<meta charset="utf8"></head><body>유연한 연구 응용을 위한 효율적인 3D 피부 모델

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plate for adherent cell culture</td>
			<td>Biologix Europe GmbH</td>
			<td>07-6024</td>
			<td>-</td>
		</tr>
		<tr>
			<td>35%&#8211;38% HCL</td>
			<td>Chempur</td>
			<td>115752837</td>
			<td>-</td>
		</tr>
		<tr>
			<td>60 mm cell culture Petri dish&#160;</td>
			<td>Nest</td>
			<td>705001</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Avidin&#8722;Sulforhodamine 101</td>
			<td>Sigma Aldrich</td>
			<td>A2348-5MG</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Bright-field inverted microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Avantor</td>
			<td>874870116</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cell culture flask T75 for adherent cells</td>
			<td>Genoplast</td>
			<td>G77080033</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Centrifuge tube 15 mL</td>
			<td>GoogLab Scientific</td>
			<td>G66010522</td>
			<td>-</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Heal Force</td>
			<td>Galaxy 170R</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Col1A2 antibody produced in rabbit</td>
			<td>Novus</td>
			<td>NBP2-92790</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Corning(R) Transwell(R) Polycarbonate</td>
			<td>Corning</td>
			<td>CLS3422-48EA</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cytokeratin 14 antibody produced in mouse</td>
			<td>Novus</td>
			<td>NBP1-79069</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DPX Mountant for histology</td>
			<td>Sigma Aldrich</td>
			<td>06522-100ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>VWR Chemicals</td>
			<td>L0102-500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Eosine Y</td>
			<td>Kolchem</td>
			<td>-</td>
			<td>0.5 % aquatic solution</td>
		</tr>
		<tr>
			<td>Eppendorf tube 1.5 mL</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Eppendorf tube 2 mL</td>
			<td>Sarstedt</td>
			<td>72.691</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethyl alcohol absolute 99.8%</td>
			<td>Avantor</td>
			<td>396480111</td>
			<td>diluted in ultrapure water to the needed concentrations&#160;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fluorescent inverted microscope&#160;</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Goat anti-mouse secondary antibody conjugated with FITC</td>
			<td>Sigma Aldrich</td>
			<td>F0257-1mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary antibody conjugated with FITC</td>
			<td>Novus</td>
			<td>NB7159</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Harris Hematoxylin</td>
			<td>Kolchem</td>
			<td>-</td>
			<td>1 mg/mL in 95% ethanol</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Laminar chamber</td>
			<td>Heal Force</td>
			<td>HFSafe-1200</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Melan-A antibody produced in mouse</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-20032</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm</td>
			<td>HM355S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Avantor</td>
			<td>810981997</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraffin pastilles</td>
			<td>Sigma Aldrich</td>
			<td>1.07164</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>1581227</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin solution</td>
			<td>Sigma Aldrich</td>
			<td>P4333</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipette tip, 1000 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.305</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipette tip, 20 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.3021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipette tip, 200 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.303</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>BASF</td>
			<td>50401036</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Serological pipette 10 mL</td>
			<td>GoogLab Scientific</td>
			<td>G33270011</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Serological pipette 25 mL</td>
			<td>GoogLab Scientific</td>
			<td>G33280011</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Serological pipette 5 mL</td>
			<td>GoogLab Scientific</td>
			<td>G33260011</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Chempur</td>
			<td>118105307</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25% solution, phenol red</td>
			<td>Sigma Aldrich</td>
			<td>25200072</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Type 1 collagen</td>
			<td>IBIDI</td>
			<td>50201</td>
			<td>-</td>
		</tr>
		<tr>
			<td>U-bottom 96-well plate</td>
			<td>Sarstedt</td>
			<td>83.3925500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma Aldrich</td>
			<td>534056</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

building up skin models for numerous applications - from two-dimensional (2d) monoculture to three-dimensional (3d) multiculture

The skin is a continuous structure with multicell interactions revealing the proper functioning and homeostasis of this complex organ. It is built from morphologically different layers: the inner layer - dermis, and the outer layer - the epidermis. On top of the epidermis, we additionally distinguish the stratum corneum&#160;(consisting of flattened dead cells - corneocytes), which provides the greatest protection against the external environment. Some of the most important passive and active functions of the skin are body protection against external factors, participation in the immunological processes, secretion, resorption, thermoregulation, and sensing1,2,3. Because it is considered one of the largest organs in the body, it is impossible to avoid contact with various pathogens, allergens, chemicals, as well as ultraviolet (UV) radiation. Thus, it is structured with many types of cells with specific functions. The main types of cells present in the epidermis are keratinocytes (almost 90% of all cells, with structural and immunological functions in the deeper parts of the epidermis, but which later undergo the keratinization process to turn to corneocytes in the top layer of the epidermis), melanocytes (only 3%-7% of the epidermal cell population, which produce the UV protective pigment melanin) and the Langerhans cells (from the immune system). In the case of the dermis, the main cells are fibroblasts (producing growth factors and proteins), dendritic cells, and mast cells (both cell types of the immune system)4,5,6. Moreover, the skin is equipped with several extracellular proteins (such as collagen type I and IV, fibronectin, and laminin; Figure 1) and protein fibers (collagen and elastin), which ensure the specific structure of the skin but also encourage cell binding, cell adhesion, and other interactions7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65773/65773fig01.jpg" /> 
Figure 1: Schematic showing the skin structure. The skin structure marked four basic cell types occurring in its individual layers and distinguished proteins of the extracellular matrix. This figure was created with MS PowerPoint. <a href="https://www.jove.com/files/ftp_upload/65773/65773fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The safety of cosmetics and pharmaceutical products is a very important issue, and protecting the health of consumers and patients is a priority8. Until recently, it was supposed to be guaranteed by numerous tests, including studies conducted on animals. Unfortunately, these often required the use of drastic methods, causing pain and suffering in animals used for research purposes (frequently mice, rats, and pigs). In 1959, the Principles of the Humane Experimental Technique (the 3Rs principle) were introduced: (1 - Replacement) replacing animals in research with in vitro, in sillico, or ex vivo models, (2 - Reduction) reducing the number of animals used for research, and (3 - Refinement) improving the well-being of the animals which are still needed for research and at the same time improving the developed alternative methods9. Furthermore, in the European Union (EU), cosmetic testing on animals is regulated by law. From September 11, 2004 the ban on animal-tested cosmetic products came into force. On March 11, 2009, the EU banned animal testing of cosmetic ingredients. The sale of cosmetic products made of newly animal-tested ingredients was not allowed; however, testing the products on animals for complex human health issues such as repeat dose toxicity, reproductive toxicity, and toxicokinetics was still acceptable. Starting from March 11, 2013, in the EU, it is illegal to sell cosmetics where the finished product or its ingredients have been tested on animals10. Therefore, currently, in cosmetology, research is carried out at three levels: in vitro (cells), ex vivo (real tissues), and in vivo (volunteers)11. In the case of pharmaceuticals, the need for animal testing remains; however, it is significantly reduced and strictly controlled12.
As alternative methods to animal testing and for the initial assessment of the effectiveness of a novel active ingredient, the in vitro skin cell cultures are used. Isolation of different types of skin cells and their cultivation in sterile laboratory conditions allows one to assess the safety and toxicity of active substances. Skin cell lines are also widely recognized models for research as the cells are sold by certified companies and the results can be comparable in different laboratories. These tests are usually performed on simple 2D models of the human skin cell monocultures. Some of the more advanced models are their co-cultures (such as keratinocytes with fibroblasts and keratinocytes with melanocytes), as well as the three-dimensional models, including scaffold-free cultures (spheres) and scaffold-based skin equivalents of the epidermis, dermis or even the full-thickness substitutes of the skin13. It is worth mentioning that apart from the last type (skin equivalents), the rest are not commercially available, and if needed, a scientist must prepare them on his/her own.
Even though plenty of these models have been maintained and are routinely sold nowadays (Table 1), additional models are constantly needed to validate most of the results. Thus, the newly engineered models ought to recreate better the real interactions taking place in the human body. When a mixture of cells of different types is used to form such models, the reproduction of the multicellular aspect of tissue in vivo can be achieved. As a result, an organotypic culture is developed (Figure 2).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Name</td>
			<td colspan="4">Description</td>
		</tr>
		<tr>
			<td rowspan="6">Normal skin</td>
			<td colspan="2">EpiSkin</td>
			<td colspan="4">Reconstructed Human Epidermis - Keratinocytes on a collagen membrane</td>
		</tr>
		<tr>
			<td colspan="2">SkinEthic RHE</td>
			<td colspan="4">Reconstructed Human Epidermis - Keratinocytes on a polycarbonate membrane</td>
		</tr>
		<tr>
			<td colspan="2">SkinEthic RHE-LC</td>
			<td colspan="4">Human Epidermal Model Langerhans Cells - Keratinocytes and Langerhans cells on a polycarbonate membrane</td>
		</tr>
		<tr>
			<td colspan="2">SkinEthic RHPE</td>
			<td colspan="4">Reconstructed Human Pigmented Epidermis - Keratinocytes and Melanocytes on a polycarbonate membrane</td>
		</tr>
		<tr>
			<td colspan="2">T-Skin</td>
			<td colspan="4">Reconstructed Human Full Thickness Skin Model - Keratinocytes on a layer of Fibroblasts, which were grown on a polycarbonate membrane</td>
		</tr>
		<tr>
			<td colspan="2">Phenion FT Skin model</td>
			<td colspan="4">Keratinocytes and Fibroblasts in hydrogel</td>
		</tr>
		<tr>
			<td rowspan="2">Skin with a disease</td>
			<td colspan="2">Melanoma FT Skin Model</td>
			<td colspan="4">Normal human-derived Keratinocytes and Fibroblasts with human Malignant Melanoma cell line A375</td>
		</tr>
		<tr>
			<td colspan="2">Psoriasis Tissue Model</td>
			<td colspan="4">Normal human Keratinocytes and Fibroblasts</td>
		</tr>
	</tbody>
</table>
Table 1: The most popular commercial skin equivalents for various studies.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65773/65773fig02.jpg" /> 
Figure 2: Complexity of different in vitro models. The relation between the complexity of different in vitro models to recreate an organism and the real interactions occurring directly in the human body. The figure has been modified from the set &#34;Microbiology and cell culture&#34; from Servier Medical Art by Servier (https://smart.servier.com/).&#160;<a href="https://www.jove.com/files/ftp_upload/65773/65773fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
One of the most important limitations of the commercial equivalents is the availability of only very general research models with a few types of cells (typically 1-2, seldom 3). Yet, there are many more cells present in the skin, and their interaction with each other can ensure better or worse tolerance of various ingredients14. The lack of some immune components can decrease its value in several kinds of research, including immunotherapy. This is a serious issue as melanoma is a life-threatening skin cancer due to the early onset of metastasis and frequent resistance to the applied treatment15. To improve the artificial skin model, researchers try to establish a co-culture of immune cells with cell lines and organoids16&#160;and this is considered a big improvement of the studied models. For example, mast cells take part in many physiological (wound healing, tissue remodeling) and pathological (inflammation, angiogenesis, and tumor progression) processes in the skin17. Thus, their occurrence in the model can significantly change the model&#39;s response to the studied compound. Finally, a lot of skin-related information is still missing, which can only be discovered by performing basic research. This is why creating and refining different artificial skin models (Table 2) is such an important endeavor. This article presents several procedures to create advanced skin models, including spheres and skin equivalents.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>In vitro skin model</td>
			<td>Attempt to recreate interactions occurring in the tissue</td>
			<td>Examples of the used cells</td>
		</tr>
		<tr>
			<td rowspan="12">2D or 3D cell culture</td>
			<td rowspan="3">Epidermis</td>
			<td>Keratinocytes</td>
		</tr>
		<tr>
			<td>Melanocytes</td>
		</tr>
		<tr>
			<td>Keratinocytes + Melanocytes</td>
		</tr>
		<tr>
			<td rowspan="3">Dermis</td>
			<td>Fibroblasts</td>
		</tr>
		<tr>
			<td>Mast cells</td>
		</tr>
		<tr>
			<td>Fibroblasts + Mast cells</td>
		</tr>
		<tr>
			<td rowspan="6">Skin</td>
			<td>Keratinocytes + Fibroblasts</td>
		</tr>
		<tr>
			<td>Keratinocytes + Mast cells</td>
		</tr>
		<tr>
			<td>Melanocytes + Fibroblasts</td>
		</tr>
		<tr>
			<td>Melanocytes + Mast cells</td>
		</tr>
		<tr>
			<td>Keratinocytes + Fibroblasts + Melanocytes</td>
		</tr>
		<tr>
			<td>Keratinocytes + Fibroblasts + Mast cells</td>
		</tr>
	</tbody>
</table>
Table 2: Examples of cell type mixture to recreate skin tissue in 2D and 3D culture.

<meta charset="utf8"></head><body>저자 스포트라이트: 비용 효율적인 3D 세포 모델을 통한 피부 모델 다양성 향상

2차원(2D) 단일 배양에서 3차원(3D) 다중 배양에 이르기까지 다양한 응용 분야를 위한 피부 모델 구축

Watch this Scientific Journal Video about Building Up Skin Models for Numerous Applications - from Two-Dimensional 2D Monoculture to Three-Dimensional 3D Multiculture at JoVE.com

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

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Research

JoVE Journal

Bioengineering

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

1.4K 조회수.  Warsaw University of Technology. 우리의 과제는 화장품 또는 의학적 사용 전에 성분의 세포 독성을 테스트하는 것입니다. 약 3가지 세포 유형으로 제한되는 상용 3D 피부 모델은 실제 피부 복잡성을 반영하지 않습니다. 또한 피부는 필수 기능을 가진 다양한 세포 유형을 가지고 있어 성분의 영향을 정확하게 평가하기가 어렵습니다.우리의 연구 결과는 제한 세포 접착 방법이 일?...

비디오: 저자 스포트라이트: 비용 효율적인 3D 세포 모델을 통한 피부 모델 다양성 향상

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.