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Cells-self Extracellular Matrices-based Bioinks to create accurate 3D diseased skin tissue models

Periodic Reporting for period 2 - ECM_INK (Cells-self Extracellular Matrices-based Bioinks to create accurate 3D diseased skin tissue models)

Reporting period: 2018-11-01 to 2020-04-30

In recent years, 3D in vitro cell models have drawn increasing interest from the scientific community contributing to the refinement of novel tools for disease modelling and drug development and also to reduce the need for animal experimentation. This has led to the surge of new human tissue bioengineering models based on 3D cellular structures that, due to the high degree of complexity of the native tissue, still don’t fully reflect its properties and composition.
Lately, bioprinting technology has captured attention as it allows a better recreation of the human tissue characteristics and the development of more reliable 3D in vitro models. Bioinks are a key element of bioprinting as they are the building pillars for a functional cellular structure in these novel models. Although several hydrogel approaches have been used for the development of more functional bioinks, considerable work is still needed to achieve formulations allowing engineered tissue to function as desired. For this purpose, ECM_INK aims to develop new bioinks that fully mimic the native environment to be recreated hypothesizing that this is mandatory to be able to build accurate skin models fully representative of the pathophysiology of diseases such as pemphigus vulgaris, dystrophic epidermolysis bullosa and squamous cell carcinoma.
Skin cells from patients suffering from dystrophic epidermolysis bullosa (DEB – 3 variants), squamous cell carcinoma and pemphigus vulgaris have been accessed either from collaborators or from a local Hospital. After optimization of the culture conditions for maximization of extracellular matrix production, it was confirmed that cells of each one of the diseases, and the variants in the case of DEB, produce a specific ECM. Therefore, the analyses already performed and those that are being carried out will allow mapping those singularities.
Additionally, different ECM extraction protocols have been trialled in order to obtain optimal protein content without losing critical native features. This was achieved by looking at the ECM contents from different perspectives and considering its subsequent use in the bioinks composition. Therefore, different ECM contents were obtained and combined with a biomaterial; depending on the chemistry of the material, the type of the extract and the amount of extract and material, different bioinks capable of being printed and forming stable hydrogel-like structures, various bioinks were obtained. So far, these were shown to influence cell behaviour and the specific effect over cell phenotype is currently being assessed. Moreover, based on the specificities of the bioinks a customised print head and an extrusion-based bioprinter were developed. This permitted higher printing versatility and precision allowing, for example, the creation of gradients of cell concentrations and materials determinant for the creation of a diseased environment within a surrounding normal tissue.
Considering the ultimate goal of the project of creating skin diseases models that can be used as in vitro platform for varied tests, a micro-bioreactor that allows for in vitro preparation, maintenance and conditioning of human skin tissues in a setting that can provide each layer of tissue with separate nourishing fluids according to its requirements was developed. The bioreactor was built with a modular design that permits its expansion for culturing more complex multilayer tissue or multi-tissue structures.
So far, the many strategies that have been followed to bioengineer in vitro 3D human tissue models mostly rely on the random culture of cells within a 3D structure without reflecting the compositional and structural complexity of the native tissues. 3D-printing technologies are expected to contribute to overcome that by allowing accurate and high speed deposition of various cells and matrices at high resolution. However, the bioinks currently available are limited in terms of providing the native-like biocues specific of a certain microenvironment. The strategy proposed in ECM_INK allows obtaining and use cell’s own ECM as those biocues thus providing the exact components of a tissue. The encouraging results bring closer the development of bioprinted engineered skin tissues (normal and diseased), where different cell types are presented with signals that promote their natural role and ultimately being representative of the native tissue. Additionally, considering the custom-made bioprinter that was developed it expected that the project will contribute to the field of 3D printing by providing the scientific grounds for advanced strategies that are capable of lead to the creation of complex bioprinted tissues including a vascular-like structure. Likewise, the developed dynamic culture system for multilayer tissue or multi-tissue structures is a highly versatile platform that is expected to allow not only the maintenance of the bioprinted tissues for a longer time period, but also to promote their maturation within a period of time compatible with the life span of an in vitro model, never achieved before.
Ultimately the models to be developed will provide unprecedented human-origin 3D in vitro systems in which a wide range of mechanistic questions regarding pemphigus vulgaris, dystrophic epidermolysis bullosa and squamous cell carcinoma can be addressed. Thus more accurate and translatable therapeutic targets can be revealed or therapy-related knowledge generated than those obtained with animal experimentation.