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.

Permeability of PEGylated immunoarsonoliposomes through in vitro blood brain barrier-medulloblastoma co-culture models for brain tumor therapy.

PURPOSE: Owing to restricted access of pharmacological agents into the brain due to blood brain barrier (BBB) there is a need: 1. to develop a more representative 3-D-co-culture model of tumor-BBB interaction to investigate drug and nanoparticle transport into the brain for diagnostic and therapeutic evaluation. 2. to address the lack of new alternative methods to animal testing according to replacement-reduction-refinement principles. In this work, in vitro BBB-medulloblastoma 3-D-co-culture models were established using immortalized human primary brain endothelial cells (hCMEC/D3). METHODS: hCMEC/D3 cells were cultured in presence and in absence of two human medulloblastoma cell lines on Transwell membranes. In vitro models were characterized for BBB formation, zonula occludens-1 expression and permeability to dextran. Transferrin receptors (Tfr) expressed on hCMEC/D3 were exploited to facilitate arsonoliposome (ARL) permeability through the BBB to the tumor by covalently attaching an antibody specific to human Tfr. The effect of anticancer ARLs on hCMEC/D3 was assessed. RESULTS: In vitro BBB and BBB-tumor co-culture models were established successfully. BBB permeability was affected by the presence of tumor aggregates as suggested by increased permeability of ARLs. There was a 6-fold and 8-fold increase in anti-Tfr-ARL uptake into VC312R and BBB-DAOY co-culture models, respectively, compared to plain ARLs. CONCLUSION: The three-dimensional models might be appropriate models to study the transport of various drugs and nanocarriers (liposomes and immunoarsonoliposomes) through the healthy and diseased BBB. The immunoarsonoliposomes can be potentially used as anticancer agents due to good tolerance of the in vitro BBB model to their toxic effect.

Evaluation of bupropion hydrochloride developmental cardiotoxic effects in chick cardiomyocyte micromass culture and stem cell derived cardiomyocyte systems.

The use of antidepressant drug bupropion hydrochloride (BPN) during pregnancy results in increased cardiovascular anomalies. In this study, BPN developmental cardiotoxic effects in in vitro system were evaluated using chick cardiomyocyte micromass (MM) culture system and mouse embryonic stem cell derived cardiomyocyte (ESDC) system. In MM system, the cardiomyocyte contractile activity significantly decreased only at BPN 200 µM, while in ESDC system BPN concentration above 75 µM resulted in decreased contractile activity. The increase in drug concentration also affected the cardiomyocyte viability and total cellular protein content in both systems, but in ESDC system the cell viability failed to attain significant difference. The drug failed to induce reactive oxygen species production in both systems, but has affected the cardiac connexin43 expression especially in MM system. We observed that BPN showed developmental cardiotoxic effects irrespective of the stage of cardiac development in both in vitro systems.

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.

The preparation of magnetically guided lipid based nanoemulsions using self-emulsifying technology.

This paper reports an easy and highly reproducible preparation route, using self-emulsifying technology, for an orally administered high quality magnetically responsive drug delivery system. Hydrophobic iron oxide nanoparticles of about 5 nm in diameter were prepared and incorporated into the lipid core of the produced oil droplets of a self-nanoemulsifying drug delivery system (MagC(18)/SNEDDS). The produced nanoemulsion exhibits colloidal stability at high ionic strengths and temperatures. The observed value of the saturation magnetization at 2 K is approximately 4.1 emu g(-1). The nanoemulsion displayed the magnetic properties of a non-interacting assembly of superparamagnetic particles and a low blocking temperature. Moreover the effect of MagC(18)/SNEDDS on biological systems in vitro was investigated in rodent fibroblasts (3T3 cells). The cytotoxicity studies show that none of the formulations tested affected cell activity significantly over the 24 h incubation. Such systems might have a potential use for oral delivery of poorly soluble compounds by extending the residence time of the formulation in the small intestine resulting in increased drug absorption values.

Biocompatibility and hemocompatibility of surface-modified NiTi alloys.

Nickel titanium (NiTi) shape memory alloys have been investigated for several years with regard to biomedical applications. However, little is known about the influences of surface modifications on the biocompatibility of these alloys. The effects of a range of surface treatments were investigated. Cytotoxicity and cytocompatibility studies with both fibroblast and endothelial cells showed no differences in the biocompatibility of any of the NiTi surfaces. The cytotoxicity and cytocompatibility of all surfaces were favorable compared to the controls. The hemolysis caused by a range of NiTi surfaces was no different from that caused by polished 316L stainless steel or polished titanium surfaces. The spreading of platelets has been linked to the thrombogenicity of materials. Platelet studies here showed a significant increase in thrombogenicity on polished NiTi surfaces compared to 316L stainless steel and pure titanium surfaces. Heat treatment of NiTi was found to significantly reduce thrombogenicity, to the level of the control. The XPS results showed a significant decrease in the concentration of surface nickel with heat treatment and changes in the surface nickel itself from a metallic to an oxide state. This correlates with the observed reduction in thrombogenicity.