KR-102959962-B1 - Method for Simultaneous Assessing of Skin and Ocular Irritation with a Microfluidic System-Based Cell Culture Chip

Abstract

The present invention relates to a method for simultaneously evaluating skin and eye irritation using a microfluidic system-based cell culture chip, and specifically, to a method for simultaneously evaluating skin and eye irritation using a microfluidic system-based model that utilizes corneal-derived epithelial cells, vascular endothelial cells, and skin keratinocytes, and can indirectly exchange substances through well-to-well channels using a hydrogel.

Inventors

• 권민서
• 채혜원
• 박병준
• 고지훈
• 이태임
• 경상윤

Assignees

• 한국콜마주식회사

Dates

Publication Date

20260508

Application Date

20251110

Claims (5)

1. (S1) A step of manufacturing a microfluidic system-based cell culture chip comprising three horizontally arranged wells, wherein the wells are connected to adjacent wells by a passage and are compartmentalized including a hydrogel wall; (S2) A step of seeding corneal-derived epithelial cells, vascular endothelial cells, and skin keratinocytes in sequence into the three horizontally arranged wells; (S3) A step of treating a target sample in a well seeded with the above corneal-derived epithelial cells or skin keratinocytes and (S4) A method for simultaneous evaluation of skin and eye irritation using a microfluidic system-based model for simultaneous evaluation of skin and eye irritation, comprising the step of confirming the effect of the above sample on cells.
2. In Article 1, A method for simultaneous evaluation of skin and eye irritation, wherein the well of step (S1) above has a cell culture area diameter of 4 to 5 mm.
3. In Article 1, A method for simultaneous evaluation of skin and eye irritation, wherein the height of the hydrogel wall in step (S1) is 300 to 900 μm.
4. In Article 1, A method for simultaneous evaluation of skin and eye irritation, wherein the hydrogel of step (S1) above is treated in a well at a concentration of 0.8 to 3 mg/ml to form a hydrogel wall.
5. In Article 1, A method for simultaneously evaluating skin and eye irritation, wherein the method for determining the effect of the sample on the cell in the above (S4) step is a measurement of cell viability or a measurement of the expression of a biomarker.

Description

Method for Simultaneous Assessing of Skin and Ocular Irritation with a Microfluidic System-Based Cell Culture Chip The present invention relates to a method for simultaneously evaluating skin and eye irritation using a microfluidic system-based cell culture chip, and specifically, to a method for simultaneously evaluating skin and eye irritation using a microfluidic system-based model that utilizes corneal-derived epithelial cells, vascular endothelial cells, and skin keratinocytes, and can indirectly exchange substances through well-to-well channels using a hydrogel. Ensuring the safety of cosmetics and their ingredients is an essential requirement for entering the global market, and major countries such as China, the United States, and Europe have legally mandated the submission of safety evidence data. Traditionally, toxicity assessments based on animal testing were primarily used, but animal testing is gradually being restricted due to ethical concerns. Furthermore, while human cell-based in vitro testing methods are being utilized as an alternative to animal testing, existing technologies have limitations: high costs for establishing and maintaining skin models, the time required for evaluation making simultaneous processing of large volumes of samples difficult, and the inability to sufficiently reproduce the complex intercellular interactions of actual skin due to simplified cell structures. Accordingly, the inventors conducted research to construct a cell model using microfluidic technology, and as a result, they constructed a simultaneous skin and eye irritation evaluation model using corneal-derived epithelial cells, vascular endothelial cells, and skin keratinocytes, which allows for the indirect exchange of substances through well-to-well channels using a hydrogel, and confirmed that the effect of a sample on cells can be verified more quickly and accurately using the simultaneous evaluation model. A brief description of each drawing is provided to help to better understand the drawings cited in the detailed description of the invention. Figure 1 is a schematic diagram of a microfluidic system-based cell culture chip according to the present invention. Figure 2 is a figure showing the cell dispensing arrangement within a cell culture chip. Figure 3 is a figure showing the method of treating the stimulus source (sample). Figure 4 is a figure showing the absorbance measurement process using a CCK assay. Figure 5 shows the absorbance measurement results using a CCK assay. Figure 6 shows the process of quantifying cell area using a fluorescence microscope with a Live/Dead staining assay. Figure 7 shows the cell viability results confirmed through a fluorescence microscope using a Live/Dead staining assay. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art. Furthermore, in describing embodiments of the present invention, detailed descriptions of related known components or functions are omitted if it is determined that such detailed descriptions would hinder understanding of the embodiments of the present invention. Additionally, while embodiments of the present invention will be described below, the technical concept of the present invention is not limited or restricted thereto and can be modified and implemented in various ways by those skilled in the art. In this specification, when a part is described as including a certain component, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. In this specification, the term "and/or" includes a combination of a plurality of related items or any one of a plurality of related items. According to an embodiment of the present invention, a method for simultaneous evaluation of skin and eye irritation using a microfluidic system-based model is provided, comprising: (S1) manufacturing a microfluidic system-based cell culture chip comprising three horizontally arranged wells, wherein the wells are connected to adjacent wells by a passage and are compartmentalized including a hydrogel wall; (S2) seeding corneal-derived epithelial cells, vascular endothelial cells, and skin keratinocytes in sequence into the three horizontally arranged wells; (S3) treating a target sample into the wells seeded with the corneal-derived epithelial cells or skin keratinocytes; and (S4) confirming the effect of the sample on the cells. The skin and eye irritation simultaneous evaluation model according to the present invention is characterized by containing corneal-derived epithelial cells, vascular endothelial cells, and skin keratinocytes within compartmentalized regions, respectively, and by allowing each well to indirectly share irritating substances through hydrogel walls, thereby enab