Although the explantation of cells from a multicellular organism into an extraorganismal envi- ronment, that is, tissue culture, had been known since the beginning of this century, it was not until the early 1940s that Earle and associates (cf. Earle and Nettleship, 1943) attempted to in- duce the neoplastic transformation in cultured mammalian cells. These now classic experiments, which were carried out by the addition of polycyclic hydrocarbons to cultures of mouse fibro- blasts, were monitored by the inoculation of treated and untreated cell cultures into host animals. Unfortunately the results of these experiments were ambiguous, since neoplasms arose in the test animals whether treated or control cultured cells were inoculated. As a result, the question of the feasibility of carcinogenesis in vitro lay dormant for almost two decades. However, the cells from these experiments have been maintained, even up to the present day, as the L cell line (cf. Jackson, 1991).
TECHNIQUES OF TISSUE CULTURE
It is not the objective of this text to present an extensive discussion of the methodology of cell and organ culture. The interested student is referred to the bibliographic references (e.g., Nar- done, 1987; Celis and Celis, 1994). However, it is important to understand the difference be- tween organ culture and cell culture. In the former, fragments of organs or even whole organs are removed from the organism, and their viability and organization are maintained in an artificial environment. While larger samples of tissue usually did not survive more than a few days, organ culture of fragments of colon (Shamsuddin et al., 1978), mouse mammary glands (Iyer and Ban- erjee, 1981), and embryonic rat pancreas (Parsa and Marsh, 1976) could be maintained for some period of time in culture. During this time the microscopic anatomy of the tissue was retained, remaining essentially identical with that seen in the organ in vivo.
On the other hand, cell culture involves the isolation and/or dispersion of cells from organs and tissues, with their subsequent cultivation as cellular populations with little regard to their original morphology and functionality. Such cell cultures may be cloned, that is, single cells are isolated and allowed to proliferate to a visible colony. Quantitative measurements and genetic studies may be performed relatively easily with cell cultures provided that the cells either are available in significant quantity or replicate quite readily in the culture environment. The term primary culture indicates those cells obtained directly from the animal and cultured in the spe- cific system under study. The terms secondary and transfer cultures refer to the subsequent cul- tivation of cells, with the inoculum taken from cells already in culture. In Figure 14.1, a cutaway
Figure 14.1 Diagram of tissue culture as organ culture (A) and cell culture (B). In the organ culture system shown, a fragment of whole tissue rests on a wire screen through which the medium permeates. For cell culture, suspensions of cells are plated on the surfaces of petri dishes, allowed to attach, and then cov- ered with appropriate media. (The example in A is taken from Trier, 1976, with permission of the author and publisher.)
diagram of an organ and a cell culture in a petri dish may be noted. The major distinction is the special structure required to maintain the tissue in its organoid form while assuring access of sufficient nutrients from the medium for maintenance of viability. Cell cultures, on the other hand, usually consist of a monolayer of cells or small cell colonies, at most only a few cells in thickness, lying entirely within the environment of the liquid medium. A variety of other config- urations for organ cultures have also been described (cf. Anderson and Jenkinson, 1998; Minuth et al., 1996). Cell cultures may also be maintained as suspensions of cells (e.g., Himmelfarb et al., 1969) or as multicellular spheroids (e.g., Korff and Augustin, 1998).
In general, cells within organ cultures maintain most of the genetic and functional capabil- ities of comparable cells in vivo. In cell culture, however, especially of tissues from certain types of animals such as rodents, the natural history of cells transferred and cultivated for long periods is that of karyotypic and biochemical changes, usually in the direction of increased aneuploidy and decreased biochemical differentiation. Certain terminologies have been developed for cer- tain types of cell cultures and their progeny (Schaeffer, 1990). A cell line arises from a primary culture at the time of the first successful secondary or subculture. The cell line cultures thus should consist of lineages of cells originally present in the primary culture. Cell lines are termed finite or continuous if the history and potential of the line is known. If this is not known, then the term line will suffice. A cell strain may be derived either from a primary culture or from a cell line by the selection or cloning of cells having specific properties or markers. Thus, descriptions of cell strains should also include any specific features of the strain. As with cell lines, the terms finite or continuous are used as prefixes if the history and potential of the culture is known; oth- erwise, the term strain by itself is sufficient. Obviously, cell lines and strains may be derived from organ cultures as well. Cells from strains or lines may be frozen at liquid air temperatures and maintained in this state for years with relatively efficient recovery of viability on thawing. A
few primary cell cultures have also been successfully maintained in this way, but organ cultures do not readily survive such treatment.
The media in which organ and cell cultures are maintained or stimulated to grow have been quite complex, consisting of numerous nutrients of known composition as well as, in most instances, numerous materials of unknown chemical composition such as serum, partial protein hydrolysates, and tissue extracts. During the last several decades, a number of chemically de- fined media have been developed for use with primary cultures as well as for the growth and maintenance of cell lines and cell strains. While the vast majority of these media are effective for cells that are derived from neoplasms or have a significant number of characteristics of neopla- sia, several such media have been utilized for normal cell lines. These include human diploid fibroblasts (Bettger et al., 1981), human epidermal keratinocytes (Tsao et al., 1982), and human lymphocytes (Shive and Matthews, 1988). A number of other cell lines may also be maintained in defined media (Taub, 1990). The defined media may contain a number of proteins, growth factors, and other nutrients that presumably occur at some level in the serum, plasmid, or other organismal fluid that is used in nondefined culture media (Barnes, 1987; Bjare, 1992).
In addition to the composition of the medium, which is critical for cell growth and mainte- nance, the matrix on which the cells are cultured has become of increasing significance during the last two decades. Prior to this time most cultures with the exception of organ cultures were maintained on glass or plastic as the substratum. In 1975, Michalopoulos and Pitot described the primary culture of parenchymal hepatic cells from rats on collagen membranes. This technology allowed considerably longer maintenance times as well as enhanced biochemical and morpho- logical differentiation of these cells in primary culture. During the next decade or more, similar or related technologies were developed to culture a variety of mesenchymal (cf. Kleinman et al.,
1981) and differentiated epithelial tissues (e.g., Montesano et al., 1983; Yang et al., 1979; Yang et al., 1982; Chambard et al., 1981). Reid and associates (Rojkind et al., 1980) demonstrated the importance of the presence of extracellular matrix molecules (Chapter 10) in maintaining epithe- lial cells in a morphologically and biochemically differentiated state for extended periods of weeks or months (cf. Reid and Jefferson, 1984). A combination of extracellular components and collagen gels could effect the same result (Lin et al., 1997). Other variations in substratum for epithelial cell culture included the “sandwich” technique, with the matrix present on both sides of the epithelial sheet (Kern et al., 1997; Beken et al., 1997), and cocultivation of hepatocytes with littoral cells from the same organ (Namieno et al., 1996; Guguen-Guillouzo et al., 1983). A number of other very sophisticated technologies have been utilized for the culture of a variety of different cell types, both in microscopic and very large instruments, the latter allowing for the culture of large numbers of cells useful as molecular biological reagents and/or the production of specific protein products.