Resazurin-based Assay Research Articles

Evaluation of Resins for Stereolithographic 3D-Printed Surgical Guides: The Response of L929 Cells and Human Gingival Fibroblasts.

Additive manufacturing is becoming increasingly important in dentistry for the production of surgical guides. The development of cost-effective desktop stereolithography (SLA) printing systems and the corresponding resins makes this novel technique accessible to dental offices and dental laboratories. The aim of the study was to reveal the response of soft tissue cells to Clear and Dental SG resins used in desktop SLA printing systems at different stages of processing. Cell activity of L929 cells and gingival fibroblasts (GF) in response to the materials was examined in indirect and direct monolayer culture models and a direct spheroid culture model based on MTT, resazurin-based toxicity assays, and live-dead staining. Overall we found that the impact of Clear and Dental SG resins on L929 and GF depends on the processing stage of the materials. Liquid Clear resin induced a stronger reduction of cell activity compared to Dental SG resin. Printing and postcuring reduced the impact on cell activity and viability. As in-house 3D printing for surgical guides is getting integrated in the digital workflow, our data suggest that careful adherence to processing guidelines—especially postcuring—is of clinical relevance.

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-α) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-α) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Screen for genes involved in radiation survival of Escherichia coli and construction of a reference database

A set of 3907 single-gene knockout (Keio collection) strains of Escherichia coli K-12 was examined for strains with increased susceptibility to killing by X- or UV-radiation. After screening with a high-throughput resazurin-based assay and determining radiation survival with triplicate clonogenic assays, we identified 76 strains (and associated deleted genes) showing statistically-significant increased radiation sensitivity compared to a control strain. To determine gene novelty, we constructed a reference database comprised of genes found in nine similar studies including ours. This database contains 455 genes comprised of 103 common genes (found 2–7 times), and 352 uncommon genes (found once). Our 76 genes includes 43 common genes and 33 uncommon (potentially novel) genes, i.e., appY, atoS, betB, bglJ, clpP, cpxA, cysB, cysE, ddlA, dgkA, dppF, dusB, elfG, eutK, fadD, glnA, groL, guaB, intF, prpR, queA, rplY, seqA, sufC,yadG, yagJ, yahD, yahO, ybaK, ybfA, yfaL, yhjV, and yiaL. Of our 33 uncommon gene mutants, 4 (12%) were sensitive only to UV-radiation, 10 (30%) only to X-radiation, and 19 (58%) to both radiations. Our uncommon mutants vs. our common mutants showed more radiation specificity, i.e., 12% vs. 9% (sensitive only to UV-); 30% vs. 16% (X-) and 58% vs. 74% (both radiations). Considering just our radiation-sensitive mutants, the median UV-radiation survival (75Jm−2) for 23 uncommon mutants was 6.84E-3 compared to 1.85E-3 for 36 common mutants (P=0.025). Similarly, the average X-radiation survival for 29 uncommon mutants was 1.08E-2, compared to 6.19E-3 for 39 common mutants (P=0.010). Comparing gene functions using MultiFun terms, uncommon genes tended to show less involvement in DNA repair-relevant categories (information transfer and cell processes), but greater involvement in seven other categories. Our analysis of 455 genes suggests cell survival and DNA repair processes are more complex than previously understood, and may be compromised by deficiencies in other processes.

Development of Respiratory Mucosal Irritation and Toxicity Screening Methods: Comparison of MTT and Colorimetric Resazurin-Based Assay

Development of Respiratory Mucosal Irritation and Toxicity Screening Methods: Comparison of MTT and Colorimetric Resazurin-Based Assay

Quantifying implant-associated biofilms: Comparison of microscopic, microbiologic and biochemical methods

Biofilm-associated infections pose severe problems in modern implant medicine. Screening for new implant materials with antibacterial properties requires reliable quantification of colonizing bacteria. There are many different methods to quantify biofilms on solid surfaces in vitro, employing different (bio-)chemical/microbiological reference parameters. It is therefore difficult to compare studies with different quantification techniques. Here, we have evaluated commonly used microscopic, microbiologic and biochemical methods to quantify bacterial biofilms, in order to clarify their comparability and applicability. Two bacterial species frequently involved in biofilm-associated infections, Staphylococcus aureus and Aggregatibacter actinomycetemcomitans, were used as model organisms; their initial adhesion and biofilm formation on titanium and on antibacterial copper were analyzed using the following methods: LIVE/DEAD fluorescence staining and confocal laser-scanning microscopy, ultrasonic or a newly developed enzymatic detachment followed by standard plate counting (CFU method), a resazurin-based assay, the BacTiter-Glo™ assay and crystal violet staining. The methods differed greatly in complexity, reliability and the applicability to initial adhesion and biofilm formation. To screen biofilm formation on a multitude of surfaces, the resazurin-based and the BacTiterGlo™ assay are well suited. LIVE/DEAD staining and confocal laser-scanning microscopy can be applied for a more detailed analysis of both, initial adhesion and biofilm formation. When using the CFU method for screening purposes, the introduced enzymatic detachment procedure is to be favored over ultrasonic detachment. There is not one single method, which is suitable for all purposes. The appropriate biofilm quantification method has to be chosen on the basis of the specific scientific question.

Amphipathic β2,2-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae

We demonstrate in the current work that small cationic antimicrobial β2,2-amino acid derivatives (Mw < 500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (< 5 μM, i.e. < 3.4 μg/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the β2,2-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these β2,2-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic β2,2-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-to-treat intracellular pathogen.

Abstract 117: Development of a Hybrid Cryogel-coated Prosthetic Vascular Graft for Delivery of Targeted Gene Therapies

Objectives: Anastomotic neo-intimal hyperplasia (ANIH) remains a limiting factor in the long-term success of prosthetic vascular grafts. Understanding of the molecular mechanisms involved in the host response to prosthetic material has set the stage for using this material to deliver siRNA and other modulators of the ANIH-associated transcriptome response, along with antithrombotic agents. To create practical and effective drug delivery from a prosthetic material, we have combined a platform of polyethylene terephthalate (PET) fabric with an applied cryogel carrying the biologic agents, resulting in a bioactive prosthetic graft system (BPGS). Methods: Hybrid grafts were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun, knitted, or woven PET. Arg-Gly-Asp peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Human aortic endothelial cell (HAoEC) adherence and proliferation were assessed with a resazurin-based assay, and confocal microscopy was used to visualize cell interaction with the graft material. Heparin activity of the material in buffer solution was measured using an anti-Xa assay. Results: SEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. HAoEC incorporated, adhered, and proliferated over 7 days for all materials (Figure 1). Anti-Xa assay confirmed continued activity of heparin from all grafts over 7 days. Conclusion: We have created a BPGS with a biocompatible cryogel polymer coating that allows for cell adherence and growth, with antithrombotic activity. This system allows application of the gel and biologic agents to PET prior to implantation, and provides flexibility in combining bioengineering and targeted gene therapy approaches to create an improved prosthetic graft adaptable to evolving strategies.

Aspiration, but not injection, decreases cultured equine mesenchymal stromal cell viability.

BackgroundRecently, equine multipotent mesenchymal stromal cells (MSC) have received significant attention as therapy for various conditions due to their proposed regenerative and immune-modulating capacity. MSC are commonly administered to the patient through a hypodermic needle. Currently, little information is available on the effect of such injection has on equine MSC immediate and delayed viability. We hypothesize that viability of equine MSC is not correlated with needle diameter during aspiration and injection.ResultsUsing a 3 mL syringe, manual injection of equine cord blood (CB) or bone marrow-derived (BM) MSC with no needle and needles ranging in size from 18 to 30 Ga did not affect immediate MSC viability. Similarly, 24 h post-injection, MSC delayed viability was not different between any of the tested needles as determined by a resazurin-based proliferation assay. Using a 3 mL syringe, aspiration of MSC through 20, 25, and 30 Ga needles resulted in significant decreases in immediate viability with no change in delayed viability when compared to aspiration without a needle. BM- and CB-MSC were observed to be of similar size with a diameter ± SD of 19.8 ± 2.7 and 20.4 ± 2.2 μm, respectively. In comparison, the smallest needles, (30 Ga) have an internal diameter of 160 μm.ConclusionsFollowing injection, needle diameter did not affect immediate or delayed viability of equine MSC. Following aspiration through needles sizes 20 Ga and smaller, immediate viability, but not delayed viability, decreased. As a result, an 18 Ga or larger needle should be utilized for aspiration of cell suspensions. In contrast, needle selection for MSC injection should be based on clinical preference and experience rather than concerns over decreasing MSC viability.

Cytotoxicity and apoptotic gene expression in an in vitro model of the blood–brain barrier following exposure to poly(butylcyanoacrylate) nanoparticles

Background Poly(butylcyanoacrylate) (PBCA) nanoparticles (NPs) loaded with doxorubicin (DOX) and coated with polysorbate 80 (PS80) have shown efficacy in the treatment of rat glioblastoma. However, cytotoxicity of this treatment remains unclear.Purpose The purpose of this study was to investigate cytotoxicity and apoptotic gene expression using a proven in vitro co-culture model of the blood–brain barrier.Methods The co-cultures were exposed to uncoated PBCA NPs, PBCA-PS80 NPs or PBCA-PS80-DOX NPs at varying concentrations and evaluated using a resazurin-based cytotoxicity assay and an 84-gene apoptosis RT-PCR array.Results The cytotoxicity assays showed PBCA-PS80-DOX NPs exhibited a decrease in metabolic function at lower concentrations than uncoated PBCA NPs and PBCA-PS80 NPs. The apoptosis arrays showed differential expression of 18 genes in PBCA-PS80-DOX treated cells compared to the untreated control.Discussion As expected, the cytotoxicity assays demonstrated enhanced dose-dependent toxicity in the DOX loaded NPs. The differentially expressed apoptotic genes participate in both the tumor necrosis factor receptor-1 and mitochondria-associated apoptotic pathways implicated in current DOX chemotherapeutic toxicity.Conclusion The following data suggest that the cytotoxic effect may be attributed to DOX and not the NPs themselves, further supporting the use of PBCA-PS80 NPs as an effective drug delivery vehicle for treating central nervous system conditions.

Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents

To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z′ factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications.

Abstract B88: Mechanisms of resistance to the Hsp90 inhibitor ganetespib in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC), which accounts for 10-17% of all breast cancers, is challenging to treat due to its heterogeneity and the absence of identified targeted therapies. Heat shock protein 90 kDa (Hsp90) is a molecular chaperone protein essential for regulating the stability and activity of several client proteins required for oncogenesis, and selective Hsp90 inhibitors have shown anti-cancer activity in multiple cancer models including TNBC. Ganetespib, a second generation Hsp90 inhibitor, has shown promising clinical activity in TNBC patients but responders develop acquired resistance. Here, we generated an in vitro model of ganetespib-resistant TNBC cells and investigated the mechanisms of resistance to ganetespib in TNBC using a 96-well based small molecule screen. Methods: Ganetespib treatment sensitivity in a panel of seven TNBC cell lines (HS578T, HCC1143, HCC1937, MDA-MB-468, MDA-MB-231, MDA-MB-453 and BT20) was assessed using a resazurin-based cell proliferation assay. HS578T cells were incubated in a high concentration of ganetespib (&amp;gt;IC50) to generate ganetespib-resistant clones. The effects on Hsp90 oncogenic client proteins (AKT and EGFR) and Hsp70 were determined by western blotting. Cross resistance to other Hsp90 inhibitors was also assessed. 96-well based small molecule screen with a library of 326 bioactive small molecule inhibitors was performed on parental HS578T and ganetespib-resistant clones in the absence and presence of ganetespib. Resazurin-based cell proliferation assay was used as an end point. Positive hits were identified as log10(percentage of proliferation in parental/resistant) ≥ 2SD. Results: TNBC cell lines showed differential sensitivity to ganetespib (IC50 range: 4.75 to 20.16 nM), with HS578T being the most sensitive and HCC1143 the most resistant. Independent ganetespib-resistant HS578T clones (CR2 and CR3) were successfully generated by incubating parental HS578T cells in ganetespib (30 nM) for 42 days. IC50 values for the ganetespib-resistant clones (CR2: 15.57 ± 1.90 nM and CR3: 20.28 ± 2.75 nM) were 3 to 4-fold higher than the parental HS578T cells (IC50: 4.79 ± 0.32 nM). The client proteins were not further down-regulated after ganetespib treatment in the ganetespib-resistant clones compared to the parental HS578T cells. Resistant clones also showed cross resistance to 1st generation (17-AAG) and 2nd generation (NVP-AUY922) Hsp90 inhibitors. The small molecule screen was reproducible as the independent runs and duplicates strongly correlated (R2 &amp;gt;0.7). The ganetespib-resistant CR3 clones showed selective sensitivity to 11/326 compounds (Z score ≥ 2), only in the presence of ganetespib when compared to the parental HS578T cells, most notable being LY2784544, a JAK2 inhibitor. Conclusion: Ganetespib-resistant HS578T cells provide a promising model to study the resistance mechanisms to ganetespib in TNBC. Our current data suggest that TNBC cells are responsive to JAK2 inhibition in the presence of ganetespib despite prior acquisition of resistant to ganetespib. Activation of a JAK2/STAT3 survival pathway may be a potential resistance mechanism to Hsp90 inhibition and combined Hsp90 and JAK2 inhibition could potentially be a useful therapeutic strategy for TNBC. Citation Format: Nuramalina H. Mumin, Sivan Bokobza, Fiona Cahill, Neele Drobnitzky, Yanyan Jiang, Luiza Madia Lourenco, Aoife Vaughan, Anika Weber, Anthony Kong, Anderson Ryan. Mechanisms of resistance to the Hsp90 inhibitor ganetespib in triple negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B88.

In vitro antileishmanial activity of Brazilian plant species

Leishmaniasis is an infectious disease still widely spread in Brazil. It is a public health problem for the country due to its magnitude and geographic expansion leading to a complex, expensive and laborious control system [1, 2]. There is an urgent need to search for new medicines to treat leishmaniasis. Natural products can provide unlimited opportunities for the discovery of new lead compounds targeting this neglected disease. In this work, we investigated the antileishmanial activity of 22 plant extracts, from seven different species belonging to Apocynaceae, Asteraceae, Ebenaceae, Primulaceae and Rutaceae families. Samples were evaluated against promastigotes from L. infantum (strain BH46). The antileishmanial activity was measured 24h after exposition to the sample using the resazurin-based viability assay [3]. The toxicity of test samples is currently being investigated using dye exclusion method for cell viability using non-infected macrophages. According to preliminary results, four extracts from Aspidosperma spp. showed antileishmanial activity. The dichloromethane (DCM) extract from the stem showed an IC50 value of 89.5 ± 4.9 µg/mL. The IC50 of the remaining active extracts is currently being determined. The antileishmanial activity of this species will be further investigated, using intra- and extracellular parasite forms from other Leishmania species. The metabolic profiling and targeted isolation of bioactive constituents are in progress aiming for the identification of new lead compounds with antileishmanial activity.

A First Approach to Evaluate the Cell Dose in Highly Porous Scaffolds By Using a Nondestructive Metabolic Method

Background:In cell-based therapies, in vitro studies on biomimetic cell–scaffold constructs can facilitate the determination of the cell dose, a key factor in guaranteeing the effectiveness of the treatment. However, highly porous scaffolds do not allow a nondestructive evaluation of the cell number. Our objective was to develop a nondestructive method for human mesenchymal stem cells dose evaluation in a highly porous scaffold for bone regeneration.Materials & measurement method:Proliferation trend of human mesenchymal stem cells on Biocoral® scaffolds was measured by a resazurin-based assay here optimized for 3D cultures. The method allows to noninvasively follow the cell proliferation on biocorals over 3 weeks with very high reproducibility.Conclusion:This reliable method could be a powerful tool in cell-based therapies for cell dose determination.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

Bronchopneumonia caused by Rhodococcus equi is an important disease of young horses throughout the world. Although early diagnosis and treatment improves the prognosis, this also increases the amount of antimicrobial usage and therefore increases the likelihood of resistance developing. The aim of this study was to determine the level of resistance to commonly prescribed antimicrobial agents of 97 virulent Rhodococcus equi isolates. Analysis of archived samples. Virulent Rhodococcus equi isolates were collected between 1991 and 2014 from clinically affected horses and from air samples collected in the breathing zone of foals. Antimicrobial susceptibility of these isolates was assessed using a disc diffusion assay with a panel of agents. The minimum inhibitory concentration (MIC) was determined for rifampicin, erythromycin, tetracycline, and neomycin using a novel resazurin-based microtitre assay. Resistance to rifampicin was detected in 3 of the isolates (2 collected in 2013 and one in 2014) by both methods. The MIC for these isolates was 64 μg/ml (n = 2) and 16 μg/ml (n = 1). All isolates collected prior to 2013 had MICs<0.125 μg/ml, which was the limit of detection in this assay. Although no isolates were resistant to tetracycline, there was a general increase in MIC in isolates collected in recent years. No isolates were resistant to either neomycin or erythromycin, with MIC values ranging between 0.25 and 2 μg/ml for neomycin and 0.125-1 μg/ml for erythromycin. The success of the macrolide-rifampicin combination relies on the synergistic action of these 2 agents. Resistance to rifampicin will reduce the therapeutic efficacy of this treatment. It is of serious concern that the resistant isolates were all recently collected. Hopefully, recent research will lead to fewer asymptomatic foals receiving antimicrobials which will in turn reduce the likelihood of ongoing development of resistance. Ethical animal research: All organisms in this study were received by the laboratory from diagnostic accessions. Sources of funding: Funding for the study was provided by Rural Industries Research and Development Corporation and the University of Melbourne. G. Herbert was the recipient of a RN McCarthy scholarship from the Faculty of Veterinary Science. Competing interests: None declared.

A comparative study of colorimetric cell proliferation assays in immune cells.

Cell proliferation assays are basic and essential techniques for assessing cellular function. Various colorimetric assays, such as MTT-, WST-1-, and resazurin-based assays, are available; however, studies directly comparing the suitability of each method for immune cell proliferation are scarce. Thus, we aimed to determine the best reagent and its optimal conditions based on variables such as cell number range, stimulation dose, kinetics, and compatibility with the cell division assay using CFSE fluorescence dye which is able to directly monitor divided cells by flow cytometry. In the absence of stimulation, MTT solubilized with SDS (MTT-SDS) and resazurin appeared to accurately reflect the cell numbers in a linear fashion. On the other hand, WST-1 exhibited a higher stimulation index following strong stimulation, whereas MTT-SDS and resazurin exhibited a better sensitivity to weak stimulation. A longer duration for stimulation did not necessarily increase sensitivity. CFSE staining revealed incremental cell division in response to anti-CD3 antibody stimulation in a dose-dependent manner. The cell numbers indirectly estimated from cell division profiles were consistent with the dose-response curve in the absorbance of MTT-SDS and resazurin. The absorbance does not increase before cell division, irrespective of T cell activation status, suggesting that these reagents reflect the cell number but not the cellular volume. Collectively, resazurin and MTT-SDS seem to be more reliable than others, and thus appear applicable in various conditions for the immune cell experiments.

Abstract 5136: The Flint Animal Cancer Center (FACC) canine tumor cell line panel: A resource for comparative and translational oncology

Abstract Purpose: Mammalian cell tissue culture has been a critical tool leading to our current understanding of cancer including many aspects of cellular transformation, growth, and response to therapies. The current use of large panels of human cancer cell lines (i.e. NCI60, GDSC) with associated phenotypic and genotypic information allows for informatics approaches and in silico screens to rapidly test hypotheses based on simple as well as complex relationships. We have assembled a panel of canine cancer cell lines to facilitate translational studies in canine cancer and report here the characteristics and some applications of this panel in comparative oncology. Methods: Canine cancer cell lines isolated from tumors have been accumulated from a number of sources over the years. Our current panel consists of 29 cell lines that have been validated as being of canine origin and “fingerprinted” by microsatellite analysis. The tumor types represented include 10 osteosarcomas, 5 melanomas, 2 mammary carcinomas, 1 hemangiosarcoma, 2 bladder carcinomas, 4 lymphoma/leukemias, 1 mast cell tumor, 2 histiocytic sarcomas, 1 thyroid carcinoma and 1 soft-tissue sarcoma. Drug sensitivity analysis was carried out using a resazurin-based bioreductive fluorometric assay. Gene expression microarray analysis was carried out using the Affymetrix GeneChip Canine 2.0 at the Genomics and Microarray Core (University of Colorado Cancer Center). Results: Drug sensitivity to the cytotoxic chemotherapeutic agents carboplatin, cisplatin, doxorubicin, lomustine, paclitaxel and vinblastine were assessed and compared to responses in the human NCI-60 panel. Results show that the human and canine cell lines show similar ranges of sensitivity to these agents. The mean GI50 values for the human and canine cell lines are significantly different for DOX, VBL, CARBO, CCNU as well as PTX. The variances in the data were also significantly different for CARBO and PTX. Overall, however, the drug sensitivity/resistance patterns in the canine and human tumor cell line panels shared general trends with regard to sensitivity and variance observed to the specific agents with the mean Log GI50 values for each agent showing a cross-species correlation (r = 0.88, p = 0.0194, Pearson). Cluster analysis of the gene expression data using the top 100 most variant genes as well as the 100 top most variant cancer genes largely separates the panel into groups with similar histiotypes. Cluster analysis of cancer genes also highlights alterations in gene expression that may contribute to pathogenesis in some cell lines such as loss of the PTEN tumor suppressor or elevated expression of receptor tyrosine kinase genes. Conclusions: The FACC canine tumor cell line panel represents a validated panel of representative cancers from dogs that can be utilized for comparative oncology studies as well as applications in drug development where use of a companion animal model is appropriate. Citation Format: Daniel L. Gustafson, Jared S. Fowles, Douglas H. Thamm, Rodney L. Page, Dawn L. Duval. The Flint Animal Cancer Center (FACC) canine tumor cell line panel: A resource for comparative and translational oncology. [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 5136. doi:10.1158/1538-7445.AM2015-5136

Increased Cytotoxicity of Vanadium to CHO-K1 Cells in the Presence of Inorganic Selenium.

The effect of selenium applied as sodium selenite (Na2SeO3) on the cytotoxicity of vanadyl sulphate (VOSO4) was examined using CHO-K1 cells. From the resazurin-based assay, it appears that Na2SeO3 at low doses (0.5 and 1 μM) can enhance 100 μM VOSO4-induced cell damage. The two-way ANOVA analysis revealed that the increased cell damage was a consequence of a synergistic interaction of 0.5 μM Na2SeO3 with VOSO4 and 1 μM Na2SeO3 with VOSO4. Observations performed with a phase-contrast microscope showed most cells to be rounded upon treatment with VOSO4 alone. In turn, a majority of cells co-treated with VOSO4 and 1 μM Na2SeO3 were elongated, and exhibited cytoplasmic vacuolization. These results warn of the potential contribution of inorganic selenium to vanadium-induced toxicity.

Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

Ex vivo non-invasive assessment of cell viability and proliferation in bio-engineered whole organ constructs

Decellularized organ scaffolds allow whole organ regeneration and study of cell behavior in three-dimensional culture conditions. Cell viability within the bio-engineered organ constructs is an essential parameter reflecting the performance of participating cells during long-term ex vivo culture, and is a prerequisite for further functional performance. Resazurin-based redox metabolic assays have been used to monitor cell viability in both two- and three-dimensional cell cultures. Here we developed a method for monitoring cell viability and proliferation in bio-engineered organ constructs using a resazurin perfusion assay. This method allows non-invasive, repetitive and rapid estimation of viable cell numbers during long-term ex vivo culture. As a proof-of-principle, we assessed the performance of two different endothelial sources and the impact of different perfusion programs on endothelial viability after re-endothelialization of decellularized lung scaffolds. The resazurin-based perfusion assay revealed changes in endothelial viability and proliferation during long-term ex vivo culture, which was consistent with histological assessment at different time points. Finally, we showed that this method could be used for assessment of proliferation and cytotoxicity after pharmacological treatment on a three-dimensional non-small cell lung cancer culture model.