Overview of Cell and Tissue Culture UNIT 12.1 Techniques The techniques for tissue and cell culture methods that must be utilized. On the other evolved from ex vivo studies of whole organs hand, it is difficult to obtain cultures of a single or tissue fragments that were kept in vitro for defined cell type with immature undifferenti- various length of time. To prolong their struc- ated tissue. The availability of multipotent tural and functional integrity in vitro, the bal- cells, such as the hematopoietic precursor cells anced salt solutions used in acute experiments from bone marrow (Metcalf, 1984; Celis, were replaced, first by complex biological me- 1998), embryonic stem cells prepared from dia (e.g., plasma, serum, or tissue extracts), then preimplantation embryos (Conn, 1990; Celis, by synthetic media containing various propor- 1998), or stem cells from immature or adult tions of biological fluids, and more recently by brain (Celis, 1998) makes it possible to conduct chemically defined media. At the same time, a lineage-specific cell differentiation using ap- variety of culture techniques have evolved, tak- propriate signaling factors. In general, primary ing into account both the goal of a particular cells undergo only a finite number of cell divi- experimental approach and the particular sions (and may not proliferate at all in culture) growth requirements of a given cell or tissue. and have a limited life span. With the exception Culture preparations vary greatly in complex- of hemopoietic cells and certain transformed ity, ranging from single isolated cells to three- cells (see discussion of Suspension Cultures dimensional histotypic cell structures. Besides under Culture Techniques), most animal cells cultures obtained directly from animal tissues are adherent (they are therefore called “anchor- (primary cultures), permanent cultures of con- age-dependent”). For growth and maintenance tinuously dividing cells have been established. in vitro, these cells must attach either to a Therefore, before utilizing cell cultures, there specifically treated artificial surface or to the is a need to select among the multitude of surface of other cells. Furthermore, for the established in vitro systems. In general, it ap- subdivision and replating of adherent cell popu- pears that with the increasing complexity of the lations (“subculturing” or “passaging”), spe- culture system, the relevance to the in vivo cific techniques are used for cell detachment. situation increases, while it becomes more dif- ficult to control the cellular and molecular vari- Continuous (Permanent) Cell Cultures ables. It is unlikely that there will ever be a Continuous cultures are composed of cells single in vitro approach adequate for all experi- that may proliferate indefinitely. Continuously mental needs. Therefore, to select a particular dividing cells may arise spontaneously in the approach, both the advantages and disadvan- process of continuous subculturing of normal tages of each in vitro approach must be consid- cells that proliferate in vitro (e.g., fibroblasts or ered. The aim of the present unit is to highlight astrocytes). Such a cell lineage is termed a cell these issues. strain. However, the majority of “immortal” cells belong to the class of transformed cells, CULTURE TYPES and an established clone of such cells is termed a cell line. Transformed cells have undergone Primary Cultures a stable heritable change and they are tu- Primary cultures are prepared with cells or morigenic (“malignant”)—i.e., they are able to tissues taken directly from the intact organism. form tumors in an appropriate recipient animal They can be maintained in vitro for only a such as nude mice (Jakoby and Pastan, 1979). limited period of time, ranging from several Historically, most cell lines were derived from days to months, depending upon the cell type tumors that occurred either spontaneously or and the culture conditions. Often, primary cul- after chemical or viral induction either in vivo tures are derived from immature cells or tissues, or in vitro. Some of these cell lines exhibit a being allowed to differentiate and mature in more or less undifferentiated phenotype, vitro. In some cases, cultures of differentiated whereas others show certain, but never all, cells are prepared from tissue taken from a characteristics of the corresponding differenti- mature organism. It is often more difficult to ated cell. Many of the currently available neural obtain viable cells from differentiated tissues cell lines belong to the latter category, such as because of the relatively harsh dissociation clones of the mouse C1300 neuroblastoma, the In Vitro Cellular Assays Contributed by Paul Honegger 12.1.1 Current Protocols in Pharmacology (1999) 12.1.1-12.1.12 Copyright © 1999 by John Wiley & Sons, Inc. Supplement 4 rat C6 glioma, the rat PC12 pheochromocy- CULTURE TECHNIQUES toma, and the rat RN22 schwannoma. A variety A number of excellent books detail the basic of strategies have been used in the search for methods for tissue and cell culture, including new cell lines exhibiting specific differentiated medium preparation, sterilization, cell han- phenotypes. An early approach was somatic dling, aseptic working techniques, and quality cell hybridization (e.g., Jakoby and Pastan, control (e.g., Davis, 1994; Freshney, 1992; Jak- 1979). By fusing transformed cells with so- oby and Pastan, 1979; Pollard and Walker, matic cells, immortal hybrid cells (hybridomas) 1990; Boulton et al., 1992). Furthermore, there were obtained that express characteristics of the is a large body of literature providing detailed normal parent cell. Cell fusion is promoted by protocols of culture techniques for a great va- chemicals, such as polyethylene glycol, or by riety of cell types, including neural cells (Bot- viruses, such as β-propiolactone-inactivated tenstein and Sato, 1985; Conn, 1990; Banker Sendai virus. For isolation of the hybridomas, and Goslin, 1991; Fedoroff and Richardson, selection media containing an inhibitor of de 1997), epithelial cells (Shaw, 1996), liver cells novo nucleotide synthesis, such as aminopterin, (Brill et al., 1994), renal cells (Handler and methotrexate, or azaserine, in addition to the Kreisberg, 1991), heart muscle and endothelial required purine and/or pyrimidine salvage pre- cells (Piper, 1990; Deli and Joo, 1996; Celis, cursor(s), such as hypoxanthine and/or thymid- 1998), keratinocytes (Daniels et al., 1996; ine, are used. For example, the fusion of normal Celis, 1998), and hemopoietic cells (Metcalf, B lymphocytes with transformed lymphocytes 1984; Celis, 1998). Because of the wide variety (myeloma cells) is a routine procedure for gen- of culture systems, it is sometimes difficult to erating immortalized hybrids that produce the select the most appropriate for study. The pur- monoclonal antibody encoded by the original pose of this unit is to provide guidance in the B lymphocyte. Also, many neuroblastoma and search for an appropriate in vitro system by glioma cell hybrids are derived this way. Fur- detailing basic aspects of culture methodolo- thermore, techniques have been developed to gies. prepare permanent cell lines by the transfection of immortalizing oncogenes (see discussion of Suspension Cultures Cell Immortalization under Culture Prepara- Suspension cultures are generally used for tion and Maintenance). However, transfection anchorage-independent cells, such as procedures are generally harsh and require a hemopoietic cells and some transformed cell relatively large number of cells. Therefore, vi- lines, and for free-floating cells or cell forma- rus-mediated transformation has been found to tions (e.g., isolated cells, aggregates, or tissue be a milder and more efficient method for this fragments). Anchorage-independent cells are purpose (Cepko, 1989). Immortalization of grown in either a semisolid medium, such as cells is attained by retroviral gene transfer— agarose, or in fluid suspension culture under e.g., using oncogene-containing retrovirus vec- continuous agitation (stirring or shaking). tors. However, with both transfection and viral Short-term suspension cultures of bulk, iso- infection, heterogeneous cell populations are lated brain cells have also been described (Ver- produced, exhibiting different sites of gene in- ity, 1995). In general, suspension cultures are tegration. To overcome this problem, a method easy to maintain and are ideal for scale-up. For was developed to generate genetically homo- the latter, culture vessels of various sizes are geneous immortalized cell lines from trans- available, ranging from small spinner flasks to genic mice. An advantage of cell lines is that industrial-size fermenters (e.g., Jakoby and they can be propagated indefinitely, providing Pastan, 1979). large quantities of a relatively homogenous cell population. Problems encountered with tu- Attached (“Monolayer”) Cell Culture morigenic and hybridoma cell lines include Monolayer cell culture is the most widely their phenotypic and functional deviation from used technique, with numerous protocol vari- the normal parent cell(s), and their abnormal ants of this approach practiced in different labo- and unstable karyotype resulting in frequent ratories (Sensenbrenner, 1977; Conn, 1990; intra- and interclonal heterogeneity. Neverthe- Jakoby and Pastan, 1979). In these cultures, the less, cell lines are invaluable for many in vitro cells grow attached to the surface of the culture approaches, and are useful complementary sys- vessel, although