Influence of surface geometry on the culture of human cell lines: a comparative study using flat, round-bottom and v-shaped 96 well plates

© 2017 Shafaie et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.In vitro cell based models have been invaluable tools for studying cell behaviour and for investigating drug disposition, toxicity and potential adverse effects of administered drugs. Within this drug discovery pipeline, the ability to assess and prioritise candidate compounds as soon as possible offers a distinct advantage. However, the ability to apply this approach to a cell culture study is limited by the need to provide an accurate, in vitro-like, microenvironment in conjunction with a low cost and high-throughput screening (HTS) methodology. Although the geometry and/or alignment of cells has been reported to have a profound influence on cell growth and differentiation, only a handful of studies have directly compared the growth of a single cell line on different shaped multiwell plates the most commonly used substrate for HTS, in vitro, studies. Herein, the impact of various surface geometries (flat, round and v-shaped 96 well plates), as well as fixed volume growth media and fixed growth surface area have been investigated on the characteristics of three commonly used human cell lines in biopharmaceutical research and development, namely ARPE-19 (retinal epithelial), A549 (alveolar epithelial) and Malme-3M (dermal fibroblastic) cells. The effect of the surface curvature on cells was characterised using a combination of a metabolic activity assay (CellTiter AQ/MTS), LDH release profiles (CytoTox ONE) and absolute cell counts (Guava ViaCount), respectively. In addition, cell differentiation and expression of specific marker proteins were determined using flow cytometry. These in vitro results confirmed that surface topography had a significant effect (p < 0.05) on cell activity and morphology. However, although specific marker proteins were expressed on day 1 and 5 of the experiment, no significant differences were seen between the different plate geometries (p < 0.05) at the later time point. Accordingly, these results highlight the impact of substrate geometry on the culture of a cell line and the influence it has on the cells' correct growth and differentiation characteristics. As such, these results provide important implications in many aspects of cell biology the development of a HTS, in vitro, cell based systems to further investigate different aspects of toxicity testing and drug delivery.Peer reviewedFinal Published versio

Living Bacterial Sacrificial Porogens to Engineer Decellularized Porous Scaffolds

Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D) hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types

New Detection Systems of Bacteria Using Highly Selective Media Designed by SMART: Selective Medium-Design Algorithm Restricted by Two Constraints

Culturing is an indispensable technique in microbiological research, and culturing with selective media has played a crucial role in the detection of pathogenic microorganisms and the isolation of commercially useful microorganisms from environmental samples. Although numerous selective media have been developed in empirical studies, unintended microorganisms often grow on such media probably due to the enormous numbers of microorganisms in the environment. Here, we present a novel strategy for designing highly selective media based on two selective agents, a carbon source and antimicrobials. We named our strategy SMART for highly Selective Medium-design Algorithm Restricted by Two constraints. To test whether the SMART method is applicable to a wide range of microorganisms, we developed selective media for Burkholderia glumae, Acidovorax avenae, Pectobacterium carotovorum, Ralstonia solanacearum, and Xanthomonas campestris. The series of media developed by SMART specifically allowed growth of the targeted bacteria. Because these selective media exhibited high specificity for growth of the target bacteria compared to established selective media, we applied three notable detection technologies: paper-based, flow cytometry-based, and color change-based detection systems for target bacteria species. SMART facilitates not only the development of novel techniques for detecting specific bacteria, but also our understanding of the ecology and epidemiology of the targeted bacteria

Novel phenyl(thio)ureas bearing (thio)oxothiazoline group as potential BACE-1 inhibitors: synthesis and biological evaluation

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