A key feature of mature epithelium is the presence of an apical-basal polarization. In vitro this is achieved by
culturing either pure populations of epithelial cells or unorganized mixtures of primary cell populations harvested from
epithelial tissues in a filter insert culture system. In addition to epithelial cells however, multiple other cell types are also
present in normal epithelium in a specific organization. Generally the organization of component cells within a tissue is
critical for normal function. The importance of the organization of different cell populations within normal and diseased
epithelium in vitro has received little attention, in part due to the lack of available methods for spatially controlling
multiple cell populations while maintaining the culture conditions necessary to generate polarized and mature epithelium.
We have developed a set of micropatterning techniques to spatially control the organization of co-cultures of epithelial
cells on filter insert membranes under the culture conditions necessary to induce epithelial cell polarization. We describe a
fibronectin microprinting method that allows arbitrary pattern generation and a parafilm insert method that allows only
simple stripe geometry patterning but does not require the photolithographic equipment that the fibronectin microprinting
method requires, and can be adopted by any epithelial biology lab. We also demonstrate that our micropatterning methods
do not alter the formation of cilia, a marker of epithelium maturation. Our methods provide a novel tool for studying
epithelial biology in polarized epithelial sheets containing multiple cell populations with controlled organization.