To study epithelial migration in vivo, different experimental wound models, both in humans and in animals, have been developed. Full-thickness wounds can be generated on the epidermis or mucosa by punch biopsy, after which the area is allowed to heal for the desired time, and removed for analysis. In full-thickness wounds, the BM is removed along with the epithelium. In the suction blister method, separation of the epidermis from the dermis takes place at the level of lamina lucida as a result of negative pressure applied using a specific device, which leaves the basement membrane intact (Kiistala 1968). Cell motility and behavior in vivo can be directly monitored with intravital microscopy in various experimental animal models. For monitoring cell migration, fluorescently labelled cells are injected locally into tissues. In transparent organs, native cells can also be monitored. (von Andrian & M"Rini 1998, Friedl & Brocker 2000)
By ex vivo epithelialization assays, it is possible to study, for example, the protein function in epithelial cell migration in gene-manipulated mouse models. Mouse skin biopsies are spread flat on tissue culture plates. Explants are grown in cell culture medium, and keratinocyte outgrowth is determined at frequent intervals from phase-contrast micrographs. The explants can be fixed and immunostained to differentiate keratinocytes from fibroblasts. Similarly, skin explants can be seeded and cultured on the membrane of Transwell tissue culture inserts, to assess cell transmigration. (Mazzalupo et al. 2002)
In vivo cell migration is a complex and poorly understood process. Two- or three-dimensional in vitro models of migration are able to provide information of the basic principles involved. Haptokinetic epithelial cell migration has been widely studied on two-dimensional migration models. In scratch assays, cells are grown to confluency, an approximately 2 mm wide area is scraped clear of cells using a pipette tip, and cells are allowed to migrate to the area. Another way of studying lateral migration is to plate the confluent cells inside a steel cylinder on a culture dish and and to remove the cylinder after the cells have adhered. The cells at the edge are allowed to migrate. Lateral migration assays can be monitored by photographing at regular intervals. Different aspects of migration can be studied by coating the culture plate with ECM proteins, on which the cells migrate. The directionality and speed of cell migration can be assessed in vitro using time-lapse video microscopy. Cells are plated on culture dishes and allowed to migrate, and the coordinates of cells are frequently recorded by image processing software. In cell-mixing experiments, different cell types can be labelled with fluorescent markers. (Friedl & Brocker 2000, Maheshwari et al. 2001)
In three-dimensional models mimicking the basement membrane structure, cells migrate from an upper chamber to a lower chamber through a porous filter. In Transwell chambers (Costar), the filter can be coated with basement membrane proteins. In Matrigel™ chambers, the filter is covered by a layer of gel prepared from murine EHS tumor, which mainly contains basement membrane components. Matrigel assay can be considered a model for quantitating the invasive potential of cells, as the cells must invade the gel as well as transmigrate to the other side of the filter (Albini 1998). In both types of transmigration assays, chemotactic agents may be added to the lower chamber to attract the cells. The migration level is quantified by counting the fixed and stained cells on the lower side of the filter by light microscopy. Three-dimensional collagen matrix models provide a defined model of studying cell migration, resembling connective tissue in vivo. Collagen solutions are polymerized, whereupon they form interconnected fibers and bundles. The cell suspensions can be added into the collagen solution before plating or on top of the polymerized collagen gel. Migration can be quantified by counting the fixed and stained cells that have invaded after a given period, or it can be monitored by computer-assisted cell tracking. (Friedl & Brocker 2000)