New 3D skin models for in vivo assay

Develops new 3D skin model is strategic for better understanding of skin physiology with the aim to provide new insights for health care and cosmetic applications In order to develop new, effective cosmetic molecules it is necessary to have effective and sensitive toxicological tests. Normally in studies conducted to develop new active ingredients we study a large number of molecules, among them many are discarded because they do not prove effective, however many molecules are discarded because of an insecure toxicological profile. few years ago an active ingredient evaluated as potentially effective is also subjected to in vivo toxicological tests. research in the cosmetic field is evolving and now er are trying to develop more effective toxicological test, able to reproduce the results of the assya tha have always been carried out in vivo.
Current research and toxicological test are still partly based on 2D cell culture. In such a setting, skin cells are cultivated in one layer in petri dishes before being exposed to selected compounds tested for potential skin applications. Conversely, human skin is organized three-dimensionally which sets quality limits for 2D screening methods. The multicellular setting of 3D models allows for a much better interaction of cells with each other and with the extracellular matrix. They are therefore much more representative of the in vivo environment of human skin.

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The project
Currently one of promising international research projects that affects the future of the animal tes in cosmetic industry is the M2Aind (Multimodal Analytics and Intelligent Sensorics for the Health Industries. This research project is a Public-Private-Partnership project led by Mannheim University of Applied Sciences (MUAS). As a first project milestone, the research partners MUAS have now published a review article entitled «In Vitro Skin Three-Dimensional Models and Their Applications» in the Journal of Cellular Biotechnology. The article describes the composition as well as principal features and functions of human skin. It discusses the setup, prerequisites, advantages, and disadvantages of currently available in vitro 3D skin models and compares them in a comprehensive overview table. The key advantages of the new approach within the M2Aind projects are a more realistic understanding of the physiological behavior of skin cells and the discovery of superior small-molecule actives. Dr Rüdiger Rudolf, coordinator of the M2Aind says «the review article also gives an outlook on prospective future developments, including the use and exploitation of novel human stem cell technologies for personalized diagnosis, therapy development, and regenerative medicine. The review serves as a guide for choosing appropriate cell models in skin pharmaceutical and cosmetics research». The article cites several innovative models to study skin physiology and in the future to develop increasingly efficient toxicological tests. Among the systems mentioned in the review: the spheroids 3D skin model, 3D hydrogen system and skin bioprinting, also the most innovative organ on chip system.

Spheroids 3D system
The spectrum of 3D cell culture models is vast and varied owing to the diverse requirements of different cell types and applications. Each model comes with its own set of advantages and limitations, and one distinct model is not suitable for all applications. Nevertheless, some methodologies have gained wider applicability than others especially for in vitro 3D culture. According to a 3D cells culture trends spheroids are among the most widely used models for 3D cell culture in vitro.
These 3D models have been used to demonstrate increased physiological representation for diverse cell types and have been extensively applied to stem cell differentiation, tumorigenesis, and drug discovery. Spheroids have an added advantage in that they can be easily manipulated into more complex in vivo-like co-culture models that incorporate multiple cell types. Cellular spheroids are simple 3D models that can be generated from a wide range of cell types, which form spheroids because of the tendency of adherent cells to aggregate. Common examples of spheroids include embryonic bodies, mammospheres, tumor spheroids. Adherent cells have a natural tendency to aggregate and form spheroids under circumstances that impede adhesion to cell culture substrates. matrix-free methods employed for generating spheroids include the use of attachment resistant cell culture surfaces, or by maintaining the cells as suspension cultures in media. Spheroids naturally mimic various aspects of solid tissues and are equipped with inherent gradients for efficient diffusion of oxygen and nutrients as well as the removal of metabolic wastes. It still needs to be determined, if skin cell spheroids can be grown to reach complete differentiation and full layer stratification as for standard hydrogel or 3D printing models. First promising results were reported, which describe a spherical skin microtissue (3D InSight Skin Microtissue, InSphero,) composed of different keratinocyte layers and a dermal fibroblast core, which produces exctracellular matrix proteins without the need for exogenous collagen. Spheroids are potentially very interesting systems also for skin research due to their simplicity, low cost and high reproducibility. Hence, they are suitable for high-throughput cell function and cytotoxicity analysis. Most spheroids can be dispersed by lysis buffer or enzymes and therefore, they are also appropriate. In vitro skin three-dimensional models and their applications for biochemical analysis Whole mount microscopic analysis of spheroids can be performed by confocal microscopy or light-sheet microscopy, while for conventional microscopy one has to section spheroids prior to imaging.

Hydrogel systems
3D in vitro hydrogel systems can be used to evaluate epidermal behavior and was developed for oncological studies and research.  The most dominant technique for creating an in vitro skin model is the use of hydrogels that serve as a scaffold for dermal fibroblasts, which is then co-cultured with keratinocytes on the surface. Collagen I, the leading class of ECM protein, is the typically used hydrogel material, and dermal fibroblast cells are usually distributed within the collagen gel to mimic the dermal layer. On the fibroblasts, keratinocytes are then seeded and appropriately stimulated, they are differentiated by creating a system very similar to the skin on which it is possible to test the toxicological effects of cosmetic active ingredients.

3D Bioprinting
Bioprinting allows the automated generation of complex tissue architectures. Different layers of a desired material, mostly in the forms of hydrogel or biodegradable scaffolds, can be printed to form such geometries. Subsequently, biomolecules or cells can be added to defined positions to form various biological structures of interest. As 3D bioprinting has the ability to manufacture the 3D structures, it has been applied to create various biomimetic structures, such as vascular-like structures or 3D neural tissues. Different bioprinted skin, complex skin tissues with dermal and epidermal layers containing keratinocytes, melanocytes and fibroblasts were generated. Currently 3D printing is only available in a few laboratories due to its high costs and complex printing system. Modern 3D bioprinting is mostly automated and therefore provides high reproducibility and highthroughput. As the scaffolds can be varied on demand and as different cell types and active molecules can be applied, bioprinting is a highly flexible method. Consequently, this system has potentials in tissue engineering as well as in cytotoxicity testing and pathophysiology of skin diseases.

Organ on chip
The future of in vitro toxicology is the on-chip organ system. this system, even if still under development, allows to study different molecules and their effects in very complex biological systems. the tests performed on these models allow to obtain representative toxicological data and are particularly suitable for toxicological screening in vitro.
Organs-on-chips are devices with one or more biocompatible microfluidic chambers (known as ‘chips’) containing multiple cell types in 3D culture; the living cells interact much as they might do in tissue as either miniature organs or miniature tumors. The chip design allows the cell cultures they contain to be continuously perfused and mechanically or electrically manipulated. The microfluidic flow can supply nutrients, drugs, immune cells, bacteria or viruses to the cell cultures inside the chips as necessary for the question to be addressed. Synthetic blood vessels can also be incorporated into the chip with real or synthetic blood flowing through. This mimics normal organ physiology or can be used to induce disease pathology at the organ and tissue level or test new compound and its toxicity. It is even possible to link chips containing different organ and tissue types; these might become especially valuable when one tissue (like the liver) processes a compound to a metabolite that causes an effect for example on kidney, brain or heart. Most of these systems are in the experimental phase of study. there are still no validated and full replacement tests of the in vivo. nevertheless, they seem to be extremely promising systems which, in the not too distant future, will allow to give a molecule a complete toxicological profile


by Elisa Brunelli, PhD Biotecnology