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``Biology of Cultured Cells''. In: Culture of Animal Cells, 5Th Edition

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``Biology of Cultured Cells''. In: Culture of Animal Cells, 5Th Edition CHAPTER3 BiologyofCulturedCells 3.1 THE CULTURE ENVIRONMENT or subculture they will need to attach and spread out on the substrate before they will start to proliferate (see Sections 13.7, The validity of the cultured cell as a model of physiological 21.9.2). Originally, it was found that cells would attach to, function in vivo has frequently been criticized. Often, the and spread on, glass that had a slight net negative charge. cell does not express the correct in vivo phenotype because Subsequently, it was shown that cells would attach to some the cell’s microenvironment has changed. Cell–cell and plastics, such as polystyrene, if the plastic was appropriately cell–matrix interactions are reduced because the cells lack treated with an electric ion discharge or high-energy ionizing the heterogeneity and three-dimensional architecture found radiation. We now know that cell adhesion is mediated by in vivo, and many hormonal and nutritional stimuli are specific cell surface receptors for molecules in the extracellular absent. This creates an environment that favors the spreading, matrix (see also Sections 8.4, 17.7.3), so it seems likely that migration, and proliferation of unspecialized progenitor cells, spreading may be preceded by the secretion of extracellular rather than the expression of differentiated functions. The matrix proteins and proteoglycans by the cells. The matrix influence of the environment on the culture is expressed adheres to the charged substrate, and the cells then bind to the via four routes: (1) the nature of the substrate on or in matrix via specific receptors. Hence, glass or plastic that has which the cells grow—solid, as on plastic or other rigid been conditioned by previous cell growth can often provide matrix, semisolid, as in a gel such as collagen or agar, or a better surface for attachment, and substrates pretreated liquid, as in a suspension culture; (2) the degree of contact with matrix constituents, such as fibronectin or collagen, or with other cells; (3) the physicochemical and physiological derivatives, such as gelatin, will help the more fastidious cells constitution of the medium; (4) the constitution of the gas to attach and proliferate. phase; and (5) the incubation temperature. The provision of With fibroblast-like cells, the main requirement is for the appropriate environment, including substrate adhesion, substrate attachment and spreading and the cells migrate nutrient and hormone or growth factor concentration, and individually at low densities. Epithelial cells may also require cell interaction, is fundamental to the expression of specialized cell–cell adhesion for optimum survival and growth and, functions (see Sections 17.1, 17.7 and Alberts et al., 2002). consequently, they tend to grow in patches. 3.2.1 Cell Adhesion Molecules 3.2 CELL ADHESION Three major classes of transmembrane proteins have been shown to be involved in cell–cell and cell–substrate adhesion Most cells from solid tissues grow as adherent monolayers, (Fig. 3.1). Cell–cell adhesion molecules, CAMs (Ca2+ and, unless they have transformed and become anchorage independent), and cadherins (Ca2+ dependent) are involved independent (see Section 18.5.1), after tissue disaggregation primarily in interactions between homologous cells. These Culture of Animal Cells: A Manual of Basic Technique, Fifth Edition, by R. Ian Freshney Copyright 2005 John Wiley & Sons, Inc. 31 32 CULTURE OF ANIMAL CELLS CAMs Cell layer Basement membrane Cadherins Connective - Ca - tissue / stroma - Ca - - Ca - Integrins Proteoglycans Receptors with intracellular Low affinity, transmembrane signaling domain and receptors, without signaling variable extracellular domain: domains: bind matrix bind fibronectin, vitronectin, proteoglycans, collagen, and laminin, collagen growth factors Fig. 3.1. Cell Adhesion. Diagrammatic representation of a layer of epithelial cells above connective tissue containing fibrocytes and separated from it by a basal lamina. CAMs and cadherins are depicted between like cells, integrins and proteoglycans between the epithelial layer and the matrix of the basal lamina. proteins are self-interactive; that is, homologous molecules more resistant to disaggregation, as they tend to have tighter in opposing cells interact with each other [Rosenman junctional complexes (desmosomes, adherens junctions, and & Gallatin, 1991; Alberts et al., 2002], and the cell–cell tight junctions) holding them together, whereas mesenchymal recognition that this generates has a signaling role in cell cells, which are more dependent on matrix interactions for behavior [Cavallaro & Christofori, 2004]. Cell–substrate intercellular bonding, are more easily dissociated. Endothelial interactions are mediated primarily by integrins, receptors cells may also express tight junctions in culture, especially for matrix molecules such as fibronectin, entactin, laminin, if left at confluence for prolonged periods on a preformed and collagen, which bind to them via a specific motif matrix, and can be difficult to dissociate. In each case, the usually containing the arginine–glycine–aspartic acid (RGD) cells must resynthesize matrix proteins before they attach or sequence [Yamada & Geiger, 1997]. Each integrin comprises must be provided with a matrix-coated substrate. one α and one β subunit, the extracellular domains of which are highly polymorphic, thus generating considerable 3.2.2 Intercellular Junctions diversity among the integrins. Both integrins and cadherins Although some cell adhesion molecules are diffusely interact with vinculin, a step in signaling to the nucleus arranged in the plasma membrane, others are organized [Bakolitsa et al., 2004]. into intercellular junctions. The role of the junctions varies The third group of cell adhesion molecules is the between mechanical, such as the desmosomes and adherens transmembrane proteoglycans, also interacting with matrix junctions, which hold epithelial cells together, tight junctions, constituents such as other proteoglycans or collagen, but which seal the space between cells, e.g. between secretory not via the RGD motif. Some transmembrane and soluble cells in an acinus or duct or between endothelial cells in a proteoglycans also act as low-affinity growth factor receptors blood vessel, and gap junctions, which allow ions, nutrients, [Subramanian et al., 1997; Yevdokimova & Freshney, 1997] and small signaling molecules such as cyclic adenosine and may stabilize, activate, and/or translocate the growth monophosphate (cAMP) to pass between cells in contact factor to the high-affinity receptor, participating in its [see Alberts et al., 2002]. Although desmosomes may be dimerization [Schlessinger et al., 1995]. distributed throughout the area of plasma membranes in Disaggregation of the tissue, or an attached monolayer contact (Fig. 3.2a), they are often associated with tight and culture, with protease will digest some of the extracellular adherens junctions at the apical end of lateral cell contacts matrix and may even degrade some of the extracellular (Fig. 3.2b). domains of transmembrane proteins, allowing cells to become As epithelial cells differentiate in confluent cultures they dissociated from each other. Epithelial cells are generally can form an increasing number of desmosomes and, if CHAPTER 3 BIOLOGY OF CULTURED CELLS 33 T JCJC D (a) (b) Fig. 3.2. Intercellular Junctions. Electron micrograph of culture of CA-KD cells, an early-passage culture from an adenocarcinoma secondary in brain (primary site unknown). Cells grown on Petriperm dish (Vivascience). (a) Desmosomes (D) between two cells in contact; mag. 28,000×. (b) Canaliculus showing tight junctions (T) and junctional complex (JC); mag. 18,500×. (Courtesy of Carolyn MacDonald). some morphological organization occurs, can form complete the cells under study (see Protocol 8.1), or by using a junctional complexes. This is one reason why epithelial preformed matrix generated by the Engelberth-Holm-Swarm cells, if left at confluence for too long, can be difficult to (EHS) mouse sarcoma, available commercially as Matrigel disaggregate. As many of the adhesion molecules within (see Section 8.4.1). Matrigel is often used to encourage these junctions depend on Ca2+ ions, a chelating agent, such differentiation and morphogenesis in culture and frequently as EDTA, is often added to the trypsin during or before generates a latticelike network with epithelial (Fig 3.3; Plate disaggregation. 12c) or endothelial cells. At least two components of interaction with the substrate 3.2.3 Extracellular Matrix may be recognized: (1) adhesion, to allow the attachment Intercellular spaces in tissues are filled with extracellular and spreading that are necessary for cell proliferation matrix (ECM), the constitution of which is determined [Folkman & Moscona, 1978], and (2) specific interactions, by the cell type, e.g., fibrocytes secrete type I collagen reminiscent of the interaction of an epithelial cell with and fibronectin into the matrix, whereas epithelial cells basement membrane, with other ECM constituents, or with produce laminin. Where adjacent cell types are different, adjacent tissue cells, and required for the expression of e.g., at the boundary of the dermis (fibrocytes) and epidermis some specialized functions (see Sections 3.4.1 and 17.7.3). (epithelial keratinocytes), both cell types will contribute to Rojkind et al. [1980], Vlodavsky et al. [1980], and others the composition of the ECM, often producing a basal lamina. explored the growth of cells on other natural
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