A novel technique for positioning multiple cell types by liquid handling.

The spatial control of cells on a surface and the patterning of multiple cell types is an important tool for fundamental biological research and tissue engineering applications. A novel technique is described for the controlled seeding of multiple cell types at specific locations on a surface without requiring the use of specialized equipment or materials. Small-volume, quasi-hemispherical drops of cell solution are deposited onto a cell culture surface immersed under barrier oil, which serves to contain the drop and prevents evaporation of the cell culture medium during the time necessary for cells to attach to the cell culture surface. Subsequent flooding with an aqueous cell-compatible buffer displaces the barrier oil, allowing the cells to grow freely across the surface. This technique offers a simple and easily implemented solution for defining the initial position of cultured cells. The coculture of multiple cell types may be carried out by incorporating different cell types in each drop. A suitable drop volume was found to be 1 microl dispensed with a standard 0.5-10 microl pipette. The drop formed resulted in a footprint diameter of approximately 2 mm. Mineral oil and silicone oil do not compromise the viability of cultured cells when used in this technique. Moreover, a surface with heparin-immobilized FGF2 is shown to retain its bioactivity following drying of the substrate and contact with mineral oil.