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

Ivanov, Delyan P.; Parker, Terry L.; Walker, David; Alexander, Cameron; Gellert, Paul; Garnett, Martin C.

doi:10.1016/j.jbiotec.2015.01.002

Cited by 55 publications

(48 citation statements)

References 70 publications

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“…Differentiation in spheroid culture includes not only conservation of morphogenic capacities and histotypic reorganization but also maintenance of functional activities and gene expression patterns. [19][20][21][22][23][24] Traditional methods for tumor spheroid generation include hanging drop, 25 liquid overlaying, spinner flasks, 26 rotary cell culture systems, 27 poly-2-hydroxyethyl methacrylate, low binding plates, 28 gel/ matrix based culture, 29,30 microencapsulation, 31 polymeric scaffolds, 30,32 and micropatterned plates. 8,[33][34][35] However, these methods are frequently associated with poor reproducibility, scalability, adaptability, and lack high-throughput formats amendable to automation and drug screening.…”

Section: Introductionmentioning

confidence: 99%

Generation of Homogenous Three-Dimensional Pancreatic Cancer Cell Spheroids Using an Improved Hanging Drop Technique

Ware

Colbert

Keshishian

et al. 2016

Tissue Engineering Part C: Methods

134

101

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In vitro characterization of tumor cell biology or of potential anticancer drugs is usually performed using tumor cell lines cultured as a monolayer. However, it has been previously shown that three-dimensional (3D) organization of the tumor cells is important to provide insights on tumor biology and transport of therapeutics. Several methods to create 3D tumors in vitro have been proposed, with hanging drop technique being the most simple and, thus, most frequently used. However, in many cell lines this method has failed to form the desired 3D tumor structures. The aim of this study was to design and test an easy-to-use and highly reproducible modification of the hanging drop method for tumor sphere formation by adding methylcellulose polymer. Most pancreatic cancer cells do not form cohesive and manageable spheres when the original hanging drop method is used, thus we investigated these cell lines for our modified hanging drop method. The spheroids produced by this improved technique were analyzed by histology, light microscopy, immunohistochemistry, and scanning electron microscopy. Results show that using the proposed simple method; we were able to produce uniform spheroids for all five of the tested human pancreatic cancer cell lines; Panc-1, BxPC-3, Capan-1, MiaPaCa-2, and AsPC-1. We believe that this method can be used as a reliable and reproducible technique to make 3D cancer spheroids for use in tumor biology research and evaluation of therapeutic responses, and for the development of bio-artificial tissues.

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Section: Introductionmentioning

confidence: 99%

Generation of Homogenous Three-Dimensional Pancreatic Cancer Cell Spheroids Using an Improved Hanging Drop Technique

Ware

Colbert

Keshishian

et al. 2016

Tissue Engineering Part C: Methods

134

101

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“…If purchased as resazurin powder and prepared in-house, the assay is thousands of times cheaper than the ATP-based kits. The resazurin assay does not kill or lyse the spheroids and the latter can be harvested for dissociation and cell counts, flow cytometry or histology 4 h after Resazurin addition (1,19). Its main limitation compared to the ATPassay is that both resazurin and the fluorescent resorufin need to distribute throughout the spheroid and back into the medium to be quantified.…”

Section: Ic50mentioning

confidence: 99%

High-Throughput Spheroid Screens Using Volume, Resazurin Reduction, and Acid Phosphatase Activity

Ivanov

Grabowska

Garnett

2017

Methods in Molecular Biology

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. (2017) High-throughput spheroid screens using volume, resazurin reduction and acid phosphatase activity. In: Cell Viability Assays. Methods in Molecular Biology, 1601 . Humana Press (Springer), pp. [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/43565/1/AAM%20Ivanov_D_Methods%20in%20Mol %20Biology%20Vol%20Res%20APH%203D_Springer.pdf Copyright and reuse:The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions. This article is made available under the Creative Commons Attribution licence and may be reused according to the conditions of the licence. For more details see: http://creativecommons.org/licenses/by/2.5/ A note on versions:The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the repository url above for details on accessing the published version and note that access may require a subscription. volume, metabolism and enzymatic activity. We provide two ImageJ macros for automated spheroid size determination -for both single images and for images in stacks. We also share an Excel template spreadsheet allowing users to rapidly process spheroid size data, analyse plate uniformity (such as edge effects and systematic seeding errors), detect outliers and calculate dose-response.The methods would be useful to researchers in preclinical and translational research planning to move away from simplistic monolayer studies and explore 3D spheroid screens for drug safety and efficacy without substantial investment in money or time.

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“…Ara-C: cytosine β-D-arabinofuranoside; CXCR4: CXC-motif-chemokine receptor 4; PERK: pancreatic endoplasmic reticulum kinase; EGF: epidermal growth factor; GBM: glioblastoma multiforme; GM-CSF: granulocyte-macrophage colony stimulating factor; GSCs: glioblastoma stem cells; GRK6: g-protein coupled receptor kinase 6; HA: hyaluronic acid; HAMA: methacrylated HA; HGF: hepatocyte growth factor; IDH1: isocitrate dehydrogenase 1; IFO: ifosfamide; MB: medulloblastoma; MMP: matrix metalloproteinase; PEG: polyethylene glycol; PDGFR: platelet-derived growth factor receptor; PI3-K: phosphoinositide 3'Kinase; RGD: l-arginine, glycine, and L-aspartic acid; SAHA: suberoylanilide hydroxamic acid (or vorinostat a HDACi); SPARC: secreted protein acidic and rich in cysteine; Src: rous sarcoma kinase; TMZ: temozolomide; VEGF: vasculature endothelial growth factor; 2D/3D: two dimensional/three dimensional sensors [54] ) and it should have a high-throughput potential.…”

Section: Cellsmentioning

confidence: 99%

“…A milestone in this context was the generation of spheroid cultures in 96 or even 384 well format from primary brain tumors that allowed the parallel testing or large sample sizes. [54][55][56] In these studies, diagnostic dyes and fluorescent proteins were used individually or in combination for probing cellular functions on the one hand and for discriminating specific cell populations on the other hand. A general protocol describing the reproducible establishment and microscopy-based analysis of spheroid cultures using fluorescent protein quantification in high throughput was described recently.…”

Section: D and 3d Model Systems In Primary Brain Tumor Researchmentioning

confidence: 99%

“…Using different combination of dyes to separate subpopulation of cells grown in co-culture combined with diagnostic flow cytometry and two-photon microscopy allowed to further refine the selective output of 3D methods. [54] However, high-throughput capabilities and accuracy of a selected read-out has to be carefully balanced and discriminating phenotypic differences at single cell level in 3D cultures in high throughput remains a formidable challenge.…”

Section: D and 3d Model Systems In Primary Brain Tumor Researchmentioning

confidence: 99%

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Dissecting brain tumor growth and metastasis in vitro and ex vivo

Grotzer¹,

Neve²,

Baumgärtner³

2016

JCMT

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Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system (CNS) tumors and greatly influence mortality and therapy-induced longterm morbidity in survivors. A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes, to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness. These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments. However, the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel, physiologically more relevant assay systems including organotypic brain slice culture. In this review, using publicly available peer-reviewed primary research and review articles, we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors. Furthermore, we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors. We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors and facilitate the development of more efficacious anti-metastatic treatments. Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system (CNS) tumors and greatly influence mortality and therapy-induced long-term morbidity in survivors. A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes, to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness. These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments. However, the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel, physiologically more relevant assay systems including organotypic brain slice culture. In this review, using publicly available peer-reviewed primary research and review articles, we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors. Furthermore, we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors. We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors ...

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

Cited by 55 publications

References 70 publications

Generation of Homogenous Three-Dimensional Pancreatic Cancer Cell Spheroids Using an Improved Hanging Drop Technique

Generation of Homogenous Three-Dimensional Pancreatic Cancer Cell Spheroids Using an Improved Hanging Drop Technique

High-Throughput Spheroid Screens Using Volume, Resazurin Reduction, and Acid Phosphatase Activity

Dissecting brain tumor growth and metastasis in vitro and ex vivo

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