FIGURE 1 Preparation of organotypic brain slice cultures. The tissue of interest is first dissected free from the neonatal brain, as illustrated for the hippocampus, and thin sections are cut under aseptic conditions using either a tissue chopper or a vibratome. A: In the roller-tube method, the slices are attached to cleaned glass coverslips in a film of clotted fibrin or chicken plasma; the slices are then placed in sealed test tubes and placed on a roller drum in a dry-air incubator. B: In the membrane method, the slices are placed on semipermeable membranes in multiwell plates so that they are at the gas-air interface and then maintained in a CO2 incubator.
In the roller-tube method, brain slices can be attached to glass coverslips using various substances, including chicken plasma clotted with thrombin, collagen, or synthetic fibrin solutions (e.g., Tisseal, Baxter International, Deerfield, IL), either alone or in combination. These substances serve as effective, tissue-friendly "glues" for holding the tissue down firmly in the roller drum. Coverslips containing the slices are placed in sealed test tubes containing culture medium, and the tubes are placed on a roller drum in an incubator at 36°C. The drum rotates at about 10 revolutions per hour. For the membrane cultures, the slices are placed directly on semipermeable membrane inserts (Millicell, Millipore, Billerica, MA) and need no adhesive substance because they remain static in the CO2 incubator. The inserts are placed in culture wells filled with medium so that the slices are at the interface between the medium and the air; this interface allows oxygen to diffuse into the slice at a sufficiently high concentration to maintain tissue viability. Similarly, in the roller-drum method, the slices are only submerged in medium for part of each cycle so that adequate oxygenation is maintained. In both methods, the slices are typically grown in a semisynthetic medium containing bovine or horse serum. Serum-free medium can also be used (Annis et al., 1990), most effectively after the cultures have become established.
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