Electromagnetic Actuated Stiring in Microbioreactor Enabling Easier Multiplexing and Flexible Device Design

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 presentedPeer reviewe

Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

Article in press. Kulsharova, G., New BIOTECHNOLOGY (2017), https://doi.org/10.1016/j.nbt.2017.12.004Poly(methyl methacrylate) (PMMA) microfluidic devices have become promising platforms for a wide range of applications. Here we report a simple method for immobilising histidine-tagged enzymes suitable for PMMA microfluidic devices. The 1-step-immobilisation described is based on the affinity of the His-tag/Ni-NTA interaction and does not require prior amination of the PMMA surface, unlike many existing protocols. We compared it with a 3-step immobilisation protocol involving amination of PMMA and linking NTA via a glutaraldehyde cross-linker. These methods were applied to immobilise transketolase (TK) in PMMA microfluidic devices. Binding efficiency studies showed that about 15% of the supplied TK was bound using the 1-step method and about 26% of the enzyme was bound by the 3-step method. However, the TK-catalysed reaction producing l-erythrulose performed in microfluidic devices showed that specific activity of TK in the device utilising the 1-step immobilisation method was approximately 30% higher than that of its counterpart. Reusability of the microfluidic device produced via the 1-step method was tested for three cycles of enzymatic reaction and at least 85% of the initial productivity was maintained. The device could be operated for up to 40 h in a continuous flow and on average 70% of the initial productivity was maintained. The simplified immobilisation method required fewer chemicals and less time for preparation of the immobilised microfluidic device compared to the 3-step method while achieving higher specific enzyme activity. The method represents a promising approach for the development of immobilised enzymatic microfluidic devices and could potentially be applied to combine protein purification with immobilisation.Peer reviewe

The tumour microenvironment modulates cancer cell intravasation

Development of three dimensional (3D) in vitro models to realistically recapitulate tumor microenvironment has the potential to improve translatability of anti-cancer drugs at the preclinical stage. To capture the in vivo complexity, these in vitro models should minimally incorporate the 3D interactions between multiple cell types, cellular structures such as vasculature and extracellular matrices. Here, we utilised microfluidic platforms to study the effect of various natural hydrogels (fibrin, collagen, Matrigel) and presence of tumor spheroids on the 3D vascularisation morphology. Various extracellular matrix (ECM) compositions impacted the vessel morphology while near the tumor spheroids the vessel diameter was considerably smaller for all different ECM compositions. Strikingly, cancer cells could enter the microvessel lumens (i.e. intravasate) only when the ECM was comprised of all the three types of hydrogels which increased the physical contact between the microvessels and the tumour spheroids. Our findings highlight the role of ECM composition in modulating the intravasation capacity of tumours

Stromal cells regulate mechanics of tumour spheroid

The remarkable contractility and force generation ability exhibited by cancer cells empower them to overcome the resistance and steric hindrance presented by a three-dimensional, interconnected matrix. Cancer cells disseminate by actively remodelling and deforming their extracellular matrix (ECM). The process of tumour growth and its ECM remodelling have been extensively studied, but the effect of the cellular tumour microenvironment (TME) has been ignored in most studies that investigated tumour-cell-mediated ECM deformations and realignment. This study reports the integration of stromal cells in spheroid contractility assays that impacts the ECM remodelling and invasion abilities of cancer spheroids. To investigate this, we developed a novel multilayer in vitro assay that incorporates stromal cells and quantifies the contractile deformations that tumour spheroids exert on the ECM. We observed a negative correlation between the spheroid invasion potential and the levels of collagen deformation. The presence of stromal cells significantly increased cancer cell invasiveness and altered the cancer cells' ability to deform and realign collagen gel, due to upregulation of proinflammatory cytokines. Interestingly, this was observed consistently in both metastatic and non-metastatic cancer cells. Our findings contribute to a better understanding of the vital role played by the cellular TME in regulating the invasive outgrowth of cancer cells and underscore the potential of utilising matrix deformation measurements as a biophysical marker for evaluating invasiveness and informing targeted therapeutic opportunities

Emergent mechanical control of vascular morphogenesis

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine

Emergent mechanical control of vascular morphogenesis

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine