Detection Of Cell Adhesion Research Articles

In Situ Imaging of Endogenous Hydrogen Peroxide Efflux from Living Cells via Bipolar Gold Nanoelectrode Array and Electrochemiluminescence Technology.

The integration of a closed bipolar electrode (c-BPE) array and electrochemiluminescence (ECL) detection received a boost in applications in the detection of cell adhesion and disease-related biomarkers. This work proposed a gold nanorod array based c-BPE-ECL system to realize an in situ image of endogenous hydrogen peroxide (H2O2) efflux from living cells and parallel analysis of endogenous H2O2 released from multiple cells by converting electrochemical signals into optical signals. The gold nanorod array with high density was prepared by a repeating chronopotentiometry procedure with anodic aluminum oxide (AAO) membrane as a template. The c-BPE array was fabricated by assembling poly(dimethylsiloxane) (PDMS) chips on both sides of the gold nanorod array. When an appropriate driving potential is applied, H2O2 generated from living cells at the sensing pole was reduced on the gold nanorod, triggering the oxidation of the ECL reagent at the reporting pole, which allowed the detection of H2O2 released from living cells. Under phorbol myristate acetate (PMA) stimulation, H2O2 released from living HeLa, HepG2, MCF-7, and LO2 cells was determined to be 47, 32.4, 25.7, and 6.3 μM, respectively. This indicated that the amount of H2O2 released from PMA-stimulated cancer cells was significantly higher than that from the stimulated normal cells. This work presented a new approach for in situ imaging of H2O2 released from living cells and could also be used to detect other electrochemically active or non-electrochemically active molecules through simple cell surface modification, which may have potential applications in cell apoptosis study and disease diagnosis.

Gold Nanowires Array-Based Closed Bipolar Nanoelectrode System for Electrochemiluminescence Detection of α-Fetoprotein on Cell Surface.

Inspired by the promising applications of a closed bipolar electrodes (c-BPEs) system in electrochemiluminescence (ECL) detection of cell adhesion and disease-related biomarkers, here, a gold nanowires array-based c-BPEs system was constructed for cell surface protein detection. Regular and uniform gold nanowires array were prepared by intermittent potentiostatic deposition. Then, two poly(dimethylsiloxane) (PDMS) chips with a hole diameter of 2 mm as a reservoir were placed at both sides of Au nanowires array to construct c-BPEs system. Thionine-functionalized silicon dioxide nanoparticles conjugated to antibody (Ab2-Th@SiO2) were used as the electrochemical probe, while [Ru(bpy)3]2+-wrapped SiO2 nanoparticles (Ru(II)@SiO2) were employed as the ECL signal readout. Taking α-fetoprotein (AFP) as model, the gold nanowires array-based c-BPEs system allowed sensitive detection of AFP at a linear range from 0.002 to 50.0 ng/mL and at least 6 living cells ascribing to the synergetic amplification effect at both sensing and reporting chambers. Besides, the amount of AFP expressed by HepG2 cells was calculated to be 6.71 pg/cell. The presented strategy with high sensitivity provided a promising and universal platform for the detection of other cancer cells and disease-related biomarkers (such as proteins, glycan, miRNA).

Non enzymatic isolation of adipose tissue and stromal vascular fraction derived cells

The therapeutic potential of the adipose tissue is also supported by a source of mesenchymal stem cells (MSCs), endothelial progenitor cells, mast cells, T- lymphocyte, B lymphocyte, and adipose-resident macrophages with repairing and regenerative ability. The purpose of this study was to compare adipose tissue derived mesenchymal stem cells (ADSCs) and stromal vascular fraction cells (SVF cells) in terms of the usage of non-enzymatic isolation, detection of cell adhesion, fibroblastoid cell formation, properties of cell population, cell culture duration until 3rd passages under in-vitro culture condition. We used 3 months old, 4 male Sprague Dawley rats (mean of live weight about 250 g) to examined the variation of the cell population, cell properties, phases of cellular formation, cell culturing time, subculturing duration, differences in confluency between SVF cells and ADSCs. In in-vitro expansion, ADSCs displayed higher rate of adhesiveness, homogenous cell population, faster proliferation and formation of fibroblast like cells compared to SVF cells. As a result, we showed that ADSCs have better adhesive ability, higher proliferative capacity in all of the 3 passages and require shorter time to reach confluency compared with SVF cells in vitro. These findings may contribute to future studies that deal with isolation and selection of stem cells from various tissues, as well as design clinical trials based on ADSCs and SVF cells.

ILF2 promotes anchorage independence through direct regulation of PTEN.

Anoikis is a specific form of programmed cell death induced by loss of contact between cells and extracellular matrices or other cells. Only tumor cells that are resistant to anoikis can survive in the state of detachment from the primary tissue during the early stages of metastasis. The ability to resist anoikis is crucial for cancer cell metastasis. ILF2 is a proto-oncogene previously studied in glioma, NSCLC, esophageal cancer and pancreatic ductal carcinoma. The results from the present study revealed that the transcription factor interleukin enhancer-binding factor 2 (ILF2) was highly expressed in non-small cell lung cancer (NSCLC) cell lines compared with in normal cell lines. ChIP and luciferase reporter gene assays demonstrated that ILF2 inhibited the expression level of the tumor suppressor gene phosphatase and tensin homolog (PTEN) by directly binding to its upstream regulatory region. Furthermore, the results from the detection of cell adhesion and apoptosis in cell suspension culture demonstrated that this mechanism enabled NSCLC cells to reduce adherence to the matrix and to survive in this abnormal state. These results suggested that ILF2 may promote the anchorage-independence of NSCLC cells through the suppression of PTEN.

In Vitro Validation of Cancer Immunotherapy Reagents Using an Impedance‐Based Platform

Immunotherapy is emerging as one of the most promising approaches in cancer treatment, allowing specific targeting of cancer cells from immune cells, mediated by surface marker expression. In vitro characterization of the efficacy and potency of cancer immunotherapy reagents is a necessary step before moving to animal models and Phase I and II clinical studies. In this study we report the validation and optimization of protocols for analyzing the efficacy of reagents designed to stimulate the immune response against cancer cells using an impedance‐based and label‐free platform. The platform allows detection of cell adhesion and cell death trough monitoring the change in conductance of microelectrodes embedded in the bottom of 96‐wells cell culture plates. Because cell adhesion alters the conductivity of the electrodes, any change in cell morphology, cell adhesion or cell number is detected as variation of an impedance‐related parameter named Cell index (Ci). The platform avoids the use of any chemical label or sample processing, allowing continuous monitoring of cell activity over prolonged time periods. Furthermore, the untouched cells are still available for secondary assays that can better clarify the biological mechanism involved in the response.To mimic tumors that are target of immunotherapy efforts we utilized several carcinoma cell lines whit different level of expression of the EpCAM membrane protein. Such antigen is overexpressed in many tumors and is currently under evaluation in clinical trials. In PC3 prostate cancer cells we measured cell cytotoxicity of Primary Blood Mononuclear Cells (PBMCs) mediated by an EpCAM/CD3 Bispecific T cell Engager (BiTE) antibody. The use of the impedance‐based platform allowed measuring the full kinetic of the cytotoxic activity under different effector:target ratios and BiTE concentrations. Apoptosis of tumor cells was confirmed trough flow cytometer analysis. We also demonstrated the relation between amount of PBMCs cytotoxic activity and EpCAM surface expression trough testing MCF7 (high EpCAM expressor) and HeLa (low EpCAM expressor).To evaluate cancer immunotherapy protocols in lymphomas and leukemia, we developed a protocol where lymphomas cells are immobilized on electrodes‐coated plates and the T‐Lymphoblast cell line TALL‐104 is used as cytotoxic effector.Taken together our results demonstrate high sensitivity of the impedance‐based approach in measuring cytotoxic activity in sophisticated high throughput set up. Furthermore, the availability of continuous recording of the Cell Index over the temporal scale allows better characterization, comparing to alternative end point assays, of construct efficacy.

Abstract LB-B16: In vitro monitoring of the efficacy of cancer immunotherapy designs trough an impedance-based platform

Abstract Immunotherapy is emerging as one of the most promising approaches in cancer treatment, allowing specific targeting of cancer cells from CD8+ T Lymphocytes based on surface marker expression. In vitro characterization of the efficacy and potency of BiTEs or CAR T-Cell constructs is a necessary step before moving to animal models and Phase I and II clinical studies. In this study we present the optimization of several protocols to analyze cancer immunotherapy reagents using an impedance-based platform. The platform allows detection of cell adhesion and cell death trough monitoring the change in conductance of microelectrodes embedded on the bottom of the culture dish. Because cell adhesion over the electrodes alters the conductivity of the surface, any change in cell morphology, adhesion or number is detected as alteration of an impedance-related parameter called Cell index (Ci). Furthermore, because the platform avoid the use of any chemical label or sample processing, it allows continuous monitoring of cell activity. Dose-responses curves of BiTEs antibodies against epithelial tumor cell lines with a range of expression of the EpCAM antigen show correlation between surface expression level and efficacy of the constructs. Decreasing ratios between effector and target cells, in combination with longer time of monitoring reveal the efficacy of such constructs also at equivalent stoichiometric ratio. Taken together our results demonstrate higher sensitivity of the impedance-based approach over established methodologies like Chromium-51 release assay and live-cell imaging, improving and speeding up the workflow for therapy optimization. Citation Format: Fabio Cerignoli, Jenny Zhu, Biao Xi, Leyna Zhao, Jeff Irelan, Yama Abassi. In vitro monitoring of the efficacy of cancer immunotherapy designs trough an impedance-based platform. [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 LB-B16.

Dielectrophoresis measurement of bovine endothelial cells adhesion on different materials

In this study, the design, fabrication, and characterization of dielectrophoresis based devices for the measurement of bovine endothelial cell adhesion on different biomaterials are conducted. During the design stage, the finite element analysis software COMSOL is used to determine a better design for the dielectrophoretic electrode. Accordingly, a dielectrophoretic device that contains several micro electrodes for producing unbalanced electrical fields is fabricated using the microelectromechanical fabrication technique. The proposed device is then used for the detection of cell adhesion on polydimethylsiloxane (PDMS) and poly lactide (PLA) substrates. The hydrophilicity measurement results reveal that PLA is more hydrophilic than PDMS. It is inferred that bovine endothelial cell (BEC) should have better adhesion on PLA than on PDMA. However, the cell detachment results do not fully agree with this inference. It is further suggested that other features of the substrate are more crucial for the adhesion of BEC than the hydrophilicity. Cell detachment experiments demonstrate that the applied electrophoresis cannot detach the adhered BECs from a PDMS substrate when the seeding time is longer than 4 h. However, the dielectrophoretic force caused by a 6 V applied potential is enough to lift those cells cultured on the PLA for 4 and 6 h respectively. When the culture time is increased to 8 h, the cells apparently stretch out and a higher voltage is required to lift and move them. The results of the cell detachment experiments may suggest that the adherence of BEC to PLA is more stable after 8 h of seeding.

Polypyrrole incorporating biomolecules

Polypyrrole (PPy) is a conducting polymer with emerging applications in biomedical engineering. We report on the synthesis and properties of PPy incorporating biomolecules using AC impedance spectroscopy. Impedance behaviour was consistent with two basic charge transfer processes, polaronic and electronic, demonstrated by two-electrode impedance spectroscopy over the frequency range 5 Hz?13 MHz. These processes were sensitive to alterations in environmental conditions such as pH and ionic strength. The reported results are likely to be a consequence of the intimate molecular association between PPy chains and incorporated molecules in the matrix as evidenced by variations in impedance spectra related to polymer loading. PPy incorporating physically entrapped biomolecules together with impedance spectroscopy could be exploited in reagentless biosensor applications. Furthermore, this could be coupled to their emerging role in cell contact and guidance applications for the detection of cell adhesion, migration or metabolite monitoring.