Nicholas Willoughby

Spectral conversion of light for enhanced microalgae growth rates and photosynthetic pigment production.

The effect of light conditions on the growth of green algae Chlorella vulgaris and cyanobacteria Gloeothece membranacea was investigated by filtering different wavelengths of visible light and comparing against a model daylight source as a control. Luminescent acrylic sheets containing violet, green, orange or red dyes illuminated by a solar simulator produced the desired wavelengths of light for this study. From the experimental results the highest specific growth rate for C. vulgaris was achieved using the orange range whereas violet light promoted the growth of G. membranacea. Red light exhibited the least efficiency in conversion of light energy into biomass in both strains of microalgae. Photosynthetic pigment formation was examined and maximum chlorophyll-a production in C. vulgaris was obtained by red light illumination. Green light yielded the best chlorophyll-a production in G. membranacea. The proposed illumination strategy offers improved microalgae growth without resorting to artificial light sources, reducing energy use and costs of cultivation.

A 3D mammalian cell separator biochip

The dissimilar cytoskeletal architecture in diverse cell types induces a difference in their deformability that presents a viable approach to separate cells in a non-invasive manner. We report on the design and fabrication of a robust and scalable device capable of separating a heterogeneous population of cells with variable degree of deformability into enriched populations with deformability above a certain threshold. The three dimensional device was fabricated in fused silica by femtosecond laser direct writing combined with selective chemical etching. The separator device was evaluated using promyelocytic HL60 cells. Using flow rates as large as 167 μL min(-1), throughputs of up to 2800 cells min(-1) were achieved at the device output. A fluorescence-activated cell sorting (FACS) viability analysis on the cells revealed 81% of the population maintain cellular integrity after passage through the device.

Adapted Ultra Scale-Down Approach for Predicting the Centrifugal Separation Behavior of High Cell Density Cultures

The work presented here describes an ultra scale‐down (USD) methodology for predicting centrifugal clarification performance in the case of high cell density fermentation broths. Existing USD approaches generated for dilute systems led to a 5– to 10‐fold overprediction of clarification performance when applied to such high cell density feeds. This is due to increased interparticle forces, leading to effects such as aggregation, flocculation, or even blanket sedimentation, occurring in the low shear environment of a laboratory centrifuge, which will not be apparent in the settling region of a continuous‐flow industrial centrifuge. A USD methodology was created based upon the dilution of high solids feed material to ∼2% wet wt/vol prior to the application of the clarification test. At this level of dilution cell‐cell interactions are minimal. The dilution alters the level of hindered settling in the feed suspensions, and so mathematical corrections are applied to the resultant clarification curves to mimic the original feed accurately. The methodology was successfully verified: corrected USD curves accurately predicted pilot‐scale clarification performance of high cell density broths of Saccharomyces cerevisiae and Escherichia coli cells. The USD method allows for the rapid prediction of large‐scale clarification of high solids density material using millilitre quantities of feed. The advantages of this method to the biochemical engineer, such as the enabling of rapid process design and scale‐up, are discussed.

Luminescent photobioreactor design for improved algal growth and photosynthetic pigment production through spectral conversion of light

Growth characteristics of two strains of microalgae in bubble column photobioreactors were investigated under different cultivation conditions. Chlorella vulgaris and Gloeothece membranacea were cultivated in luminescent acrylic photobioreactors at different seed culture densities. Luminescent acrylic photobioreactors in blue, green, yellow, orange, and red colours capable of spectral conversion of light were used. The results indicated that the red luminescent photobioreactor enhanced biomass production in both strains of microalgae while pigmentation was induced under different light colours. Green light promoted chlorophyll production in C. vulgaris however chlorophyll production in G. membranacea cultures was less influenced by the light condition or culture density. Phycobiliproteins were the dominant pigments in G. membranacea and red light favoured synthesis of these pigments.

Experimental measurement of particle size distribution and voidage in an expanded bed adsorption system

This paper presents an experimental analysis of matrix bead size distribution and voidage variations with axial height in an expanded bed adsorption system. Use of a specially constructed expanded bed with side ports has enabled sampling from within the expanded bed along the vertical axis. Particles removed from within the bed were measured for their size distributions. Residence time distribution studies were used to estimate bed voidage. Measurements of axial and radial particle size distributions and axial voidage distribution have been made at different flow rates. Particle size was found to be radially constant, indicating constant stratification in the column. The particle size was found to decrease with increasing axial height. Voidage increased with axial height from a settled bed value of 0.39 to approaching unity for high liquid velocities and increased at a constant axial position with increased flowrate. This information provides key insight into bed stability and data for the improved modeling of this important unit operation.

Large‐scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA

The effect of bacterial‐cell centrifugation and handling on the initial stages of plasmid processing was investigated. Escherichia coli cells containing either a 6 or 20 kb plasmid were grown in 75‐ and 450‐litre bioreactors, and the process yield of the early recovery stages was characterized in terms of SC pDNA (supercoiled plasmid DNA) recovered. In all cases, the cells were totally recovered using either a continuous‐feed, intermittent‐solids‐discharge, disc‐stack centrifuge or a continuous‐feed, batch‐discharge, solid‐bowl centrifuge. The cells were then either processed immediately or stored frozen. The centrifugation method considerably affected the yield of SC pDNA, and there was evidence that the intermittent discharge of cells from a centrifuge operating at high speed led to a sediment containing lysed cells and degraded pDNA. This led to estimated plasmid yield losses of up to 40% as compared with cells recovered from laboratory or solid‐bowl centrifuges, where there is evidently no cell stress on discharge. By inference, the cell stress on feed to either of the continuous centrifuges studied was not implicated in product loss. Freezing of the recovered cells gives a convenient hold stage prior to further processing. In all cases, this extra freeze–thaw stage led to loss of SC pDNA, and this was in addition to the loss attributed to cell lysis during centrifugation discharge. Only average yields can be gained from pilot plant‐scale studies; separate laboratory‐based experiments indicated that this loss of SC pDNA is determined by the time and temperature for which the resuspended cells are held.

Elasticity of human embryonic stem cells as determined by atomic force microscopy

The expansive growth and differentiation potential of human embryonic stem cells (hESCs) make them a promising source of cells for regenerative medicine. However, this promise is off set by the propensity for spontaneous or uncontrolled differentiation to result in heterogeneous cell populations. Cell elasticity has recently been shown to characterize particular cell phenotypes, with undifferentiated and differentiated cells sometimes showing significant differences in their elasticities. In this study, we determined the Youngs modulus of hESCs by atomic force microscopy using a pyramidal tip. Using this method we are able to take point measurements of elasticity at multiple locations on a single cell, allowing local variations due to cell structure to be identified. We found considerable differences in the elasticity of the analyzed hESCs, reflected by a broad range of Youngs modulus (0.05-10 kPa). This surprisingly high variation suggests that elasticity could serve as the basis of a simple and efficient large scale purification/separation technique to discriminate subpopulations of hESCs.

A scalable label-free approach to separate human pluripotent cells from differentiated derivatives

The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the devices utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells.

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.

PASSIVE COMPONENTS AND FIBER-BASED DEVICES VIII

Recent advances in the field of ultrafast laser inscription provide ample evidence underscoring the potential of this technique in fabricating novel and previously unthinkable 2D and 3D photonic and optofluidic platforms enabling current sensor, diagnostics, monitoring and biochemical research to scale new heights. In addition to meeting the demands for compact, active waveguide devices designed for diverse applications such as optical metrology, non-linear microscopy and astrophotonics, this technology facilitates the integration of microfluidics with integrated optics which creates a powerful technology for the manufacture of custom lab-on-chip devices with advanced functionality. This paper highlights the capabilities of ultrafast laser inscription in fabricating novel 3D microfluidic devices aimed for biomedical applications.