibroblasts) and epidermal layer bio-ink (8% Gelalin, (w / v) and human umbilical vein endothelial cells) are loaded into two different extrusion nozzles respectively. S4: Bioprinting of cross-scale vascularized skin models in the order of vascular layer, dermis, and epidermis; S5: The printed skin model is placed in a 37°C culture environment. The inner phase gelatin dissolves to form sub-millimeter hollow channels. Endothelial cells adhere to the channel wall and quickly form the vascular endothelial layer. Coaxial printing allows the dispersed cell spheres in the outer phase to be deposited around the sub-millimeter vascular channels with the ink during the printing process. They also sprout under the induction of the post-culture process and connect with the main blood vessels, thereby simultaneously realizing the cross-scale integration of sub-millimeter blood vessels and micron-level capillary networks. S6: The model obtained in S5 was subjected to immersion culture for 5 days and gas-liquid interface culture for 21 days in sequence to obtain a cross-scale vascularized skin model.

[0042] This invention constructs a skin model with a physiologically similar hierarchical structure through the differentiated formulation and spatially localized deposition of three types of bio-inks: the epidermis is composed of keratinocytes, simulating the skin barrier function; the dermis contains fibroblasts and extracellular matrix analogs, providing mechanical support and a matrix microenvironment; the vascular layer contains a multi-scale vascular network integrated within the dermis, simulating the in vivo nutrient transport and metabolic clearance pathways. The introduction of the multi-scale vascular network allows the culture medium to be perfused through sub-millimeter-level main channels and then diffused to surrounding tissues via a micrometer-level capillary network, solving the common problem of long-term cell survival due to hypoxia and nutrient deficiency in traditional skin models. More importantly, in existing avascular skin models, drugs mainly enter the model through passive diffusion, which is fundamentally different from the process of distribution to the skin via blood circulation in vivo. The vascularized skin model constructed in this invention allows for drug delivery or perfusion through vascular channels, more realistically simulating the process of drugs reaching the target tissue of the skin via blood circulation, making the efficacy and toxicity evaluation results closer to the actual in vivo situation. Furthermore, this model aligns with the strategic goals of the Ministry of Industry and Information Technology's pharmaceutical industry development plan to improve R&D efficiency and reduce reliance on animal testing, and can serve as a substitute or supplementary tool for animal testing.

[0043] In other words, this invention, through a three-level synergistic system of microscale angiogenesis unit pre-formation, coaxial sacrificial bioprinting, and post-culture directional budding and anastomosis, has for the first time achieved cross-scale, functional integration from micron-scale capillary plexuses to sub-millimeter-scale perfusionable vascular trunks in the same model, fundamentally breaking through the bottleneck of the disconnect between microvascular self-assembly and trunk vascular directional construction technology in existing skin models. At the structural biomimetic level, this invention utilizes microscale vascularized cell spheres formed by the self-assembly of endothelial cells and stromal cells through co-culture as angiogenesis units. These spheres are uniformly dispersed in the outer phase of the vascular layer using bio-ink, and an inner phase channel is constructed using a gelatin-based sacrificial material via coaxial printing. After printing, the gelatin in the inner phase is rapidly dissolved at 37 °C to form a hollow sub-millimeter-sized main channel with a diameter of 200-500 μm. Simultaneously, the endothelial cells suspended in the inner phase adhere to the inner wall of the channel, forming a complete endothelial layer. Crucially, the pre-anchored microscale vascularized cell spheres in the outer phase are precisely confined within a 100-300 μm range around the main channel. During post-culture, they are induced by the hypoxic gradient of the microenvironment and soluble signaling factors to directionally sprout towards the main channel and ultimately establish functional luminal anastomoses with the endothelialized channel wall. The resulting cross-scale vascular network covers a range from 5 to 20 μm. The continuous diameter distribution from μm capillaries to sub-millimeter trunks; at the functional biomimetic level, the introduction of a multi-scale vascular network enables the skin model to simulate the stepwise nutrient transport and metabolic clearance from blood vessels to interstitial fluid and then to cells in vivo. Active perfusion through the trunk channels allows the culture medium to diffuse through the capillary network to the surrounding dermis and epidermis, significantly improving material exchange efficiency and effectively solving the problem of central hypoxia and necrosis caused by limited nutrient supply in traditional three-dimensional skin models. Simultaneously, the drug evaluation method shifts from passive diffusion to active perfusion administration via the vascular pathway, more realistically replicating the pharmacokinetic process of drugs circulating in the bloodstream to the target skin tissue; at the process synergy level, this invention designs three biomimetic approaches tailored to the different biological and material requirements of the epidermis, dermis, and vascular layer. The bio-ink system achieves precise control over the spatial deposition of multiple materials and at multiple scales through a composite process of coaxial printing and extrusion printing. In particular, microscale vascularized cell spheres are anchored in situ around the sacrificial channels during the printing process, and can spontaneously complete the entire process of budding, ligation and maturation in post-culture without additional micro-nano processing or exogenous growth factor gradient modification. It combines process simplicity and batch-to-batch reproducibility. In terms of application prospects, the multi-scale vascularized skin model constructed by this invention can serve as an in vitro alternative platform for evaluating the permeability of skin drugs, screening for toxicity, verifying efficacy and studying the mechanisms of vascular-related skin diseases. It is in line with the Ministry of Industry and Information Technology's pharmaceutical industry development plan, which aims to improve R&D efficiency and reduce reliance on animal experiments. It has broad industrialization value in the fields of drug development, cosmetic safety evaluation and regenerative medicine.In summary, this invention achieves significant technological advancements in cross-scale vascular network construction, biomimetic functional reproduction, multi-material collaborative manufacturing, and industrial application potential through the deep integration of microscale angiogenesis units and coaxial sacrificial printing technology, overcoming long-standing technical challenges in this field.

[0044] The multi-scale vascularized skin model prepared by this invention significantly outperforms existing technologies in terms of structural biomimicry, functional integration, and long-term culture capability. This model can be widely applied in scenarios such as research on skin disease mechanisms, evaluation of transdermal drug absorption, toxicity screening, and efficacy verification. It is expected to greatly reduce reliance on animal experiments, aligning with the strategic needs of the pharmaceutical industry's development plan. It provides a novel technological path for the manufacture of highly biomimetic and highly functional skin models, and also offers a more reliable and precise tool platform for drug development and regenerative medicine.

[0045] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing a multi-scale vascularized skin model, characterized in that, Includes the following steps: S1: Constructing microscale angiogenesis units; S2: Formulate various bio-inks for constructing the vascular layer, dermis, and epidermis, respectively; S3: Using coaxial printing and extrusion printing processes, bio-inks of the vascular layer, dermis layer and epidermis layer are deposited according to a predetermined spatial configuration. The coaxial printing process is used to construct sub-millimeter-scale vascular trunks with endothelialized cavities. The bio-ink of the vascular layer contains the microscale angiogenesis units. S4: A hollow submillimeter-scale vascular channel is formed inside the vascular layer through a sacrificial material removal process; through a post-culture process, the microscale angiogenesis unit is induced to sprout and connect with the channel of its adjacent submillimeter-scale vascular trunk, thereby forming a cross-scale vascular network from the submillimeter-scale trunk to the micrometer-scale capillary, resulting in a cross-scale vascularized skin model.

2. The method for preparing a multi-scale vascularized skin model according to claim 1, characterized in that, In S1, the step of constructing the microscale angiogenesis unit includes: Endothelial cells and stromal cells were co-cultured to allow them to self-assemble into microscale vascularized cell spheres with budding potential; The endothelial cells are human umbilical vein endothelial cells, and the matrix cells are fibroblasts.

3. The method for preparing a multi-scale vascularized skin model according to claim 2, characterized in that, In S2, the vascular layer bio-ink comprises a first structural material, a first cellular component, and the microscale angiogenesis unit; The dermal bio-ink contains a second structural material and fibroblasts; The epidermal bio-ink contains a third structural material and keratinocytes.

4. The method for preparing a multi-scale vascularized skin model according to claim 3, characterized in that, In S2, the first structural material comprises gelatin, methacrylamide gelatin, decellularized extracellular matrix, and a photoinitiator; the first cell component comprises human umbilical vein endothelial cells. In the dermal bio-ink, the concentration of gelatin is 7%-10% (w / v), the concentration of methacrylamide gelatin is 8%-15% (w / v), the concentration of decellularized extracellular matrix is ​​1%-3% (w / v), the concentration of photoinitiator is 0.25% (w / v), and the concentration of microscale vascularized cell spheroids is 1×10⁻⁶. 4 -5×10 4 The concentration of cells / mL and human umbilical vein endothelial cells was 5 × 10⁶. 6 -10×10 6 per mL.

5. The method for preparing a multi-scale vascularized skin model according to claim 4, characterized in that, In the vascular layer bio-ink, the density of microscale angiogenesis units is configured such that the capillary network formed after budding can functionally match the submillimeter-scale vascular channels.

6. The method for preparing a multi-scale vascularized skin model according to claim 4, characterized in that, The second structural material is GelMA, dECM, and LAP; the concentration of dECM in the dermal bio-ink is 1%-3% (w / v), the concentration of LAP is 0.25% (w / v), and the concentration of GelMA is 8%-15% (w / v); the concentration of fibroblasts is 0.5 × 10⁻⁶. 6 -2×10 6 cells / mL; The third structural material is gelalin; in the epidermal bio-ink, the concentration of gelalin is 7%-10% (w / v), and the concentration of keratinocytes is 1×10⁻⁶. 6 -6×10 6 per mL.

7. The method for preparing a multi-scale vascularized skin model according to claim 6, characterized in that, In S3, in the coaxial printing process, the structural material containing the microscale angiogenesis unit is used as the outer phase fluid, and the mixture containing endothelial cells and sacrificial material is used as the inner phase fluid.

8. The method for preparing a multi-scale vascularized skin model according to claim 6, characterized in that, In S4, the specific operation of forming a hollow sub-millimeter-level vascular channel inside the vascular layer through the sacrificial material removal process is as follows: the obtained skin model is placed in an incubator at 37°C, and the inner phase of the skin model dissolves to form a sub-millimeter-level hollow channel.

9. The method for preparing a multi-scale vascularized skin model according to claim 6, characterized in that, In S6, the post-culture process includes sequential immersion culture for promoting the growth and proliferation of skin cells and gas-liquid interface culture for promoting epidermal differentiation, skin barrier function and cross-scale vascular network formation. In S6, the immersion culture lasts for 3-5 days; the gas-liquid interface culture lasts for 14-21 days.

10. A multi-scale vascularized skin model, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 9.

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