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Prof Dr Rüdiger Rudolf about the develpoment of Organoids based on complex 3D cell cultures for the screening of new bioactive compounds for skin care and biomedicine.

Organoids – a drug screening approach driving technology development

Up to now, most compound and drug screening campaigns rely on either two-dimensional (2D) cell cultures or animal research. Both, however, suffer from major drawbacks. First, 2D cell cultures might be easy and cheap to perform even in high throughput and they can use cells of human origin, but flat culture dishes represent very unnatural environments due to their rigidity and the limitation of contact formation to one side of the cell surface. Furthermore, penetration of drugs as well as the distribution of waste products, oxygen, and nutrients is certainly completely different in such 2D cultures as compared to the three-dimensional arrangement as it is found in human tissue. With respect to animal models, these often do not mimic human physiology, are difficult and expensive to perform, and are ethically more and more critical. For that reason and owing to several recent technological advancements, a paradigm shift is currently taking place in a wide range of fundamental and applied research. Indeed, 3D cell cultures of increasing complexity are being developed. These might be based on cell lines for their ease and reproducibility of culturing or, more recently, on primary human material, such as cultures derived from induced pluripotent stem cells or cancer cells obtained from biopsies. Intriguingly, many of these 3D cultures are now able to mimic not only the composition of cell types found in organs but also important aspects of their functions, and thus are often referred to as ‘organoids’. Currently, organoid technologies are considered the gold standard for drug testing, in particular to model complex organ functions or to develop personalized medicine, and as such they have been coined ‘technology of the year 2017’ by Nature Methods. Clearly, 3D cell culture and organoid technologies are currently amongst the most explorative and dynamic fields in the biomedical sciences and, owing to their three-dimensionality and novelty, the toolset for their production, probing, and analysis is far from being complete. Indeed, this discrepancy of biomedical relevance on the one hand and the partial lack of straightforward methods on virtually all levels of 3D cell culture work on the other hand, render the organoid field a major driving force for technology development.

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Fig. 2: Microscopic structure of a taste bud. Confocal image of a mouse taste bud section. Cell nuclei can be seen in blue, taste and apical epithelial cells in green, and stem and lower epithelial cells in red. The papilla has a height of about 100 µm.

Joining forces for applied 3D taste and skin research

It is precisely at this interface between biomedical/bioeconomy-based compound search, a need for 3D culture systems to obtain novel relevant test models, and the development of pertinent technologies, where BRAIN and Mannheim University of Applied Sciences (MUAS) chose to join forces in a BMBF-funded PPP termed “Multimodal Analysis and Intelligent Sensorics in the Health Industries” (acronym M2Aind). This project, running in a first phase since January 2017 until the end of 2020 and potentially being prolonged for another term of four years, has a total budget with BMBF and industry funds of more than 6.5 M€. It aims at creating industry-driven highly integrated applications that mostly involve the setup of complex 3D cell culture models and their subsequent live cell and ex vivo analytics, using primarily mass spectrometry, infrared- and fluorescence-microscopy approaches. Further sensor systems, including Raman scanning, and the development of more efficient compound production pathways are further branches of the project finally flowing into the highly integrated applications. In this framework, two of the product development lines followed at BRAIN are being addressed in M2Aind, i.e. nutraceutical and skin cosmetics. In both areas, considerable BRAIN-internal efforts have led to major advancements, including biosensor-expressing or other genetically modified human cells as well as adequate read-outs in 2D cultures and product-oriented hypotheses concerning known or novel mechanistic processes at the level of cellular biology.

A tasty project: going live in 3D

Taste or gustatory sense, is largely relying on so called taste buds located in discrete zones of the tongue. Taste buds show a highly conserved anatomical design with a supporting cup, basal progenitors and apical taste cells. The latter, depending on their molecular repertoire convert specific taste modalities like salty, sour, sweet, umami, and bitter (perhaps there more…) into intracellular signals that are ultimately relayed into yet poorly understood outputs to the central nervous system that interprets these signals and converts it into our sensation of a corresponding taste modality. In the field of nutraceutical development, it is state of the art, to hire professional taste testers in order to characterize novel compounds with regard to the gustatory qualities. With the aim of replacing this routine by a standardized and molecularly well-controlled in vitro test system, BRAIN has established cell lines derived from human taste buds and has characterized them in detail in 2D cell cultures. While this revealed high fidelity responses to certain taste modalities, others were still lacking, and there has been a wish to create a more physiological and representative model – in 3D. Here, the cooperation with Mannheim University of Applied Sciences kicked in. Joining the vast taste-related expertise and unique cellular systems from BRAIN with the 3D cell culture and analytical capabilities of MUAS, it was possible to upgrade the taste cell culture from 2D to 3D with different culture approaches including spheroid- and cup-like chip-based systems from another M2Aind partner, 300MICRONS. Analytical methods to characterize these cultures, including ex vivo and live cell 3D analytics, were developed and their application revealed the conservation of critical features, such as response to standard essences, but also revealed profound alterations of certain features like cell morphology and alignment. Novel forms of fixing the 3D cultures in place during perfusion had to be developed as well as ways to handle the spatial constraints given by the optical settings of the microscope. Special co-culture systems with other taste-bud relevant cell types are currently being explored to further increase the similarity to the physiological system. A long-term goal is the modelling of a taste bud organoid that is able to monitor certain taste modalities in a standardized manner, thus allowing for in vitro screens of novel essences.

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Fig. 3: Characteristics of skin cell spheroid cultures. Confocal images of keratinocyte spheroid culture sections show stratification of differentiation markers (left panel, differentiated cells in green) and mixed occurrence of apoptosis (right panel, apoptotic cells in green). Red signals indicate either less differentiated cells (left) or cell nuclei (right). Spheroid diameter in both cases about 300 µm.

To swell or not to swell…

Another focus of the cooperation between BRAIN and MUAS consists in the establishment of 3D models for studying the molecular mechanisms underlying skin development, swelling, and wrinkling, and to use this knowledge for the search of novel compounds with cosmetic or curative value. Stimulated by the ban of animal testing of cosmetics and the profound differences in skin structure between human and many mammals (including, in particular, rodents), dermatological skin research has gone through decades of 3D model development and these have yielded all kinds of highly stratified, often air lifted, skin equivalents. However, processes like swelling and, primarily, the cell-to-cell based and differentiation-status dependent reactions of individual cells to osmotic challenges has been extremely difficult to study in these models, which is essentially due to their intrinsic setup. Thus, using several human keratinocyte cell lines created by BRAIN, spheroid-shaped 3D models have been developed at MUAS. Analysis of these 3D cultures confirmed a highly reproducible stratification into basal and more differentiated zones as it can be found in human skin. Intriguingly, live cell imaging of such spheroids made from biosensor-carrying keratinocytes, recapitulated anticipated physiological responses such as swelling under hypo-osmotic conditions and subsequent regulated volume decrease. Combining these biosensor read-outs with adequate genetic models will now allow to assign these features to specific molecular mechanisms. Further downstream, it can be envisaged to harness such models for the identification of novel compounds.

Data handling – lost in space

The brave new world of 3D cell culture comes with clear advancements of our models in terms of physiological relevance and thus, hopefully, of predictive strength. However, adding a third dimension to cell biology inevitably potentiates the amounts of data to be made, stored, and analyzed. This has been and is currently triggering enormous joint efforts of academics and industries to solve these immediately imminent issues. Indeed, the business of 3D cell cultures and organoids has quickly moved from a modest megabytes monthly data production to an open-ended situation, that is currently typically settling in the terabytes range. This is primarily due to novel high-end analytical approaches, such as lightsheet microscopy, that allows to observe live cell behavior in 3D at fast rate and at single cell resolution. Apparently, under such conditions, safe and efficient data storage is becoming increasingly important, but efficient data analysis of complex cellular information is equally critical and needs extensive computational work. More than just computing power, it is also extremely challenging to reliably identify relevant features in an automated manner. We have recently been able to solve some of these issues adopting and adapting open source solutions, but considerable efforts will be necessary to quantify highly complex traits such as fast live cell signal transduction responses upon stimulation in the complexity of multicellular 3D cultures. An amazing flight into the galaxies of 3D cell culture and organoids has just started. It will certainly bring many challenges to be solved, anticipated or not. But it will definitely also lead us to novel magic worlds of technologies, biologies, and compounds. Please fasten seat belts.

M2Aind - Multimodal Analysis and Intelligent Sensorics in the Health Industries
Rudolf Ruediger

Prof Dr Rüdiger Rudolf

Prof Dr Rüdiger Rudolf is a Professor of Biosensorics at Mannheim University of Applied Sciences (MUAS) and head of the “Molecular Human Organoid and Tissue Analysis” impulse project under the umbrella of the M2Aind ( innovation partnership. Within this project and in cooperation with Prof Dr Mathias Hafner, Dr Tiziana Cesetti, Elena von Molitor MSc and Dr Mario Vitacolonna he investigates the use of 3D cell culture technologies, 3D live cell analysis, tissue clearing, 3D microscopy, biosensors and automated substance testing quantification in the fields of bioeconomy and biomedicine with a focus on taste and skin related applications.

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