Human leucocytes processed by fast-rate inertial microfluidics retain conventional functional characteristics.

The manufacturing of clinical cellular therapies is a complex process frequently requiring manipulation of cells, exchange of buffers and volume reduction. Current manufacturing processes rely on either low throughput open centrifugation-based devices, or expensive closed-process alternatives. Inertial focusing (IF) microfluidic devices offer the potential for high-throughput, inexpensive equipment which can be integrated into a closed system, but to date no IF devices have been approved for use in cell therapy manufacturing, and there is limited evidence for the effects that IF processing has on human cells. The IF device described in this study was designed to simultaneously separate leucocytes, perform buffer exchange and provide a volume reduction to the cell suspension, using high flow rates with high Reynolds numbers. The performance and effects of the IF device were characterized using peripheral blood mononuclear cells and isolated monocytes. Post-processing cell effects were investigated using multi-parameter flow cytometry to track cell viability, functional changes and fate. The IF device was highly efficient at separating CD14+ monocytes (approx. 97% to one outlet, approx. 60% buffer exchange, 15 ml min-1) and leucocyte processing was well tolerated with no significant differences in downstream viability, immunophenotype or metabolic activity when compared with leucocytes processed with conventional processing techniques. This detailed approach provides robust evidence that IF devices could offer significant benefits to clinical cell therapy manufacture.

Limitation of spiral microchannels for particle separation in heterogeneous mixtures: Impact of particles' size and deformability.

Spiral microchannels have shown promising results for separation applications. Hydrodynamic particle-particle interactions are a known factor strongly influencing focusing behaviors in inertial devices, with recent work highlighting how the performance of bidisperse mixtures is altered when compared with pure components in square channels. This phenomenon has not been previously investigated in detail for spiral channels. Here, we demonstrate that, in spiral channels, both the proportion and deformability of larger particles (13 µm diameter) impact upon the recovery (up to 47% decrease) of small rigid particles (4 µm). The effect, observed at low concentrations (volume fraction <0.0012), is attributed to the hydrodynamic capture of beads by larger cells. These changes in particles focusing behavior directly impede the efficiency of the separation-diverting beads from locations expected from measurements with pure populations to co-collection with larger cells-and could hamper deployment of technology for certain applications. Similar focusing behavior alterations were noted when working with purification of stem cell end products.

Off-Chip-Controlled Droplet-on-Demand Method for Precise Sample Handling.

We present a simple, stable, and highly reproducible off-chip-controlled method for generating droplets-on-demand. To induce the droplet generation, externally pre-programmed positive pressure pulses are applied to the dispersed phase input while the continuous phase channel remains at constant input pressure. By controlling solely one fluid phase, the method allows for connecting multiple independent dispersed-phase channels to a single continuous channel. Experimental results show that the method allows for a droplet generation frequency of 33 Hz and a high reproducibility of droplets with standard deviations less than 5% of the mean value. Moreover, utilization of the off-chip-controlled method results in the simplicity in chip design and allows rapid (∼5 min) and cost-efficient (0.5 USD) prototyping of the device.

Correction: Deformability-induced lift force in spiral microchannels for cell separation.

Correction for 'Deformability-induced lift force in spiral microchannels for cell separation' by Ewa Guzniczak et al., Lab Chip, 2020, 20, 614-625.

Purifying stem cell-derived red blood cells: a high-throughput label-free downstream processing strategy based on microfluidic spiral inertial separation and membrane filtration.

Cell-based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability-based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high-throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products.

Deformability-induced lift force in spiral microchannels for cell separation.

Cell sorting and isolation from a heterogeneous mixture is a crucial task in many aspects of cell biology, biotechnology and medicine. Recently, there has been an interest in methods allowing cell separation upon their intrinsic properties such as cell size and deformability, without the need for use of biochemical labels. Inertial focusing in spiral microchannels has been recognised as an attractive approach for high-throughput cell sorting for myriad point of care and clinical diagnostics. Particles of different sizes interact to a different degree with the fluid flow pattern generated within the spiral microchannel and that leads to particles ordering and separation based on size. However, the deformable nature of cells adds complexity to their ordering within the spiral channels. Herein, an additional force, deformability-induced lift force (FD), involved in the cell focusing mechanism within spiral microchannels has been identified, investigated and reported for the first time, using a cellular deformability model (where the deformability of cells is gradually altered using chemical treatments). Using this model, we demonstrated that spiral microchannels are capable of separating cells of the same size but different deformability properties, extending the capability of the previous method. We have developed a unique label-free approach for deformability-based purification through coupling the effect of FD with inertial focusing in spiral microchannels. This microfluidic-based purification strategy, free of the modifying immuno-labels, allowing cell processing at a large scale (millions of cells per min and mls of medium per minute), up to high purities and separation efficiency and without compromising cell quality.

Sensors for Fetal Hypoxia and Metabolic Acidosis: A Review.

This article reviews existing clinical practices and sensor research undertaken to monitor fetal well-being during labour. Current clinical practices that include fetal heart rate monitoring and fetal scalp blood sampling are shown to be either inadequate or time-consuming. Monitoring of lactate in blood is identified as a potential alternative for intrapartum fetal monitoring due to its ability to distinguish between different types of acidosis. A literature review from a medical and technical perspective is presented to identify the current advancements in the field of lactate sensors for this application. It is concluded that a less invasive and a more continuous monitoring device is required to fulfill the clinical needs of intrapartum fetal monitoring. Potential specifications for such a system are also presented in this paper.

Impact of poloxamer 188 (Pluronic F-68) additive on cell mechanical properties, quantification by real-time deformability cytometry.

Advances in cellular therapies have led to the development of new approaches for cell product purification and formulation, e.g., utilizing cell endogenous properties such as size and deformability as a basis for separation from potentially harmful undesirable by-products. However, commonly used additives such as Pluronic F-68 and other poloxamer macromolecules can change the mechanical properties of cells and consequently alter their processing. In this paper, we quantified the short-term effect of Pluronic F-68 on the mechanotype of three different cell types (Jurkat cells, red blood cells, and human embryonic kidney cells) using real-time deformability cytometry. The impact of the additive concentration was assessed in terms of cell size and deformability. We observed that cells respond progressively to the presence of Pluronic F-68 within first 3 h of incubation and become significantly stiffer (p-value < 0.001) in comparison to a serum-free control and a control containing serum. We also observed that the short-term response manifested as cell stiffening is true (p-value < 0.001) for the concentration reaching 1% (w/v) of the poloxamer additive in tested buffers. Additionally, using flow cytometry, we assessed that changes in cell deformability triggered by addition of Pluronic F-68 are not accompanied by size or viability alterations.

High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing.

Stem cell products, including manufactured red blood cells, require efficient sorting and purification methods to remove components potentially harmful for clinical application. However, standard approaches for cellular downstream processing rely on the use of specific and expensive labels (e.g. FACS or MACS). Techniques relying on inherent mechanical and physical properties of cells offer high-throughput scalable alternatives but knowledge of the mechanical phenotype is required. Here, we characterized for the first time deformability and size changes in CD34+ cells, and expelled nuclei, during their differentiation process into red blood cells at days 11, 14, 18 and 21, using Real-Time Deformability Cytometry (RT-DC) and Atomic Force Microscopy (AFM). We found significant differences (p < 0.0001; standardised mixed model) between the deformability of nucleated and enucleated cells, while they remain within the same size range. Expelled nuclei are smaller thus could be removed by size-based separation. An average Young's elastic modulus was measured for nucleated cells, enucleated cells and nuclei (day 14) of 1.04 ± 0.47 kPa, 0.53 ± 0.12 kPa and 7.06 ± 4.07 kPa respectively. Our identification and quantification of significant differences (p < 0.0001; ANOVA) in CD34+ cells mechanical properties throughout the differentiation process could enable development of new routes for purification of manufactured red blood cells.

Multiplexed single-mode wavelength-to-time mapping of multimode light.

When an optical pulse propagates along an optical fibre, different wavelengths travel at different group velocities. As a result, wavelength information is converted into arrival-time information, a process known as wavelength-to-time mapping. This phenomenon is most cleanly observed using a single-mode fibre transmission line, where spatial mode dispersion is not present, but the use of such fibres restricts possible applications. Here we demonstrate that photonic lanterns based on tapered single-mode multicore fibres provide an efficient way to couple multimode light to an array of single-photon avalanche detectors, each of which has its own time-to-digital converter for time-correlated single-photon counting. Exploiting this capability, we demonstrate the multiplexed single-mode wavelength-to-time mapping of multimode light using a multicore fibre photonic lantern with 121 single-mode cores, coupled to 121 detectors on a 32 × 32 detector array. This work paves the way to efficient multimode wavelength-to-time mapping systems with the spectral performance of single-mode systems.

Antimicrobial efficacy and safety of mucoadhesive exopolymer produced by Acinetobacter haemolyticus.

This study evaluated five extracellular polymers of bacterial origin possessing mucoadhesive properties for their antimicrobial properties and toxicological characteristics. Of the five tested mucoadhesive biopolymers, the extracellular polymer produced by a strain of Acinetobacter haemolyticus exhibited broad antimicrobial efficacy towards Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7 and Bacillus subtilis. Significant (p<0.05) inhibition of gram negative bacterial pathogens followed by gram positives were observed with the biopolymer at a dose of 40-60µg ml-1 at ambient temperature. The cytotoxicity under in vitro conditions and oral toxicity in murine models was also evaluated. The biopolymer did not elicit either haemolytic activity or toxicity in RAW 264.7 cell lines. Haemotological, histopathological and general examinations indicated no adverse effects in Swiss albino mice fed with the biopolymer (120mg kg-1 body weight-1 day1) over a period of 30 days. These results suggested that the biopolymer was well tolerated without any signs of toxicity and may have several potential biomedical applications where disinfection is desired.

Solid-phase microextraction to determine micropollutant-macromolecule partition coefficients.

Aqueous micropollutants such as estradiol can have a large environmental impact-even at low concentrations. Part of understanding this impact involves determining the extent to which the micropollutants interact with macromolecules in water. In environmental samples, relevant macromolecules to which micropollutants bind are referred to as dissolved organic matter, and the most common examples of these in freshwater and coastal seawater are fulvic and humic acids. In living organisms, the most common macromolecules that affect bioavailability of a drug (or toxin) are proteins such as albumin. Using [2, 4, 6, 7 - (3)H]estradiol as an example compound, this protocol uses solid-phase microextraction and scintillation detection as analytical tools to quantify the amount of radiolabeled micropollutant available in solution. The measured free concentration after exposure to various concentrations of macromolecule (dissolved organic matter or protein) or micropollutant is used to determine the partition coefficient in the case of micropollutant-macromolecule interactions. The calibration and preparatory studies take at least 8 d, and the steps to determine the partition coefficient can be completed within 3 d. The protocol could be modified such that nonlabeled compounds are studied; instead of detection of activity by a liquid scintillation counter (LSC), the compounds can be quantified using gas chromatography-mass spectrometry (GC-MS) or liquid chromatography (LC)-MS(/MS).

Deformability Assessment of Waterborne Protozoa Using a Microfluidic-Enabled Force Microscopy Probe.

Many modern filtration technologies are incapable of the complete removal of Cryptosporidium oocysts from drinking-water. Consequently, Cryptosporidium-contaminated drinking-water supplies can severely implicate both water utilities and consumers. Existing methods for the detection of Cryptosporidium in drinking-water do not discern between non-pathogenic and pathogenic species, nor between viable and non-viable oocysts. Using FluidFM, a novel force spectroscopy method employing microchannelled cantilevers for single-cell level manipulation, we assessed the size and deformability properties of two species of Cryptosporidium that pose varying levels of risk to human health. A comparison of such characteristics demonstrated the ability of FluidFM to discern between Cryptosporidium muris and Cryptosporidium parvum with 86% efficiency, whilst using a measurement throughput which exceeded 50 discrete oocysts per hour. In addition, we measured the deformability properties for untreated and temperature-inactivated oocysts of the highly infective, human pathogenic C. parvum to assess whether deformability may be a marker of viability. Our results indicate that untreated and temperature-inactivated C. parvum oocysts had overlapping but significantly different deformability distributions.

Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts.

Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 µg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 µg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 µg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall.

Enhancing Cryptosporidium parvum recovery rates for improved water monitoring.

Water monitoring is essential to ensure safe drinking water for consumers. However existing methods have several drawbacks, particularly with regard to the poor recovery of Cryptosporidium due to the inability to efficiently elute Cryptosporidium oocysts during the established detection process used by water utilities. Thus the development of new inexpensive materials that could be incorporated into the concentration and release stage that would control Cryptosporidium oocysts adhesion would be beneficial. Here we describe improved filter performance following dip-coating of the filters with a "bioactive" polyacrylate. Specifically 69% more oocysts were eluted from the filter which had been coated with a polymer than on the naked filter alone.

Megasonic sonication for cost-effective and automatable elution of Cryptosporidium from filters and membranes.

Sample processing is a highly challenging stage in the monitoring of waterborne pathogens. This step is time-consuming, requires highly trained technicians and often results in low recovery rates of pathogens. In the UK but also in other parts of the world, Cryptosporidium is the only pathogen directly tested for in routine operational monitoring. The traditional sampling process involves the filtration of 1000L of water, semi-automated elution of the filters and membranes with recovery rates of about 30-40% typically. This paper explores the use of megasonic sonication in an attempt to increase recovery rates and reduce both the time required for processing and the number of labour-intensive steps. Results demonstrate that megasonic energy assisted elution is equally effective as the traditional manual process in terms of recovery rates. Major advantages are however offered in terms of reduction of the elution volume enabling the current centrifugation stage to be avoided. This saves time, equipment and staff costs and critically removes the step in the process that would be most challenging to automate, paving the way thereby for highly effective automated solutions to pathogens monitoring.

Exploitation of Nanotechnology for the Monitoring of Waterborne Pathogens: State-of-the-Art and Future Research Priorities.

Contaminated drinking water is one of the most important environmental contributors to the human disease burden. Monitoring of water for the presence of pathogens is an essential part of ensuring drinking water safety. In order to assess water quality it is essential to have methods available to sample and detect the type, level and viability of pathogens in water which are effective, cheap, quick, sensitive, and where possible high throughput. Nanotechnology has the potential to drastically improve the monitoring of waterborne pathogens when compared to conventional approaches. To date, there have been no reviews that outline the applications of nanotechnology in this area despite increasing exploitation of nanotechnology for this purpose. This review is therefore the first overview of the state-of-the-art in the application of nanotechnology to waterborne pathogen sampling and detection schemes. Research in this field has been centered on the use of engineered nanomaterials. The effectiveness and limitations of nanomaterial-based approaches is outlined. A future outlook of the advances that are likely to emerge in this area, as well as recommendations for areas of further research are provided.

Impact of microfluidic processing on bacterial ribonucleic acid expression.

Bacterial transcriptomics is widely used to investigate gene regulation, bacterial susceptibility to antibiotics, host-pathogen interactions, and pathogenesis. Transcriptomics is crucially dependent on suitable methods to isolate and detect bacterial RNA. Microfluidics offer ways of creating integrated point-of-care systems, analysing a sample from preparation, and RNA isolation to detection. A critical requirement for on-chip diagnostics to deliver on their promise is that mRNA expression is not altered via microfluidic sample processing. This article investigates the impact of the use of microfluidics upon RNA expression of bacteria isolated from blood, a key step towards proving the suitability of such systems for further development.

Angry pathogens, how to get rid of them: introducing microfluidics for waterborne pathogen separation to children.

The purpose of this paper is to present a new approach for introducing to a non-scientific audience a major public health issue: access to safe drinking water. Access to safe drinking water is a privilege in developed countries and an urgent need in the third world, which implies always more efficient and reliable engineering tools to be developed. As a major global challenge it is important to make children aware of this problem for understanding (i) what safe drinking water is, (ii) how ingenious techniques are developed for this purpose and (iii) the role of microfluidics in this area. This paper focuses on different microfluidic-based techniques to separate and detect pathogens in drinking water that have been adapted to be performed by a young audience in a simplified, recreational and interactive way.

Application of microfluidics in waterborne pathogen monitoring: a review.

A review of the recent advances in microfluidics based systems for the monitoring of waterborne pathogens is provided in this article. Emphasis has been made on existing, commercial and state-of-the-art systems and research activities in laboratories worldwide. The review separates sample processing systems and monitoring systems, highlighting the slow progress made in automated sample processing for monitoring of pathogens in waterworks and in the field. Future potential directions of research are also highlighted in the conclusions.