Topographical imaging of macroporous microcarriers using laser scanning confocal microscopy

Macroporous microcarriers are used for the high density cultivation of mammalian cells. The large pores in the interior of these beads provide space for cell growth. However, it has been observed that the cell distribution in the interior of microcarriers is often uneven. In some cases, the pores appear to be unoccupied. Questions thus arise as to whether all pores are open to the bead surface, and whether the pores allow cell entry and proliferation. To examine the accessibility and geometrical features of the pores, laser scanning confocal microscope was used to optically section fluorescein isothiocyanate stained microcarriers. Three dimensional images were reconstructed by combining the sections for examination of the openness as well as the connectivity of the pores. By employing image processing software, the solid structure of the beads was removed from reconstructed three dimensional images to allow for direct visualization of the pores. A wide variety of geometries was observed; some pores were interconnected while others were segregated from each other. It was observed that while many pores were open to the surface through sufficiently large channels for cell entrance, a large number of pores was also either closed to the exterior or accessed the surface only through constricted channels which might impede cell entry. This confocal microscopic technique for direct evaluation of pore structure of macroporous beads is noninvasive and requires minimal sample preparation. It can easily be adapted for in situ observation of cell behavior as well as find application for structural assessment for various biomaterials.