The authors are grateful for the financial support given by the Warsaw University of Technology from the program &#39;Excellence Initiative - Research University&#39; in the form of two grants: POB BIB BIOTECHMED-2 start (no. 1820/2/ZO1/POB4/2021) and the Rector&#39;s grant for Student Research Groups (SKIN-ART, no. 1820/116/Z16/2021). Moreover, the Authors would like to acknowledge the support received from Prof. Joanna Cie&#347;la and the Chair of Drug and Cosmetics Biotechnology&#160;as well as the Biotechnology Science Club &#39;Herbion&#39;&#160;at the Faculty of Chemistry, Warsaw University of Technology. Special thanks go to Dr. Micha&#322; Stepulak for providing the compound Pluronic F-127.

Efficient 3D Skin Models for Flexible Research Application

The authors declare no conflicts of interest.

This article presents the methodology that can be applied to prepare one&#39;s own advanced artificial skin models. It is a good solution whenever the planned research needs strictly defined research models that may turn out to be unavailable on the market or very expensive. As mentioned earlier, several commercial skin equivalents are available on the market (e.g., EpiSkin, EpiDerm&#160;FT). However, their cost (&#8364;100-&#8364;400 per piece) and delivery time (a few days-weeks) may encourage the researcher to attempt to prepare such a model on their own. The proposed procedures are easy to perform even for inexperienced scientists, and at the same time, allow to obtain very advanced skin models. It is worth emphasizing that the decision on the cellular composition of a given model is fully dependent on the researcher. Apart from the created model, it can be further developed and improved, which opens up completely new research perspectives. In the case of commercial models, it is necessary to buy a different equivalent.
Although the 3D cell cultures may be advanced with multiple cell types, easy to handle and accessible, they are still just artificial models which cannot fully recreate the complexity and functionality of the tissue (e.g., immunological functions, vascularization). That is why, in most studies, several models are required to confirm the obtained results. Some advantages and disadvantages of these models were gathered in Table 9, as well as their limitations. On the other hand, commercial models guarantee high qualitative standards with reproducibility of experiments and comparability of data between the laboratories. To implement the use of a new compound for research, it will certainly be necessary to purchase the appropriate commercial equivalent. But at the preparatory stage, such a self-made 3D model of the skin (multicell type sphere or equivalent) can help to reduce the number of experiments needed to be carried out on a commercial equivalent. The goal of producing and using the described models is not to bypass the need to apply certified research models but to facilitate research and reduce related expenses.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compared pair of models</td>
			<td colspan="3">Advantages</td>
			<td colspan="3">Disadvantages</td>
		</tr>
		<tr>
			<td rowspan="8">cell culture vs. animals</td>
			<td colspan="3">Minimalized animal suffering</td>
			<td colspan="3">Limited information on the influence of a tested factor on the whole body</td>
		</tr>
		<tr>
			<td colspan="3">High experiment standardization - better reproducibility of the results</td>
			<td colspan="3">A single model is not enough to reflect the processes occurring in the body</td>
		</tr>
		<tr>
			<td colspan="3">No side effects for the whole organism</td>
			<td colspan="3">-</td>
		</tr>
		<tr>
			<td colspan="3">Better control over the conditions of the experiment</td>
			<td colspan="3">-</td>
		</tr>
		<tr>
			<td colspan="3">Possibility of automatization (e.g., bioprinting)</td>
			<td colspan="3">-</td>
		</tr>
		<tr>
			<td colspan="3">Lower costs</td>
			<td colspan="3">-</td>
		</tr>
		<tr>
			<td colspan="3">The small size of the sample needed</td>
			<td colspan="3">-</td>
		</tr>
		<tr>
			<td colspan="3">Limited amount of waste generated</td>
			<td colspan="3">-</td>
		</tr>
		<tr>
			<td rowspan="5">3D vs. 2D cultures</td>
			<td colspan="3">Better reflect the full organism</td>
			<td colspan="3">Time-consuming culture</td>
		</tr>
		<tr>
			<td colspan="3">Possibility to create a functional tissue</td>
			<td colspan="3">Higher costs</td>
		</tr>
		<tr>
			<td colspan="3">Possibility to create a model tailored to the needs of the carried research</td>
			<td colspan="3">Spontaneous formation of a 3D structure is almost not possible</td>
		</tr>
		<tr>
			<td colspan="3">-</td>
			<td colspan="3">Lack of standardized tests to quantify the effects of various compounds</td>
		</tr>
		<tr>
			<td colspan="3">-</td>
			<td colspan="3">Limited access to different 3D cultures available on the market</td>
		</tr>
		<tr>
			<td rowspan="5">cell line vs. primary cells</td>
			<td colspan="3">Certificated and approved models&#160;</td>
			<td colspan="3">Only a limited number of cell lines are available</td>
		</tr>
		<tr>
			<td colspan="3">High experiment standardization - better reproducibility of the results</td>
			<td colspan="3">Limited possibility to obtain several types of cells from the same donor</td>
		</tr>
		<tr>
			<td colspan="3">Longer life span</td>
			<td colspan="3">May possess changed properties from the native cells&#160;</td>
		</tr>
		<tr>
			<td colspan="3">Rather quick proliferation rate</td>
			<td colspan="3">Frequently disturbed functionality of cells</td>
		</tr>
		<tr>
			<td colspan="3">Less sensitive to several activities (e.g., freezing, centrifugation)</td>
			<td colspan="3">-</td>
		</tr>
	</tbody>
</table>
Table 9: Comparison of the usage of different models in research - advantages vs. disadvantages
Several articles describe how to prepare 3D skin models (apart from review articles summarizing commercially available models14,43,44, they are usually focused on a single methodology to obtain spheres45 or equivalents46).
In this article, two methodologies were described for the sphere formation with skin cells. The hanging drop method is widely used, but its repeatability and stability may be insufficient in some cases. Most steps require specific actions, such as high-speed work due to the evaporation of water from droplets during transfer. Gentle movements are also recommended, as lack of such a skill may result in cell aggregate damage31,32. Thus, an easier method for sphere preparation is focused on limiting cell adhesion. The absence of a good surface for cell attachment promotes higher interactions between cells. As a consequence, cell aggregates are generated. Its repeatability is much higher as there is no necessity for sphere transferring. With these methods, the optimal number of skin cells to create a sphere was established at 1 x 104 cells/sphere.
Next, procedures describing the preparation of skin equivalents were shown. Their appearance and functionality in research may strongly depend on the elements from which they are constructed, including cells (Table 2), scaffolds and media. The 3D scaffolds used for the preparation of artificial skin can be divided into synthetic hydrogels and those formed from natural sources. Depending on the used material and its properties to compose the hydrogel, the necessity to additionally supplement the medium may occur. Synthetic hydrogels require incorporating bioactive molecules (proteins, enzymes, and growth factors) into the synthetic hydrogel network to mediate specific cell functions47. The main approaches presented in the literature for achieving controlled delivery of growth factors to hydrogels include direct loading, electrostatic interaction, covalent binding, and the use of carriers48. Hydrogels formed from natural sources such as ECM proteins and polymers can generate fluid pathways throughout the 3D scaffold, accelerating the distribution of nutrients; thus, there is no need for additional supplementation of the medium. Investigations have shown that small molecules (like cytokines and growth factors) and macromolecules (including glycosaminoglycans and proteoglycans) can be transported through the ECM by diffusion47. However, the molecular diffusion of oxygen, nutrients, and other bioactive molecules may be hindered by the properties of the ECM hydrogel itself. Lower diffusion was correlated with the higher thickness of the hydrogel but also with a very high concentration of collagen37. In this study, to create the skin equivalent, a low collagen concentration equal to 2 mg/mL was used, which suggests that the molecular diffusion through the hydrogel should be good and rapid. Thus, no additional supplementation to the medium at this stage nor to the hydrogel itself was provided. To mimic the dermis, mast cells and fibroblasts (1:10) were embedded into the collagen hydrogel. Next, melanocytes and keratinocytes (1:15) were seeded onto the hydrogel and the whole equivalent was cultured in the medium. It is worth mentioning that the basic medium is composed of several amino acids, inorganic acids, and vitamins, and it is additionally supplemented with serum (consisting of multiple: growth and attachment factors for cells, lipids, hormones, nutrients, and energy sources, carriers, binding and transfer proteins, etc.). To achieve the proper structure of the epidermis, different supplements to the medium should be added at a certain time. The most important stimulator to initiate epidermal differentiation is calcium, as it activates intracellular signaling. Ascorbic acid stimulates a similar signaling pathway as the one mediated by calcium, but its effect is also accompanied by enhanced ascorbate transport and prevention of hydrophilic antioxidant depletion41. Furthermore, the differentiation of cells was improved when other components were added to the medium (such as caffeine, hydrocortisone, triiodothyronine, adenine, and cholera toxin)41,44. It is important that the prepared models should always be checked for the presence of a given cell type in the appropriate layer. The presence of all four types of skin cells was confirmed in the structure of the created equivalent by H&#38;E staining.
The most common problem encountered is the delicacy and intuition in the handling of the obtained models. Some difficulties may be connected with the cell sphere formation as well as with the hydrogel preparation. During the cell culture, several other problems can also occur; these include microbial infections, low proliferation rate of cells, aging of primary cells used in the models, maximum cultivation time of 2D and 3D models reconstructed from primary cells vs. cell lines, etc. In Table 10, some practical advice were gathered on what to do when one of the following problems is encountered.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Common problems in cell culture</td>
			<td colspan="4">Suggestions</td>
		</tr>
		<tr>
			<td>Microbial infection</td>
			<td colspan="4">If a microbial infection occurs in one of the flasks/dishes with cells, it is better to remove the infected culture as fast as possible (not to contaminate the remaining flasks/dishes with cells). Refreeze a new vial with cells.&#160; If the infection returns, it is good to try to widen the spectra of the applied antibiotics and increase their concentration.</td>
		</tr>
		<tr>
			<td>Low proliferation rate of cells</td>
			<td colspan="4">Some cells have a long doubling time. To stimulate their proliferation, several cell-specific growth factors can be added to the basic medium. Also increasing the concentration of FBS or L-glutamine in the basal medium may help to stimulate the growth of the cells.</td>
		</tr>
		<tr>
			<td>Aging of primary cells used in the models</td>
			<td colspan="4">After a few passages, the primary cells enter senescence and stop dividing. To overcome this problem in the models, it is recommended to use the cells from as early passage as possible to build the model.&#160;</td>
		</tr>
		<tr>
			<td>Maximum cultivation time of 2D and 3D models reconstructed from primary cells vs. cell lines</td>
			<td colspan="4">The time of cultivation of a model depends strongly on the type of used cells. With primary cells, the time of cultivation will be shorter due to their short life span.</td>
		</tr>
		<tr>
			<td>Difficulties in the cell sphere formation</td>
			<td colspan="4">Some cells may require a longer time for sphere formation. If after a few more days the spheres have not been formed, collect the cells from the sample and check their viability with, for example, trypan blue staining.</td>
		</tr>
		<tr>
			<td>Problems with sphere stability</td>
			<td colspan="4">If the spheres are not stable and get destroyed while handling, try to create spheres from a lower number of cells. Make sure to always gently transfer the dishes in which the spheres are growing.&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Difficulties with the hydrogel preparation&#160;</td>
			<td colspan="4">Check if the proportion of the ingredients (water, PBS [10x], NaOH, collagen type 1) was correct. The stock solution of collagen is usually very dense, thus make sure to slowly pipet it. Air bubbles disturb the morphology of the hydrogel, thus reverse pipetting of the gel may help with this issue.&#160;</td>
		</tr>
	</tbody>
</table>
Table 10: Cell culture troubleshooting
The established models after fabrication can be used in multiple fields, beginning with (1) cytotoxicity and genotoxicity experiments of novel compounds with biological activity for use in drugs and cosmetics49, (2) experiments with various factor stimulation50, (3) basic research increasing our knowledge about skin cells, their biological functions, interactions with other cells and the environment51,52, (4) research on selected disease entities where a specific type of cell can be introduced into the created model (cancer cells, cells with a mutation in a given gene, etc.14,53) and many more. It is needless to say that the application of these models stays in agreement with the 3Rs principle for more ethical use of animals in product testing and scientific research and does not violate the prohibition law of cosmetic product testing on animals.

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.

<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

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.

Author Spotlight: Enhancing Skin Model Diversity with Cost-Effective 3D Cellular Models

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

Our challenge is to test ingredients'cytotoxicity prior to cosmetic or medical use. The commercial 3-D skin models limited to around three cell types don't reflect real skin complexity. And skin has diverse cell types with essential functions, posing a challenge in accurately assessing ingredient impact.Our findings indicate that the limiting cell adhesion method produced more well-shaped spheres compared to the commonly-used methods. Additionally, it demands less manipulation, ensuring greater sphere stability. In our skin equivalent, we use up to four types of skin cells, unlike the commercial models that typically incorporate a maximum of three cell types.Common to these cell models lack accuracy in representing real interaction. While commercial 3-D skin equivalents offer better tissue representation, their cost limits widespread use. Consequently, our self-established models allow for initial cost-effective experiments on a more realistic platform.Our protocols outline affordable, straightforward ways to create customized 3-D skin models, encompassing spheres and equivalents. In contrast to commercial options, our models offer adjustable appearance and composition tailored to the study's requirements. The method is accessible, not demanding specialized skills or equipment, making them replicable even by novice researchers.Our aim is to highlight the simplicity of creating various 3-D cellular models, while emphasizing the value of certified commercial models in research. We stress the significance of using realistic models, such as spheres and equivalents. Showcasing how the cells make models, can aid in pre-selecting the testing conditions before employing certified models.

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

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.

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

building-up-skin-models-for-numerous-applications-from-two

Research

JoVE Journal

Bioengineering

Preparing a Cell Suspension from the Adherent Skin Cell Cultures

To begin, remove the respective medium from the culture flasks containing the keratinocytes, fibroblasts, and melanocytes. Gently wash the cells with five to 10 milliliters of PBS. Now add 0.5 to two milliliters of Trypsin-EDTA solution to the flask, depending on its size.Incubate the flask at 37 degrees Celsius. Use a microscope to control the detachment of cells from the surface. Suspend the detached cells in double the volume of Trypsin Neutralizer to deactivate Trypsin.Then transfer the suspension into a 15-milliliter tube. Now pipette about 20 microliters of the skin cell suspension into a 1.5-milliliter tube. With the help of a hemocytometer, count the cells Next, centrifuge the tube at 300 g for five minutes at room temperature.Then pipette out most of the supernatant and resuspend the cell pellet in the small amount of remaining liquid. Add an equal volume of fresh medium to the resuspended cells. In a separate 15-milliliter tube, prepare cell suspension.The primary cells of keratinocytes, melanocytes, fibroblasts, and mast cells presented their typical morphology when grown in their respective media.

Preparation of Skin Cell Spheres for 3D Multiculture

To begin the hanging drop method, pipette 20 microliters of a pre-prepared skin cell suspension onto the lid of a Petri dish. Now, cover the lid with the bottom half of the Petri dish and gently flip it to create the hanging drops. Add sterile water to the Petri dish to prevent evaporation of the full-grown medium from droplets.Incubate the droplets for 48 to 72 hours at 37 degrees Celsius. Next, fill the wells of a new plate with the full growth medium. Using sterile cut pipette tips, transfer the cell spheres to the wells of the multi-well plate.Incubate the transferred spheres for one day in the multi-well plate at 37 degrees Celsius before experimentation. For the limiting cell adhesion method, first add 100 microliters of 1%surfactant solution in PBS into each well of a U-bottom plate. Next, incubate the plate with the solution at 37 degrees Celsius for 24 hours.Now, prepare the cell suspension in the desired cell density. Pipette out the surfactant solution from the wells, then seed 50 microliters of the cell suspension into each well. Finally, incubate the plate at 37 degrees Celsius for 24 hours to reach cell aggregates.Spheres consisting of 10, 000 cells were easier to manipulate, as they were visible in the wells. The bigger spheres showed decreased stability. Different cell types formed spheres of different colors.The perfectly rounded spheres were prone to losing their shape during the transfer in the hanging drop method. Accurate handling was required to reduce the deformation resulting in intact cell aggregates. Inaccuracy and inexperience resulted in sphere damage.

Preparation of Full-Thickness Skin Equivalents for 3D Multiculture

To begin, mix water, 10 times concentrated PBS, and one molar sodium hydroxide in a tube. Next, pipette 500 microliters of the appropriately dense dermal cell solutions to a two milliliter tube. Centrifuge the solution at 300g for three minutes at room temperature.Once centrifugation is complete, pipette out the supernatant and gently resuspend the cells in a mixture of water, PBS, and sodium hydroxide. Now, add 200 microliters of the collagen solution to the mixture. Pipette the suspension to mix it well.Next, pipette 500 microliters of the prepared mixture into an insert in a 24 well plate. For a model without the stratum corneum, pipette 500 microliters of the mixture to a well without an insert. After incubating the plate for 10 minutes at room temperature, transfer it to an incubator for 30 minutes.Pipette 500 microliters of PBS buffered into each well to rinse the hydrogel surface. Next, mix 200 microliters of the keratinocyte suspension with an equal volume of melanocyte suspension in the supplemented DMEM medium. Gently pipette 500 microliters of the total cell suspension into each well.Incubate the plates at 37 degrees Celsius for two to five days. Replace the medium every 48 hours and use an optical microscope to monitor the cell growth. An equivalent 3D model with distinguishable dermis and epidermis was created, which could be monitored in real time.Keratinocytes in different stages of cell growth were observed, which are indicators of a living equivalent. Mast cells with recognizable granules were also seen.

1.4K Views.  Warsaw University of Technology.  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.

Efficient 3D Skin Models for Flexible Research Application

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