Impedance-based Real-time Cell Analysis Research Articles

The Effect of Enzymatic Treatment with Mutanase, Beta-Glucanase, and DNase on a Saliva-Derived Biofilm Model

Introduction: The dental biofilm matrix is an important determinant of virulence for caries development and comprises a variety of extracellular polymeric substances that contribute to biofilm stability. Enzymes that break down matrix components may be a promising approach to caries control, and in light of the compositional complexity of the dental biofilm matrix, treatment with multiple enzymes may enhance the reduction of biofilm formation compared to single enzyme therapy. The present study investigated the effect of the three matrix-degrading enzymes mutanase, beta-glucanase, and DNase, applied separately or in combinations, on biofilm prevention and removal in a saliva-derived in vitro-grown model. Methods: Biofilms were treated during growth to assess biofilm prevention or after 24 h of growth to assess biofilm removal by the enzymes. Biofilms were quantified by crystal violet staining and impedance-based real-time cell analysis, and the biofilm structure was visualized by confocal microscopy and staining of extracellular DNA (eDNA) and polysaccharides. Results: The in vitro model was dominated by Streptococcus spp., as determined by 16S rRNA gene amplicon sequencing. All tested enzymes and combinations had a significant effect on biofilm prevention, with reductions of >90% for mutanase and all combinations including mutanase. Combined application of DNase and beta-glucanase resulted in an additive effect (81.0% ± 1.3% SD vs. 36.9% ± 21.9% SD and 48.2% ± 14.9% SD). For biofilm removal, significant reductions of up to 73.2% ± 5.5% SD were achieved for combinations including mutanase, whereas treatment with DNase had no effect. Glucans, but not eDNA decreased in abundance upon treatment with all three enzymes. Conclusion: Multi-enzyme treatment is a promising approach to dental biofilm control that needs to be validated in more diverse biofilms.

Local administration of shikonin improved the overall survival in orthotopic murine glioblastoma models with temozolomide resistance

BackgroundGlioblastoma is a type of intracranial malignancy. Shikonin, a Chinese traditional medicine, has been shown to have anti-tumor efficacy toward human glioblastoma cells in vitro. However, shikonin cannot easily cross the blood–brain barrier. To address this issue, we evaluated the anti-tumor effects of direct intracranial infusion of shikonin in in vivo orthotopic syngeneic murine glioblastoma models using C57BL/6 mice. Materials and methodsThe cytotoxic effects of shikonin against murine glioblastoma cells, SB28 and CT-2A, were reported resistance to temozolomide, were evaluated using an allophycocyanin-conjugated annexin V and propidium iodide assay with flow cytometry. Impedance-based real-time cell analysis (RTCA) was used to analyze the inhibitory effects of shikonin on growth and proliferation. To evaluate the anti-tumor activity of shikonin in vivo, we used orthotopic syngeneic murine glioblastoma models with SB28 and CT-2A cells. ResultsIn flow cytometry-based cytotoxic assays, shikonin induced apoptosis. RTCA indicated that shikonin decreased the cell index of murine glioblastoma cells, SB28 and CT-2A, in a dose-dependent manner (p < 0.0001 for both cell lines), while temozolomide did not (p = 0.91 and 0.82, respectively). In murine glioblastoma models, SB28 and CT-2A, direct intracranial infusion of shikonin, as a local chemotherapy, improved the overall survival of mice in a dose-dependent manner compared with control groups (p < 0.0001 and p = 0.02, respectively). While temozolomide did not (p = 0.48 and 0.52, respectively). ConclusionsThe direct intracranial infusion of shikonin has potential as a local therapy for patients with glioblastoma.

Differential effects of resolvin D1 and resolvin E1 on cementoblast function.

Resolvins are endogenous mediators of the resolution of inflammation. They are derived from omega-3 polyunsaturated fatty acid precursors. Resolvin D1 (RvD1) and Resolvin E1 (RvE1) are the best-characterized members for actively promoting periodontal regeneration in experimental animal models. Here, we evaluated the efficacy of RvD1 and RvE1 on cementoblasts, the key cells involved in dental cementum regeneration and the attachment of the tooth to the alveolar bone. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1-1000ng/mL) of RvD1 and RvE1. Cell proliferation was measured using an electrical impedance-based real-time cell analyzer. Mineralization was evaluated with von Kossa staining. The mRNA expression of mineralized tissue-associated markers of bone sialoprotein (BSP), Type I collagen (COL I), osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RunX2), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (NF-κB) (RANK), receptor activator of NF-κB ligand (RANKL), and extracellular matrix-degrading enzymes [matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and their tissue inhibitors (TIMP-1, TIMP-2)], RvE1 receptor (ChemR23) and RvD1 receptor (ALX/PFR2), cytokines (tumor necrosis factor-alpha {TNF-α}, interleukin {IL}-1β, IL-6, IL-8, IL-10, IL-17), oxidative stress enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPX), and cyclooxygenase-2 (Cox-2)] were analyzed using quantitative polymerase chain reaction (qPCR). Both RvD1 and RvE1 (10-100ng/mL) significantly increased the proliferation of cementoblasts and mineralized nodules at all concentrations (p<0.05). RvE1 increased BSP, RunX2, and ALP compared with the RvD1 dose and time-dependently, while RvD1 and RvE1 differentially regulated COL-I. RvE1 increased OPG mRNA expression, whereas RANK-RANKL mRNA expression decreased by RvE1. MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 expressions were reduced by RvE1 compared with RvD1. Treatment of cementoblasts with RvD1 and RvE1 differentially affected cytokine and oxidative stress enzymes while significantly increasing their receptor expressions (ChemR23 and ALX/PFR2). RvD1 and RvE1 regulate proliferation, mineralization, and gene expression in cementoblasts using similar pathways while differentially affecting tissue degradation, suggesting a targeted therapeutic approach for cementum turnover during periodontal regeneration.

In vitro determination of the remineralizing potential and cytotoxicity of non-fluoride dental varnish containing bioactive glass, eggshell, and eggshell membrane.

This study compares in vitro remineralization potential and cytotoxicity of fluoride-free varnish combinations containing bioactive glass, eggshell, and membrane powder and fluoride varnish formulations on artificial caries lesions. Artificial caries lesions were formed in two windows on third molars. One of the windows was coated with one of the following varnish formulations: FV (fluoride varnish), F-BAGV (fluoride and bioactive glass containing varnish), BAGV (bioactive glass containing varnish), EPV (eggshell powder containing varnish), EMP-EPV (eggshell membrane protein and eggshell powder containing varnish), STMP-EMP-EPV (sodium trimetaphosphate-treated eggshell membrane protein and eggshell powder containing varnish). The samples were remineralized, then investigated under scanning electron microscopy, and elemental analyses were performed by X-ray dispersive analysis (EDX). In addition, the traditional colorimetric tetrazolium-based reduction assay (XTT) and the modern impedance-based real-time cell analysis system (RTCA) were used to investigate their cytotoxicity. The varnish applied area's Ca/P ratio was lower than stoichiometric hydroxyapatite except for EPV (1.66) and STMP-EMP-EPV (1.67) groups. Undiluted extracts of all varnishes, 1:2 dilutions of FV and F-BAGV groups were cytotoxic in XTT assay. In RTCA, the normalised cell index of the EMP-EPV and STMP-EMP-EPV groups was higher than the control group. Bioactive glass, eggshell, eggshell membrane proteins and STMP-treated eggshell and eggshell membrane protein containing varnish have similar remineralizing effect to fluoride-containing varnish on demineralized enamel. Integrating biological or bioactive components instead of fluoride into the dental varnishes might reduce cytotoxicity.

Exploiting the immunogenic potential of standard of care radiation or cisplatin therapy in preclinical models of HPV-associated malignancies

BackgroundWhile radiation and chemotherapy are primarily purposed for their cytotoxic effects, a growing body of preclinical and clinical evidence demonstrates an immunogenic potential for these standard therapies. Accordingly, we sought...

Prolonged Exposure to Oxaliplatin during HIPEC Improves Effectiveness in a Preclinical Micrometastasis Model.

Simple SummaryAbsence of survival benefits when adding hyperthermic intraperitoneal chemotherapy (HIPEC) with oxaliplatin to cytoreductive surgery in peritoneal metastasis from colorectal cancer has recently been shown in the randomized controlled PRODIGE 7 trial. We therefore aimed to investigate the effects of this treatment modality in a preclinical micrometastasis model. Cancer cells were incubated with either patient samples obtained during HIPEC procedures or with defined oxaliplatin-containing solutions prepared according to clinically established HIPEC protocols. Our results demonstrate a limited effectiveness of short-term HIPEC in simulations with oxaliplatin to eliminate micrometastases, although we used platinum-sensitive cell lines for our model. Since these results are in line with findings from current research, our studies might offer further convincing evidence and potential explanations for HIPEC futility observed in clinical application.Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) was considered a promising treatment for patients with peritoneal metastasis from colorectal cancer. However, the recently published randomized controlled PRODIGE 7 trial failed to demonstrate survival benefits through the addition of short-term oxaliplatin-based HIPEC. Constituting a complex multifactorial treatment, we investigated HIPEC in a preclinical model concerning the elimination of minimal tumor residues, thereby aiming to better understand the size of effects and respective clinical trial results. Patient samples of peritoneal perfusates obtained during HIPEC treatments and oxaliplatin-containing solutions at clinically relevant dosages, conforming with established HIPEC protocols, were assessed regarding their ability to eliminate modelled ~100 µm thickness cancer cell layers. Impedance-based real-time cell analysis and classical end-point assays were used. Flow cytometry was employed to determine the effect of different HIPEC drug solvents on tumor cell properties. Effectiveness of peritoneal perfusate patient samples and defined oxaliplatin-containing solutions proved limited but reproducible. HIPEC simulations for 30 min reduced the normalized cell index below 50% with peritoneal perfusates from merely 3 out of 9 patients within 72 h, indicating full-thickness cytotoxic effects. Instead, prolonging HIPEC to 1 h enhanced these effects and comprised 7 patients’ samples, while continuous drug exposure invariably resulted in complete cell death. Further, frequently used drug diluents caused approximately 25% cell size reduction within 30 min. Prolonging oxaliplatin exposure improved effectiveness of HIPEC to eliminate micrometastases in our preclinical model. Accordingly, insufficient penetration depth, short exposure time, and the physicochemical impact of drug solvents may constitute critical factors.

Real-Time Analysis of Antiproliferative Effects of Mouthwashes Containing Alcohol, Sodium Fluoride, Cetylpyridinium Chloride, and Chlorhexidine In Vitro.

Objectives In this study, the cytotoxic responses of six different over-the-counter mouthwashes on L929 cells were analyzed by two different techniques: the traditional colorimetric tetrazolium-based reduction assay (MTT) and the modern impedance-based real-time cell analysis (RTCA) system to investigate their biocompatibility in vitro. Thus, the investigation of the antiproliferative effects of the specified materials via different techniques is vital to reach this goal. Materials and Methods First, L929 mouse fibroblasts were exposed to the dilutions of mouthwashes for 2 minutes. After incubation, the tetrazolium reduction method was used to assess the metabolic viability of cells measured by colorimetric MTT assay and morphological inspection of cells was performed via phase-contrast microscopy. Furthermore, the effect of each mouthwash on the proliferation, morphology, and adhesion of L929 cells was monitored continuously by a noninvasive and label-free RTCA system for 140 h. Results Our data showed that all of the mouthwashes had varying cytotoxic effects on fibroblasts compared to the control group in MTT assay. In addition to that, RTCA technology has provided the growth kinetic profiles that can be used to analyze if the treatment is causing antimitotic or DNA-damaging effect on cells. Thus, analysis via this system can tell us the mechanism of toxicity behind the cell growth inhibition in vitro. Here, we found that only mouthwash 1 moderately maintained the viability of the L929 cells, yet displaying antimitotic effects and the other mouthwashes (mouthwash 2-mouthwash 6) showed toxicity via DNA-damaging effects. Conclusions Of the six types of mouthwash tested, the most biocompatible result was obtained from a mouthwash containing alcohol (i.e., mouthwash 1). On the other hand, sodium fluoride- (NaF-) and cetylpyridinium chloride- (CPC-) containing mouthwash (i.e., mouthwash 2) showed the most cytotoxic effect.

Toxicity of abrasive nanoparticles (SiO2, CeO2, and Al2O3) on Aliivibrio fischeri and human bronchial epithelial cells (16HBE14o-)

SiO2, CeO2, and Al2O3 nanoparticles (NPs) are used as abrasive particles in chemical and mechanical planarization (CMP), a key process in semiconductor fabrication. The CMP process generates high volumes of effluents containing abrasive NPs. Since no regulations are available limiting the release of these NPs, there are concerns about their potential environmental and health risks. This study investigates four industry relevant model CMP slurries including colloidal silica (c-SiO2), fumed silica (f-SiO2), cerium oxide (CeO2), and aluminum oxide (Al2O3) for their inhibition on bioluminescence activity of the marine bacterium, Aliivibrio fischeri. Additionally, the cytotoxicity of the slurries on human bronchial epithelial cells (16HBE14o-) was evaluated using a novel impedance-based real-time cell analysis (RTCA) system. The results showed that f-SiO2 and CeO2 slurries had no acute toxicity on A. fischeri at concentrations up to 1136 and 909 mg/L, respectively. A concentration of 1364 mg/L of c-SiO2 and Al2O3 led to 37.6% and 28.4% inhibition on microbial activity after 30 min of exposure. High concentrations of c-SiO2 and f-SiO2 slurries (250 and 500 mg/L) led to cell death in the RTCA assay. This study further demonstrated that the toxicity of the model CMP slurries was due to the abrasive NPs but not to other additives in the slurry. In contrast, CeO2 and Al2O3 slurries were not inhibitory or only showed limited inhibitory effect on the viability and proliferation of 16HBE14o- cells after 24 h of exposure. These results indicate that the abrasive NPs used in CMP are not likely to cause acute environmental and health risks at environmentally relevant concentrations.

Differences of time-dependent microRNA expressions in breast cancer cells.

MicroRNA (miRNA) expression is a dynamic process in the cell, and the proper time period for post-transcriptional regulation might be critical due to the gene-on/-off expression times of the cell. Here, we investigated the effect of different time-points on proliferation, invasion and miRNA expression profiles of human breast cancer cell lines MCF-7 (non-metastatic, epithelium-like breast cancer cell line with oestrogen receptor (ER) positive (+) and human breast cancer cell lines MDA-MB-435 (metastatic, invasive, ER negative (-). For this purpose, MCF-7 and MDA-MB-435 cells were seeded different number in E-plate 16 for proliferation experiment using an electrical impedance-based real-time cell analyzer system (RTCA) for 168 h. Similarly, invasion potential of MCF-7 and MDA-MB-435 were determined by RTCA for 90 h. Total RNAs including miRNAs were isolated at 2, 4, 6, 12, 24, 48 h from the MCF-7 and MDA-MB-435 cells. Afterward, the quantitative 84 miRNA expressions of MCF-7 and MDA-MB-435 were analyzed by Fluidigm Microfluidic 96.96 Dynamic Array. The results of these study demonstrated that both proliferation potential and invasion capacity of MDA-MB-435 is higher than MCF-7 as time-dependent manner. Furthermore, we detected that up/down expressions of 32 miRNAs at all time points in MDA-MB-435 compared to MCF-7 (at least ten-fold increased). Because of the high number of miRNAs, we more closely evaluated the expression of six of them (miR-100–5p, miR-29a-3p, miR-130a-3p, miR-10a-5p, miR-10b-5p, miR-203a), and determined that their levels were dramatically changed by at least 50-fold at different time points of the experiment (p < 0.01). The expression levels of five of these miRNAs (miR-100–5p, miR-10a-5p, miR-10b-5p, miR-130a-3p, and miR-29a-3p) started to increase from the fourth hour and continued to increase until the 48th hour in MDA-MB-435 cells compared to MCF-7 cells (p < 0.01). Simultaneously, the expression of one of these miRNAs (miR-203a) decreased from the sixth hour to the 48th hour in MDA-MB-435 as compared to MCF-7. We determined pathways associated with target genes using mirPath - DIANA TOOLS. Small RNAs including miRNA are essential regulatory molecules for gene expressions. In the literature, gene expressions have been published as burst and pulse in the form of discontinuous transcription. The data of the research suggested that time-dependent changes of miRNA expressions can be affected target gene transcriptional fluctuations in breast cancer cell and can be base for the further studies.

Abstract 2145: Ex vivo phenotyping and potency monitoring of CD19 CAR T cells with a combined flow cytometry and impedance-based real time cell analysis workflow

Abstract Immunotherapy harnesses the immune system to attack cancer cells. While immunotherapy holds tremendous promise for cancer treatment, significant challenges remain in clinical translation. Further development is still required to expand treatment to more cancers and minimize adverse effects. T-cell differentiation and exhaustion influence the establishment and persistence of CAR-expressing T cells. Evaluation of the potency of CAR T cells is essential for effective immunotherapies. Here, we study the potency of CAR-T cells using a combined impendence-based Real-Time Cell Analysis (RTCA) and flow cytometry cell analysis workflow for ex vivo cytolytic potency monitoring of CD19 CAR T cells (CART19). CART19 cell killing of CD19 expressing HEK-293 cells with different E:T (Effector to Target) ratio was monitored by the xCELLigience RTCA system. Concurrently, T cell cytokine production was measured on the NovoCyte Quanteon. This workflow provides a thorough examination of CART cell function, RTCA data demonstrates that CART19 killing is highly potent as well as being antigen-specific and dose-dependent. In conjunction, more cytokine was produced with higher numbers of effector CART19 cells. It was also observed that CART19 cells express stem memory cell (TSCM) markers, expressing activation and exhaustion markers after expansion. Co-culture with HEK-293-CD19 also results in increased expression of CD25, decreased expression of CD127 and upregulation of exhaustion markers, PD-1, TIM-3, and LAG-3. In summary, this study demonstrates the combination of RTCA and flow cytometry cell analysis is a powerful workflow for immunotherapy research and can be employed for QC in CAR T cell manufacturing. Citation Format: Li Zhao, Ming Lei, Peifang Ye, Yan Lu, Xiaoping Ji, Yu Yan, Lauren Jachimowicz, Garret Guenther, Nancy Li. Ex vivo phenotyping and potency monitoring of CD19 CAR T cells with a combined flow cytometry and impedance-based real time cell analysis workflow [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2145.

In-situ monitoring of the unstable bacterial adhesion process during wastewater biofilm formation: A comprehensive study

The initial bacterial adhesion phase is a pivotal and unstable step in the formation of biofilms. The initiation of biofilm formation is an unstable process caused by the reversible adhesion of bacteria, which is always time-consuming and yet to be elucidated. In this study, impedance-based real time cell analysis (RTCA) was employed to comprehensively investigate the initial bacterial adhesion process. Results showed that the time required for the unstable adhesion process was significantly (p < 0.05) reduced by increasing the initial concentration of bacteria, which is mainly attributed to the large deposition rate of bacteria at high concentrations. In addition, the unstable adhesion process is also regulated by shear stress, derived in this work from orbital shaking. Shear stress improves the reversibility of unstable bacterial attachment. Furthermore, attachment characteristics during the unstable phase vary between different species of bacteria (Sphingomonas rubra, Nakamurella multipartita and mixed bacteria). The S. rubra strain and mixed culture were more prone to adhere to the substratum surface during the unstable process, which was attributed to the smaller xDLVO energy barrier and motility of species in comparison with N. multipartita. Meanwhile, the molecular composition of extracellular polymeric substances (EPS) in the initial attachment phase presented a significant difference in expressed proteins, indicating the important role of proteins in EPS that strengthen bacterial adhesion. Overall, these findings suggest that during the biofilm reactor start-up process, seed sludge conditions, including the bacterial concentration, composition and hydraulics, need to be carefully considered.

Resveratrol targets PD-L1 glycosylation and dimerization to enhance antitumor T-cell immunity.

New strategies to block the immune evasion activity of programmed death ligand-1 (PD-L1) are urgently needed. When exploring the PD-L1-targeted effects of mechanistically diverse metabolism-targeting drugs, exposure to the dietary polyphenol resveratrol (RSV) revealed its differential capacity to generate a distinct PD-L1 electrophoretic migration pattern. Using biochemical assays, computer-aided docking/molecular dynamics simulations, and fluorescence microscopy, we found that RSV can operate as a direct inhibitor of glyco-PD-L1-processing enzymes (α-glucosidase/α-mannosidase) that modulate N-linked glycan decoration of PD-L1, thereby promoting the endoplasmic reticulum retention of a mannose-rich, abnormally glycosylated form of PD-L1. RSV was also predicted to interact with the inner surface of PD-L1 involved in the interaction with PD-1, almost perfectly occupying the target space of the small compound BMS-202 that binds to and induces dimerization of PD-L1. The ability of RSV to directly target PD-L1 interferes with its stability and trafficking, ultimately impeding its targeting to the cancer cell plasma membrane. Impedance-based real-time cell analysis (xCELLigence) showed that cytotoxic T-lymphocyte activity was notably exacerbated when cancer cells were previously exposed to RSV. This unforeseen immunomodulating mechanism of RSV might illuminate new approaches to restore T-cell function by targeting the PD-1/PD-L1 immunologic checkpoint with natural polyphenols.

Ex Vivo Phenotyping and Potency Monitoring of CD19 CAR T Cells With a combined Flow Cytometry and Impedance-based Real-Time Cell Analysis Workflow

Abstract Cancer immunotherapy is increasingly utilized for cancer treatment. CARs (Chimeric antigen receptors) targeting CD19 have produced strong antitumor responses in hematologic malignancies, but tumor regression has seldom occurred when CARs targeting other antigens. The state of T-cell differentiation and T cell exhaustion influences the engraftment and persistence of CAR-expressing T cells following adoptive transfer. Here we report the use of a combined impendence-based Real-Time Cell Analysis (RTCA) and flow cytometry cell analysis workflow for ex vivo cytolytic potency monitoring of CD19 CAR T cell, as well as the examination of phenotypic and functional responses to antigen exposure over time. CD19 CAR T cell killing of HEK293 CD19 with different E:T ratio was monitored by RTCA system. Cytokine productions were measured by NovoCyte® Quanteon™ flow cytometer after 24 hrs. Co-incubation with HEK293 CD19, phenotypic and functional responses of CD19 CAR T cells towards exposure of different antigen levels were systematically examined over time. Both assays reveal that the CD19 CAR T cells show high dose-dependent killing activities against HEK293 CD19 but not against HEK293. Using the multi-color Flow Cytometry immunophenotyping, CD4 dim and CD8 dim populations are identified as effective killing cells which are enriched in central memory (CD45RA+CCR7−) cells while normal CD4+ or CD8+ T cells consist of predominant naïve-like (CD45RA+CCR7+) T cells after co-incubation with HEK293 CD19 cells. Exhaustion marker (PD-1, LAG-3, TIM-3) are overexpressed in normal CD4+ and CD8+ populations but not in CD4 dim and CD8 dim populations with CD19 antigen exposure. After antigen exposure, T cell activation levels change significantly.

Establishment of oral squamous cell carcinoma cell line and magnetic bead-based isolation and characterization of its CD90/CD44 subpopulations.

In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44+/CD90−, CD44−/CD90−, CD44+/CD90+, CD44−/CD90−). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90+) and epithelial (CD90−) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44+ cells exhibited stronger migratory capacity than CD44− subpopulations. CD44+ subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44− subpopulations. Furthermore, co-cultivation experiments revealed that CD44−/CD90+ cells supported growth of epithelial tumor cells (CD44+/CD90−). On the contrary, factors released by CD44+/CD90+ type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44−/CD90+, but this subpopulation had a low migratory capacity.

Effects for Sequential Treatment of siAkt and Paclitaxel on Gastric Cancer Cell Lines.

Real-time screening of cellular response on the drugs could provide valuable insights for the early detection of therapeutic efficiency and the evaluation of disease progression. Cancer cells have the ability to vary widely in response to stress in a manner to adjust the signaling pathway to promote the survival or having a resistance to stimulation. Cell-based label-free technologies using electronic impedance sensor have strategies for constructing the signature profiles of each cells. To achieve exquisite sensitivity to substantially change of live-cell response have an important role that predict the potential of therapeutic effects. In this study, we use an impedance-based real-time cell analysis system to investigate dynamic phenotypes of cells described as a cellular index value. We show that gastric cancer cells generated characteristic kinetic patterns that corresponded to the treatment order of therapeutics. The kinetic feature of the cells offers insightful information that cannot be acquired from a conventional single end-point assay. Furthermore, we employ a 'sequential treatment strategy' to increase cytotoxic effects with minimizing the use of chemotherapeutics. Specifically, treatment of paclitaxel (PTX) after down-regulating Akt gene expression using RNAi reduces the cell proliferation and increases apoptosis. We propose that the sequential treatment may exhibit more effective approach rather than traditional combination therapy. Moreover, the dynamic monitoring of cell-drug interaction enables us to obtain a better understanding of the temporal effects in vitro.

Particle-induced cell migration assay (PICMA): A new in vitro assay for inflammatory particle effects based on permanent cell lines

Inflammation is a decisive pathophysiologic mechanism of particle toxicity and accumulation of neutrophils in the lung is believed to be a crucial step in this process. This study describes an in vitro model for investigations of the chemotactic attraction of neutrophils in response to particles using permanent cell lines. We challenged NR8383 rat macrophages with particles that were characterized concerning chemical nature, crystallinity, and size distribution in the dry state and in the culture medium. The cell supernatants were used to investigate migration of differentiated human leukemia cells (dHL-60 cells). The dose range for the tests was determined using an impedance-based Real-Time Cell Analyzer. The challenge of NR8383 cells with 32–96μgcm−2 coarse and nanosized particles resulted in cell supernatants which induced strong and dose-dependent migration of dHL-60 cells. Quartz caused the strongest effects – exceeding the positive control “fetal calf serum” (FCS) several-fold, followed by silica, rutile, carbon black, and anatase. BaSO4 served as inert control and induced no cell migration. Particles caused NR8383 cells to secrete chemotactic compounds. The assay clearly distinguished between the particles of different inflammatory potential in a highly reproducible way. Specificity of the test is suggested by negative results with BaSO4.

Characterization of the murine myeloid precursor cell line MuMac-E8.

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies.

Senescence affects endothelial cells susceptibility to dengue virus infection.

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.

Cytotoxicity and genotoxicity of phenazine in two human cell lines

Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9–123μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2′-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24h IC50: 11μM; 48h IC50: 7.8μM) observed as low as 1.9μM. T24 cells experienced a minor antiproliferative effect (BrdU 24h IC50: 47μM; 48h IC50: 17μM). IC50 values for HepG2 proliferation and viability were 54–77% lower compared to T24 cells. In both cell lines, IC50 values for proliferation were 66–90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9–30.8μM) experienced no significant genotoxic effects, while T24 cells (7.7–123μM) experienced significant genotoxicity at ⩾61.5μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.

The pan-deacetylase inhibitor panobinostat suppresses the expression of oncogenic miRNAs in hepatocellular carcinoma cell lines

Deacetylase inhibitors (DACi) are a new class of drugs with a broad spectrum of mechanisms that favor their application in cancer therapy. Currently, the exact mechanisms and cellular effects of DACi have not been fully elucidated. In addition to their effects on histone acetylation, DACi can interfere with gene expression via miRNA pathways. Treatment with panobinostat (LBH589), a novel potent DACi, led to the highly aberrant modulation of several miRNAs in hepatocellular carcinoma (HCC) cell lines as shown by miRNA array analysis. Among them, hsa-miR-19a, hsa-miR-19b1 and the corresponding precursors were down-regulated by panobinostat in TP53(-/-) Hep3B and TP53(+/+) HepG2 cell lines; hsa-miR30a-5p mature form only was suppressed in both HCC cell lines, as confirmed by further RT-qPCR analysis. In HCC cell lines, panobinostat caused the upregulation of the predicted miRNA targets APAF1 and Beclin1 protein levels. Transfection with oligonucleotides mimicking these miRNAs led to an increase in the viability rate of both cell lines as analyzed by impedance-based real-time cell analysis. In addition, transfecting miRNA mimicking oligonucleotides resulted in the decrease of APAF1, Beclin1 and PAK6 at the protein level, proving the regulating influence of the investigated miRNAs on gene final products. The overexpression of the above mentioned oncomiRs in Hep3B and HepG2 cell lines leads to cell proliferation and downregulation of cell death associated proteins. In our model, panobinostat exerts its anti-cancer effect by suppressing these miRNAs and restoring the expression of their corresponding tumor suppressor targets.