Developing optimal input design strategies in cancer systems biology with applications to microfluidic device engineering

<p>Abstract</p> <p>Background</p> <p>Mechanistic models are becoming more and more popular in Systems Biology; identification and control of models underlying biochemical pathways of interest in oncology is a primary goal in this field. Unfortunately the scarce availability of data still limits our understanding of the intrinsic characteristics of complex pathologies like cancer: acquiring information for a system understanding of complex reaction networks is time consuming and expensive. Stimulus response experiments (SRE) have been used to gain a deeper insight into the details of biochemical mechanisms underlying cell life and functioning. Optimisation of the input time-profile, however, still remains a major area of research due to the complexity of the problem and its relevance for the task of information retrieval in systems biology-related experiments.</p> <p>Results</p> <p>We have addressed the problem of quantifying the information associated to an experiment using the Fisher Information Matrix and we have proposed an optimal experimental design strategy based on evolutionary algorithm to cope with the problem of information gathering in Systems Biology. On the basis of the theoretical results obtained in the field of control systems theory, we have studied the dynamical properties of the signals to be used in cell stimulation. The results of this study have been used to develop a microfluidic device for the automation of the process of cell stimulation for system identification.</p> <p>Conclusion</p> <p>We have applied the proposed approach to the Epidermal Growth Factor Receptor pathway and we observed that it minimises the amount of parametric uncertainty associated to the identified model. A statistical framework based on Monte-Carlo estimations of the uncertainty ellipsoid confirmed the superiority of optimally designed experiments over canonical inputs. The proposed approach can be easily extended to multiobjective formulations that can also take advantage of identifiability analysis. Moreover, the availability of fully automated microfluidic platforms explicitly developed for the task of biochemical model identification will hopefully reduce the effects of the 'data rich-data poor' paradox in Systems Biology.</p

Synthetic biology: advancing biological frontiers by building synthetic systems

Advances in synthetic biology are contributing to diverse research areas, from basic biology to biomanufacturing and disease therapy. We discuss the theoretical foundation, applications, and potential of this emerging field

Cell Dynamics in Three-dimensional (3D) Culture Environments

A three-dimensional (3D) cell culture system provides an effective platform to study cell dynamics in in vivo-mimicking conditions and thus plays an important role in understanding cell biology, organ function, and disease model. This dissertation investigates cell dynamics in a variety of 3D environments including solid and liquid matrix. We study cell dynamics in 3D hydrogel microparticles and show that cells exhibit significant differences with that from 2D monolayer culture, including cell cycle, survival, morphology and the sensitivity to inflammation. We further develop a 3D printed cell-laden hybrid hydrogel construct to investigate colon cancer cell dynamics in physiologically relevant bowel environment. Such system enables in vivo-mimicking cell environment and offers an effective platform to uncover inflammation mechanisms in bowel area. Long-term cell culture in 3D solid matrix, however, is challenged by nutrient delivering problems. We thus engineer a novel leaf-inspired artificial microvascular network to support the long-term cell growth. Apart from the 3D solid environment, we also investigate cell dynamics cultured in 3D fluidic environment and study the regulatory roles of shear stress in circulating cancer cells. Cancer cells are circulated in suspension for mimicking cancer metastasis through blood stream and a previously unrecognized role of circulatory shear stress in regulating cancer cell dynamics is revealed. The research presented in this dissertation introduces a comprehensive study of cell dynamics in 3D environments and paves a new avenue to establish physiologically relevant model systems for tissue engineering and artificial functional organs

Microtechnologies for Cell Microenvironment Control and Monitoring

A great breadth of questions remains in cellular biology. Some questions cannot be answered using traditional analytical techniques and so demand the development of new tools for research. In the near future, the development of highly integrated microfluidic analytical platforms will enable the acquisition of unknown biological data. These microfluidic systems must allow cell culture under controlled microenvironment and high throughput analysis. For this purpose, the integration of a variable number of newly developed micro- and nano-technologies, which enable control of topography and surface chemistry, soluble factors, mechanical forces and cell-cell contacts, as well as technology for monitoring cell phenotype and genotype with high spatial and temporal resolution will be necessary. These multifunctional devices must be accompanied by appropriate data analysis and management of the expected large datasets generated. The knowledge gained with these platforms has the potential to improve predictive models of the behavior of cells, impacting directly in better therapies for disease treatment. In this review, we give an overview of the microtechnology toolbox available for the design of high throughput microfluidic platforms for cell analysis. We discuss current microtechnologies for cell microenvironment control, different methodologies to create large arrays of cellular systems and finally techniques for monitoring cells in microfluidic devices.E.A.-H. acknowledges funding from the Basque Government, Department of Education, for predoctoral fellowship 2016. M.G.-H. acknowledges funding from the University of the Basque Country UPV/EHU, PIF16/204 predoctoral fellowship "call for recruitment of research personnel in training". J.E.-E. acknowledges funding from the University of the Basque Country UPV/EHU, postdoctoral fellowship ESPPOC 16/65 "Call for recruitment and specialization of Doctor Researchers 2016". M.M.D.P. and L.B.-D., acknowledge funding support from University of the Basque Country UPV/EHU, UFI11/32, and from Gobierno Vasco under Grupos Consolidados with Grant No. IT998-16. F.B.-L. acknowledges funding support from the Ramon y Cajal Programme (Ministerio de Economia y Competitividad), Spain. F.B.-L. and L.B.-D. acknowledge funding support from the European Union's Seventh Framework Programme (FP7) for Research, Technological Development and Demonstration under Grant agreement No. 604241 as well as Gobierno Vasco, Dpto. Industria, Innovacion, Comercio y Turismo under ELKARTEK 2015 with Grant No. KK-2015/0000088

Developing Droplet Based 3D Cell Culture Methods to Enable Investigations of the Chemical Tumor Microenvironment

Adaptation of cancer cells to changes in the biochemical microenvironment in an expanding tumor mass is a crucial aspect of malignant progression, tumor metabolism, and drug efficacy. In vitro, it is challenging to mimic the evolution of biochemical gradients and the cellular heterogeneity that characterizes cancer tissues found in vivo. It is well accepted that more realistic and controllable in vitro 3D model systems are required to improve the overall cancer research paradigm and thus improve on the translation of results, but multidisciplinary approaches are needed for these advances. This work develops such approaches and demonstrates that new droplet-based cell-encapsulation techniques have the ability to encapsulate cancer cells in droplets for standardized and more realistic 3D cell culture and cancer biology applications. Three individual droplet generating platforms have been designed and optimized for droplet-based cell encapsulation. Each has its own advancements and challenges. Together, however, these technologies accomplish medium to high-throughput generation (10 droplets/second to 25,000 droplets/second) of biomaterial droplets for encapsulation of a range of cell occupancies (5 cells/droplet to 400 cells/droplet). The data presented also demonstrates the controlled generation of cell-sized small droplets for biomolecule compartmentalization, droplets with diameters ranging between 100-400 μm depending on device parameters, and the generation of instant spheroids. Standardized assays for analyzing cells grown within these new 3D environments include proliferation assays of cells grown in mono- and co-cultures, the generation of large and uniform populations of scaffold supported multicellular spheroids, and a new system for culturing encapsulated cells in altered environmental conditions

Microscale methods to investigate and manipulate multispecies biological systems

The continuing threats from viral infectious diseases highlight the need for new tools to study viral interactions with host cells. Understanding how these viruses interact and respond to their environment can help predict outbreaks, shed insight on the most likely strains to emerge, and determine which viruses have the potential to cause significant human illness. Animal studies provide a wealth of information, but the interpretation of results is confounded by the large number of uncontrolled or unknown variables in complex living systems. In contrast, traditional tissue culture approaches have provided investigators a valuable platform with a high degree of experimental control and flexibility, but the static nature of flask-based cell culture makes it difficult to study viral evolution. Serial passaging introduces un-physiological perturbations to cell and virus populations by drastically reducing the number of species with each passage. Low copy, high fitness viral variants maybe eliminated, while in vivo these variants would be essential in determining the virus’ evolutionary fate. Bridging technologies are urgently needed to mitigate the unrealistic dynamics in static flask-based cultures, and the complexity and expense of in vivo experiments. This thesis details the development of a continuous perfusion platform capable of more closely mimicking in vivo cell-virus dynamics, while surpassing the experimental control and flexibility of standard cell culture. First, a microfluidic flow through acoustic device is optimized to enable efficient and controllable separation of cells and viruses. Repeatable isolation of cell and virus species is demonstrated with both a well-characterized virus, Dengue Virus (DENV), and the novel Golden Gate Virus. Next, a platform is built around this device to enable controllable, automated, continuous cell culture. Beads are used to assess system performance and optimize operation. Subsequently, the platform is used to culture both murine hybridoma (4G2) and human monocyte (THP-1) cell lines for over one month, and demonstrate the ability to manipulate population dynamics. Finally, we use the platform to establish a multispecies culture with THP-1 cells and Sindbis Virus (SINV). This work integrates distinct engineering feats to create a platform capable of enhancing existing cell virus studies and opening the door to a variety of high-impact investigations

Digital Microfluidics for Isothermal Nucleic Acid Amplification: Exploring Sensing Methodologies

Digital Microfluidics (DMF) has recently emerged as a promising candidate for nucleic acid amplification for molecular diagnostics, by virtue of its precise control over unit droplets without the need of any propulsion devices, ease of integration with chemical/biological reac-tions and multiplex assay capabilities. Nevertheless, current scientific research is still far from accomplishing the full potential of the technique, so new, innovative nanotechnology/biotech-nology hybrid approaches are necessary. As such, the purpose of this work is to contribute for the paradigm shift of nucleic acid amplification from central laboratories to point-of-care (POC) by designing and fabricating DMF devices compatible with isothermal nucleic acid amplifica-tion (loop-mediated isothermal amplification - LAMP). For biological validation of the devices, detection of cancer biomarker c-Myc is performed, and further real-time amplification moni-toring is attempted through several methodologies, namely fluorescence, impedance and elec-trochemical measurements. The DMF devices produced herein enable optimal temperature control, crucial for LAMP reactions, and further allow for a novel methodology of reagent mix-ing, based on dual actuation with back-and-forth motion and actuation frequency tuning. Such innovations lead to successful amplification of 0.5 ng/μL or 90 pg of c-Myc in one hour, in line with the range reported in the literature, and further monitoring of the LAMP reaction profile by microscopy-based fluorescence measurements. Impedimetric and electrochemical method-ologies did not meet the tight criteria required for biomarker detection, yet the developments achieved herein open the path for other applications. Lastly, the dielectric layer (key element of a DMF device) was optimized to assure long reactions (up to two hours) without device degradation.A microfluídica digital (MFD) surgiu como uma tecnologia promissora para amplificação de ácidos nucleicos em diagnóstico molecular, permitindo controlo sobre gotas unitárias sem necessidade de dispositivos de propulsão, facilidade de integração com reações químicas/bi-ológicas e capacidade de realização de ensaios simultâneos. Contudo, a investigação científica atual ainda está longe de atingir o máximo potencial da técnica, pelo que são necessárias abordagens novas, inovadoras e híbridas de nanotecnologia e biotecnologia. Como tal, o pro-pósito deste trabalho é contribuir para a mudança de paradigma da amplificação de ácidos nucleicos de laboratórios centralizados para ponto-de-atendimento (PDA) através do desenho e fabricação de dispositivos de MFD compatíveis com amplificação isotérmica de ácidos nu-cleicos (loop-mediated istothermal amplification - LAMP). Para validação biológica dos dispo-sitivos, será detetado o biomarcador de cancro c-Myc, e testada a monitorização da amplifica-ção em tempo real através de várias metodologias, nomeadamente medidas de fluorescência, impedância ou medidas eletroquímicas. Os dispositivos MFD produzidos permitem um con-trolo ótimo da temperatura, crucial para reações LAMP, e introduzem uma metodologia para mistura de reagentes, com movimentos em vaivém e ajuste da frequência de atuação. Tais inovações conduziram à amplificação de 0.5 ng/μL ou 90 pg de c-Myc em uma hora, em linha com o intervalo relatado na literatura, permitindo ainda monitorização do perfil da reação LAMP através de medidas de fluorescência mediadas por microscopia. As metodologias impe-dimétricas e eletroquímicas não cumpriram os exigentes critérios requeridos para deteção de biomarcadores, no entanto, os desenvolvimentos alcançados abrem caminho para outras apli-cações. Por último, a camada dielétrica (elemento-chave de um dispositivo MFD) foi otimizada para assegurar reações mais longas (até duas horas) sem degradação do dispositivo