A list of Publications in Scientific Journals detailing research where Alvetex® has been used.
The publications below are hosted on the websites of various scientific journals.
Please note Reinnervate is now ReproCELL Europe Ltd.
A list of Publications in Scientific Journals detailing research where Alvetex® has been used.
The publications below are hosted on the websites of various scientific journals.
Ugbode CI, Smith I, Whalley BJ, Hirst WD, Rattray M.
J Neurochem. 2017 May 9. doi: 10.1111/jnc.14064.
Sonic HedgeHog (SHH) signalling is already known to reduce astrocyte reactivity after CNS injury in vivo. In this study, the authors used two SHH pathway agonists, purmorpharmine (Pur) and smoothened agonist (SAG) to characterise the effects of SHH signalling upon astrocytes grown in vitro in both conventional 2D and Alvetex 3D cultures, with a focus on neuroprotection. Following agonist treatment, primary astrocytes isolated from E15 mouse embryo cerebral cortices exhibited cell elongation, increased cell proliferation and decreased expression of the reactivity marker GFAP in 2D. In 2D astrocyte-neuron co-cultures, treatment with SAG resulted in a decrease in neuronal firing frequency, as determined by multi-electrode array (MEA). To better characterise this observation, astrocytes were cultured and pre-treated with SAG as a monoculture in a 3D Alvetex Scaffold insert, after which SAG was washed out and the Alvetex insert was suspended in a culture well already containing neurons grown in 2D. After allowing sufficient time for the astrocytes to condition the medium, the insert was removed and the neurons were treated with kainate to induce injury. Medium conditioning with SAG-treated astrocytes grown in 3D Alvetex inserts conferred cytotoxic protection upon the neurons, as assessed by MAP-2 expression levels. This experimental set-up illustrates the suitability of 3D Alvetex inserts for multi-phasic co-culture and for the in vitro characterisation of functionally-significant cell-cell interactions observed in vivo.
Niu N, Mercado-Uribe I, Liu J.
Oncogene. 2017 Apr 24. doi: 10.1038/onc.2017.72.
In this study, the authors characterised polyploid giant cancer cells (PGCC) derived from mitosis-arrested ovarian cancer cell lines. When grown in 2D, PGCCs were capable of exhibiting stem cell characteristics, as evidenced by the expression of OCT4, SOX2 and NANOG and the loss of Xist expression. YAP, a critical factor in regulating the tumour-suppressor pathway, had a distinct nuclear localisation in PGCCs, which, when disrupted by the addition of dobutamine, prevented the differentiation of PGCCs into either endoderm or neural-like cells. Daughter cells arising from PGCCs by budding exhibited morphologies reminiscent of various cell lineages and differentiated when cultured in either chondrogenic, adipogenic or osteogenic medium, as indicated by positive staining for sulphate chondroitin, oil red O and osteogenin, respectively. In xenografts, tumours obtained from daughter cells where smaller and more heterogenous than those from un-arrested cancer cells, which mimics clinical observations post-chemotherapy in humans. When grown on Alvetex Scaffold, PGCCs remained at the surface of the substrate and exhibited an epithelial-like morphology, rather than invading into the substrate as seen with un-arrested cancer cells. Taken together, this study shows how the use of 2D, 3D and animal models can be combined to assess specific aspects of cell expression and behaviour to gain a more complete understanding of cancer cell regulation under chemotherapy.
Wood C.L., Divieti Pajevic P., Gooi J.H.
Bone Reports. 2017 Jun 6:74-80. doi:10.1016/j.bonr.2017.02.007
In this study, the authors investigated the cross-talk between bone and muscle, and more specifically the effect of osteocyte-conditioned medium on myogenic differentiation. MLO-Y4 osteocytes were cultured in Alvetex scaffold in either static or perfused conditions, using the Alvetex perfusion plate, and the resulting conditioned medium was added to C2C12 myoblasts. A significant decrease in the number and length of myofibers was observed after 6 days of treatment with medium from either static or perfused cultures and the expression of several myogenic regulatory factors (eg. Myf5, Myogenin, Myh1) was also decreased. Several cytokines relevant to muscle formation were detected in the conditioned medium, with differential amounts of individual cytokines between static and perfused medium generally supporting a greater inhibitory activity of perfused conditioned medium, as exemplified by a decrease in IL-1β, IL-3, IL-4, IL-10, MIG and MIP-1a in perfused culture conditioned medium compared to its static culture equivalent.
These results demonstrate how Alvetex technologies can be successfully combined to provide several physiological cues in vitro, i.e. both three dimensionality and mechanical stimulation, with benefits to protein expression and the understanding of cell-cell interaction mechanisms.
Shannon S, Jia D, Entersz I, Beelen P, Yu M, Carcione C, Carcione J, Mahtabfar A, Vaca C, Weaver M, Shreiber D, Zahn JD, Liu L, Lin H, Foty RA.
BMC Cancer. 2017 Feb 10; 17(1):121. doi: 10.1186/s12885-017-3107-x.
Continuous dispersal of individual glioblastoma cells away from the main tumour mass is a challenge to the long-term efficacy of resection surgery.
In this study, the authors investigated the activity of the MEK inhibitor PD032901 on the growth and mechanical properties of hanging-drop aggregates generated from human primary glioblastoma cells, as well as on the actin organisation, detachment under shear stress and dispersal of individual cells. Of particular note, some of these aggregates were transferred on top of Alvetex scaffold that had been previously seeded with normal human astrocytes, and the ability of the aggregated glioblastoma cells to detach and disperse through the astrocyte culture was quantified by spinning disk confocal microscopy. In the absence of PD0325901, glioblastoma cells penetrated into the astrocyte-seeded scaffold for up to 30 microns deep in the space of 48h, and this was significantly reduced in the presence of the MEK inhibitor.
This study demonstrates how Alvetex 3D co-culture capabilities can further the applications of other 3D culture methods and enable the use of more complex in vitro models of cancer cell invasion through neighbouring tissue.
Clarke KE, Tams DM, Henderson AP, Roger MF, Whiting A, Przyborski SA.
Neurochem Int. 2016 Dec 20. pii: S0197-0186(16)30507-1. doi: 10.1016/j.neuint.2016.12.009.
In this study, the authors present a novel 3D model which enables the investigation of neurite outgrowth in vitro, by culturing neurospheres derived from pluripotent stem cells onto Alvetex Scaffold inserts. Stem cells were differentiated with the synthetic retinoid EC23 in 2D, as evidenced by decreased expression of the pluripotency markers SSEA3, Nanog and Oct-4, as well as increased expression of the neuronal differentiation markers A2B5 and Pax6 and of the mature neuronal protein NF-H. Differentiated neurospheres were also transferred onto the top surface of Alvetex scaffold inserts. Only a small minority of individual cells within the neurospheres migrated into the scaffold while neurites extended throughout the material. Neurites could be tracked using various techniques and their length and number extending through the 3D material could be quantified from imaging beneath the scaffold membrane. In experiments where U-118 MG glioma cells were co-cultured in the scaffold prior to neurosphere transfer, 3D neurite extension into the scaffold was drastically halted. Such suppression of neurite outgrowth was consistent with the detectable expression of inhibitory CSPGs expressed by U-118 MG cells grown in Alvetex Scaffold. This inhibitory effect could be rescued by addition of the ROCK inhibitor Y-27632.
These results demonstrate that Alvetex 3D Cell Culture technology can further the use and complexity of established differentiation in vitro models. The technology can be developed to replicate aspects of certain neuropathological disorders, allowing the more convenient acquisition of functional endpoints for testing drug candidates.
Sheng Yao, Bosung Kim, Xiling Yue, Maria Y. Colon Gomez, Mykhailo V. Bondar, and Kevin D. Belfield.
ACS Omega, 2016, 1 (6), pp 1149–1156. DOI: 10.1021/acsomega.6b00289
Although two-photon fluorescence microscopy (2PFM) is already widely used to image ex-vivo tissue up to several millimetres depths, its potential use for microscopy-aided clinical surgery is currently untapped.
In this study, the authors synthesised four bis-thienylbenzothiadiazole chromophores and assessed their performance as candidate near-infrared (NIR) dyes for 2PFM. Their absorption/fluorescence spectra and quantum yields were found to be roughly constant in toluene, tetrahydrofuran and methylene chloride, suggesting that the dyes might also maintain their properties in aqueous solvents, i.e. in a biologically-relevant environment. Out of the four candidates, the one exhibiting the highest figure of merit was encapsulated in Pluronic micelles and added to 3T3 fibroblast grown in both 2D and Alvetex 3D substrate, whereby fluorescence was maintained and could be imaged up to a depth of 100 microns in Alvetex using confocal microscopy.
This study demonstrate the utility of Alvetex substrate for early studies destined to be applied in living tissues.
Natividad Gomez-Roman, Katrina Stevenson, Lesley Gilmour, Graham Hamilton and Anthony J Chalmers
Neuro Oncology (2016) First published online: August 30, 2016. doi: 10.1093/neuonc/now164
In this study, the authors utilised Alvetex 3D cell culture technology to develop models of glioblastoma that would better recapitulate the in vivo behaviour of this common type of brain tumor. Although 2D models of glioblastoma already exist, they are unable to accurately predict drug sensitivity in vivo, which greatly impairs efforts to bring about new therapeutic agents. Cell lines derived from patient resections were grown in stem-cell promoting medium on either ECM-coated 2D plates or in ECM-coated Alvetex. Alvetex-grown cells exhibited a differential gene expression profile, notably in members of the EGFR and VEGF pathways and stem cell markers. They responded to presence of VEGF by an increase in proliferation and to withdrawal of VEGF by increased differentiation, which is consistent with the hypothesised role of VEGF in maintaining a stem cell character in vivo and is relevant to cancer treatment as drug resistance is thought to be associated with cancer stem-like cells. Also in accordance with in vivo data and in opposition to results from 2D cultures, cells grown in 3D in Alvetex were not radiosensitised by erlotinib treatment but were radiosensitised by either bevacizumab or temozolomide. Finally, upon injection into mice to assess tumorigenicity, cells from 3D cultures resulted in a greater number of symptomatic mice. Taken together, these results give strong evidence that Alvetex 3D culture models are better able to predict cell responses to therapeutic candidates before pre-clinical and clinical in vivo testing than current 2D models, with great potential benefit to cancer research and ultimately human health.
Scherr AL, Gdynia G, Salou M, Radhakrishnan P, Duglova K, Heller A, Keim S, Kautz N, Jassowicz A, Elssner C, He YW, Jaeger D, Heikenwalder M, Schneider M, Weber A, Roth W, Schulze-Bergkamen H, Koehler BC.
Cell Death Dis. 2016 Aug 18; 7(8):e2342. doi: 10.1038/cddis.2016.233.
This study highlights the role of the anti-apoptotic protein Bcl-xL in the incidence and treatment of colorectal cancer (CRC). After demonstrating using human CRC tissue and mouse knock-out mutants that Bcl-xL is upregulated in CRC and that its absence results in fewer tumor lesions, the authors cultured HT29 cells in Alvetex Scaffold and exposed them to ABT-737, a BH3 mimetic which competitively binds both Bcl-xL and Bcl-2. IHC-stained sections of the Alvetex-grown cultures revealed a greater amount of PARP cleavage and LDH medium activity in ABT-373-treated cells compared to untreated controls, although numbers of Ki67-expressing cells were unchanged. Crucially, when the authors repeated this ABT-737 treatment in human ex vivo CRC lesions slices, similar results were observed. This study demonstrates that data obtained using Alvetex can be replicated in human samples and that Alvetex 3D cell culture technology is therefore a physiologically-relevant model to study human disease in vitro.
Simkova D, Kharaishvili G, Korinkova G, Ozdian T, Suchánková-Kleplová T, Soukup T, Krupka M, Galandakova A, Dzubak P, Janikova M, Navratil J, Kahounova Z, Soucek K, Bouchal J.
Oncotarget. 2016 Jul 7. doi: 10.18632/oncotarget.10471.
Asporin, an aspartic acid-rich protein belonging to the small leucine-rich proteoglycan family, is involved in the development and pathogenesis of cartilage and bone tissues, thanks to its ability to bind both calcium and collagen types I and II, leading to ECM mineralisation. Although high expression of asporin is also detected in a number of cancers (eg. breast, lung, ovary, GI) and correlates with increased invasiveness in vitro, its relation to prognosis seems largely dependent on stage and subtype. In this report, the authors investigated the role of asporin on breast cancer cell invasion in a number of 3D matrices, including Alvetex Scaffold. Both human breast carcinoma Hs578T cells and human primary cancer-associated fibroblasts exhibited up-regulated asporin protein expression when grown in Alvetex Scaffold compared to 2D. Neither was asporin up-regulated in Hs578T cells grown in collagen gels, leading the authors to speculate that the stiffness of Alvetex Scaffold better mimics the physical environment of solid tumours.
Manghera M, Ferguson-Parry J, Douville RN
Neurobiol Dis. 2016 Oct;94:226-36. doi: 10.1016/j.nbd.2016.06.017.
This study investigates the interplay of human endogenous retrovirus-K (ERVK) and TAR DNA binding protein 43 (TDP-43), a regulator of RNA processing and protein homeostasis, in amyotrophic lateral sclerosis (ALS). The authors specifically compared astrocytes to neurons in vitro and detected differences in aggregation and clearance of ERVK/TDP-43 granules between both cell types. RenCell CX cells, grown and differentiated into neurons using the Alvetex Scaffold 12-well plate format, consistently expressed ERVK-reverse transcriptase, regardless of treatment with TNF-a and/or MG132 proteasome inhibitor, as shown by confocal microscopy. These results highlight differences in the robustness of protein degradation pathways in different CNS cell types and their potential sensitivity to protein-aggregation diseases.
Edmondson R, Adcock AF, Yang L.
PLoS One. 2016 Jun 28;11(6):e0158116. doi: 10.1371/journal.pone.0158116.
Although 2D cell-based assays have been a very useful and widespread model for drug toxicity testing in the pharmaceutical industry, the growing realisation of their limitations is leading to an increasing interest in 3D assays. However, commercially-available 3D in vitro assays vary greatly in their chemical and physical characteristics, potentially leading to model-specific changes in cell behaviour and drug response. In this report, the authors investigated the effects of three such 3D models, i.e. two basement-membrane gelling agents and Alvetex Scaffold 96-well plate format, on the morphology, proliferation and drug sensitivity of the LNCaP and DU145 prostate cancer cell lines. The results highlighted several model-specific, cell-specific and drug-specific differences between 2D and 3D models. While both gelling agents gave mostly similar results throughout, cells grown in Alvetex Scaffold exhibited cell-specific morphology, as well as drug sensitivity and protein expression profiles that could not consistently be matched to either the gelling agents or the 2D set-up. This report exemplifies the uniqueness of Alvetex Scaffold, which provides three-dimensionality of culture without the diffusional encumbrance or chemical signalling of a gelling system.
Patrick Terrence Brooks, Mikkel Aabech Rasmussen and Poul Hyttel.
J Stem Cell Res Ther 2016, 6:4 http://dx.doi.org/10.4172/2157-7633.1000337
In this study, the authors have characterised a 3D in vitro model of neural differentiation utilising human iPSCs grown on Alvetex. After an initial period of differentiation into neuroepithelial sheets on 2D conventional plastic using BMP and TGFβ inhibitors, the cultures were transferred onto laminin-coated Alvetex Scaffold inserts and maintained in hypoxic conditions (5% O2) for up to one month. The neural tissue equivalent obtained grew several hundred microns thick within and above the scaffold. In-depth morphological characterisation by confocal microscopy revealed neural tube-like structures (NTLS) formation within the tissue mass, with SOX2 and Ki67 positive neural stem cells concentrated at the edge of the NTLS lumen, while neuroprogenitor cells and radiating glial-like cells were found at the NTLS periphery. Other neural markers, such as βIII-tubulin and Tau, were more widely expressed throughout the tissue construct, with MAP2 being expressed preferentially in cells growing on the underside of the scaffold. TEM imaging further confirmed the high cohesion and maturity of the cultures, with clear example of tight junctions, basal bodies and primary cilia.
This model of extended neural differentiation demonstrate how the Alvetex insert format can enable the long-term differentiation of tissue constructs in vitro. This is of benefit not only in providing the research community with a more in vivo-like model of neural development, but could also be of use as a test of the differentiation capacity of iPSC lines.
Ugbode CI, Hirst WD, Rattray M.
Neurochem Res. 2016 Aug; 41(8):1857-67. doi: 10.1007/s11064-016-1911-3. Epub 2016 Apr 21.
This study investigates the effect of in vitro culture conditions on the marker expression profile of astrocytes and how well the resulting cells approximate astrocytes in vivo. In vitro astrocytes were obtained from either embryonic or postnatal mouse brain tissue and grown on both 2D glass coverslips and 3D Alvetex inserts coated with poly-L-ornithine. Using western blotting and qPCR to examine the expression levels of markers of astrocytic character (GLAST, GS, SMC3, NG2) and of reactive gliosis associated with CNS injury (GFAP, GLT-1, LCN2, Serpina3n, Cx43), the authors demonstrated that, although postnatal astrocytes only show very few differences between 2D and 3D, embryonic astrocytes grown in 3D exhibit lower levels of GFAP, GLAST, LCN2, serpina3n and Cx43, supporting the conclusion that culture in Alvetex can lead to astrocytes adopting a phenotype more representative of uninjured CNS tissue. These results highlights one of the advantages of using Alvetex 3D cell culture technology over conventional 2D culture to construct physiologically-relevant in vitro models.
Pasquale Marrazzo, Silvia Maccari, Annarita Taddei, Luke Bevan, John Telford, Marco Soriani, Alfredo Pezzicoli
PLOS ONE Published: April 21, 2016 http://dx.doi.org/10.1371/journal.pone.0153985
The usefulness of animal systems to model human infectious diseases can be greatly limited by host-pathogen species-specificity, while human tissue biopsies are only amenable to short-term experiments due to tissue degradation. In this study, the authors describe a 3D in vitro human bronchial equivalent model, consisting of two types of commercially-sourced cells, i.e. normal human lung fibroblasts cultured within the thickness of Alvetex Scaffold and overlaid by a thin collagen I coating as a growth surface for normal human trachea-bronchial cells. After three weeks of air-liquid interface culture, advanced imaging (immunostaining and SEM) was performed. The epithelium was found to exhibit a columnar morphology, nuclear distribution and mucus secretion much closer to the in vivo tissue than that obtained in transwell-grown cells. The integrity of the Alvetex-cultured epithelium was demonstrated by the presence of ZO-1, laminin and integrin alpha-6, while markers for basal cells and club cells were also detected. The authors tested the validity of this model for microbial infection by introducing the Non-Typeable Haemophilus influenza strain Fi176, isolated from a clinical otitis media sample, and recorded infiltration within the mucus, epithelium and stromal layers 40h after infection. Interestingly, bacterial aggregates and vesicles were also observed, which is reminiscent of infection progression in vivo.
These results demonstrate how Alvetex technology can enable the development of complex in vitro models that incorporate multiple cell types and successfully replicate both the morphology and expression profile of in vivo tissue, which in turn allows for the in vitro study of physiological processes traditionally restricted to in vivo models or tissues biopsies.
Rifaey HS, Villa M, Zhu Q, Wang YH, Safavi K, Chen IP.
J Endod. 2016 May; 42(5):760-5. doi: 10.1016/j.joen.2016.02.001. Epub 2016 Mar 16.
Caiazza F, Murray A, Madden SF, Synnott NC, Ryan EJ, O’Donovan N, Crown J, Duffy MJ.
Endocr Relat Cancer. 2016 Apr; 23(4):323-34. doi: 10.1530/ERC-16-0068. Epub 2016 Mar 1.
James Jenkins, Ruslan I. Dmitriev, Karl Morten, Kieran W. McDermott, Dmitri B. Papkovsky
Acta Biomaterialia 2015 doi:10.1016/j.actbio.2015.01.032 Published 31 January 2015
Alvetex porous membrane scaffolds are widely used materials for three-dimensional cell cultures and tissue models. Additional functional modification of such scaffolds can potentially extend their use and operational performance. In this paper Alvetex microporous polystyrene-based scaffolds were impregnated with a phosphorescent O2-sensitive dye PtTFPP, optimized for live cell fluorescence microscopy and characteristics for the stable and robust response to pO2 in phosphorescence enabling imaging of O2 distribution in 3D cell cultures. The modified scaffolds possessed high brightness, convenient spectral intensity and lifetime imaging modes (>twofold response over 21/0% O2). They are suitable for prolonged use under standard culturing conditions without affecting cell viability, and for multi-parametric imaging analysis of cultured cells and tissue samples. The coated Alvetex membranes were cultured with cancer cells (HCT116), multicellular aggregates (PC12) and rat brain slices and showed that they can inform on tissue oxygenation at different depths and cell densities, changes in respiration activity, viability and responses to drug treatment. Using this method multiplexed with staining of dead cells (CellTox Green) and active mitochondria (TMRM), we demonstrated that decreased O2 (20–24 µM) in scaffold corresponds to highest expression of tyrosine hydroxylase in PC12 cells. Such hypoxia is also beneficial for action of hypoxia-specific anti-cancer drug tirapazamine (TPZ). Thus, oxygen sensitive alvetex scaffolds allow for better control of conditions in 3D tissue cultures, and are useful for a broad range of biomaterials and physiological studies.
Sevim Yildiz Arslan, Yanni Yu, Joanne E. Burdette, Mary Ellen Pavone, Thomas J. Hope, Teresa K. Woodruff, J. Julie Kim
Endocrinology Published 30 Jan 2015 doi: 10.1210/en.2014-1840
The endocervix plays an important role in conception and protection from pathogens. It is sensitive to changing concentrations of the sex hormones and alters the consistency of the mucus it secretes in response to these signals. This article reports on the development of a novel three dimensional (3D) model of human endocervix comprising both epithelial cells and stromal cells. Cells derived from primary sources were first explanted in conventional cultured and then seeded onto Alvetex Scaffold membranes and maintained for up to 28 days. Suspensions of two million cells were seeded into each 12-well insert containing Alvetex Scaffold. The cells remained viable and formed 3D cultures composed of epithelial and stromal cells that were treated with estradiol or progesterone over the growth period. Treatment by the hormones resulted in increased cell growth and proliferation. Cells expressed the expected repertoire of hormone receptors and produced both neutral and acid mucins. The article demonstrates the compatibility of Alvetex technology to support an endocervical 3D in vitro model that reacts to changing hormonal conditions. Alvetex Scaffold was further shown to be compatible with standard analytical assays such as cell viability, immunocytochemistry and histochemical methods. In summary, the paper describes a robust and novel human 3D culture model of the endocervix which showed physiological responses to menstrual hormones.
Emma J. Davies, Meng Dong, Matthias Gutekunst, Katja Närhi, Hanneke J. A. A. van Zoggel, Sami Blom, Ashwini Nagaraj, Tauno Metsalu, Eva Oswald, Sigrun Erkens-Schulze, Juan A. Delgado San Martin, Riku Turkki, Stephen R. Wedge, Taija M. af Hällström, Julia Schueler, Wytske M. van Weerden, Emmy W. Verschuren, Simon T. Barry, Heiko van der Kuip & John A. Hickman
Nature-Scientific Reports 5, Article number: 17187 (2015) doi:10.1038/srep17187 Published online: 09 December 2015
Precision-cut tissue slices are morphologically-correct arrangements of multiple cell types in their original extracellular environment, but are limited in their use by their rapid degradation in vitro. This paper aimed to identify optimal in vitro cultures conditions for precision-cut slices from cell-line derived and patient-derived xenografts, as well as primary patient tumour samples, with specific attention to viability and stress over time.
The authors identified that maintaining slices at the air-liquid interface with the help of a filter and under atmospheric oxygen conditions resulted in the best tissue maintenance for up to 96h after cutting, as assessed by H&E staining for morphology, Ki67/cleaved caspase-3 double staining for viability and expression profile of a range of stress markers. Interestingly, a vertical gradient of cell viability was observed within the slices, whereby cells at the filter edge exhibit higher levels of hypoxic stress, as suggested by increased HIF1alpha expression.
In order to further characterise the performance of filter supports, the authors compared Millipore filters to Alvetex Strata (void size 5 microns) in slices cut from the breast carcinoma cell-line MCF7-derived xenografts and found that, although HIF1α expression was still present at the filter side in both systems, less stress biomarkers expression was detected in Alvetex Strata at 48h after cutting.
This paper demonstrates the suitability of the small-void size Alvetex Strata 3D substrate for the maintenance of precision-cut tissue slices, thus enabling scientists to extend their use of this valuable study model.
Lara Stevanato, Caroline Hicks, John D. Sinden
Journal of Visualized Experiments (98), e52410, doi:10.3791/52410 (2015).
The human neural stem cell line CTX0E03 is a therapeutically-relevant cell line currently undergoing clinical trials related to Stroke-induced conditions and this paper investigates the effect of 3D culture on the ability of this cell line to differentiate in vitro. The authors describe methods to culture and differentiate CXT0E03 cells on laminin-coated Alvetex scaffolds, as well as how to measure the length of axon processes stained with anti-beta3-tubulin antibodies and to perform RNA extraction followed by cDNA reverse transcription and real-time PCR of MiRNA targets. Their results demonstrate that significantly longer axon processes are obtained after both one week and three weeks of differentiation in Alvetex scaffolds compared to 2D. MiRNA expression changes indicative of cell differentiation are also detected earlier when the CTX0E03 cells are grown in Alvetex scaffolds compared to 2D. This study demonstrates the feasibility of high-quality imaging and RNA extraction from cultures grown in Alvetex scaffolds, as well as its beneficial effect on stem cell differentiation in vitro.
Gabriela Martínez-Revollar, Erika Garay, Dolores Martin-Tapia, Porfirio Nava, Miriam Huerta, Esther Lopez-Bayghen, Noemí Meraz-Cruz, José Segovia, Lorenza González-Mariscal
Experimental Cell Research, Volume 339, Issue 1, 15 November 2015, Pages 67-80 doi:10.1016/j.yexcr.2015.10.006
Triple negative breast cancers (ER-/PR-/HER2-), although representing only 15% of all breast cancers, exhibit resistance to existing hormonal therapy and are subsequently associated with the lowest patient survival rate. This paper used the triple-negative cell line MDA-MB-231 to characterises in details the morphology, protein expression and migratory activity of two cell sub-populations, i.e. fibroblasts-like cells and semi-epithelial cells, with fibroblast-like cells already being known to express cancer stem cell markers and therefore being of greater interest to understanding mechanisms of tumorigenesis. After culturing cells from both sub-types in Alvetex Scaffold 12-well inserts for a period of 10 days, the authors double-stained the cultures as wholemounts with phalloidin-rhodamine and DAPI. Depth colour-coded images taken using confocal microscopy clearly demonstrated the greater migratory activity of fibroblast-like cells compared to semi-epithelial cells in 3D. This difference in cell behaviour in 3D correlated well with decreased levels of cell-cell junction proteins observed in 2D and increased tumorigenicity in vivo. This report demonstrates how Alvetex 3D technology is uniquely well-suited to bridge the in vitro 3D model gap between 2D systems and in vivo animal models.
Imogen Smith, Marcus Haag, Christopher Ugbode, Daniel Tams, Marcus Rattray, Stefan Przyborski, Angela Bithell, Benjamin J. Whalley
19 October 2015 J Neuroscience Letters doi:10.1016/j.neulet.2015.10.044
In this study, the authors characterise the morphological and electrophysiological features of embryonic mouse cortical neuroglial cells grown on 2D and in 3D Alvetex Scaffold. Embryonic day 14 mouse cortical cells grown on punch-holed 6mm diameter Alvetex Scaffold discs for 14-21 days expressed GFAP and betaIII-tubulin, indicating the presence of both glial and neuronal cells. Although these markers were also present in 2D cultures, cell morphology was markedly different between 2D and 3D substrates, with more cell processes and less cell flattening being noted in Alvetex. Both 2D and 3D cells also exhibited spontaneous action potential firing, which responded adequately to the GABA and glutamate antagonists BMI and CNQX. A positive correlation was found between burst incidence and signal power in 2D cultures, while this was only true in BMI-treated Alvetex cultures. Although the rate of firing was less in 3D than in 2D, the authors speculated this might be due to the limited range of MEA recording. These results highlight the profound effect of 3D cell culture on the morphology of individual cells, with functional consequences at the level of multicellular networks.
David S. Hill, Neil D.P. Robinson, Matthew P. Caley, Mei Chen, Edel A. O’Toole, Jane L. Armstrong, Stefan Przyborski, and Penny E. Lovat
September 1, 2015 doi: 10.1158/1535-7163 MCT-15-0394 Molecular Cancer Therapeutics
S Shannon, C Vaca, D Jia, I Entersz, et al.
PloS One, 2015 doi: 10.1371/journal.pone.0135951
In this study, Shannon and colleagues report the effect of dexamethasone on the dispersal of primary human glioblastoma cells. They use a variety of modern molecular and cellular techniques to investigate this process, including the use of Alvetex Scaffold to assess the ex vivo dispersal of tumour cells. They provide a detailed method describing the seeding of the GFP-labelled tumour cells onto the scaffold and their subsequent culture for 48 hours to allow the cells to infiltrate the 3D structure of Alvetex and disperse. After two days, the scaffolds were mounted onto microscope slides and cover-slipped. Confocal microscopy was used to capture images at successive focal planes at 1 micron intervals to generate a z stack. Differential interference contrast (DIC) microscopy was used to analyse each z stack and measure the z-axis position of cells within the tissue scaffold. The results of the Alvetex Scaffold study are shown in Figure 6 and demonstrate that dexamethasone decreased the dispersal of tumour cells in an ex vivo 3D model. In summary, this paper provides a good example of where Alvetex technology has been applied to assess the action of a drug on tumour cell motility. This technique could be developed further to form a general assay to study tumour cell dispersal and be used to assess the action of drugs on specific tumour tissues ex vivo as part of an applications to personal medicine.
Bajcsy P, Simon M, Florczyk SJ, Simon CG Jr, Juba D, Brady MC.
J Microsc. 2015 Dec; 260(3):363-76. doi: 10.1111/jmi.12303.
In this study from the US National Institute of Standards and Technology, the authors tackle the issue of how to validate segmentation algorithms for large confocal Z-stack data sets, to specifically evaluate their accuracy, precision and efficiency in assessing the effect of 3D in vitro culture on the shape of stem cells. After culturing primary human bone marrow stem cells in ten types of 3D scaffolds, including Alvetex as representative of porous substrates, and staining the cells for actin by immunofluorescence, the authors applied six candidate segmentation algorithmic sequences and compared them for accuracy against manually-contoured cells, for precision against manually-segmented projections, and for efficiency as measured by execution time. This process also identified rejected, missed or inaccurately-segmented cells for each test substrate. The two best performing candidate algorithms incorporated both imaging and geometric criteria, as well as either minimum error thresholding or topological stable state thresholding, and a morphological step that ensured removal of voxels of low intensity.
This study demonstrate that Alvetex is being recognised as a benchmark material for the evaluation of porous 3D cell culture substrates.
Han Xie, Tong Cao, José Viana Gomes, Antônio Hélio Castro Neto, Vinicius Rosa
Carbon 93, 2015 doi:10.1016/j.carbon.2015.05.071
In this report the authors investigate the differentiation of periodontal ligament stem cells (PDLSCs) on graphene substrates. They test the growth of cells on two- and three-dimensional (3D) graphene supports and use Alvetex polystyrene scaffolds as an established control for 3D cell culture. With specific reference to the use of Alvetex, the investigators successfully demonstrate the 3D culture of PDLSCs and the application of a range of analytic methods to monitor cell behavior including: 1) using scanning electron microscopy they visualized cells growing on Alvetex Scaffold; 2) cell viability and proliferation were assessed using a conventional MTS assay; 3) induction of osteogenic differentiation was monitored by staining for alizarin red; 4) gene expression was measured by quantitative real time PCR. The paper provides a good illustration of the versatility of Alvetex technology and its compatibility with a range of conventional analytical methods.
Jordan M. Spatz, Marc N. Wein, Jonathan H. Gooi, Yili Qu, Jenna L. Garr, Shawn Liu, Kevin J. Barry, Yuhei Uda, Forest Lai, Christopher Dedic, Mercedes Balcells-Camps, Henry M. Kronenberg, Philip Babij, Paola Divieti Pajevic
JOURNAL OF BIOLOGICAL CHEMISTRY JBC Papers in Press. Published on May 7, 2015 Manuscript M114.628313
Osric Forrest, Sarah Ingersoll, Marcela Preininger, Julie Laval, Milton Brown and Rabindra Tirouvanziam
Journal of immunology vol.194 (1 Supplement) 192.12. Published on May 1, 2015
F Caiazza, P M McGowan, M Mullooly, A Murray, N Synnott, N O’Donovan, L Flanagan, C J Tape, G Murphy, J Crown and M J Duffy
British Journal of Cancer, 8 April 2015 doi:10.1038
This paper describes an investigation into the action of an inhibitory cross-domain humanized monoclonal antibody known as D1(A12) targeting the matrix metalloproteinase, ADAM-17, in cultured human triple negative breast cancer cells. To create a more biologically relevant in vitro model, the authors have used Alvetex Scaffold to support three dimensional (3D) of the cancer cells. A selection of triple negative breast cancer cell lines were grown on Alvetex for up to 7 days. Data for the cell lines HCC1143 and HCC1937 are shown in the main paper. The investigators visualized the gross distribution and density of cells on Alvetex using Neutral Red staining. Treatment by the inhibitory antibody resulted in a noticeable reduction in Neutral Red staining that was quantified using ImageJ software. Discs of Alvetex stained by Neutral Red were subsequently fixed and processed for histological analysis. The structure of individual 3D cultured cancer cells was visualized by conventional haematoxylin and eosin (H&E) staining. This paper provides a good demonstration of how Alvetex technology can be used to support 3D growth of tumour cells during treatment with anti-cancer agents. It also uses alternative methods to visualize the 3D cultures.
Imogen Smith, Vasco Silveirinha, Jason L. Stein, Luis de la Torre-Ubieta, Jonathan A. Farrimond, Elizabeth M. Williamson and Benjamin J. Whalley
JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE Published online: 25 Feb 2015 doi: 10.1002/term.2001
This paper demonstrates the use of the Alvetex platform to culture 3D differentiated human neural stem cells. The cells formed spontaneously active, functional neuronal networks which were not seen in an otherwise comparable 2D culture system. The 3D neural networks responded reproducibly to pharmacological treatments revealing functional glutamatergic synapses. Further imaging analysis revealed a neuronal and glial population, where markers of maturity were apparent in the former. Microrarray analysis of the cultures of the 3D and 2D neuronal cultures showed substantial differences in the gene expression profile of genes coding for neuronal function, extracellular matrix and cytoskeleton. The authors conclude that culturing differentiated neural stem cells in Alvetex offer significant advantages over conventional 2D culture including cost savings and enhanced physiological relevance for pharmacological and toxicological assay used by neuroscientists.
Schammim Ray Amith, Jodi Marie Wilkinson, Larry Fliegel
27 January 2016
J Biochimie Open
2 (2016) 16-23 doi: 10.1016/j.biopen.2016.01.001 This paper describes methods allowing the investigation of Na+/H+ exchanger isoform 1(NHE 1) function, which is of particular relevance to drug resistance in triple-negative breast cancers. By promoting the general acidification of the extracellular environment, NHE1 activity results in increased degradation of extracellular matrix and facilitates cancer cell invasion. The authors used matrigel-coated Alvetex Scaffold in a 6-well insert format to assess whether the disruption of NHE1 expression in MDA-MB-231 cells would affect their invasive behaviour. After 7 days of Alvetex Scaffold 3D culture, MDA-MD-231 cells expressing wild-type NHE1 were present throughout the scaffold, as detected by histological staining of paraffin-embedded sections . NHE1-knock-out MDA-MB-23 cells, however, were predominantly present at the surface of the Alvetex Scaffold, with only occasional cells present within the depth of the substrate. By taking advantage of the thickness of Alvetex Scaffold, this report presents a straightforward assay which enables scientists to study cell invasion through a substantial depth of 3D substrate.
Ruslan I. Dmitriev, Sergey M. Borisov, Alina V. Kondrashina, Janelle M. P. Pakan, Ujval Anilkumar, Jochen H. M. Prehn, Alexander V. Zhdanov, Kieran W. McDermott, Ingo Klimant, Dmitri B. Papkovsky
Cellular and Molecular Life Sciences (2015) 72:367–381 doi:10.1007/s00018-014-1673-5 Published January 2015
Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce the novel anionic probe PA2 for the analysis of neural cells and neural tissues. PA2 efficiently stains rat brain slices and permits detailed O2 imaging experiments. In this study, brain slices were prepared and maintained as viable preparations on alvetex scaffolds. Analysis revealed age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O2 in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing oxygen levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O2 in 3D tissue models supported on Alvetex membranes.
Christa MacDonald, David Benjamin Finlay, Anower Jabed, Michelle Glass, E. Scott Graham Journal of Neuroscience Methods doi:10.1016/j.jneumeth.2014.09.006 Published 30 December 2014
In this study, the authors present a new method to generate biological samples that can be used as controls for in situ hybridisation (ISH) studies. Currently, ISH is limited due to inefficient probe penetration and loss of sample during preparation. In this paper researchers describe a novel approach using Alvetex Scaffold to create 3D tissue-like structures suitable for sectioning on a cryostat. These samples can subsequently be used as material for positive controls to verify gene expression by ISH. Cells are first transfected with the gene of interest, then cultured in 3D on Alvetex Scaffold, samples are then fixed, embedded and sectioned, and subsequently prepared for ISH using the riboprobe of interest. Sectioning increases probe penetration and further enhances the opportunity for good hybridisation. The authors demonstrated this technique using HEK cells transfected with CB1 and NeuN to optimise hybridisation stringency conditions. These conditions were then applied to samples of brain tissue analysed by ISH. This method can be adapted to generate positive controls for ISH for any gene of interest and it is especially useful where access to precious tissue is limited. In relation to Alvetex, this method further demonstrates the versatility of the material and its broad range of applications.
Gema Vallés, Fátima Bensiamar, Lara Crespo, Manuel Arruebo, Nuria Vilaboa, Laura Saldaña
doi: 10.1016/j.biomaterials.2014.10.028 Published online October 28, 2014
Valles and colleagues report on the role of the physical microenvironment in the modulation of signalling between mesenchymal stem cells and macrophages. Three dimensional culture of mesenchymal stem cells in Alvetex Scaffold stimulates the secretion of anti-inflammatory molecules that have a differential effect on co-cultured macrophages compared to cells grown in conventional cell culture on flat two dimensional substrates. The role of specific factors involved in this signalling process, namely IL-6 and MCP-1, was identified using immunological inhibition. In effect, the local inflammatory environment provided in 3D co-cultures induces a decrease in monocyte migration compared conventional monolayer cultures. These data highlight the importance of the three dimensional topography of the microenvironment in the regulation of paracrine factors and soluble-factor guided communication between two different cell populations.
Bruno Christian Koehler, Anna-Lena Scherr, Stephan Lorenz, Christin Elssner, Nicole Kautz, Stefan Welte, Dirk Jaeger, Toni Urbanik, Henning Schulze-Bergkamen
PLoS ONE 9(9): e106571. doi: 10.1371/journal.pone.0106571 Published September 05, 2014
Siwei Li, Bram G Sengers, Richard OC Oreffo and Rahul S Tare
Journal of Biomaterials Applications August 20, 2014 DOI: 10.1177/0885328214548604
This study investigates the chondrogenic expression profiles of human articular chondrocytes (HACs) and differentiated human bone-marrow-derived mesenchymal stem cells (MSCs) in a bid to assess their utility as cell-based therapy for cartilage repair following injury or degeneration. Both HACs and MSCs were cultured in Alvetex scaffold presented as 12-well inserts in 12-well plates, and compared with more conventional scaffold-free pellet cultures. Although both Alvetex-grown and pellet-grown MSCs cultures exhibited a high level of Col10a1 expression, a marker of hypertrophic differentiation which is only present at low level in HACs, Alvetex-grown cultures also showed expression of the chondrogenic markers Sox9, Aggrecan and Col2a1 at levels similar to that seen in HACS. Pellet-grown cultures, by comparison, failed to express such high levels of the same chondrogenic markers. The authors speculated that the stiffness and the high porosity of Alvetex might have aided the improved MSCs chondrogenic differentiation by giving physical cues and allowing improved solutes transport.
Wei-wei Hu, Zhe Wang, Shan-shan Zhang, Lei Jiang, Jing Zhang, Xiangnan Zhang, Qun-fang Lei, Hyun-Joo Park, Wen-jun Fang, Zhong Chen
Biomaterials 2014 doi: 10.1016/j.biomaterials.2014.05.026 Published 2 June 2014
Note: Hu and colleagues present work that describes the development and application of water-repellent fractal tripalmitin surfaces upon which they cultured primary rat astrocytes. Cells grown on these substrates form a more three dimensional in vivo-like phenotype. Such cell architecture was confirmed in comparison to the cells growing on Alvetex® Scaffold. In this case, Alvetex Scaffold was used as a form of positive control to create a three dimensional micro-environment in which the astrocytes could form in vivo-like morphologies. When cultured on Alvetex Scaffold, the astrocytes developed a more sophisticated structure: they formed more numerous and longer processes, they developed numerous filopodia extensions, and they increased cell-to-cell interactions. The paper also demonstrates the use of confocal microscopy to image the GFAP-immunostained astrocytes growing in Alvetex Scaffold.
Zuzana Pernicová, Eva Slabáková, Radek Fedr, Šárka Šimečková, Josef Jaroš, Tereza Suchánková, Jan Bouchal, Gvantsa Kharaishvili, Milan Král, Alois Kozubík and Karel Souček
Molecular Cancer 2014, 13:113
doi:10.1186/1476-4598-13-113 Published: 20 May 2014
Lara Stevanato and John D Sinden
Stem Cell Research & Therapy 2014, 5:49
doi:10.1186/scrt437 Published: 11 April 2014
Ruslan I. Dmitriev, Alina V. Kondrashina, Klaus Koren, Ingo Klimant, Alexander V. Zhdanov, Janelle M. P. Pakan, Kieran W. McDermott and Dmitri B. Papkovsky
Biomaterials Science (28 Jan 2014) doi: 10.1039/c3bm60272a
Faye F. Liu, Cheng Peng, Beate I. Escher, Emmanuelle Fantino, Cindy Giles, Stephen Were, Lesley Duffy, Jack C. Ng Journal of Hazardous Materials Volume 261, 15 October 2013, Pages 701-710. doi:10.1016/j.jhazmat.2013.01.027
The usability of 2D in vitro models of toxicity following chronic air exposure to volatile chemicals is greatly limited, either by the lack of viability of air-exposed cells grown in 2D substrates or by the interference of diffusion effects in medium-submerged 2D cultures. In this study, the authors tested two 3D models of in vitro air exposure, by growing the human lung cell line A549 in hanging drops and in Alvetex Scaffold, which both provide a better nutrient support with a small air-liquid diffusion barrier.
When comparing 2D cells periodically exposed to air to 3D Alvetex-grown cells exposed to air via their apical surface, the Alvetex-grown cells were three times less sensitive to benzene after 24h exposure, as assessed by MTS viability assay. They were also less sensitive to low doses of benzene (0.25mL/mL) when compared to 3D hanging drop cultures. The authors speculated that this apparent greater resistance to benzene might in part be due to the robust cell growth in Alvetex Scaffold.
This report introduces a novel application for Alvetex Scaffold by taking advantage of the capillary action exerted by its porous structure to maintain cell viability at the air-liquid interface.
Bruno Christian Koehler, Anna-Lena Scherr, Stephan Lorenz, Toni Urbanik, Nicole Kautz, Christin Elssner, Stefan Welte, Justo Lorenzo Bermejo, Dirk Jäger, Henning Schulze-Bergkamen
Public Library of Science. PLoS ONE 8(10): e76446. doi:10.1371/journal.pone.0076446 Published: October 3, 2013
Alaadin Alayoubi, Saeed Alqahtani, Amal Kaddoumi, Sami Nazzal
The AAPS Journal (August 2013) doi: 10.1208/s12248-013-9525-z
Sheena Pinto, Chloé Michela, Hannah Schmidt-Glenewinkel, Nathalie Harder, Karl Rohr, Stefan Wild, Benedikt Brorse, and Bruno Kyewskia,
PNAS. 26 August 2013. doi: 10.1073
Ruchi Sharma, Safia Z. Barakzai, Sarah E. Taylor, F. Xavier Donadeu
Journal of Tissue Engineering and Regenerative Medicine. First published online 30 July 2013. doi: 10.1002/term.1788
Jessica M. Stiles, Clarissa Amaya, Steven Rains, Dolores Diaz, Robert Pham, James Battiste, Jaime F. Modiano, Victor Kokta, Laura E. Boucheron, Dianne C. Mitchell, Brad A. Bryan
Public Library of Science. March 2013 | Volume 8 | Issue 3 | e60021 PLoS ONE 8(3): e60021. doi:10.1371/journal.pone.0060021
C Dähn, N Hewitt, D Maltman, G Talas, S Przyborski, S Heinz, A Nörenberg, K Scheller, J Braspenning
Z Gastroenterol, 2012; 50 – P2_05 doi: 10.1055/s-0031-1295802
Alexandra Burkard, Caroline Dähn, Stefan Heinz, Anne Zutavern, Vera Sonntag-Buck, Daniel Maltman, Stefan Przyborski, Nicola J. Hewitt, and Joris Braspenning
Xenobiotica, 2012, doi: 10.3109/00498254.2012.675093
S Galavotti, S Bartesaghi, D Faccenda, M Shaked-Rabi, S Sanzone, A McEvoy, D Dinsdale, F Condorelli, S Brandner, M Campanella, R Grose, C Jones, P Salomoni
Oncogene, 2012, doi: 10.1038/ocn.2012.111
Schutte, M., Fox. B., Baradez, M., Devonshire, A., Minguez., J., Bokhari. M., Przyborski. S., Marshall. D., (2011)
Assay and Drug Development Technologies doi: 10.1089/adt.2011.0371
Rajan. N., Elliott. R., Clewes. O., Mackay. A., Reis-Filho. J.S., Burn. J., Langtry. J., Sieber-Blum. M., Lord. C.J., Ashworth. A., (2011)
Oncogene doi: 10.1038/onc.2011.133
Neofytou. E.A., Chang. E., Patlola. B., Joubert. L.M., Rajadas. J., Gambhir. S.S., Cheng. Z., Robbins. R.C., Beygui. R.E., (2011)
Journal Biomedical Materials Research Part A doi: 10.1002/jbm.a.33113
Knight, E., Murray, B., Carnachan, R., Przyborski, S.A., (2011).
Methods in Molecular Biology, 695, 323-40.
Toxicology In Vitro, Volume 24, Issue 7, October 2010, Pages 1962-1970. doi:10.1016/j.tiv.2010.08.007
Hepatotoxicity is an important aspect of pharmaceutical drug safety and a major cause of attrition in the drug discovery pipeline. In order to correctly detect and validate early gene expression changes indicative of hepatic cell stress, the authors have evaluated a panel of routinely-used reference genes, before monitoring gene expression changes in both the hepatocarcinoma cell line HepG2 and primary rat hepatocytes following treatment with acetaminophen (APAP).
By comparing the response of primary rat hepatocytes grown in both conventional 2D tissue culture plastic and in 3D Alvetex Scaffold to 4mM APAP exposure, similar stress markers were detected to be differentially regulated in 3D versus 2D, although expression levels of Jun and Myc were up-regulated to a different extent in both culture systems.
This study demonstrates the feasibility of Alvetex 3D culture for mRNA extraction and gene expression analysis to study early changes associated with drug exposure in hepatocytes.
Maltman, D., Przyborski, S.A., (2010)
Biochemical Society Transactions, 38(4), 1072-5
Carnachan, R.J., Bokhari, M., Maatta, A., Cameron, N.R., Przyborski, S.A. (2008).
American Chemical Society, Division of Polymer Chemistry, 49, 418-419.
Bokhari, M., Carnachan, R., Cameron, N.R., Przyborski, S.A. (2007).
Journal of Anatomy, 211, 567-76.
Bokhari, M., Carnachan, R., Przyborski, S.A., Cameron, N.R. (2007).
Journal of Materials Chemistry, 17, 4088-4094.
Bokhari, M., Carnachan, R., Cameron, N.R., Przyborski, S.A. (2007).
Biochemical and Biophysical Research Communications, 354, 1095-1100.
Carnachan, R.J., Bokhari, M., Przyborski, S.A., Cameron, N.R. (2006).
Soft Matter, 2, 608-616.
Barbetta, A., Carnachan, R.J., Smith, K.H., Zhao, C., Cameron, N.R., Kataky, R., Hayman, M., Przyborski, S.A., Swan, M. (2005).
Macromolecular Symposia, 226, 203-211.
Hayman, M.W., Smith, K.H., Cameron, N.R., Przyborski, S.A. (2005).
Journal of Biochemical and Biophysical Methods, 62, 231-240.
Hayman, M.W., Smith, K.H., Cameron, N.R., Przyborski, S.A. (2004).
Biochemical and Biophysical Research Communications, 314, 483-488.