Bioprocess microfluidics: applying microfluidic devices for bioprocessing

Scale-down approaches have long been applied in bioprocessing to resolve scale-up problems. Miniaturized bioreactors have thrived as a tool to obtain process relevant data during early-stage process development. Microfluidic devices are an attractive alternative in bioprocessing development due to the high degree of control over process variables afforded by the laminar flow, and the possibility to reduce time and cost factors. Data quality obtained with these devices is high when integrated with sensing technology and is invaluable for scale-translation and to assess the economical viability of bioprocesses. Microfluidic devices as upstream process development tools have been developed in the area of small molecules, therapeutic proteins, and cellular therapies. More recently, they have also been applied to mimic downstream unit operations

Segmentation of phase contrast microscopy images based on multi-scale local Basic Image Features histograms

Phase contrast microscopy (PCM) is routinely used for the inspection of adherent cell cultures in all fields of biology and biomedicine. Key decisions for experimental protocols are often taken by an operator based on typically qualitative observations. However, automated processing and analysis of PCM images remain challenging due to the low contrast between foreground objects (cells) and background as well as various imaging artefacts. We propose a trainable pixel-wise segmentation approach whereby image structures and symmetries are encoded in the form of multi-scale Basic Image Features local histograms, and classification of them is learned by random decision trees. This approach was validated for segmentation of cell versus background, and discrimination between two different cell types. Performance close to that of state-of-the-art specialised algorithms was achieved despite the general nature of the method. The low processing time ( < 4 s per 1280 × 960 pixel images) is suitable for batch processing of experimental data as well as for interactive segmentation applications

Possible mechanisms of CO₂ reduction by H₂ via prebiotic vectorial electrochemistry

Methanogens are putatively ancestral autotrophs that reduce CO2 with H2 to form biomass using a membrane-bound, proton-motive Fe(Ni)S protein called the energy-converting hydrogenase (Ech). At the origin of life, geologically sustained H+ gradients across inorganic barriers containing Fe(Ni)S minerals could theoretically have driven CO2 reduction by H2 through vectorial chemistry in a similar way to Ech. pH modulation of the redox potentials of H2, CO2 and Fe(Ni)S minerals could in principle enable an otherwise endergonic reaction. Here, we analyse whether vectorial electrochemistry can facilitate the reduction of CO2 by H2 under alkaline hydrothermal conditions using a microfluidic reactor. We present pilot data showing that steep pH gradients of approximately 5 pH units can be sustained over greater than 5 h across Fe(Ni)S barriers, with H+-flux across the barrier about two million-fold faster than OH–-flux. This high flux produces a calculated 3-pH unit-gradient (equating to 180 mV) across single approximately 25-nm Fe(Ni)S nanocrystals, which is close to that required to reduce CO2. However, the poor solubility of H2 at atmospheric pressure limits CO2 reduction by H2, explaining why organic synthesis has so far proved elusive in our reactor. Higher H2 concentration will be needed in future to facilitate CO2 reduction through prebiotic vectorial electrochemistry

Integration and Application of Optical Chemical Sensors in Microbioreactors

The quantification of key variables such as oxygen, pH, carbon dioxide, glucose, and temperature provides essential information for biological and biotechnological applications and their development. Microfluidic devices offer an opportunity to accelerate research and development in these areas due to their small scale, and the fine control over the microenvironment, provided that these key variables can be measured. Optical sensors are well-suited for this task. They offer non-invasive and non-destructive monitoring of the mentioned variables, and the establishment of time-course profiles without the need for sampling from the microfluidic devices. They can also be implemented in larger systems, facilitating cross-scale comparison of analytical data. This tutorial review presents an overview of the optical sensors and their technology, with a view to support current and potential new users in microfluidics and biotechnology in the implementation of such sensors. It introduces the benefits and challenges of sensor integration, including, for example, their application for microbioreactors. Sensor formats, integration methods, device bonding options, and monitoring options are explained. Luminescent sensors for oxygen, pH, carbon dioxide, glucose and temperature are showcased alongside other optical detection methods, such as Raman and surface plasmon resonance. Areas where further development is needed are highlighted with the intent to guide future development efforts towards analytes for which reliable, stable, or easily integrated detection methods are not yet available

Quantification of the oxygen uptake rate in a dissolved oxygen controlled oscillating jet-driven microbioreactor

BACKGROUND: Microbioreactors have emerged as a new tool for early bioprocess development. The technology has advanced rapidly in the last decade and obtaining real-time quantitative data of process variables is nowadays state of the art. In addition, control over process variables has also been achieved. The aim of this study was to build a microbioreactor capable of controlling dissolved oxygen (DO) concentrations and to determine oxygen uptake rate in real time. RESULTS: An oscillating jet driven, membrane-aerated microbioreactor was developed without comprising any moving parts. Mixing times of ∼7 s, and kLa values of ∼170 h−1 were achieved. DO control was achieved by varying the duty cycle of a solenoid microvalve, which changed the gas mixture in the reactor incubator chamber. The microbioreactor supported Saccharomyces cerevisiae growth over 30 h and cell densities of 6.7 gdcw L−1. Oxygen uptake rates of ∼34 mmol L−1 h−1 were achieved. CONCLUSION: The results highlight the potential of DO-controlled microbioreactors to obtain real-time information on oxygen uptake rate, and by extension on cellular metabolism for a variety of cell types over a broad range of processing conditions. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry

Electromagnetic Actuated stirring in Microbioreactors enabling easier multiplexing & flexible device design

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.The development of a novel electromagnetically (EM) actuated stirring method, for use in microbioreactors, is reported. Mixing in microbioreactors is critical to ensure even distribution of nutrients to microorganisms and cells. Magnetically driven stirrer bars or peristaltic mixing are the most commonly utilised mixing methods employed in completely liquid-filled microbioreactors. However the circular reactor shape required for mixing with a stirrer bar and frequently used for peristaltically mixed microbioreactors presents difficulties for bubble-free priming in a microfluidic bioreactor. Moreover the circular shape and the hardware required for both types of mixing reduces the potential packing density of multiplexed reactors. We present a new method of mixing, displaying design flexibility by demonstrating mixing in circular and diamond-shaped reactors and a duplex diamond reactor and fermentation of the gram-positive bacteria S. carnosus in a diamond-shaped microbioreactor system. The results of the optimisation of this mixing method for performing fermentations alongside both batch and continuous culture fermentations are presented

Oxygen transfer characteristics of miniaturized bioreactor systems.

Since their introduction in 2001 miniaturized bioreactor systems have made great advances in function and performance. In this article the dissolved oxygen (DO) transfer performance of submilliliter microbioreactors, and 1-10 mL minibioreactors was examined. Microbioreactors have reached k(L) a values of 460 h(-1) , and are offering instrumentation and some functionality comparable to production systems, but at high throughput screening volumes. Minibioreactors, aside from one 1,440 h(-1) k(L) a system, have not offered as high rates of DO transfer, but have demonstrated superior integration with automated fluid handling systems. Microbioreactors have been typically limited to studies with E. coli, while minibioreactors have offered greater versatility in this regard. Further, mathematical relationships confirming the applicability of k(L) a measurements across all scales have been derived, and alternatives to fluorescence lifetime DO sensors have been evaluated. Finally, the influence on reactor performance of oxygen uptake rate (OUR), and the possibility of its real-time measurement have been explored

Conscious coupling: The challenges and opportunities of cascading enzymatic microreactors

The continuous production of high value or difficult to synthesize products is of increasing interest to the pharmaceutical industry. Cascading reaction systems have already been employed for chemical synthesis with great success, allowing a quick change in reaction conditions and addition of new reactants as well as removal of side products. A cascading system can remove the need for isolating unstable intermediates, increasing the yield of a synthetic pathway. Based on the success for chemical synthesis, the question arises how cascading systems could be beneficial to chemo-enzymatic or biocatalytic synthesis. Microreactors, with their rapid mass and heat transfer, small reaction volumes and short diffusion pathways, are promising tools for the development of such processes. In this mini-review, the authors provide an overview of recent examples of cascaded microreactors. Special attention will be paid to how microreactors are combined and the challenges as well as opportunities that arise from such combinations. Selected chemical reaction cascades will be used to illustrate this concept, before the discussion is widened to include chemo-enzymatic and multi-enzyme cascades. The authors also present the state of the art of online and at-line monitoring for enzymatic microreactor cascades. Finally, the authors review work-up and purification steps and their integration with microreactor cascades, highlighting the potential and the challenges of integrated cascades

Quantifying cell-generated forces: Poisson's ratio matters

Quantifying mechanical forces generated by cellular systems has led to key insights into a broad range of biological phenomena from cell adhesion to immune cell activation. Traction force microscopy (TFM), the most widely employed force measurement methodology, fundamentally relies on knowledge of the force-displacement relationship and mechanical properties of the substrate. Together with the elastic modulus, the Poisson’s ratio is a basic material property that to date has largely been overlooked in TFM. Here, we evaluate the sensitivity of TFM to Poisson’s ratio by employing a series of computer simulations and experimental data analysis. We demonstrate how applying the correct Poisson’s ratio is important for accurate force reconstruction and develop a framework for the determination of error levels resulting from the misestimation of the Poisson’s ratio. In addition, we provide experimental estimation of the Poisson’s ratios of elastic substrates commonly applied in TFM. Our work thus highlights the role of Poisson’s ratio underpinning cellular force quantification studied across many biological systems

Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies