A multiwell electrochemical biosensor for real-time monitoring of the behavioural changes of cells in vitro.

We report the development of a multiwell biosensor for detecting changes in the electrochemical open circuit potential (OCP) generated by viable human cells in vitro. The instrument features eight culture wells; each containing three gold sensors around a common silver/silver chloride reference electrode, prepared using screen-printed conductive inks. The potential applications of the device were demonstrated by monitoring rheumatoid synovial fibroblasts (RSF) and HepG2 hepatocarcinoma cells in response to chemical and biological treatments. This technology could provide an alternative to conventional end-point assays used in the fields of chemotherapy, toxicology and drug discovery.

Electrochemical monitoring of rat mammary adenocarcinoma cells: an in vitro assay for anticancer drug selection.

We report the application of an electrochemical biosensor (Oncoprobe; Marks & Clerk, Manchester, UK) to determine whether changes in the open circuit potential (OCP) of rat mammary adenocarcinoma cells (MTLn3) treated in vitro with four cytotoxic anticancer drugs could predict their effects in vivo. MTLn3 cells were seeded onto sensors, then exposed to each anticancer compound (cisplatin, doxorubicin, paclitaxel, or vinblastine), and monitored for 44 hours. Electrochemical monitoring in vitro detected OCP responses to all four drugs, with cisplatin and doxorubicin producing greater changes over a shorter period than vinblastine and paclitaxel. Syngeneic MTLn3 cells were used to generate palpable tumors in 50 female Fischer 344 rats. Animals were divided into five equal groups; on day 12 four of the groups received an anticancer drug, and one received a saline control. Fourteen days later the animals were killed, and primary tumor weights were determined. Tumors from cisplatin- and doxorubicin-treated rats were significantly reduced in weight compared to the control, paclitaxel-, and vinblastine-treated groups. The anticancer drug-induced changes observed through real-time electrochemical monitoring of MTLn3 cells in vitro correlated well with the in vivo animal model, unlike the conventional end-point assays of lactate dehydrogenase release and Alamar Blue.

Cell separation with horizontal cell electrophoresis under near-isopycnic conditions on a "density cushion".

This report describes an improvement made to the horizontal cell electrophoresis methodology. It involves using two liquid layers differing in density to produce an interface described as a "density cushion". The electrophoretic system that employed an anti-convective porous matrix to separate red blood cells (RBC) and charged dyes effectively was found to be unsuitable for some other mammalian cells. The "density cushion" method was found to be more versatile and applicable to studies on the separation of a variety of cell types. The experiments described show the differences between the electrophoretic mobilities of a human eosinophilic leukaemia cell line (Eol-1) and RBC, both with and without the modification of the cell surface properties.

Effect of histamine on the production of matrix metalloproteinases-1, -3, -8 and -13, and TNFalpha and PGE(2) by human articular chondrocytes and synovial fibroblasts in vitro: a comparative study.

Histamine has many regulatory activities and is well recognised for its importance in allergic and inflammatory disorders. Recently, histamine has been implicated in the pathophysiological processes of both rheumatoid and osteoarthritis, where human articular chondrocytes (HACs) and rheumatoid synovial fibroblasts (RSFs) are reported to express histamine receptors. This study has demonstrated H(1) and H(2) histamine receptors using immunohistochemistry on HACs and RSFs in vitro and has compared the effects of histamine (20 microM) on both cell types with regard to the production of matrix metalloproteinases (MMPs-1, -3, -8 and -13), the proinflammatory cytokine tumour necrosis factor alpha (TNFalpha) and prostaglandin E(2) (PGE(2)). On incubation with histamine, HACs showed increased production of MMP-3, MMP-13, TNFalpha and PGE(2) (statistical significance P=0.02, 0.005, 0.008 and 0.03, respectively, student's t-test), but MMP-1 expression was unaffected. In contrast, the RSF showed a histamine-induced increase in MMP-1 ( P=0.028) and an approximate 10-fold level of MMP-3 and PGE(2) release over that of HACs, each being stimulated by histamine ( P=0.02 and 0.032, respectively, student's t-test). However, MMP-8, MMP-13 and TNFalpha were not detected for RSF cultures. Our results show that histamine modifies the behaviour of both HACs and RSFs in vitro, but different effects were observed for the production of specific MMPs and TNFalpha by the two cell types.

Histamine, histamine receptors (H1 and H2), and histidine decarboxylase expression by chondrocytes of osteoarthritic cartilage: an immunohistochemical study.

Degeneration and loss of articular cartilage are the characteristic features of osteoarthritis (OA), with the appearance of fibrillations, cell clusters, matrix depletion, and changes in matrix composition all apparent. Histamine has a recognised role in allergic and inflammatory reactions, and is reported to affect several aspects of chondrocyte behaviour. The immunohistochemical (IHC) studies reported here have demonstrated histamine (H), both H1 and H2 receptors, and the histamine-producing enzyme histidine decarboxylase (HDC) in a variable proportion of human articular chondrocytes in OA cartilage specimens. Such observations were especially evident within the degenerative, superficial zone, and more so in late-stage disease. By contrast, "normal" age-matched cartilage specimens showed relatively little immunopositive staining for histamine and HDC. These findings strongly suggest that histamine and H-receptor expression by HAC in OA cartilage is potentially an important contributor to the atypical, aberrant phenotype of OA chondrocytes.