Oxygen-permeable microwell device maintains islet mass and integrity during shipping

Islet transplantation is currently the only minimally invasive therapy available for patients with type 1 diabetes that can lead to insulin independence; however, it is limited to only a small number of patients. Although clinical procedures have improved in the isolation and culture of islets, a large number of islets are still lost in the pre-transplant period, limiting the success of this treatment. Moreover, current practice includes islets being prepared at specialized centers, which are sometimes remote to the transplant location. Thus, a critical point of intervention to maintain the quality and quantity of isolated islets is during transportation between isolation centers and the transplanting hospitals, during which 20-40% of functional islets can be lost. The current study investigated the use of an oxygen-permeable PDMS microwell device for long-distance transportation of isolated islets. We demonstrate that the microwell device protected islets from aggregation during transport, maintaining viability and average islet size during shipping.Darling M Rojas-Canales, Michaela Waibel, Aurelien Forget, Daniella Penko, Jodie Nitschke, Fran J Harding, Bahman Delalat, Anton Blencowe, Thomas Loudovaris, Shane T Grey, Helen E Thomas, Thomas W H Kay, Chris J Drogemuller, Nicolas H Voelcker, and Patrick T Coate

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

Background: CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas. Results: A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate. Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species

Exploring the mesenchymal stem cell niche using high throughput screening

In the field of stem cell technology, future advancements rely on the effective isolation, scale-up and maintenance of specific stem cell populations and robust procedures for their directed differentiation. The stem cell microenvironment - or niche - encompasses signal inputs from stem cells, supporting cells and from the extracellular matrix. In this context, the contribution of physicochemical surface variables is being increasingly recognised. This paradigm can be exploited to exert control over cellular behaviour. However, the number of parameters at play, and their complex interactions, presents a formidable challenge in delineating how the decisions of cell fate are orchestrated within the niche. Additionally, in the case of mesenchymal stem cells (MSC), more than one type of stem cell niche has been identified. By employing high throughput screening (HTS) strategies, common and specific attributes of each MSC niche can be probed. Here, we explore biological, chemical and physical parameters that are known to influence MSC self-renewal and differentiation. We then review techniques and strategies that allow the HTS of surface properties for conditions that direct stem cell fate, using MSC as a case study. Finally, challenges in recapturing the niche, particularly its three dimensional nature, in surface-based HTS formats are discussed.Soraya Rasi Ghaemi, Frances J. Harding, Bahman Delalat, Stan Gronthos, Nicolas H. Voelcke

Fabrication of silicon nanowire arrays by near-field laser ablation and metal-assisted chemical etching

We present an elegant route for the fabrication of ordered arrays of vertically-aligned silicon nanowires with tunable geometry at controlled locations on a silicon wafer. A monolayer of transparent microspheres convectively assembled onto a gold-coated silicon wafer acts as a microlens array. Irradiation with a single nanosecond laser pulse removes the gold beneath each focusing microsphere, leaving behind a hexagonal pattern of holes in the gold layer. Owing to the near-field effects, the diameter of the holes can be at least five times smaller than the laser wavelength. The patterned gold layer is used as catalyst in a metal-assisted chemical etching to produce an array of vertically-aligned silicon nanowires. This approach combines the advantages of direct laser writing with the benefits of parallel laser processing, yielding nanowire arrays with controlled geometry at predefined locations on the silicon surface. The fabricated VA-SiNW arrays can effectively transfect human cells with a plasmid encoding for green fluorescent protein

Nanotopography mediated osteogenic differentiation of human dental pulp derived stem cells

Advanced medical devices, treatments and therapies demand an understanding of the role of interfacial properties on the cellular response. This is particularly important in the emerging fields of cell therapies and tissue regeneration. In this study, we evaluate the role of surface nanotopography on the fate of human dental pulp derived stem cells (hDPSC). These stem cells have attracted interest because of their capacity to differentiate to a range of useful lineages but are relatively easy to isolate. We generated and utilized density gradients of gold nanoparticles which allowed us to examine, on a single substrate, the influence of nanofeature density and size on stem cell behavior. We found that hDPSC adhered in greater numbers and proliferated faster on the sections of the gradients with higher density of nanotopography features. Furthermore, greater surface nanotopography density directed the differentiation of hDPSC to osteogenic lineages. This study demonstrates that carefully tuned surface nanotopography can be used to manipulate and guide the proliferation and differentiation of these cells. The outcomes of this study can be important in the rational design of culture substrates and vehicles for cell therapies, tissue engineering constructs and the next generation of biomedical devices where control over the growth of different tissues is required.Akash Bachhuka, Bahman Delalat, Soraya Rasi Ghaemi, Stan Gronthos, Nicolas H. Voelcker and Krasimir Vasile

Synergistic influence of collagen I and BMP 2 drives osteogenic differentiation of mesenchymal stem cells: a cell microarray analysis

Abstract not availableSoraya Rasi Ghaemi, Bahman Delalat, Xavier Cetó, Frances J. Harding, Jonathan Tuke, Nicolas H. Voelcke

Antibacterial properties of silver dendrite decorated silicon nanowires

Silicon nanowires fabricated through Ag-assisted chemical etching were found to be effective bacterial-traps with strong antibacterial properties resulting from Ag-nanoclusters.</p

"Thunderstruck": plasma-polymer-coated porous silicon microparticles ss a controlled drug delivery system

Published: February 2, 2016Controlling the release kinetics from a drug carrier is crucial to maintain a drug’s therapeutic window. We report the use of biodegradable porous silicon microparticles (pSi MPs) loaded with the anticancer drug camphothecin, followed by a plasma polymer overcoating using a loudspeaker plasma reactor. Homogenous “Teflon-like” coatings were achieved by tumbling the particles by playing AC/DC’s song “Thunderstruck”. The overcoating resulted in a markedly slower release of the cytotoxic drug, and this effect correlated positively with the plasma polymer coating times, ranging from 2-fold up to more than 100-fold. Ultimately, upon characterizing and verifying pSi MP production, loading, and coating with analytical methods such as time-of-flight secondary ion mass spectrometry, scanning electron microscopy, thermal gravimetry, water contact angle measurements, and fluorescence microscopy, human neuroblastoma cells were challenged with pSi MPs in an in vitro assay, revealing a significant time delay in cell death onset.Steven J. P. McInnes, Thomas D. Michl, Bahman Delalat, Sameer A. Al-Bataineh, Bryan R. Coad, Krasimir Vasilev, Hans J. Griesser, and Nicolas H. Voelcke

High-throughput assessment and modeling of a polymer library regulating human dental pulp-derived stem cell behavior

The identification of biomaterials that modulate cell responses is a crucial task for tissue engineering and cell therapy. The identification of novel materials is complicated by the immense number of synthesizable polymers and the time required for testing each material experimentally. In the current study, polymeric biomaterial-cell interactions were assessed rapidly using a microarray format. The attachment, proliferation, and differentiation of human dental pulp stem cells (hDPSCs) were investigated on 141 homopolymers and 400 diverse copolymers. The copolymer of isooctyl acrylate and 2-(methacryloyloxy)ethyl acetoacetate achieved the highest attachment and proliferation of hDPSC, whereas high cell attachment and differentiation of hDPSC were observed on the copolymer of isooctyl acrylate and trimethylolpropane ethoxylate triacrylate. Computational models were generated, relating polymer properties to cellular responses. These models could accurately predict cell behavior for up to 95% of materials within a test set. The models identified several functional groups as being important for supporting specific cell responses. In particular, oxygen-containing chemical moieties, including fragments from the acrylate/acrylamide backbone of the polymers, promoted cell attachment. Small hydrocarbon fragments originating from polymer pendant groups promoted cell proliferation and differentiation. These computational models constitute a key tool to direct the discovery of novel materials within the enormous chemical space available to researchers.Soraya Rasi Ghaemi, Bahman Delalat, Stan Gronthos, Morgan R. Alexander, David A. Winkler, Andrew L. Hook, and Nicolas H. Voelcke

Ordered Silicon Pillar Arrays Prepared by Electrochemical Micromachining: Substrates for High-Efficiency Cell Transfection

Ordered arrays of silicon nano- to microscale pillars are used to enable biomolecular trafficking into primary human cells, consistently demonstrating high transfection efficiency can be achieved with broader and taller pillars than reported to date. Cell morphology on the pillar arrays is often strikingly elongated. Investigation of the cellular interaction with the pillar reveals that cells are suspended on pillar tips and do not interact with the substrate between the pillars. Although cells remain suspended on pillar tips, acute local deformation of the cell membrane was noted, allowing pillar tips to penetrate the cell interior, while retaining cell viability.Frances J. Harding, Salvatore Surdo, Bahman Delalat, Chiara Cozzi, Roey Elnathan, Stan Gronthos, Nicolas H. Voelcker and Giuseppe Barillar