Conventional 2D Monolayer Research Articles

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films.

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Hepatotoxicity of perfluorooctanoic acid and two emerging alternatives based on a 3D spheroid model

Perfluorooctanoic acid (PFOA) toxicity is of considerable concern due to its wide application, environmental persistence, and bioaccumulation. In the current study, we used a scaffold-free three-dimensional (3D) spheroid model of mouse liver cells (AML12) to explore the toxicity of PFOA and emerging alternatives (HFPO-DA and PFO4DA). Comparing the short-term (24 and 72 h treatment) toxicity of PFOA between conventional 2D monolayer cells and 3D spheroids, we found that spheroids had higher EC50 values and lower ROS levels after treatment, indicating their greater resistance to PFOA. Cell viability (i.e., adenosine triphosphate (ATP) content and lactate dehydrogenase (LDH) leakage) and liver-specific function (i.e., albumin secretion) were stable in spheroids through 28 day of culture. However, under 100 and 200 μM-PFOA treatment for 28 day, ROS levels, LDH leakage, and caspase3/7 activity all increased significantly. As a sensitive parameter, ROS showed a significant increase at 21 day, even in the 50 μM-PFOA group. Consistent with the elevation of ROS and caspase3/7, the expressions of oxidative stress- and apoptosis-related genes, including Gsta2, Nqo1, Ho-1, caspase3, p53, and p21, were induced in dose- and time-dependent manners after PFOA exposure. The peroxisome proliferator-activated receptor alpha (PPARα) pathway was also activated after treatment, with significant induction of its target genes, Fabp4 and Scd1. Similar to PFOA, both HFPO-DA and PFO4DA activated the PPARα pathway, induced ROS levels, and initiated cell damage, though at a relatively lower extent than that of PFOA. Our results imply that the 3D spheroid model is a valuable tool in chronic toxicological studies.

Generation of functional hepatocyte 3D discoids in an acoustofluidic bioreactor.

Ultrasonic standing wave systems have previously been used for the generation of 3D constructs for a range of cell types. In the present study, we cultured cells from the human hepatoma Huh7 cell line in a Bulk Acoustic Wave field and studied their viability, their functions, and their response to the anti-cancer drug, 5 Fluorouracil (5FU). We found that cells grown in the acoustofluidic bioreactor (AFB) expressed no reduction in viability up to 6 h of exposure compared to those cultured in a conventional 2D system. In addition, constructs created in the AFB and subsequently cultured outside of it had improved functionality including higher albumin and urea production than 2D or pellet cultures. The viability of Huh7 cells grown in the ultrasound field to 5FU anti-cancer drug was comparable to that of cells cultured in the 2D system, showing rapid diffusion into the aggregate core. We have shown that AFB formed 3D cell constructs have improved functionality over the conventional 2D monolayer and could be a promising model for anti-cancer drug testing.

Advanced In Vitro HepaRG Culture Systems for Xenobiotic Metabolism and Toxicity Characterization.

Several HepaRG three-dimensional (3D) in vitro model systems have been developed to improve the predictability of xenobiotic metabolism and toxicity. In this review, we present a detailed summary and critique of the performance of various HepaRG 3D models compared to the conventional 2D monolayer culture. HepaRG 3D models can be broadly categorized into (1) scaffold-free, (2) scaffold-based, and (3) bioartificial liver (BAL) models. With respect to the scaffold-free configurations, the hanging drop model closely mimics the normal physiological function and metabolic profile of the liver. The micromold model is suitable for high-throughput multiplexed assays and exhibits higher accuracy when predicting drug-induced liver toxicity risk in both acute and chronic culture. Scaffold- and BAL-based models also present improved precision and accuracy for hepatotoxic drug screening in addition to allowing improved model control to closely mimic physiological assay conditions. Overall, all 3D HepaRG models exhibit improved cellular function, metabolic activity, and toxicity screening ability compared to the conventional 2D monolayer culture. These improvements reported in 3D models may be due to a higher degree of differentiation and cell polarity. Nevertheless, the expression and functions of various phase II, phase III, and nuclear receptors need to be further characterized in these 3D models.

3D hydrogel breast cancer models for studying the effects of hypoxia on epithelial to mesenchymal transition.

Solid tumors are 3D assemblies of cancer cells, together with multiple stromal cell types within an extracellular matrix. Yet, the vast majority of cell-based studies to characterize oncogenesis and discovery of new anti-cancer drugs is conducted using conventional 2D monolayer culture systems, where cells are grown on plastic substratum under normoxic environments. In current study, we generated 3D breast cancer cell culture platform consists of photocrosslinkable hydrogels and encapsulated isogenic primary (21PT) and a metastatic (21MT-2) breast cancer cell lines derived from the primary tumor and pleural effusion from the same patient. We demonstrated that hypoxia decreased cellular assembly size and density, and promoted epithelial to mesenchymal transition (EMT) process, without affecting cell viability. Next, we showed hypoxia enhanced breast cancer cell migration, and expression and secretion of lysyl oxidase (LOX), which is copper-dependent amine oxidase and has the primary function to drive the crosslinking of collagen and elastin and is regulated by hypoxia. Furthermore, to recapitulate in vivo situation, we generated breast cancer and lung cells (derived from the same patient) contact model by stacking 3D hydrogel constructs with breast cancer cells onto lung mesenchymal cells (LMC) laden-hydrogel and then showed breast cancer cells migrated towards LMC during hypoxia. Lastly, as a validation of this model for future screen of therapeutic agents, we demonstrated that LOX inhibitor exhibited a significant decrease in breast cancer cell viability, migration, and EMT. Taken together, these results validate the use of hydrogels based models to examine hypoxia related EMT in breast cancer cells.

Enhanced Differentiation Potential of Primary Human Endometrial Cells Cultured on 3D Scaffolds.

Novel approaches for culturing primary human cells in vitro are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Conventional 2D monolayer cultures of endometrial epithelial and stromal cells fail to replicate the complex 3D architecture of tissue. A fully synthetic scaffold that mimics the microenvironment of the human endometrium can ultimately provide a robust platform for investigating tissue physiology and, hence, take significant steps toward tackling female infertility and IVF failure. In this work, emulsion-templated porous polymers (known as polyHIPEs) were investigated as scaffolds for the culture of primary human endometrial epithelial and stromal cells (HEECs and HESCs). Infiltration of HEECs and HESCs into cell-seeded polyHIPE scaffolds was assessed by histological studies, and phenotype was confirmed by immunostaining. Confocal microscopy revealed that the morphology of HEECs and HESCs is representative of that found in vivo. RNA sequencing was used to investigate transcriptome differences between cells grown on polyHIPE scaffolds and in monolayer cultures. The differentiation status of HEECs and HESCs grown in polyHIPE scaffolds and in monolayer cultures was further evaluated by monitoring the expression of endometrial marker genes. Our observations suggest that a 3D cell culture model that could approximate native human endometrial architecture and function can be developed using tailored polyHIPE scaffolds.

Impact of RUNX2 gene silencing on the gemcitabine sensitivity of p53‑mutated pancreatic cancer MiaPaCa‑2 spheres.

Recently, it has been well‑recognized that the response toward anticancer drugs differs between two‑ and three‑dimensional (2D and 3D) invitro cancer cell growth models. In the present study, we have demonstrated that, similar to the conventional 2D monolayer culture systems which often lack invivo physiological insights, RUNX2 gene silencing increases the gemcitabine (GEM) sensitivity of the 3D spheres generated from p53‑mutated pancreatic cancer MiaPaCa‑2 cells. According to our results, MiaPaCa‑2 cells, but not p53‑wild‑type pancreatic cancer SW1990 cells efficiently formed sphere structures in serum‑free sphere‑forming medium. Although GEM treatment caused a marked induction of TAp73/TAp63 in MiaPaCa‑2 spheres accompanied by the transcriptional activation of p53 family‑target genes such as p21WAF1 and NOXA, only 20% of cells underwent cell death. Under these experimental conditions, mutant p53 expression level was increased in response to GEM and RUNX2 remained unchanged at the protein level regardless of GEM exposure, which may suppress the pro‑apoptotic activity of TAp73/TAp63. Notably, RUNX2 gene silencing markedly augmented GEM‑mediated cell death of MiaPaCa‑2 spheres compared to that of non‑depleted ones. Expression analyses revealed that forced depletion of RUNX2 further stimulated GEM‑induced upregulation of TAp63 as well as its downstream target genes such as p21WAF1 and NOXA. In summary, our observations strongly indicated that, similarly to 2D monolayer culture, RUNX2 gene silencing increased GEM sensitivity of MiaPaCa‑2 spheres and highlighted the therapeutic potential of RUNX2 in pancreatic cancer with p53 mutation.

Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix

Cancer stem cells (CSCs), being tumor-initiating with self-renewal capacity and heterogeneity, are most likely the cause of tumor resistance, reoccurrence and metastasis. To further investigate the role of CSCs in tumor biology, there is a need to develop an effective culture system to grow, maintain and enrich CSCs. Three-dimensional (3D) cell culture model has been widely used in tumor research and drug screening. Recently, researchers have begun to utilize 3D models to culture cancer cells for CSCs enrichment. In this study, glioma cell line was cultured with 3D porous chitosan (CS) scaffolds or chitosan-hyaluronic acid (CS-HA) scaffolds to explore the possibility of glioma stem cells (GSCs)-like cells enrichment, to study the morphology, gene expression, and in vivo tumorigenicity of 3D scaffolds cells, and to compare results to 2D controls. Results showed that glioma cells on both CS and CS-HA scaffolds could form tumor cell spheroids and increased the expression of GSCs biomarkers compared to conventional 2D monolayers. Furthermore, cells in CS-HA scaffolds had higher expression levels of epithelial-to-mesenchymal transition (EMT)-related gene. Specifically, the in vivo tumorigenicity capability of CS-HA scaffold cultured cells was greater than 2D cells or CS scaffold cultured cells. It is indicated that the chemical composition of scaffold plays an important role in the enrichment of CSCs. Our results suggest that CS-HA scaffolds have a better capability to enrich GSCs-like cells and can serve as a simple and effective way to cultivate and enrich CSCs in vitro to support the study of CSCs biology and development of novel anti-cancer therapies.

A journey from the endothelium to the tumor tissue: distinct behavior between PEO-PCL micelles and polymersomes nanocarriers

Polymeric nanocarriers must overcome several biological barriers to reach the vicinity of solid tumors and deliver their encapsulated drug. This study assessed the in vitro and in vivo passage through the blood vessel wall to tumors of two well-characterized polymeric nanocarriers: poly(ethyleneglycol-b-ε-caprolactone) micelles and polymersomes charged with a fluorescent membrane dye (DiO: 3,3'-dioctadecyloxacarbo-cyanine perchlorate). The internalization and translocation from endothelial (human primary endothelial cells HUVEC) to cancer cells (human tumor cell line HCT-116) was studied in conventional 2D monolayers, 3D tumor spheroids, or in an endothelium model based on transwell assay. Micelles induced a faster DiO internalization compared to polymersomes but the latter crossed the endothelial monolayer more easily. Both translocation rates were enhanced by the addition of a pro-inflammatory factor or in the presence of tumor cells. These results were confirmed by early in vivo experiments. Overall, this study pointed out the room for the improvement of polymeric nanocarriers design to avoid drug losses when crossing the blood vessel walls.

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.—Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

Abstract 779: Blockade of the IL-6/JAK/STAT3 signaling pathway inhibits pancreatic tumor cell growth in 3D spheroid cultures and in xenograft models

Abstract Emerging evidence has demonstrated the importance of tumor-associated inflammation in the development and progression of pancreatic ductal adenocarcinoma. Specifically, constitutive activation of the inflammation-related IL-6/JAK/STAT3 signaling pathway has been reported in pancreatic tumors and has been suggested to be a poor prognostic factor for overall survival in patients with advanced disease. The aim of this study was to assess the effects of inhibition of IL-6/JAK/STAT3 signaling on pancreatic cell growth in vitro and tumor growth in vivo. INCB039110, a JAK1 selective inhibitor currently in multiple Phase 2 clinical trials, was used to block the IL-6/JAK/STAT3 signaling pathway in cells. We show that while inhibition of IL-6/JAK/STAT3 signaling by INCB039110 showed no effects on the proliferation of pancreatic cell lines grown under conventional 2D monolayer cell culture conditions, this inhibitor demonstrated growth inhibitory activity against the pancreatic tumor cell lines, Hs700T and BxPC-3, in a 3D-spheroid culture system. Importantly, addition of cytokines that stimulated JAK/STAT3 signaling in these cells significantly promoted the growth of the spheroids, and this could be completely reversed by INCB039110. Furthermore, JAK1 inhibition enhanced the cytotoxicity induced by gemcitabine in both Hs700T and BxPC-3 spheroids in a combination study. The results were confirmed with another novel Jak1 selective inhibitor, INCB052793 which is currently in a Phase 1 clinical trial in patients with advanced malignancies. Since elevated serum IL-6 and its correlation with cachexia has been observed in pancreatic cancer, we tested and found that INCB039110 prevented weight loss in an IL-6 induced cachexia model in mice. Finally, we show that INCB039110 blocked tumor growth in human pancreatic xenograft models in mice at clinically relevant doses, both as monotherapy and in combination with cytotoxic agents such as gemcitabine. Thus, we demonstrate that pharmacological blockade of the IL-6/JAK/STAT3 signaling pathway with a JAK1 selective inhibitor can have both tumor intrinsic and extrinsic effects resulting in the inhibition of pancreatic tumor cell growth and the modulation of inflammation-associated cachexia. These data suggest that pancreatic cancer patients may potentially benefit from JAK1 inhibitors in the clinic. Citation Format: Jun Li, Eian Caulder, Margaret Favata, Melody Diamond, Beth Rumberger, Holly Koblish, Taisheng Huang, Chu-Biao Xue, Wenqing Yao, Jordan Fridman, Peggy Scherle, Mike Liu, Reid Huber, Kris Vaddi. Blockade of the IL-6/JAK/STAT3 signaling pathway inhibits pancreatic tumor cell growth in 3D spheroid cultures and in xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 779. doi:10.1158/1538-7445.AM2015-779

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Human neural stem cell-derived cultures in three-dimensional substrates form spontaneously functional neuronal networks.

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research. Copyright © 2015 John Wiley & Sons, Ltd.

Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor β1

Blood–brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)β1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFβ1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit.

PCaAnalyser: a 2D-image analysis based module for effective determination of prostate cancer progression in 3D culture.

Three-dimensional (3D) in vitro cell based assays for Prostate Cancer (PCa) research are rapidly becoming the preferred alternative to that of conventional 2D monolayer cultures. 3D assays more precisely mimic the microenvironment found in vivo, and thus are ideally suited to evaluate compounds and their suitability for progression in the drug discovery pipeline. To achieve the desired high throughput needed for most screening programs, automated quantification of 3D cultures is required. Towards this end, this paper reports on the development of a prototype analysis module for an automated high-content-analysis (HCA) system, which allows for accurate and fast investigation of in vitro 3D cell culture models for PCa. The Java based program, which we have named PCaAnalyser, uses novel algorithms that allow accurate and rapid quantitation of protein expression in 3D cell culture. As currently configured, the PCaAnalyser can quantify a range of biological parameters including: nuclei-count, nuclei-spheroid membership prediction, various function based classification of peripheral and non-peripheral areas to measure expression of biomarkers and protein constituents known to be associated with PCa progression, as well as defining segregate cellular-objects effectively for a range of signal-to-noise ratios. In addition, PCaAnalyser architecture is highly flexible, operating as a single independent analysis, as well as in batch mode; essential for High-Throughput-Screening (HTS). Utilising the PCaAnalyser, accurate and rapid analysis in an automated high throughput manner is provided, and reproducible analysis of the distribution and intensity of well-established markers associated with PCa progression in a range of metastatic PCa cell-lines (DU145 and PC3) in a 3D model demonstrated.

AlgiMatrix™ Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies

BackgroundThree-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening.MethodsCancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids.ResultsIC50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.ConclusionThe results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.

Development of thermoresponsive poly(propylene-g-N-isopropylacrylamide) non-woven 3D scaffold for smart cell culture using oxyfluorination-assisted graft polymerisation

Growing cells on 3D scaffolds is far superior to the conventional 2D monolayer culture method. In this study, a novel 3D thermoresponsive poly(propylene-g-N-isopropylacrylamide) (PP-g-PNIPAAm) non-woven fabric (gNWF) was developed for cell culture using oxyfluorination-assisted graft polymerisation (OAGP). New polar functional groups were detected on the oxyfluorinated NWF (oNWF), and PNIPAAm was confirmed in the gNWF by attentuated total-reflectance Fourier transform infrared (ATR-FTIR) and scanning X-ray photoelectron spectroscopy (S-XPS). Scanning electron microscopy (SEM) revealed a rough surface morphology and confinement of the PNIPAAm graft layer to the surface of the fibres in the gNWF. The OAGP method did not affect the crystalline phase of bulk PP, however, twin-melting thermal peaks were detected for the oNWF and gNWF indicating crystal defects. Contact angle studies showed that the surface of the gNWF exhibited a thermoresponsive behaviour. Hepatocyte cells attached onto gNWF disks in a bioreactor at 37°C and remained viable for 10 days in culture. Upon cooling the cell culture media to 20°C, cells were spontaneously released as 3D multi-cellular constructs without requiring destructive enzymes. The development of 3D thermoresponsive scaffolds capable of non-invasive 3D cell culture could provide a more reliable in vitro model for cells.

In vivo biomarker expression patterns are preserved in 3D cultures of Prostate Cancer

Here we report that Prostate Cancer (PCa) cell-lines DU145, PC3, LNCaP and RWPE-1 grown in 3D matrices in contrast to conventional 2D monolayers, display distinct differences in cell morphology, proliferation and expression of important biomarker proteins associated with cancer progression. Consistent with in vivo growth rates, in 3D cultures, all PCa cell-lines were found to proliferate at significantly lower rates in comparison to their 2D counterparts. Moreover, when grown in a 3D matrix, metastatic PC3 cell-lines were found to mimic more precisely protein expression patterns of metastatic tumour formation as found in vivo. In comparison to the prostate epithelial cell-line RWPE-1, metastatic PC3 cell-lines exhibited a down-regulation of E-cadherin and α6 integrin expression and an up-regulation of N-cadherin, Vimentin and β1 integrin expression and re-expressed non-transcriptionally active AR. In comparison to the non-invasive LNCaP cell-lines, PC3 cells were found to have an up-regulation of chemokine receptor CXCR4, consistent with a metastatic phenotype. In 2D cultures, there was little distinction in protein expression between metastatic, non-invasive and epithelial cells. These results suggest that 3D cultures are more representative of in vivo morphology and may serve as a more biologically relevant model in the drug discovery pipeline.

Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes

The use of fish primary cells and cell lines offer an in vitro alternative for assessment of chemical toxicity and the evaluation of environmental samples in ecotoxicology. However, their uses are not without limitations such as short culture periods and loss of functionality, particularly with primary tissue. While three-dimensional (spheroid) technology is now established for in vitro mammalian toxicity studies, to date it has not been considered for environmental applications in a model aquatic species. In this study we report development of a reproducible six-well plate, gyratory-mediated method for rainbow trout (Oncorhynchus mykiss) hepatocyte spheroid culture and compare their functional and biochemical status with two-dimensional (2D) monolayer hepatocytes. Primary liver spheroid formation was divided into two stages, immature (1-5 days) and mature (≥6 days) according to size, shape and changes in functional and biochemical parameters (protein, glucose, albumin and lactate dehydrogenase). Mature spheroids retained the morphological characteristics (smooth outer surface, tight cell-cell contacts) previously described for mammalian spheroids as demonstrated by light and scanning electron microscopy. Glucose production and albumin synthesis were significantly higher in mature spheroids when compared to conventional 2D monolayer cultures (P < 0.01) and increased as spheroids matured (P < 0.01). Basal lactate dehydrogenase (LDH) leakage significantly decreased during spheroid formation and was significantly lower than 2D cultures (P < 0.01). It is therefore suggested that mature spheroids can maintain a high degree of functional, biochemical and morphological status over-time in culture that is superior to conventional 2D models and can provide realistic organotypic responses in vitro. Trout spheroids that take ~6-8 days to reach maturity would be suitable for use in acute toxicological tests and since it is possible to culture individual spheroids for over a month, there is potential for this work to lead towards in vitro bioaccumulation alternatives and to conduct high throughput screens of chronic exposure. This is an important step forward for developing alternative in vitro tools in future fish ecotoxicological studies.

Development of a Novel Antifouling Platform for Biosensing Probe Immobilization from Methacryloyloxyethyl Phosphorylcholine-Containing Copolymer Brushes

The immobilization of thiol-terminated poly[(methacrylic acid)-ran-(2-methacryloyloxyethyl phosphorylcholine)] (PMAMPC-SH) brushes on gold-coated surface plasmon resonance (SPR) chips was performed using the "grafting to" approach via self-assembly formation. The copolymer brushes provide both functionalizability and antifouling characteristics, desirable features mandatorily required for the development of an effective platform for probe immobilization in biosensing applications. The carboxyl groups from the methacrylic acid (MA) units were employed for attaching active biomolecules that can act as sensing probes for biospecific detection of target molecules, whereas the 2-methacryloyloxyethyl phosphorylcholine (MPC) units were introduced to suppress unwanted nonspecific adsorption. The detection efficiency of the biotin-immobilized PMAMPC brushes with the target molecule, avidin (AVD), was evaluated in blood plasma in comparison with the conventional 2D monolayer of 11-mercaptoundecanoic acid (MUA) and homopolymer brushes of poly(methacrylic acid) (PMA) also immobilized with biotin using the SPR technique. Copolymer brushes with 79 mol % MPC composition and a molecular weight of 49.3 kDa yielded the platform for probe immobilization with the best performance considering its high S/N ratio as compared with platforms based on MUA and PMA brushes. In addition, the detection limit for detecting AVD in blood plasma solution was found to be 1.5 nM (equivalent to 100 ng/mL). The results have demonstrated the potential for using these newly developed surface-attached PMAMPC brushes for probe immobilization and subsequent detection of designated target molecules in complex matrices such as blood plasma and clinical samples.