Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money

In vitro co-culture model of medulloblastoma and human neural stem cells for drug delivery assessment

Physiologically relevant in vitro models can serve as biological analytical platforms for testing novel treatments and drug delivery systems. We describe the first steps in the development of a 3D human brain tumour co-culture model that includes the interplay between normal and tumour tissue along with nutrient gradients, cell-cell and cell-matrix interactions. The human medulloblastoma cell line UW228-3 and human foetal brain tissue were marked with two supravital fluorescent dyes (CDCFDASE, Celltrace Violet) and cultured together in ultra-low attachment 96-well plates to form reproducible single co-culture spheroids (d = 600 μm, CV% = 10%). Spheroids were treated with model cytotoxic drug etoposide (0.3–100 μM) and the viability of normal and tumour tissue quantified separately using flow cytometry and multiphoton microscopy. Etoposide levels of 10 μM were found to maximise toxicity to tumours (6.5% viability) while stem cells maintained a surviving fraction of 40%. The flexible cell marking procedure and high-throughput compatible protocol make this platform highly transferable to other cell types, primary tissues and personalised screening programs. The model's key anticipated use is for screening and assessment of drug delivery strategies to target brain tumours, and is ready for further developments, e.g. differentiation of stem cells to a range of cell types and more extensive biological validation

Characterization and petrologic interpretation of olivine-rich basalts at Gusev Crater, Mars

Rocks on the floor of Gusev crater are basalts of uniform composition and mineralogy. Olivine, the only mineral to have been identified or inferred from data by all instruments on the Spirit rover, is especially abundant in these rocks. These picritic basalts are similar in many respects to certain Martian meteorites (olivine-phyric shergottites). The olivine megacrysts in both have intermediate compositions, with modal abundances ranging up to 20–30%. Associated minerals in both include low-calcium and highcalcium pyroxenes, plagioclase of intermediate composition, iron-titanium-chromium oxides, and phosphate. These rocks also share minor element trends, reflected in their nickel-magnesium and chromium-magnesium ratios. Gusev basalts and shergottites appear to have formed from primitive magmas produced by melting an undepleted mantle at depth and erupted without significant fractionation. However, apparent differences between Gusev rocks and shergottites in their ages, plagioclase abundances, and volatile contents preclude direct correlation. Orbital determinations of global olivine distribution and compositions by thermal emission spectroscopy suggest that olivine-rich rocks may be widespread. Because weathering under acidic conditions preferentially attacks olivine and disguises such rocks beneath alteration rinds, picritic basalts formed from primitive magmas may even be a common component of the Martian crust formed during ancient and recent times.Additional co-authors: PR Christensen, BC Clark, JA Crisp, DJ DesMarais, T Economou, JD Farmer, W Farrand, A Ghosh, M Golombek, S Gorevan, R Greeley, VE Hamilton, JR Johnson, BL Joliff, G Klingelhöfer, AT Knudson, S McLennan, D Ming, JE Moersch, R Rieder, SW Ruff, PA de Souza Jr, SW Squyres, H Wnke, A Wang, A Yen, J Zipfe

Control of aggregation temperatures in mixed and blended cytocompatible thermoresponsive block co-polymer nanoparticles

A small library of thermoresponsive amphiphilic copolymers based on polylactide-block-poly((2-(2-methoxyethoxy)ethyl methacrylate)-co-(oligoethylene glycol methacrylate)) (PLA-b-P(DEGMA)-co-(OEGMA)), was synthesised by copper-mediated controlled radical polymerisation (CRP) with increasing ratios of OEGMA:DEGMA. These polymers were combined in two ways to form nanoparticles with controllable thermal transition temperatures as measured by particle aggregation. The first technique involved the blending of two (PLA-b-P(DEGMA)-co-(OEGMA)) polymers together prior to assembling NPs. The second method involved mixing pre-formed nanoparticles of single (PLA-b-P(DEGMA)-co-(OEGMA)) polymers. The observed critical aggregation temperature Tt did not change in a linear relationship with the ratios of each copolymer either in the nanoparticles blended from different copolymers or in the mitures of pre-formed nanoparticles. However, where co-polymer mixtures were based on (OEG)9MA ratios within 5-10 mole% , a linear relationship between (OEG)9MA composition in the blends and Tt was obtained. The data suggest that OEGMA-based copolymers are tunable over a wide temperature range given suitable co-monomer content in the linear polymers or nanoparticles. Moreover, the thermal transitions of the nanoparticles were reversible and repeatable, with the cloud point curves being essentially invariant across at least three heating and cooling cycles, and a selected nanoparticle formulation was found to be readily endocytosed in representative cancer cells and fibroblasts

3D inkjet printing of tablets exploiting bespoke complex geometries for controlled and tuneable drug release

A hot melt 3D inkjet printing method with the potential to manufacture formulations in complex and adaptable geometries for the controlled loading and release of medicines is presented. This first use of a precisely controlled solvent free inkjet printing to produce drug loaded solid dosage forms is demonstrated using a naturally derived FDA approved material (beeswax) as the drug carrier and fenofibrate as the drug. Tablets with bespoke geometries (honeycomb architecture) were fabricated. The honeycomb architecture was modified by control of the honeycomb cell size, and hence surface area to enable control of drug release profiles without the need to alter the formulation. Analysis of the formed tablets showed the drug to be evenly distributed within the beeswax at the bulk scale with evidence of some localization at the micron scale. An analytical model utilizing a Fickian description of diffusion was developed to allow the prediction of drug release. A comparison of experimental and predicted drug release data revealed that in addition to surface area, other factors such as the cell diameter in the case of the honeycomb geometry and material wettability must be considered in practical dosage form design. This information when combined with the range of achievable geometries could allow the bespoke production of optimized personalised medicines for a variety of delivery vehicles in addition to tablets, such as medical devices for example

Synthesis, characterisation and evaluation of hyperbranched N-(2-hydroxypropyl) methacrylamides for transport and delivery in pancreatic cell lines in vitro and in vivo

Hyperbranched polymers have many promising features for drug delivery, owing to their ease of synthesis, multiple functional group content, and potential for high drug loading with retention of solubility. Here we prepared hyperbranched N-(2-hydroxypropyl)methacrylamide (HPMA) polymers with a range of molar masses and particle sizes, and with attached dyes, radiolabel or the anticancer drug gemcitabine. Reversible addition-fragmentation chain transfer (RAFT) polymerisation enabled the synthesis of pHPMA polymers and a gemcitabine-comonomer functionalised pHPMA polymer pro-drug, with diameters of the polymer particles ranging from 7-40 nm. The non-drug loaded polymers were well-tolerated in cancer cell lines and macrophages, and were rapidly internalised in 2D cell culture and transported efficiently to the centre of dense pancreatic cancer 3D spheroids. The gemcitabine-loaded polymer pro-drug was found to be toxic both to 2D cultures of MIA PaCa-2 cells and also in reducing the volume of MIA PaCa-2 spheroids. The non-drug loaded polymers caused no short-term adverse effects in healthy mice following systemic injection, and derivatives of these polymers labelled with 89Zr-were tracked for their distribution in the organs of healthy and MIA PaCa-2 xenograft bearing Balb/c nude mice. Tumour accumulation, although variable across the samples, was highest in individual animals for the pHPMA polymer of ∼20 nm size, and accordingly a gemcitabine pHPMA polymer pro-drug of ∼18 nm diameter was evaluated for efficacy in the tumour-bearing animals. The efficacy of the pHPMA polymer pro-drug was very similar to that of free gemcitabine in terms of tumour growth retardation, and although there was a survival benefit after 70 days for the polymer pro-drug, there was no difference at day 80. These data suggest that while polymer pro-drugs of this type can be effective, better tumour targeting and enhanced in situ release remain as key obstacles to clinical translation even for relatively simple polymers such as pHPMA

Media actors' perceptions of their roles in reporting food incidents

Background: Previous research has shown that the media can play a role in shaping consumer perceptions during a public health crisis. In order for public health professionals to communicate well-informed health information to the media, it is important that they understand how media view their role in transmitting public health information to consumers and decide what information to present. This paper reports the perceptions of media actors from three countries about their role in reporting information during a food incident. This information is used to present ideas and suggestions for public health professionals working with media during food incidents. Methods: Thirty three semi-structured interviews with media actors from Australia, New Zealand and the United Kingdom were conducted and analysed thematically. Media actors were recruited via purposive sampling using a sampling strategy, from a variety of formats including newspaper, television, radio and online. Results: Media actors said that during a food incident, they play two roles. First, they play a role in communicating information to consumers by acting as a conduit for information between the public and the relevant authorities. Second, they play a role as investigators by acting as a public watchdog. Conclusion: Media actors are an important source of consumer information during food incidents. Public health professionals can work with media by actively approaching them with information about food incidents; promoting to media that as public health professionals, they are best placed to provide the facts about food incidents; and by providing angles for further investigation and directing media to relevant and correct information to inform such investigations. Public health professionals who adapt how they work with media are more likely to influence media to portray messages that fit what they would like the public to know and that are in line with public health recommendations and enable consumers to engage in safe and health promoting behaviours in response to food incidents