CN-116286602-B - 3D (three-dimensional) stratum corneum model and construction method and application thereof

Abstract

The invention discloses a construction method and application of a 3D (three-dimensional) stratum corneum model, wherein the 3D stratum corneum model consists of a top surface, a middle layer and a bottom surface, the top surface and the bottom surface are rugged, the top surface and the bottom surface are composed of apoptotic cells and/or cell derivatives tightly connected with a photocurable gel, the middle layer is the photocurable gel, and the photocurable gel is used as a framework to provide a pore structure for the apoptotic cells and/or cell derivatives. The construction method is that the photocurable gel material is loaded with the apoptotic keratinocyte and/or cell derivative to form a 3D stratum corneum model under the condition of light irradiation. The 3D horny layer model constructed by the invention can be effectively applied to in vitro evaluation of the effect of regulating microecology of cosmetics and the effect of testing bacteriostasis of biological agents such as antibiotics, and has the advantages of low cost, high construction speed, high stability and capability of screening active substances with high flux.

Inventors

• WU JIANXIN
• HUANG QING
• WANG PAN

Assignees

• 中国药科大学

Dates

Publication Date

20260508

Application Date

20230309

Priority Date

20220623

Claims (3)

1. 1. An application of a 3D stratum corneum model in-vitro evaluation of efficacy of cosmetics or raw materials thereof; The 3D horny layer model consists of a top surface, an intermediate layer and a bottom surface, wherein the surfaces of the top surface and the bottom surface are uneven and are formed by apoptotic cells tightly connected through GMHA gel, the intermediate layer takes the GMHA gel as a framework, the GMHA gel framework forms a plurality of gaps, and apoptotic cells are distributed in the gaps; the GMHA structural formula is as follows: ; the 3D horny layer model is constructed by a construction method comprising the following steps of photocuring GMHA and apoptotic HaCaT cells and sebaceous gland cells; The efficacy refers to the efficacy of regulating microecology, and the method of application comprises the following steps: (1) Inoculating a plurality of facial bacteria on the 3D stratum corneum model to simulate facial microecology, wherein the plurality of facial bacteria are selected from one or more of propionibacterium acnes, staphylococcus epidermidis and staphylococcus aureus; (2) Coating a sample to be tested on the 3D stratum corneum model inoculated with the facial bacteria; (3) After a period of time, the number of bacteria or the relative abundance of each of the bacteria of the flora on the 3D stratum corneum model was examined and the results obtained were used to evaluate the test samples.
2. 2. The method of claim 1, wherein the pore size of the voids formed by the GMHA gel skeleton is 50-150 μm.
3. 3. The method according to claim 1, wherein the thickness of the intermediate layer is 10 μm to 1mm.

Description

3D (three-dimensional) stratum corneum model and construction method and application thereof Technical Field The invention relates to a horny layer model and a construction method and application thereof, in particular to a 3D horny layer model for colonization of various microorganisms and a construction method and application thereof. Background In the process of developing an effective skin care product for regulating microecology, the evaluation of the effect of regulating microecology is required for active raw materials or cosmetics. In the past, efficacy evaluation is performed by establishing a sterile mouse model, but along with the proposal and implementation of the animal experiment 3R principle, the establishment of a proper in vitro model to replace an animal model for evaluating the application of cosmetic raw materials or products in the aspect of regulating microecology is very necessary and has great application. The skin is used as the largest organ of human body, and the surface of the skin is living with a plurality of different kinds of microorganisms, and beneficial microorganisms are physical barriers for preventing pathogen invasion and can protect the body from external invasion. Skin physiological states are classified into three types, moist, greasy and dry, according to the different skin environments and physicochemical properties of different parts of the human body. Although the skin lacks nutrition, is meta-acid and dry, 10 6 bacteria/cm 2 still live on the skin, most bacteria colonize the superficial part of the stratum corneum, and the cross-linked proteins and lipids of keratinocytes are used as nutrition sources. In the study of micro-ecology using in vitro models, the effect on the growth and interaction of microorganisms when converting from the skin environment to in vitro models must be considered. At present, in vitro research on micro-ecology mainly uses a 3D epidermis model, a human primary keratinocyte is differentiated to form human epidermis equivalent to construct the 3D epidermis model, the method is mainly applied to cosmetic safety evaluation in cosmetic industries, and along with the rise of biological printing technology, research on constructing a novel epidermis model by using a 3D biological printing technology is gradually increased. Normally bacteria stay on the skin surface and symbiotic bacteria do not interfere with the skin homeostasis, and when the skin barrier is broken, the bacteria cross the epidermal barrier and cause an inflammatory response. Researchers have used healthy human plantar callus to build a stratum corneum model, which can show that the interactions among microbiome members and the composition and diversity of inoculated microbiota remain relatively stable, but the model manufacturing raw materials are derived from human tissues and are difficult to widely apply, and the stable growth of anaerobic propionibacterium acnes cannot be maintained. If the 3D epidermis model is used for evaluating the microecological efficacy of cosmetics, the time is long, the cost is high, the difference between model batches is difficult to control, and the model cannot be applied to large-scale screening of active substances, so that a 3D horny layer model for bacterial colonization, which can simulate the physiological state of the skin horny layer, needs to be developed and improved. At present, an in-vitro method for evaluating the micro-ecological regulating efficacy of raw materials and finished products of cosmetics by establishing a 3D horny layer model is not reported in related patents and documents. The 3D horny layer model established by the invention can be simply and quickly established, and can also better maintain the composition and diversity of human skin microorganisms. The current method for in vitro evaluation and regulation of micro-ecology mainly uses a 3D epidermis model to inoculate microorganisms as a model, the 3D epidermis model used in the method uses human primary keratinocytes to differentiate to form human epidermis equivalents, and the microorganisms are directly inoculated on the 3D epidermis model for regulation of micro-ecology efficacy evaluation. The 3D epidermis model used at present has higher price, long model making time and more than ten days of differentiation of human primary keratinocytes to form the 3D epidermis model. Therefore, the method has the problems of high cost, low efficiency and time consumption, and is difficult to realize high-flux screening of active ingredients. Disclosure of Invention The invention aims to provide a 3D horny layer model which can rapidly, stably and high-flux screen active substances and can be used for colonization by various microorganisms, and the invention also aims to provide a construction method of the 3D horny layer model, and the invention also aims to provide application of the 3D horny layer model in-vitro evaluation of the efficacy of cosmetics or raw materia