ORGANOTYPIC CULTURES

These cultures are used to develop certain tissues or tissue models for example skin equivalents have been created by culturing dermis, epidermis and intervening layer of collagen simultaneously. Similarly models have been developed for prostrate, breast etc. Organotypic culture involves the combination of cells in a specific ratio to create a component of an organ.

Dispersed cell cultures clearly lose their histologic characteristics after disaggregation and, although cells within a primary explant may retain some of the histology of the tissue, this will soon be lost because of flattening of the explants with cell migration and some degree of central necrosis due to poor oxygenation. Retention of histologic structure, and its associated differentiated properties, may be enhanced at the air/medium interface, where gas exchange is optimized and cell migration minimized, as distinct from the substrate/medium interface, where dispersed cell cultures and primary outgrowths are maintained. This so-called organ culture will survive for up to 3 weeks, normally, but cannot be propagated. An alternative approach, with particular relevance to tissue engineering, is the amplification of the cell stock by generation of cell lines from specific cell types and their subsequent recombination in organotypic culture. This allows the synthesis of a tissue equivalent or construct on demand for basic studies on cell-cell and cell-matrix interaction and for in vivo implantation. The fidelity of the construct in terms of its real tissue equivalence naturally depends on identification of all the participating cell types in the tissue in vivo and the ability to culture and recombine them in the correct proportions with the correct matrix and juxtaposition. So far this has worked best for skin [Michel et al., 1999, Schaller et al., 2002], but even then, melanocytes have only recently been added to the construct, and islet of Langerhans cells are still absent, as are sweat glands and hair follicles, although some progress has been made in this area [Regnier et al., 1997; Laning et al., 1999].

There are a great many ways in which cells have been recombined to try to simulate tissue, ranging from simply allowing the cells to multilayer by perfusing a monolayer [Kruse et al., 1970] to highly complex perfused membrane (Membroferm [Klement et al., 1987]) or capillary beds [Knazek et al., 1972]. These are termed histotypic cultures and aim to attain the density of cells found in the tissue from which the cells were derived. It is possible, using selective media, cloning, or physical separation methods , to isolate purified cell strains from disaggregated tissue or primary culture or at first subculture. These purified cell populations can then be combined in organotypic culture to recreate both the tissue cell density and, hopefully, the cell interactions.