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 effect of dimensionality on growth and differentiation of neural progenitors from different regions of fetal rat brain in vitro: 3-dimensional spheroid versus 2-dimensional monolayer culture.

Three-dimensional (3-D) spheroids are widely used for culturing cells. However, 2-dimensional (2-D) monolayer cultures have also been adopted for culture and used in a broad range of cell biology studies. To address the effect of dimensionality on the growth and differentiation of neuroprogenitor cells in 3-D spheroids and 2-D monolayer cultures, cells were isolated from cerebral cortex, cerebella and brainstem of fetal rat brain then cultured in serum-free DMEM/F12 medium or DMEM with 10% FBS. The growth and differentiation of neuroprogenitor cells from three brain regions in spheroids was compared with that in monolayer cultures, and the differentiation components of neuroprogenitor cells were compared with in vivo brain sections. Neuroprogenitor cells in spheroids proliferate actively over 10 days in culture as showed by Ki67 incorporation and increase in spheroid diameter. More neuroprogenitor cells underwent neuronal differentiation in spheroids than in monolayer cultures. In comparison with fixed rat brain sections, the neuron to astrocyte ratio, as shown by neurofilament to glial fibrillary acidic protein immunoreactivity, in spheroids is similar to that found in adult rat tissue sections. Our results suggest that the spheroid culture system mimics the in vivo cytoarchitecture to a greater extent and more closely reflects the cellular composition in adult brain tissue. This supports the notion that the intercellular niche in spheroids is more favorable for the survival and differentiation of neuronal precursors, while the cues in monolayer cultures may favor glial cell survival. It is therefore concluded that dimensionality plays a significant role in determining cellular behavior in vitro.

Neurotrophin-3 gene transduction of mouse neural stem cells promotes proliferation and neuronal differentiation in organotypic hippocampal slice cultures.

BACKGROUND: The transplantation of neural stem cells (NSCs) has been accepted as a promising therapeutic strategy for central nervous system disorders. However, the beneficial effect of NSC transplantation upon functional recovery is limited due to the unfavorable microenvironment (niche) at the site of trauma or degenerative disease in the brain. Combination of transplantation of NSCs with neurotrophins may overcome the hurdles of impaired cell survival and neuronal differentiation. MATERIAL/METHODS: In the current study, the neurotrophin-3 (NT-3) gene was transduced into cultured mouse embryonic cortical NSCs via an AAV vector (NSC-NT-3). The effect of NT-3 over-expression on cell proliferation and differentiation in NSCs was observed by immunohistochemistry, cell culture and organotypic hippocampal slice cultures.
RESULTS: The characteristics of self-renewal and multiple differentiation of NSCs were well-preserved. Cells in the NSC-NT-3 group proliferated faster and differentiated into more ß-tubulin III-positive neurons compared to the control group in vitro. Furthermore, cells in the NSC-NT-3 group survived in a significantly higher percentage and undertook neuronal differentiation preferably in organotypic hippocampal slice cultures. CONCLUSIONS: Our results suggest that the transduction of NT-3 into NSCs could effectively promote NSCs survival, proliferation, and neuronal differentiation in vitro without change of the stemness of NSCs. This work also offers evidence to better understand the safety and efficiency of combined treatment with NT-3 and NSCs for the central nervous system disorders.

Organotypic slices culture model for cerebellar ataxia: potential use to study Purkinje cell induction from neural stem cells.

Cerebellar ataxia is a high profile cerebella disorder currently without viable treatment. In order to establish an in vitro model of cerebellar ataxia for evaluating the potential of neural stem cells (NSC) to repair damage caused by cerebella disorders, organotypic slices of cerebellum were cultured using Stoppini method. Penitrem A (4 mg/kg) was administered intraperitoneally (IP) to postnatal day 6 neonatal Wistar rats to produce Purkinje cell deficient slices suitable to host transplanted NSCs. The survival and differentiation of lipophilic fluorochrome chloromethylbenzamido dialkylcarbocyanine (CM-DiI) labeled cerebellar NSCs isolated from E14 rat brains were investigated after addition to the slices. Analysis indicated that cerebellar cytoarchitecture was well preserved and Purkinje cells survived for over three weeks with fewer Purkinje cells in slices cultured from Penitrem A treated neonates compared to controls. The transplanted NSCs remained viable when cultured within the cerebellar slices and although the majority remained undifferentiated. Some were DiI/2',3'-cyclic nucleotide 3' phosphodiesterase (CNPase) double positive but no DiI/calbindin double labeled cells were found. Our results show that this model could be used to assess the potential for NSC repair of the damaged cerebellum and dissect the possible underlying mechanisms by observing the interaction between NSCs and Purkinje cells in an easily accessible 3-D environment in vitro.

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.

Toxin-produced Purkinje cell death: a model for neural stem cell transplantation studies.

Purkinje cell loss is the hallmark of the cerebellar ataxias. Here the fungal neurotoxin Penitrem A was used to create partially Purkinje-cell-deficient cerebella in neonate and young adult rats suitable for use in neural stem cell transplantation studies. I.p. administration of Penitrem A to P3, P6 and 11-week old rats caused noticeable tremor in all treated animals that lasted between 1 and 3 days and was more immediate in the rat pups than in the 11-week old rats. Quantification of cresyl violet stained sections showed that Purkinje cells were preferentially lost in the cerebellar vermis and specifically in folia VI to IX (P<0.001-0.05). No change occurred in Purkinje cell number in folia I-III and folium X. These results were confirmed by the loss of calbindin binding cells in the Purkinje cell layer and the appearance of enlarged vacuolated mitochondria. The results of the present study show that the Penitrem A can remove Purkinje cells in the immature rat cerebellum and thus provide a potential model to study the micro-environmental cues in vivo for the differentiation of Purkinje cells from transplanted and/or intrinsic neural stem cells.

Cytochrome P450 Cyp4x1 is a major P450 protein in mouse brain.

A novel cytochrome P450, CYP4x1, was identified in EST databases on the basis of similarity to a conserved region in the C-helix of the CYP4A family. The human and mouse CYP4x1 cDNAs were cloned and found to encode putative cytochrome P450 proteins. Molecular modelling of CYP4x1 predicted an unusual substrate binding channel for the CYP4 family. Expression of human CYP4x1 was detected in brain by EST analysis, and in aorta by northern blotting. The mouse cDNA was used to demonstrate that the Cyp4x RNA was expressed principally in brain, and at much lower levels in liver; hepatic levels of the Cyp4x1 RNA were not affected by treatment with the inducing agents phenobarbital, dioxin, dexamethasone or ciprofibrate, nor were the levels affected in PPARalpha-/- mice. A specific antibody for Cyp4x1 was developed, and shown to detect Cyp4x1 in brain; quantitation of the Cyp4x1 protein in brain demonstrated approximately 10 ng of Cyp4x1 protein.mg(-1) microsomal protein, showing that Cyp4x1 is a major brain P450. Immunohistochemical localization of the Cyp4x1 protein in brain showed specific staining of neurons, choroids epithelial cells and vascular endothelial cells. These data suggest an important role for Cyp4x1 in the brain.

Angiotensin II may mediate apoptosis via AT1-receptors in the rat cardiac conduction system.

INTRODUCTION: Apoptosis and angiotensin II (Ang II) have been suggested as possible causes of arrhythmias. In addition, Ang II via Ang II type I (AT(1)-) receptors, has been demonstrated to induce cardiomyocyte apoptosis. The transgenic m(Ren-2)27 (TG) rat carries the additional Ren-2 gene, the expression of which results in an increase in cardiac Ang II, thus potentially affecting the cell growth/death equilibrium. In this study we have investigated the effect of Ang II, via AT(1)-receptors, on mediating apoptosis in a cardiac conduction system (SA node and AV nodes). MATERIALS AND METHODS: Heart sections from male two-day, one-week and two-week TG and Sprague-Dawley (SD) rats were stained with Masson Trichrome to localise the SA and AV nodes. The sections containing SA or AV nodes were processed for quantitation of apoptotic nuclei and AT(1)-receptors. RESULTS: The number of apoptotic nuclei/mm(2) in the SA and AV nodes were found to decrease from two days to two weeks in both the TG and the SD rats, and the number of apoptotic nuclei/mm(2) in the TG groups was significantly higher than that of the SD groups for all ages (p<0.05). The number of AT(1)-receptors/mm(2) in the SA node were found to decrease with increasing age, whereas the number of AT(1)-receptors/mm(2) in the AV node was increased in both TG and SD rats and the number of AT(1)-receptors/mm(2) in the three TG groups was significantly more than that of the three SD groups (p<0.05). DISCUSSION AND CONCLUSION: As a consequence of the additional renin gene in the TG rats, which results in the alteration of the local renin-angiotensin system, the numbers of AT(1)-receptors/mm(2) and apoptotic nuclei/mm(2) are increased. The number of apoptotic nuclei/mm(2) and AT(1)-receptors/mm(2) in the SA node decrease with maturation, whereas, the number of AT(1)-receptors in the AV node increase. Thus, there may be a correlation between Ang II and apoptosis in the SA node, which does not appear to be present in the AV node.

Volume of nerve fibers in the stress-induced bladder of adult rats following capsaicin treatment.

INTRODUCTION: We have investigated the volume of nerve fibers in the rat urinary bladder following systematic exposure to cold-restraint stress and capsaicin treatment. MATERIALS AND METHODS: Adult Wistar albino rats were either exposed to cold-restraint stress (vehicle group) or treated with capsaicin before exposure to cold-restraint stress (capsaicin group). In the control group, animals were neither exposed to cold-restraint stress nor given capsaicin. From each group, samples of bladder were prepared for morphological investigation and stereological evaluation of the volume of nerve fibers. RESULTS: Stress exposure was associated with urothelial degeneration, a higher incidence and degranulation of mast cell profiles in the mucosa, and an increased volume of nerve fibers in the muscular layer of the bladder wall. Capsaicin treatment prevented the stress-induced degenerative changes. In the capsaicin group, the volume of nerve fibers in the muscular layer was also significantly smaller than that in the stress group. CONCLUSIONS: Exposure of adult rats to capsaicin prevented the stress-induced degeneration of the bladder and changed the volume of capsaicin-sensitive fibers in muscular layer. We conclude that capsaicin and related compounds may be useful in treating stress-induced bladder problems.

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.

Least-mean-squares algorithm to determine submicrometer particle diameter, volume fraction, and size distribution width by elastic light scattering.

A computationally fast method to determine values and their uncertainty for particulate system volume median diameter, volume fraction, and size distribution width is presented. These properties cannot be obtained for submicrometer particulate by diffraction-based methods. The technique relies on a least-mean-squares method applied over a prespecified size range and distribution width. Prespecifying the range significantly reduces the number of calculations required to determine the particulate parameters from experimental data, allowing the practical evaluation of large data sets. The solution method that was developed has significant advantages over ratio-style calculations that are more commonly performed, the primary of which is a simple method to determine errors in the measurement parameters. We evaluated the predicted performance for a specific experimental system for various levels of noise, with monodisperse and log-normal distributions, by analyzing synthetic data with the algorithm. Results were a quantitative statement of system accuracy. In addition, synthetic log-normal data evaluated with monodisperse models revealed significant and systematic errors in the predicted volume median diameter. These errors indicate that, in general, systems with a significant size distribution width must be analyzed with a model that includes this size distribution. Finally, calibrated polystyrene spheres were measured with an experimental system that used four simultaneous scattering measurements, and all diameters were within the reported uncertainty.

Design methodology for a Fabry-Perot interferometer used as a concentration sensor.

We present the design methodology for a sensor that can nonintrusively monitor target gas concentration levels in a power plant exhaust flow. The measurement is based on radiative emission by rovibrational transitions that are well isolated from emission features of other constituents and requires both moderate spectral resolution (typically 1 nm or below) and relatively high optical throughput. A Fabry-Perot interferometer provides this capability, and its conceptual design is discussed at length. High-temperature radiative emission of nitric oxide in a background of water was used as a sample system for the design of a prototype Fabry-Perot interferometer. Predictions for the instrument are a minimum resolvable NO column density of 100 parts per million times meter based on a simple background subtraction scheme with a gas temperature of 800 K. Improved order sorting can dramatically lower this minimum. The prototype instrument was calibrated and tested with a laboratory simulator; results are presented and compared with predictions.