CN-121975723-A - Construction method of in-vitro full-layer skin model based on dynamic skin chip

CN121975723ACN 121975723 ACN121975723 ACN 121975723ACN-121975723-A

Abstract

The invention discloses a method for constructing an in-vitro full-layer skin model based on a dynamic skin chip, which belongs to the technical field of biology and relates to a method for constructing an in-vitro full-layer skin model based on a dynamic skin chip. The invention provides a way of nutrient infusion and metabolic waste removal by simulating the structure and the function of an in-vivo blood circulation system based on a dynamic skin chip, and constructs and obtains an in-vitro full-layer skin model which is complete and has physiological functions, and the model can simulate the permeation process and the metabolic process of a medicine in human skin, thereby providing an effective platform for accurately evaluating the curative effect and the safety of the medicine.

Inventors

CHU CHENGXING
XU MINGYANG
LIU HUAYAN
BAI JIYU
ZHAO MING
YANG JIANBO
ZHOU LUPING
ZHANG JING
XU LIANG
MA KANGLE

Assignees

浙江工业大学
中国科学院基础医学与肿瘤研究所(筹)

Dates

Publication Date: 20260505
Application Date: 20260327

Claims (10)

1. The method for constructing the in-vitro full-layer skin model based on the dynamic skin chip is characterized by comprising the steps of injecting a fibroblast suspension and a 3D epithelial cell culture medium into the dynamic skin chip for culture, constructing a dermis layer, inoculating keratinocytes above the dermis layer for differentiation culture, constructing a epidermis layer, and then obtaining the in-vitro full-layer skin model through dynamic differentiation treatment, wherein the temperature of the dynamic differentiation treatment is 35-38 ℃, the shaking angle of the dynamic differentiation treatment is 10-20 ℃, and the shaking frequency of the dynamic differentiation treatment is 10-15 times/min.
2. The method for constructing an in vitro full-thickness skin model based on dynamic skin chips according to claim 1, wherein the fibroblast suspension is a mixture of fibroblasts and collagen scaffold.
3. The method for constructing an in vitro full-thickness skin model based on a dynamic skin chip according to claim 2, wherein the preparation method of the collagen scaffold comprises centrifuging collagen, taking supernatant, mixing the supernatant with a DMEM culture solution, and adjusting pH to be neutral to obtain the collagen scaffold.
4. The method for constructing an in vitro full-thickness skin model based on a dynamic skin chip according to claim 3, wherein the volume ratio of the supernatant to the DMEM culture solution is 1:0.1-0.5.
5. The method for constructing an in vitro full-thickness skin model based on a dynamic skin chip according to claim 1, wherein the fibroblast density in the fibroblast suspension is 5×10 4 cells/mL-7×10 4 cells/mL.
6. The method for constructing an in vitro full-thickness skin model based on dynamic skin chips according to claim 1, wherein the volume ratio of the fibroblast suspension to the 3D epithelial cell culture medium is 1:1-5.
7. The method for constructing an in vitro full-thickness skin model based on dynamic skin chips according to claim 1, wherein the seeding density of keratinocytes is 1 x 10 5 cells/mL-5×10 5 cells/mL.
8. The method for constructing the in-vitro full-layer skin model based on the dynamic skin chip, which is disclosed in claim 1, is characterized in that the dynamic skin chip comprises a base carrier, a micro-channel, a culture hole and a liquid storage channel, wherein the micro-channel is arranged in the base carrier, the culture hole is vertically communicated with the micro-channel in the middle area of the micro-channel, and the liquid storage channel is vertically communicated with the micro-channel at two ends of the micro-channel.
9. An in vitro full-thickness skin model constructed by the construction method of any one of claims 1 to 8.
10. Use of the in vitro full-thickness skin model according to claim 9 for drug screening, cosmetic safety assessment or skin damage repair mechanism research.

Description

Construction method of in-vitro full-layer skin model based on dynamic skin chip Technical Field The invention relates to the technical field of biology, in particular to a method for constructing an in-vitro full-layer skin model based on a dynamic skin chip. Background In the field of biotechnology, in vitro skin models have an indispensable position for skin-related research. Whether drug development, cosmetic safety evaluation, skin physiological and pathological mechanism exploration, skin injury repair research and the like depend on a reliable in-vitro skin model. However, there are a number of issues to be addressed in the existing in vitro skin model construction techniques. At present, common skin model construction modes are mainly divided into two types. One type is simple epidermal or dermal cell culture. In this way, only a single type of skin cells are cultured, severely lacking the complex structure and function of real skin. For example, real human skin has a rich network of blood vessels that are responsible for not only transporting oxygen and various nutrients to skin cells, but also for the important role of metabolic waste removal. However, in the model of simple culture, there is no simulated vascular structure at all, which makes it impossible for cells to acquire substance exchange conditions similar to the in vivo environment. Moreover, the lack of a dynamic fluid environment is also a major drawback of such models. In a real physiological environment, dynamic change fluid exists around cells, and factors such as chemical substance concentration, fluid shearing force and the like play a key role in the growth, differentiation and function maintenance of the cells. However, the simple culture model cannot simulate these key factors, greatly affecting the normal physiological activities of cells, and making it difficult to maintain the cell viability at a high level for a long period of time. Another common type of construction is layered construction of the epidermis and dermis layers. While this approach attempts to mimic the layering properties of the skin to some extent, in practice, direct contact of the dermis with the epidermis layer is not truly achieved. In human real skin, the epidermis and dermis are closely connected, and there is a wide and complex interaction between them. Fibroblasts in the dermis layer are capable of secreting a variety of growth factors and extracellular matrix components that are critical to the proliferation, differentiation and maintenance of normal physiological function of the epidermis cells. Meanwhile, the epidermis cells also feed back signals to the dermis layer to regulate the behaviors of the dermis layer cells. However, the existing hierarchically-structured models fail to achieve direct contact of the epidermis with the dermis, such that these important physiological processes cannot be effectively simulated, thereby limiting the model's ability to reproduce true skin functions. Currently, researchers have simulated signaling between epidermis and dermis by layering epidermis and dermis and attempting to add some factors that promote intercellular interactions. However, this approach does not address the fundamental problem of the dermis failing to directly contact the epidermis, and the added factors do not fully mimic the complex intercellular interaction environment in vivo. Furthermore, the layered construction of an efficient simulation of vascular structure and dynamic fluid environment with a lack of epidermis and dermis layers results in a large gap in simulating real skin function. In the aspect of skin injury repair research, the physiological process of skin injury and repair cannot be truly reproduced by the model, so that the related research is difficult to obtain substantial breakthrough. Therefore, developing an in vitro skin model construction method capable of highly simulating the structure and function of real human skin is an urgent need in the art. Disclosure of Invention The invention aims to provide a method for constructing an in-vitro full-layer skin model based on a dynamic skin chip, which simulates the structure and the function of an in-vivo blood circulation system based on the dynamic skin chip, so as to construct the in-vitro full-layer skin model with the dermis layer and the epidermis layer in direct contact, thereby providing a more accurate test model and an experimental platform for drug research and development or skin injury repair research and reducing research and development risks and cost. The technical scheme adopted by the invention for achieving the purpose is as follows: A construction method of an in-vitro full-layer skin model based on a dynamic skin chip comprises the steps of injecting a fibroblast suspension and a 3D epithelial cell culture medium into the dynamic skin chip for culture, constructing a dermis layer, inoculating keratinocytes above the dermis layer for differentiation cu