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.

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

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

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.

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 ...

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2.2K 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.

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

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...