Common principles and best practices for engineering microbiomes

Despite broad scientific interest in harnessing the power of Earth's microbiomes, knowledge gaps hinder their efficient use for addressing urgent societal and environmental challenges. We argue hat structuring research and technology developments around a design-build-test-learn (DBTL) cycle will advance microbiome engineering and spur new discoveries on the basic scientific principles governing microbiome function. In this Review, we present key elements of an iterative DBTL cycle for microbiome engineering, focusing on generalizable approaches, including top-down and bottom-up design processes, synthetic and self-assembled construction methods, and emerging tools to analyze microbiome function. These approaches can be used to harness microbiomes for broad applications related to medicine, agriculture, energy, and the environment. We also discuss key challenges and opportunities of each approach and synthesize them into best practice guidelines for engineering microbiomes. We anticipate that adoption of a DBTL framework will rapidly advance microbiome-based biotechnologies aimed at improving human and animal health, agriculture, and enabling the bioeconomy

Analysis of methods for physical and biological characterization and validation of microphysiological systems (MPSs)

Microphysiological systems (MPSs), also known as 'Organ-On-a-Chip' (OCC), have revolutionized the way of understanding biology. These systems, which include three-dimensional co-culture and microfluidic technology, aim to mimic human physiology with in-vitro culture systems. Their purpose is to increase the knowledge of biological processes, as well as to provide an effective diagnostic tool for the analysis of different drugs. Standardization of MPSs implies the robustness and reproducibility of these devices and is desirable for their industrialization, production and regularization. However, due to the early stage of academic and commercial development of this technology, no standardization procedure exists in the literature to date. Therefore, experts recommend focusing on the characterization or qualification of these devices. This characterization and qualification of microphysiological systems involves testing the different elements that make up the device to ensure that their configuration mimics the physiology of the human structures represented, behaving and providing values as similar as possible to those of the tissues in vivo. It is in this context that this thesis attempts to develop a characterization protocol applicable to any 'Organ-On-a-Chip' system based on the tests carried out and compiled in the literature of devices in the experimental or commercialization phase. Specifically, a characterization and qualification procedure is presented in which the membrane permeability is monitored in real time depending on device elements such as the presence or not of cell culture, the application or not of microfluids, among others. The choice of the assays to be performed, from among those described in the protocol, will depend on the elements of the OCC to be characterized.Els sistemes microfisiològics (MPSs), també coneguts com a "Organ-On-a-Chip" (OCC), han revolucionat la manera d'entendre la biologia. Aquests sistemes, que inclouen el co-cultiu tridimensional i la tecnologia microfluídica, pretenen imitar la fisiologia humana amb els sistemes de cultiu in-vitro. El seu objectiu és augmentar el coneixement dels processos biològics, així com proporcionar una eina de diagnòstic eficaç per a l'anàlisi de diferents fàrmacs. L'estandardització de MPSs implica la robustesa i reproductibilitat d'aquests dispositius i és desitjable per a la seva industrialització, producció i regularització. No obstant això, a causa de la primera etapa del desenvolupament acadèmic i comercial d'aquesta tecnologia, no existeix cap procediment d'estandardització en la literatura fins a data d’avui. Per tant, els experts recomanen centrar-se en la caracterització o qualificació d'aquests dispositius. Aquesta caracterització i qualificació de sistemes microfisiològics implica provar els diferents elements que componen el dispositiu per assegurar que la seva configuració imiti la fisiologia de les estructures humanes representades, comportant-se i proporcionant valors el més similars possible als dels teixits in-vivo. És en aquest context que aquesta tesi intenta desenvolupar un protocol de caracterització aplicable a qualsevol sistema "Organ-On-a-Chip" basat en les proves realitzades i compilades en la literatura de dispositius en la fase experimental o de comercialització. Concretament, es presenta un procediment de caracterització i qualificació en el qual la permeabilitat de la membrana es controla en temps real depenent dels elements del dispositiu com la presència o no del cultiu cel·lular, l'aplicació o no de microfluids, entre d'altres. L'elecció dels assaigs a realitzar, d'entre els descrits en el protocol, dependrà dels elements de l'OCC que el caracteritzin.Los sistemas microfisiológicos (MPSs), también conocidos como "Organ-On-a-Chip" (OCC), han revolucionado la forma de entender la biología. Estos sistemas, que incluyen el co-cultivo tridimensional y la tecnología microfluídica, pretenden imitar la fisiología humana con sistemas de cultivo in vitro. Su finalidad es aumentar el conocimiento de los procesos biológicos, así como proporcionar una herramienta de diagnóstico eficaz para el análisis de diferentes fármacos. La estandarización de los MPSs implica la robustez y reproducibilidad de estos dispositivos y es deseable para su industrialización, producción y regularización. Sin embargo, debido a la temprana etapa de desarrollo académico y comercial de esta tecnología, hasta la fecha no existe en la literatura ningún procedimiento de estandarización. Por ello, los expertos recomiendan centrarse en la caracterización o cualificación de estos dispositivos. Esta caracterización y cualificación de los sistemas microfisiológicos implica probar los diferentes elementos que componen el dispositivo para asegurar que su configuración imita la fisiología de las estructuras humanas representadas, comportándose y proporcionando valores lo más similares posibles a los de los tejidos in vivo. Es en este contexto en el que esta tesis trata de desarrollar un protocolo de caracterización aplicable a cualquier sistema 'Organ-On-a-Chip' basado en las pruebas realizadas y recopiladas en la literatura de dispositivos en fase experimental o de comercialización. En concreto, se presenta un procedimiento de caracterización y cualificación en el que se monitoriza en tiempo real la permeabilidad de la membrana en función de elementos del dispositivo como la presencia o no de cultivo celular, la aplicación o no de microfluidos, entre otros. La elección de los ensayos a realizar, de entre los descritos en el protocolo, dependerá de los elementos del OCC a caracterizar.Outgoin

Insight into the large-scale upstream fermentation environment using scaled-down models

Scaled‐down models are small‐scale bioreactors, used to mimic the chemical (pH, nutrient and dissolved oxygen) and physical gradients (pressure, viscosity and temperature) known to occur in the large‐scale fermenter. Conventionally, before scaling up any bioprocess, small‐scale bioreactors are used for strain selection, characterisation and optimisation. The typical small‐scale environment is homogenous, hence all the cells held within the small‐scale bioreactor can be assumed to experience the same condition at any point in time. However, for the large‐scale bioreactor, this is not the case, due to its inhomogeneous environment. Three different scaled‐down models are reviewed here, and the results suggest that a bacterium responds to changes in its environment rapidly and the magnitude of response to environmental oscillations is organism‐specific. The reaction and adaption of a bacterium to an inhomogeneous environment in most cases result in productivity and quality losses. This review concludes that consideration of fermentation gradients should be paramount when researchers screen for high yielding mutants in bioprocess development and doing this would help mitigate performance loss on scale‐up

SUITE an Innovative Bioreactor Platform for in vitro Experiments

In-vitro cell cultures are a fundamental step in preclinical drug testing and are of great interest to the pharmaceutical industry. The most common method for culturing cells is in cell culture incubators. These are large and cumbersome and all mechanical stimuli are absent. They are nevertheless used ubiquitously and their results quoted as "standards" of in-vitro protocols. Several alternative culture methods have been proposed, and many systems are currently available commercially. Indeed, systems and devices for maintaining cells and tissues in controlled physical conditions, or bioreactors, have become an important tool in many areas of research. This is not only due to the growing interest in tissue engineering but also because it is now being increasingly recognised that cells respond not only to their biochemical, but also to their physical environment, and both cues are necessary to create a biomimetic habitat. However most bioreactors for cell culture and tissue engineering are cumbersome and only provide a few cues such as flow or strain, allowing limited control and flexibility. Since drug testing involves a large number of tests on identical cell cultures, a single well culture is inadequate and costly both in time and money. The High Throughput Screening (HTS), is a methodology for scientific experimentation widely used in drug discovery, based on a brute-force approach to collect a large amount of experimental data in less time and using less animals. The parallel nature of HTS makes it possible to collect a large amount of data from a small number of experiments and in a very short time. HTS, however, suffers from a significant problem that may affect the relevance of tests: the environment discrepancy problem. Another problem related with the actual drug testing and tissue engineering experiments is the enormous number of animals that have to be scarified every year. The aim of this study was to develop a generic platform or SUITE (Supervising Unit for In-vitro TEsting) for cell, tissue and organ culture composed of two main components: a universal control unit and an array of bioreactor chambers. The platform provides a biomimetic habitat to cells and tissues since the environment in the chambers is controlled and regulated to provide biomechanical and biophysical stimuli similar to those found in-vivo. In this work I describe how a new concept of cell culture bioreactor was developed by integrating different technologies and research fields. The data extracted using this new cell culture approach is more predictive of the in vivo response with respect to the multi-well approach, particularly for drug related studies. The starting point was a thorough analysis of currently used in-vitro methods; their pros and cons were assessed to exploit their advantages and overcome or circumvent their disadvantages. As far as the culture chamber is concerned, the approach was to use the methods and materials commonly employed in microfluidic fabrication, but at scales compatible with classical culture systems such as petri-dishes and multiwells. This renders the bioreactors more amenable to use by biologists and enables the use of cell densities comparable with classic systems as well as the use of conventional assaying techniques. In most cases, the cell culture chambers are thus made out of PDMS (Polydimethylsiloxane), using soft-moulding with micro- or mini-machined masters, or what we call Soft Milli-molding. A system on a plate Multi Compartmental Modular Bioreactor (MCmB) was developed using this technology. The MCmB is a modular chamber for high throughput multi compartmental bioreactor experiments. It is designed to be used in a wide range of applications and with various cell types. A precise stimulus application is also very important to better understand the correlation between physical variables and pathologies allowing a more accurate study of the tissue physiology and pathologies. For this reason in these thesis three additional stimulation chambers for vascular and articular cartilage stimulation respectively were also designed and tested. The control system was developed to be user-friendly, flexible and expandable to include new stimuli and was based on modular components, including motors and sensors. Importantly a single software interface was designed to allow data acquisition and monitoring of several chambers in series or in parallel. Using SUITE, high throughput experiments can be performed in an in vivo-like simulated environment for a long time to simulate different physiological or pathological scenarios or for toxicity testing of cells, tissues or in-vitro organ models

Characterizing Mechanical Regulation of Bone Metastatic Breast Cancer Cells

Breast cancer most frequently metastasizes to the skeleton. Bone metastatic cancer is incurable and induces wide-spread bone osteolysis, resulting in significant patient morbidity and mortality. Mechanical stimuli in the skeleton are an important microenvironmental parameter that modulates tumor formation, osteolysis, and tumor cell-bone cell signaling, but which mechanical signals are the most beneficial and the corresponding molecular mechanisms are unknown. This work focused on bone matrix deformation and interstitial fluid flow based on their well-known roles in bone remodeling and in primary breast cancer. The goal of our research was to establish a platform that could define the relationship between applied dynamic mechanical forces and the resulting phenotype in bone metastatic breast cancer cells. To achieve this goal, we employed a high-throughput, multi-modal in vitro mechanical loading bioreactor to apply forces to 3D in vitro bone-mimetic scaffolds, thereby recapitulating the physiological skeletal mechanical environment. We combined this with multi-physics micro-CT-based computational simulation models to estimate the internal mechanical microenvironment during in vitro experimentation

System for the Application of Hydrostatic Pressure and Mechanical Strain to Cell Cultures

Pressure and stretch are two of the primary forces thatresult in mechanotransductory eventswhichregulatecertainaspects ofhuman health and disease. Laboratory systems such as the commercially available Flexercell® system and a variety of custom-made setups are currently used in research to systematically apply stretch and hydrostatic pressure independently, or in conjunction to cell and tissue cultures. However, these systems do not allow for the decoupling of pressure and stretch under the same culture conditions. The present study aims to design, fabricate, and calibratea device that can apply pressure and stretch simultaneously, as well as independently to cells in culture. Moreover, in order to characterize the mechanical behaviorof the cell substrate, equibiaxial mechanical testing was conducted on the substrate and the resulting data wereused to generate a finite element simulation of the device. Moreover, an analytical approach was used in an attempt to validate the simulation. To our knowledge, this is the first system that can definitively distinguish between the mechanotransductive events activated in response to pressure and stretch. Using this novel device, MYP3 cells cultured onfibronectin-coated substrates were exposed to pressure and stretch, together or independently. The cells exhibited the morphology characteristic of healthy urothelialcells. The extracellular ATP data indicated an increase in ATP release in response to mechanical stimuli. Caspase-1 activity decreased in response to mechanical stimuli. The present study was successful in creating a unique device capable of applying pressure and stretch, together and independently, to cells in culture to allow examination of the relative contributions of these stimuli in various mechanobiological events

High-throughput platforms for the screening of new therapeutic targets for neurodegenerative diseases

Despite the recent progress in the understanding of neurodegenerative disorders, a lack of solid fundamental knowledge on the etiology of many of the major neurodegenerative diseases has made it difficult to obtain effective therapies to treat these conditions. Scientists have been looking to carry out more-human-relevant studies, with strong statistical power, to overcome the limitations of preclinical animal models that have contributed to the failure of numerous therapeutics in clinical trials. Here, we identify currently existing platforms to mimic central nervous system tissues, healthy and diseased, mainly focusing on cell-based platforms and discussing their strengths and limitations in the context of the high-throughput screening of new therapeutic targets and drugs.This work had the financial support of Fundação para a Ciência e Tecnologia ( FCT ) through National Funds and, when applicable, co-financed by the FEDER through the PT2020 Partnership Agreement under the 4293 Unit I&D. D.N. Rocha acknowledges FCT for her PhD grant

Environmental Control in Flow Bioreactors

The realization of physiologically-relevant advanced in vitro models is not just related to the reproduction of a three-dimensional multicellular architecture, but also to the maintenance of a cell culture environment in which parameters, such as temperature, pH, and hydrostatic pressure are finely controlled. Tunable and reproducible culture conditions are crucial for the study of environment-sensitive cells, and can also be used for mimicking pathophysiological conditions related with alterations of temperature, pressure and pH. Here, we present the SUITE (Supervising Unit for In Vitro Testing) system, a platform able to monitor and adjust local environmental variables in dynamic cell culture experiments. The physical core of the control system is a mixing chamber, which can be connected to different bioreactors and acts as a media reservoir equipped with a pH meter and pressure sensors. The chamber is heated by external resistive elements and the temperature is controlled using a thermistor. A purpose-built electronic control unit gathers all data from the sensors and controls the pH and hydrostatic pressure by regulating air and CO2 overpressure and flux. The system’s modularity and the possibility of imposing different pressure conditions were used to implement a model of portal hypertension with both endothelial and hepatic cells. The results show that the SUITE platform is able to control and maintain cell culture parameters at fixed values that represent either physiological or pathological conditions. Thus, it represents a fundamental tool for the design of biomimetic in vitro models, with applications in disease modelling or toxicity testin