Selection of chromosomal DNA libraries using a multiplex CRISPR system.

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function

Scalable production of 3D microtissues using novel microfluidic technologies

Tissue engineering approaches are widely studied with the goal to replace or repair human tissues. However, while studies are often promising in a laboratory environment, there remain difficulties in the translation of laboratory-based studies towards clinical applications due to low in vivo efficiency and/or complex impractical procedures.An interesting strategy for improving therapy effectiveness is by evolving from conventional 2D cell culture to more biomimetic 3D cell culture approaches. While therapy efficiency can be greatly improved using 3D cell culture, current 3D microtissue production techniques are often non-scalable batch processes, limiting clinical and industrial translation. A continuous production method is needed in order to improve the microtissue production rate and improve the feasibility of clinical application.Microfluidics offers the possibility to evolve microtissue production towards a continuous process. Using conventional on-chip microfluidics, microtissues can be produced in a controlled and continuous manner by cell encapsulation in hollow microcapsules. However, conventional on-chip microfluidics offers challenges such as complex multistep processes, the use of potentially harmful oils and surfactants and often low throughputs, which are currently hampering widespread clinical and industrial translation of microfluidically produced microtissues. There is therefore a need to evolve microfluidics towards a clean, fast and single step scalable approach to fulfill the clinical requirements for tissue engineering approaches that take advantage of 3D microtissues.This thesis describes multiple microfluidic solutions that focus on overcoming these challenges hampering the widespread clinical and industrial use of microtissues. A reusable, cleanroom-free, multifunctional microfluidic device is developed using standard cutting and abrasion technology, which allows the production of microtissue-laden microcapsules in a single step-manner. This on-chip process is then evolved towards an off-chip jetting approach which allows for the production of microtissue-laden microcapsules in an ultra-high throughput manner (&gt;10 ml/min) without the need of potentially harmful oils and surfactants. This in-air microfluidic approach is also utilized for mass production of microtissues in larger compartmentalized hydrogels, which are used for the production of large clinical-sized tissues. A multitude of microtissues are formed using these described microfluidic technologies such as human mesenchymal stem cell spheroids, chondrocyte spheroids, fibroblast spheroids, cholangiocyte and cholangiocarcinoma organoids, lumen-forming embryoid bodies, contracting cardiospheres, and clinical sized cartilage tissues.To summarize, this thesis introduces multiple microfluidic systems for scalable microcapsule and microtissue production with the aim to remove the hurdles towards clinical and industrial translation of 3D microtissues.<br/

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring.

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research

In vitro cancer models: a closer look at limitations on translation

In vitro cancer models are envisioned as high-throughput screening platforms for potential new therapeutic discovery and/or validation. They also serve as tools to achieve personalized treatment strategies or real-time monitoring of disease propagation, providing effective treatments to patients. To battle the fatality of metastatic cancers, the development and commercialization of predictive and robust preclinical in vitro cancer models are of urgent need. In the past decades, the translation of cancer research from 2D to 3D platforms and the development of diverse in vitro cancer models have been well elaborated in an enormous number of reviews. However, the meagre clinical success rate of cancer therapeutics urges the critical introspection of currently available preclinical platforms, including patents, to hasten the development of precision medicine and commercialization of in vitro cancer models. Hence, the present article critically reflects the difficulty of translating cancer therapeutics from discovery to adoption and commercialization in the light of in vitro cancer models as predictive tools. The state of the art of in vitro cancer models is discussed first, followed by identifying the limitations of bench-to-bedside transition. This review tries to establish compatibility between the current findings and obstacles and indicates future directions to accelerate the market penetration, considering the niche market.This work is supported by FROnTHERA (NORTE-01-0145-FEDER-000023) and the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement No. 668983 —FoReCaST. N. Antunes thanks the funds provided by FCT under the doctoral program in Tissue Engineering, Regenerative Medicine and Stem Cells (PD/BD/143050/2018). SCK also records the support of FCT through the BREAST-IT project (PTDC/BTM-ORG/28168/2017)

The bioprinting roadmap

This bioprinting roadmap features salient advances in selected applications of the technique and highlights the status of current developments and challenges, as well as envisioned advances in science and technology, to address the challenges to the young and evolving technique. The topics covered in this roadmap encompass the broad spectrum of bioprinting; from cell expansion and novel bioink development to cell/stem cell printing, from organoid-based tissue organization to bioprinting of human-scale tissue structures, and from building cell/tissue/organ-on-a-chip to biomanufacturing of multicellular engineered living systems. The emerging application of printing-in-space and an overview of bioprinting technologies are also included in this roadmap. Due to the rapid pace of methodological advancements in bioprinting techniques and wide-ranging applications, the direction in which the field should advance is not immediately clear. This bioprinting roadmap addresses this unmet need by providing a comprehensive summary and recommendations useful to experienced researchers and newcomers to the field

Marine Biotechnology: A New Vision and Strategy for Europe

Marine Board-ESF The Marine Board provides a pan-European platform for its member organisations to develop common priorities, to advance marine research, and to bridge the gap between science and policy in order to meet future marine science challenges and opportunities. The Marine Board was established in 1995 to facilitate enhanced cooperation between European marine science organisations (both research institutes and research funding agencies) towards the development of a common vision on the research priorities and strategies for marine science in Europe. In 2010, the Marine Board represents 30 Member Organisations from 19 countries. The Marine Board provides the essential components for transferring knowledge for leadership in marine research in Europe. Adopting a strategic role, the Marine Board serves its Member Organisations by providing a forum within which marine research policy advice to national agencies and to the European Commission is developed, with the objective of promoting the establishment of the European Marine Research Area

Progress and challenges in large-scale expansion of human pluripotent stem cells

The constant supply of high cell numbers generated by defined, robust, and economically viable culture processes is indispensable for the envisioned application of human pluripotent stem cells (hPSCs) and their progenies for drug discovery and regenerative medicine. To achieve required cell numbers and to reduce process-related risks such as cell transformation, relative short batch-like production processes at industry- and clinically-relevant scale(s) must be developed and optimized. Here, we will review recent progress in the large-scale expansion of hPSCs with particular focus on suspension culture, which represents a universal strategy for controlled mass cell production. Another focus of the paper relates to bioreactor-based approaches, including technical aspects of bioreactor technologies and operation modes. Lastly, we will discuss current challenges of hPSC process engineering for enabling the transition from early stage process development to fully optimized hPSC production scale operation, a mandatory step for hPSCs’ industrial and clinical translation

Exometabolomics and MSI: deconstructing how cells interact to transform their small molecule environment

Metabolism is at the heart of many biotechnologies from biofuels to medical diagnostics. Metabolomic methods that provide glimpses into cellular metabolism have rapidly developed into a critical component of the biotechnological development process. Most metabolomics methods have focused on what is happening inside the cell. Equally important are the biochemical transformations of the cell, and their effect on other cells and their environment; the exometabolome. Exometabolomics is therefore gaining popularity as a robust approach for obtaining rich phenotypic data, and being used in bioprocessing and biofuel development. Mass spectrometry imaging approaches, including several nanotechnologies, provide complimentary information by localizing metabolic processes within complex biological matrices. Together, the two technologies can provide new insights into the metabolism and interactions of cells

Drug Discovery from Natural Products for Pancreatic Cancer

Since ancient times, natural products (NPs) have been used as anti-infectives, anti-inflammatories, antioxidants, analgesics and antitumorals and many compounds derived from NPs are in clinical use. The use of plants in traditional medicine for multiple purposes is well known, and throughout recent history, metabolites of microbial origin have had an extraordinary impact on the welfare of humanity. There is an outstanding diversity of chemical structures that nature, and especially microorganisms, are able to produce, due to millenniums of evolution. Since only a small amount of the world’s biodiversity has been evaluated for potential biological activity, many more useful natural lead compounds await discovery, the challenge being how to access this natural chemical diversity. However, the validation and selection of primary screening assays, both phenotypic and target-based, are vital to guaranteeing a selection of extracts or molecules with relevant pharmacological action. The screening of antitumor agents against pancreatic cancer (PC) involves the use of established cell lines, cancer stem cells and spheroids that mimic the patient’s tumor. Improvements in the discovery of natural products along with the emergence of new technologies in cancer screening assays, promise the discovery of new and valuable drugs to tackle pancreatic cancer in the coming years