It has been demonstrated that slices of developing brain tissue (e. g. rat hippocampus) can be grown for extended periods of time (weeks) on an silicon-based microelectrode array, which was manufactured for the purpose of multiple site, long term electrophysical recordings (and stimuations) of neurons and neuronal connections (during development and after maturation) and for sensitive and early detection of functional neurotoxic effects of know and novel compounds in comparison with established biomarkers for structural and neurodegenerative changes. The results were obtained in parallel with studies of slice cultures grown by conventional techniques on semiporous membranes, which also served to establish standardized protocols for reproducible exposure paradigms and quantitative image analysis with monitoring of induced neuronal cell death and structural changes. We have shown that brain slices growing on the array can be exposed to chemicals and that the electrical neuronal activity can be measured by connecting the electrodes of the array to conventional electrophysiological recording equipment. The brain tissue can be stimulated electrically through the array or chemically by adding chemicals to the culture medium.The method can be used to study acute and long-term physiological actions of drugs, toxins and other biologically active substances on cultured, organotypically organized brain slices, as well as for on-line and long-term studies of developmental and regenerative processes in brain tissues.
The result includes proof of biocompatibility and long-term support of developing brain tissue slices and hence the basic feasiability of the silicon-based microelectrode array, manufactured by Neuchatel-IMT. Included is also the demonstration by Odense-IMB of intimate structural contact between the uninsulated tips of the microelectrodes and neuronal processes in the cultured brain slices, which comply with the electrophysiological results (sensitivity and signal to noise ratios), obtained by Copenhagen-DMP. The result combines the fields of neurobiology/cell biology (A14), toxicology (A38),microelectronics (D23) and manufacturing technology. The culturing of brain tissue slices for extended periods of time on microelectrode arrays with pointed electrodes penetrating deep into and firmly positioned inside the cultured slice is unique, and must in terms of qualities of electrophysiological recordings be considered superior to the growth of brain slices on devices with planar or minor pointed electrodes available on the market. The result includes a series of procedures some of which are modifications of existing and published procedures for obtaining and maintenance of tissue for organotypic slice cultures of the mammalian brain.
Using the procedures it is possible to harvest sections of brain from newborn (to 2 week old) rats and place the sections in an incubator for maturation. After about one week the organotypic culture is ready to be placed on a microelectrode array and be maintained on the array for up to a week at present. During this period the tissue on the array can be transferred to a set-up and connected to standard electrophysiological equipment. Thus the physiology of the tissue and the effect of drugs and chemicals including toxins can be studied in vitro for several days with the existing prototype and protocol.