Overview of Cell and Tissue Culture Techniques 12.1.12

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 difficult 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 specific treatment of the cul- Overview of Cell tems for studying normal cells. ture vessel surface (e.g., with polylysine or and Tissue Culture polyornithine) may be necessary for better cell Techniques 12.1.2

Supplement 4 Current Protocols in Pharmacology attachment and/or attainment of more physi- with three-dimensional cultures, oxygen and ological growth conditions. The growth condi- nutritional gradients are critical, limiting the tions are also influenced by the cell density. size of the individual aggregate to a diameter Techniques using different cell densities range less than 400 µm. Moreover, in certain prepa- from single-cell microcultures to high-density rations, such as aggregate cultures of fetal liver mass cell cultures. The yield in monolayer cells, an additional diffusion barrier is formed cultures is limited by the available surface to by a peripheral epitheloid cell layer. This prob- which the cells may attach. Therefore, for scal- lem is not encountered with aggregate cultures ing up, cells may be grown in roller bottles of fetal brain cells, which can be maintained for (cylindrical bottles partially filled with medium months at a highly differentiated stage. Tumor and rotated around their horizontal axis at 40 cells or explants grown in sponge-gel matrices to 50 rpm to provide continuous medium sup- form three-dimensional structures (called ply), in capillary perfusion systems (in which spheroids) and exhibit tissue-specific drug re- medium is circulated through tightly packed sponses (Celis, 1998). artificial capillaries), or attached to particulate microcarriers that can be maintained in suspen- Explant Cultures sion culture (Conn, 1990). With a high-cell- Explant cultures are prepared either from density culture, essential metabolites may be intact organs, such as dorsal root ganglia, sym- rapidly depleted, causing density-dependent pathetic ganglia, parasympathetic ciliary gan- growth inhibition and eventually cell death. glia, or from small tissue fragments that do not One advantage of monolayer cultures is their exceed 1 mm in any dimension. This approach ready accessibility for direct microscopic ex- is particularly useful for neural tissues which, amination, as well as for morphological, immu- due to their structural and functional complex- nocytochemical, and electrophysiological ity, are most difficult to reconstitute in vitro. studies. On the other hand, it is relatively diffi- Explants may be maintained in stationary cul- cult to sample monolayer cultures for ultras- ture on coverglasses or coverslips (Crain, 1976; tructural or biochemical analyses without de- Bornstein, 1995; Fedoroff and Richardson, stroying the culture. Furthermore, with these 1997), or they may be placed on porous and cultures, the cells are attached to an artificial transparent membranes (e.g., Millipore Mil- substrate rather than to the natural extracellular licell-CM; Falcon Cyclopore; Whatman Ano- matrix, limiting direct cell-cell interactions. disc) that remain in contact with liquid medium in such a way that a thin film of liquid surrounds Three-Dimensional (Aggregate) Cell the tissue (Stoppini et al., 1991). Often, the Cultures attachment of explants is enhanced by substrate Aggregate cultures are prepared from disso- coating using collagen, laminin, or polylysine. ciated cells allowed to reaggregate under con- As a further variant, brain slices embedded in trolled conditions and continuous gyratory agi- a plasma clot on glass coverslips are maintained tation to form regular, spherical cell structures. in a roller tube, which allows for alternating Such cultures permit a maximum of cell-cell exposure of the tissue to air and liquid medium interactions and thus the development of a natu- (Gähwiler, 1981). Explants (or microexplants ral cell matrix and histotypic cell formations. from minced tissue) may also be maintained in Aggregate cultures prepared from fetal cells nonadherent organ culture by using continuous show a particularly high capacity for cellular gyratory agitation to keep the tissue in suspen- reorganization and maturation. Although it has sion. Stationary as well as free-floating ex- been shown by Moscona, who invented this plants preserve their cytoarchitectonic and particular cell culture technique (Moscona, gross anatomic cellular organization to a large 1965), that immature cells of any tissue are able extent, although structural and functional ab- to reaggregate and mature into histotypic struc- normalities relative to the in vivo situation tures, this method is most suitable for immature occur. Roller tube slice cultures decrease in neural cells (Conn, 1990; Fedoroff and thickness to one to three cell layers within a few Richardson, 1997). One advantage of aggre- days of culture, but generally retain their histo- gate cultures is that they provide large numbers typic organization. This preparation is particu- of highly reproducible replicates, from which larly well suited for direct microscopic obser- aliquots are readily sampled for multidiscipli- vation and electrophysiological studies. As a nary studies. On the other hand, aggregate cul- rule, explant cultures are the best approxima- tures do not permit the direct microscopic ob- tion of the in vivo situation with respect to the In Vitro Cellular servation of cells during culture. Furthermore, organ-specific cellular organization and matu- Assays 12.1.3

Current Protocols in Pharmacology Supplement 4 ration. For example, cerebellar explants de- Certain immature tissues, such as those from velop normal architectural arrangements of brain or liver, may be dissociated into single cortical laminae and deep nuclei, and hippo- cells by mechanical means only. This may be campal explants form functional synaptic net- accomplished by simple trituration using large- works in culture, as well as normal dendritic bore and/or fire-polished (constricted) glass arborizations of pyramidal and granule cells. pipets, and/or by passage through fine nylon Explant cultures also offer a unique system for filter mesh or stainless steel screen of defined the study of adjacent brain regions in coculture, pore size. For mechanical dissociation, the tis- such that they are able to form interacting af- sue is maintained in Ca2+/Mg2+-free balanced ferent and efferent projections under in vitro salt solution. In combination with proteolytic conditions (Fedoroff and Richardson, 1997). enzymes, Ca2+ chelators such as EDTA are used The main disadvantages of explant cultures are to disrupt intercellular and cell-matrix junc- the small amount of tissue available and the tions. However, some proteolytic enzymes, relatively low number and limited reproduci- such as collagenase, require Ca2+, and DNase bility of replicate cultures, making it difficult requires Mg2+. To limit enzymatic digestion, to use them for biochemical and molecular specific protease inhibitors or serum are used. biology studies. After dissociation, the resulting cell suspension is examined to assess cell yield (by counting CULTURE PREPARATION AND the number of cells obtained from the original MAINTENANCE mass of tissue using a hemocytometer or an The technical requirements for culture electronic cell counter) and viability (by exclu- preparation and maintenance vary greatly. Pri- sion of dyes such as trypan blue). mary hemopoietic cells, and many permanent cell lines, are particularly easy to obtain and Cell Separation and Purification cultivate. A large number of cell lines originat- Cell separation and purification may be nec- ing from various tissues and species, including essary to prepare cultures of defined cellular human tumor cell lines (Hay et al., 1994) have composition, or to eliminate contaminating been characterized. Many can be purchased cells (e.g., erythrocytes or fibroblasts) and de- from commercial suppliers, including the bris. Cells may be separated either immediately American Type Culture Collection (ATCC) and after dissociation, or after culture initiation. the European Collection of Animal Cell Cul- Dissociated cells with differences in size and/or tures of the Centre for Applied Microbiology buoyant density are separated by centrifugal and Research (ECACC/CAMR). In some elutriation (Celis, 1998) or by differential cen- cases, cell cultures have to be established from trifugation (Pretlow and Pretlow, 1983). For the original tissues, requiring more elaborate density gradient centrifugation, solutions of protocols for culture preparation and mainte- Percoll, Ficoll, and/or sucrose can be used. nance. Alternatively, cell separation or purification may be achieved with antibodies that recognize Tissue Dispersion cell-specific surface markers. Thus, isolated Tissue dispersion is required for preparing cells may be separated using techniques such most types of primary cell cultures (Jakoby and as fluorescence-activated cell sorting (FACS), Pastan, 1979). Suspensions of dissociated cells selective immunoadsorption (immunopan- can be obtained mechanically (by cutting, ning), or immunomagnetic separation. Alterna- mincing, shearing, or sieving), chemically (by tively, unwanted cells may be selectively de- omission of divalent cations with or without the stroyed by complement-mediated lysis. Cells addition of chelating agents), or enzymatically may also be separated by exploiting differences (by proteolytic separation of cells using en- in their attachment to artificial substrata using zymes such as trypsin, papain, dispase, col- glass or plastic surfaces with or without special lagenase, pronase, hyaluronidase, DNase, or a coating. For example, fibroblasts and macro- mixture of several of these enzymes). In some phages attach to culture surfaces more rapidly instances, a combination of dissociation tech- than other cells. Oligodendrocytes derived niques is used. The choice of a particular pro- from adult brains adhere relatively poorly to tocol depends upon the nature, developmental tissue culture plastic as compared to astrocytes stage, amount of tissue, and the intended use of or microglia, whereas microglial cells exhibit Overview of Cell the cultures. In all cases, it is important to keep stronger adherence to plastic as compared to and Tissue Culture the tissue moist and to minimize physical and astrocytes (Fedoroff and Richardson, 1997). Techniques chemical trauma to the tissue and isolated cells. Cell separation by differential adhesivity may 12.1.4

Supplement 4 Current Protocols in Pharmacology be possible also in more advanced cultures. For phate, cationic liposomes, or electroporation. example, highly purified glial subtypes can be Easily transfectable cells are, for example, isolated from confluent cultures of mixed glial mouse L cells, as well as BHK, COS, and Vero cells using a shaking procedure (Conn, 1990; cell lines. Retroviral transduction by infection Fedoroff and Richardson, 1997). During culti- with genetically modified viruses (using vation, the enrichment of a specific cell type retroviruses such as Rous sarcoma virus) is can be obtained using a selective growth me- generally better suited to generate stably inte- dium, taking advantage of known cell type– grated genes in cells of diverse origin (Cepko, specific nutritional requirements. For example, 1989; Markowitz et al., 1988). However, this pure astroglial cultures can be obtained by use requires that the recipient cells be mitotically of a selective medium containing sorbitol in- active. Nondividing cells, such as neurons, may stead of glucose, which eliminates neurons, be transfected through nontoxic mutants of oligodendrocytes, and microglia from mixed other viruses such as adenovirus or herpes virus brain cell cultures (Fedoroff and Richardson, (Fedoroff and Richardson, 1997). Transfection 1997; Wiesinger et al., 1991). Microglia can be of cells with the myc oncogene usually pro- removed chemically using defined medium duces continuously dividing cells exhibiting containing 5 mM L-leucine methyl ester (Thiele different intermediate stages of differentiation. and Lipsky, 1985). On the other hand, cultures Transfection of the SV40 tsA58 strain large T of highly enriched brain microglia can be ob- antigen generates conditionally immortal cell tained by enhancing microglial proliferation lines which continuously proliferate at the per- with colony-stimulating factor (CSF-1), while missive temperature (33°C), but are capable of eliminating the co-cultured astrocytes by star- differentiation at the nonpermissive tempera- vation (Fedoroff and Richardson, 1997). Sub- ture (39.5°C). Conditionally immortalized cell stituting lactate for glucose in the growth me- lines that are genetically homogeneous can be dium selects for oligodendrocyte precursors derived from transgenic mice harboring coding against differentiated oligodendrocytes, astro- sequences of the tsA58 SV40 large T antigen. cytes, and microglial cells (Fedoroff and This strategy has proven to be applicable to Richardson, 1997), and substituting D- for L- various cell types, including fibroblasts, thymic valine minimizes the proliferation of fi- stromal cells, and astrocytes (Baba et al., 1997; broblasts, since only cells of ectodermal origin Jat et al., 1991). express amino acid isomerase necessary to me- tabolize D-amino acids (Gilbert and Migeon, Cloning 1975). Cell type-specific toxins may be of use Cloning procedures establish a new culture in some cases for selective cell elimination; for from the progeny of a single parent cell (e.g., example, excitotoxins, such as kainic acid, may Jakoby and Pastan, 1979; Fedoroff and be used to selectively kill neurons. Overgrow- Richardson, 1997). By definition, a cell clone ing cell populations (e.g., fibroblasts or astro- is a cell population derived from a single an- cytes) are often eliminated by applying antimi- cestral cell. For cloning by dilution plating, a totic agents, such as cytosine arabinoside or cell suspension is sufficiently diluted to permit 5-fluoro-2-deoxyuridine. However, if concen- either the distribution of single cells to separate trations used are too high, they may also be dishes, or the formation of isolated colonies of toxic also to some nondividing cell types cells in the same dish. Alternatively, cells can (Banker and Goslin, 1991). be cloned by the physical isolation of single cells, for example by using the glass-shard Cell Immortalization method (Richelson, 1976). Often, isolated cells Immortal cell lines are generated from pri- require particular media conditions for survival mary cells by integrating immortalizing onco- and/or growth. The conditioning of the medium genes into their genome. With this approach, by the cell (“autoconditioning”) is facilitated many immortalized cell lines have been de- by using small volumes of medium and small rived, particularly from neural precursor cell culture dishes (e.g., microwell plates). Growth populations (Gage et al., 1995). The most com- conditions may be further improved by the monly used immortalizing oncogenes are either addition of specific survival and/or growth- members of the myc oncogene family, or a stimulating factors, conditioned medium from temperature-sensitive (ts) mutant of the simian higher density cultures, or by the use of “feeder virus 40 (SV40) large T antigen. Transfection layer” supports—i.e., monolayer cells culti- of DNA is accomplished using various en- vated to confluency and then irradiated— In Vitro Cellular hancers such as DEAE-dextran, calcium phos- which, in vivo, are normally in association with Assays 12.1.5

Current Protocols in Pharmacology Supplement 4 the cells to be cloned, such as stromal cell feeder tiation (either with broad or narrow specificity). layers for cloned epithelial cells. The attach- Transferrin is an absolute requirement for many ment of anchorage-dependent cells to the cell types, and other proteins such as albumin feeder layer is facilitated by the addition of may have beneficial effects in serum-free cul- polymerized collagen or fibrin. For cloning by ture conditions. Antibiotics are often used for isolated cell colonies, the cloning and culture the elimination or suppression of contaminat- procedure must be repeated several times to ing microorganisms (Jakoby and Pastan, 1979). assure purity. While a combination of penicillin and strepto- mycin is frequently used, an alternative is pro- Passaging vided by gentamicin, which prevents the Subculturing or passaging of cells is often growth of mycoplasma as well as gram-positive necessary to reduce the density of growing cell and gram-negative bacteria. Most antibiotics, cultures and is accomplished by transferring but particularly the fungicidal or fungistatic them to new culture vessels (e.g., Fedoroff and antibiotics such as amphotericin B and nystatin, Richardson, 1997). To subdivide cultures of are relatively toxic to most cell types and there- anchorage-dependent cells, the cells must first fore not recommended for continuous applica- be detached from their substrate. Methods for tion. Finally, phenol red is often used as a visual the detachment and dispersal of cultured cells indicator of the pH. include mechanical shearing (shaking or scrap- ing with a silicone rubber spatula), use of diva- Osmolality and pH lent-cation chelators such as EDTA, and pro- The optimum range of osmotic pressures for teolytic enzymes, such as trypsin, collagenase, a particular cell type is relatively narrow. Opti- or pronase. Often, a combination of these meth- mal clonal growth in established cell lines is ods is optimal. Usually, a balanced salt solution often found at 300 ± 25 mosmol/kg (Jakoby and is used for subculturing, and care is exercised Pastan, 1979), although certain primary cell to avoid any harsh physical manipulation and cultures and cell lines may require up to 340 to minimize the duration of the procedure. mosmol/kg. Cells should be maintained at relatively constant osmolality, particularly in the CRITICAL PARAMETERS absence of serum and macromolecular medium components. Changes in osmolality due to Culture medium evaporation should be avoided. The specific nutritional requirements for in- The optimal pH for cellular growth is usu- dividual cell types are only partially known, ally 7.2 to 7.4. However, in high-density cul- and, in many cases, the addition of poorly ture, cells may generate sufficient acidic meta- defined biological preparations, such as serum bolites to cause a considerable shift to lower pH or tissue extracts, is still needed for successful values. This may require more frequent replen- culturing. Nevertheless, chemically defined ishment of the medium, or use of a buffer with media (Bottenstein and Sato, 1985) are suitable a pK on the acidic side of the operational pH to for a variety of culture systems, and with further increase the buffering capacity. Individual cell progress in the identification of factors promot- types may exhibit specific pH preferences (e.g., ing cell survival, growth, and maturation, de- astrocytes appear to prefer slightly alkaline fined culture media will eventually become media, whereas neurons survive better under available for most in vitro systems. Medium slightly acidic culture conditions). Also, the pH components required by all or several culture optimum of a given cell type may be different systems are listed in Table 12.1.1 and include for growth and for other (differentiated) bio- essential ions and trace elements, essential logical functions. For buffering, the CO2/bicar- amino acids, and often additional amino acids, bonate system, which is similar to the natural such as Cys, Gln, and Tyr which are synthesized buffering system in blood, is most frequently in specialized cells, and in some cases also Gly, used. In some cases, organic buffers such as Pro, and others. Also included are essential fatty HEPES (up to 25 mM) are used alone or to- acids, metabolic substrates, and often addi- gether with inorganic buffer systems, such as tional metabolites such as pyruvate, choline, phosphate. Some commercially available inositol, nucleosides, and putrescine. Various HEPES may be cytotoxic and, in the presence vitamins and cofactors are typically necessary of DMSO, HEPES may enter cells and be Overview of Cell along with hormones and other specific factors cytotoxic. and Tissue Culture stimulating survival, growth, and/or differen- Techniques 12.1.6

Supplement 4 Current Protocols in Pharmacology Table 12.1.1 Media Components for Cell Culture

Components Examples

+ + 2+ 2+, – 2− − − Essential ions Na , K , Ca , Mg Cl , SO4 , H2PO4 , HCO3 3+ 2+ 2+ 2+ 2+ 2+ 2+ 6− 2− 2− Trace elements Fe , Zn , Cd , Cu , Mn , Ni , Sn , Mo7O24 , SeO3 , SiO3 Essential amino acids Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val Essential fatty acids Linoleic acid, linolenic acid Metabolic substrates D-Glucose; in some cases also D-galactose Vitamins and cofactors Biotin, cobalamine, folate, nicotinamide, pantothenate, pyridoxal, thiamine, riboflavin, α-tocopherol, retinol, ascorbate, lipoic acid Hormones Insulin, hydrocortisone, triiodothyronine; in some cases also progesterone, estradiol Specific factors stimulating survival, With broad specificity, e.g., EGF, FGF, IGF, LIF, and PDGF; or with narrow growth, and/or differentiation specificity, e.g., interleukins, chemokines, and neurotrophins

Gas Phase with open culture systems to minimize the A mixture of air and CO2 is used most evaporation of culture medium. frequently as the gas phase in conventional incubators (e.g., Jakoby and Pastan, 1979). This Specific Culture Conditions provides the oxygen necessary for aerobic cell metabolism as well as the CO2 needed for Cell adhesion maintenance of the proper pH in the growth In vivo, many cells are in contact with an medium when using the CO2/bicarbonate buff- extracellular matrix (ECM) formed by specific ering system. Although the amount of oxygen proteins and polysaccharides. Furthermore, in air (∼18%) is adequate for many culture within each tissue, cells usually form tight con- systems, O2 regulation may be necessary in tacts with neighboring cells through special- certain cases. Ideally, for a given culture type, ized cellular junctions. It is thought that the the pO2 optimum has to be determined using a ECM plays an important role in morphogene- CO2/O2 incubator, but in practice this is rarely sis, cellular development, and function. De- done. The partial pressure of oxygen in normal pending on the developmental stage, a particu- body fluids is significantly less than that of air, lar cell type may encounter different types of and some cells may function better at compa- ECM, which may greatly influence the cellular rable (reduced) oxygen levels. Other cell types, responsiveness to hormones and growth factors such as neurons, exhibit a relatively high oxy- and thus the cellular functions. Therefore, dis- gen requirement. However, high partial pres- persed cell cultures of anchorage-dependent sure of oxygen may increase the risk of oxygen cells often depend on attachment factors and/or radical formation and cell damage. The pres- ECM components for optimal cell adhesion ence of selenium, a component of the enzyme and function (e.g., Ronnett, 1995). Some types glutathione peroxidase, and/or α-tocopherol (a of cells can adhere to and grow on borosilicate strong antioxidant) in the culture medium can glass or on polystyrene plastic chemically decrease the detrimental effects of high oxygen treated to decrease hydrophobicity by creating concentrations. Using the CO2/bicarbonate an overall negatively charged surface compa- buffer system, it is necessary to adjust the bi- rable to glass. Others may require the presence carbonate concentration to the CO2 tension in of positively charged surfaces that can be ob- the incubator. Thus, the requirement for Na- tained, for example, by coating with polymers HCO3 is 2.2 g/liter with 5% CO2 and 3.7 g/liter such as polylysine or polyornithine. Again, with 10% CO2. In addition to their roles in pH others may need more specific attachment to buffering, CO2 and bicarbonate are involved in ECM components such as collagen subtypes, a number of biosynthetic reactions, so that at fibronectin, or laminin. Once attached, some low cell density an extracellular source must be cells may eventually synthesize their own ECM provided. High humidity (>98%) in the incu- molecules, whereas in other cases it may be bator atmosphere is necessary when working advantageous to use culture dishes with perma- In Vitro Cellular Assays 12.1.7

Current Protocols in Pharmacology Supplement 4 nent ECM coating. However, in vivo, the ECM Contamination composition is tissue-specific and develop- Contamination of cultures by bacteria or ment-dependent. fungi is usually easy to detect by direct inspec- tion. In contrast, infections by mycoplasma and Cell density viruses, or cross-contamination with another In general, cell density has a profound effect cell type, can be detected only by the use of on the survival, growth and/or function of cul- specialized techniques. Most problems with tured cells. Excessively high or low cell densi- infections stem from improper culture tech- ties may have detrimental effects. For example, niques or contaminated culture equipment such primary neurons in monolayer culture require as culture hoods, culture vessels, pipets, filtra- a minimal cell density 104 cells/cm2, with maxi- tion units, and incubators. Mycoplasma and mal density at ∼1.5 × 106 cells/cm2. Also, the viruses are able to pass through the 0.2-µm seeding requirement for oligodendrocytes is filters used for the sterilization of liquids, and relatively stringent, whereas astrocytes and mi- therefore are most frequently introduced by croglia survive at relatively low density (Fe- medium supplementation with biological doroff and Richardson, 1997). A minimum preparations such as serum or tissue extracts. number of cells per culture vessel is required Mycoplasma may also be accidentally intro- for medium conditioning, which occurs due to duced directly by the operator. Insidious infec- the accumulation of metabolic intermediates tions by mycoplasma, viruses, or foreign cells (e.g., amino acids, CO2, pyruvate, and lactate), are most problematic when using cell lines. detoxifying molecules, and endogenous Therefore, a number of precautions should be macromolecular factors stimulating cell sur- taken in advance. First, cell lines should be vival, growth, and development. On the other obtained only from certified sources and stock hand, if the cell density is too high, adverse cultures should be prepared from early pas- effects will result from the depletion of essen- sages and kept frozen in liquid nitrogen. Sec- tial metabolites, the accumulation of waste ondly, cultures should be handled individually products, and drastic shifts in pH. With the to avoid cellular cross-contamination and exception of some transformed cell lines, most tested regularly for mycoplasma and other pos- cells cease multiplication at high population sible contaminants. The continuous use of an- density—a phenomenon termed density-de- tibiotics is not recommended, particularly with pendent inhibition. The use of suspension cul- cell lines, since they may hide latent infections. tures in industrial quantities may require fer- On the other hand, antibiotics are usually re- menters built for continuous feeding, with feed- quired for the initiation of primary cultures and back loops for automatic control of nutrient may be used in acute cell culture experiments. supply and pH. Three-dimensional cell cultures Besides the contamination of cultures due and explant cultures naturally provide optimal to improper handling, there is also the potential (tissue-like) cell densities and cell-cell interac- risk of exposing workers to pathogens residing tions. However, the size of the cellular forma- in cultures. Therefore, it is important to apply tions must be limited to 300 to 400 µm diameter the rules of good laboratory practice, to use the to ensure a sufficient supply of oxygen, since required safety containment facilities, and to the critical oxygen diffusion distance is ∼150 follow the official safety guidelines. Particular to 200 µm. Anoxic necrosis can occur in the measures are needed when working with tu- center of multicellular structures in stationary morigenic or transformed cells, with patho- culture. This may be prevented by continuous genic viruses, and human cell cultures (e.g., agitation, which reduces glucose and oxygen Jakoby and Pastan, 1979). gradients between medium and cells. Toxic Chemicals TROUBLESHOOTING Toxic chemicals in the growth medium may Problems with cell or tissue cultures are seriously affect cell growth and viability. The readily apparent when increased cell death, most frequent source of toxic chemicals is the slow growth, extreme pH shifts, or turbid me- water used for culture preparation. Therefore, dium is observed. Sometimes, problems are only highly purified water should be used, e.g., detected only by careful analysis of the cul- water which has been passed through a Milli-Q tures. However, identification of the problem or Nanopure water purification system with Overview of Cell may be difficult because of the large number of added organic- and pyrogen-removal car- and Tissue Culture variables that need to be considered. tridges. Toxic compounds may also be intro- Techniques duced by the recycling of cultureware. If glass- 12.1.8

Supplement 4 Current Protocols in Pharmacology ware is used, all organic residues from deter- teria for culture characterization (e.g., Ronnett, gent washes must be removed prior to use for 1995; Jakoby and Pastan, 1979). Routinely, culturing. Toxic chemicals may also be intro- growth is estimated by measuring the rate of duced through impure medium components. In incorporation of radiolabeled thymidine, by the this case, cultures maintained in serum-free tetrazolium salt (MTT) assay, and/or by direct media are more susceptible to toxic contami- cell count. To determine cell type-specific dif- nants because of the lack of macromolecules ferentiation, various criteria or endpoints are that may bind and inactivate such chemicals. used, including morphological, immunocyto- Water and biological preparations may contain chemical, biochemical, genetic, and electro- also endotoxins (lipopolysaccharides from the physiological properties. Dispersed cell cul- membranes of gram-negative bacteria). Serum tures are particularly accessible for direct ob- also contains complement factors that may servation under a phase-contrast inverted stage damage cultured cells. Complement can be microscope, making it possible to monitor inactivated by incubating the serum in a water gross morphological changes over time in cul- bath at 56°C for 30 min prior to use (also see ture. More critical morphological analysis by APPENDIX 2A). high-resolution light microscopy and transmis- Alterations in materials or methods are fre- sion electron microscopy is feasible with most quent causes of culture problems, though they of the conventional culture preparations, al- are often difficult to identify. Media or media though embedding and sectioning of the cul- components may slowly degrade during stor- tures is required (Fedoroff and Richardson, age. Different batches of media components, 1997). For immunocytochemical analyses, nu- particularly serum, may vary in their composi- merous monoclonal and polyclonal antibodies tion. Cell lines may alter their characteristics are available to probe for cell type–specific with repeated subculturing, and methods may antigens (Fedoroff and Richardson, 1997), in- vary between individual co-workers or may cluding cell surface components, cytoskeletal change slightly over the years. It is therefore proteins, and specific intracellular enzymes, important to maintain strict rules for working proteins, or metabolites. Biochemical assays with cell cultures. Commercially available ma- can be used to probe for cell type–specific terials should always be purchased from the protein expression, specific enzyme activities, same supplier to avoid variations. The shelf life receptor-binding properties, characteristics of of all products must be strictly respected, and plasma membrane channels and transporters, batches of products (e.g., serum) that are known or synthesis of specific metabolites. Also, spe- to be highly variable should be tested prior to cific gene expression is studied at the transcrip- use. When working with cell lines, stock cul- tional level. In certain culture systems, the tures should be prepared and stored with great specificity of certain phenotypic markers may care. Cell cultures should be regularly sub- be limited because they are either coexpressed jected to quality controls, evaluating their vi- in different cell types, or are aberrantly ex- ability, growth characteristics, and phenotypes. pressed. The latter condition has been observed in transformed cells and in some cell types ANTICIPATED RESULTS grown in isolation. Therefore, for the pheno- Cell and tissue culture offers the possibility typic characterization or identification of a of studying biological processes at the cellular, given cell type, a different set of criteria is subcellular, and molecular levels, in a system usually needed. less complex than the whole organism. These techniques increase the accessibility of cells Functional Studies and their environment for manipulation and Any of the immunocytochemical, bio- investigation. On the other hand, complemen- chemical, or molecular criteria for charac- tary studies are usually needed to verify the terizing a culture may also be useful for the physiological relevance of observations in vi- study of normal cell functions. The type of cell tro. Tissue and cell culture have found numer- culture suitable for functional studies depends ous applications in research, clinical diagnosis, largely on the degree of functional integrity toxicology, and industrial biotechnology and needed relative to the in vivo situation. For gene targeting. example, simple cell lines (e.g., CHO cells) are particularly suitable for the pharmacological Culture Characterization study of channels or receptor complexes after Growth characteristics and phenotype ex- the genes have been stably transfected into In Vitro Cellular pression of cultures are the most common cri- these cells. On the other hand, to study signal Assays 12.1.9

Current Protocols in Pharmacology Supplement 4 transduction or other regulatory mechanisms produce biologically active factors, such as involved in cell function, a culture system specific neurotrophic factors or hormones, can closely mimicking the in vivo tissue would be encapsulated in a porous polymer fiber and appear to be the most appropriate. Electro- implanted in a host tissue (Tseng et al., 1997). physiological studies in neural tissues also require neuronal interactions comparable to the TIME CONSIDERATIONS in vivo situation. Therefore, the complex ex- Cell and tissue culture techniques have the plant cultures, which retain the greatest degree reputation of being particularly laborious and of original histotypic organization, are best time-consuming. However, with the steady in- suited for this purpose. Furthermore, cell cul- crease of culture applications, and increasing ture systems of different complexities are in- demand, standardized methods have been de- dispensable in many research areas—for exam- veloped which greatly reduce the time neces- ple in developmental studies (including cell sary for culture preparation and maintenance. lineage studies), for the analysis of functional Also, most of the components and equipment variations of cytosolic free calcium concentra- needed for cell culture are now commercially tions, for the elucidation of cell-matrix interac- available, and the use of disposable plastic tions, for the identification and study of trophic materials reduces the need for specialized factors, and for the investigation of pathogenic washing and sterilizing procedures. Neverthe- mechanisms such as microglial reactivity, less, in some cases, special equipment is still neurotoxicity, oxygen radical damage, sene- needed (e.g., for aggregate cell cultures), and scence, and mechanisms of cell death. some techniques still are particularly tedious and labor-intensive, such as some explant-cul- Screening Procedures ture techniques. In contrast, the preparation and Relatively simple cell culture systems can growth of most primary cell cultures, as well as be used for bioassays and for drug screening work with cell lines, have become common using defined cellular targets. Various cell lines laboratory practice. When cell lines are used, it are employed for the detection and quantifica- is easiest to regularly purchase fresh certified tion of bioactive substances such as cytokines, cells instead of storing them. Using exclusively neuroactive agents, or toxins. Three-dimen- freshly obtained or disposable materials—in- sional histocultures of tumor explants provide cluding cells, media, culture dishes, and test systems to evaluate the efficacy and phar- pipets—saves time and avoids the need for macokinetics of chemotherapy drugs. Culture specialized equipment for media preparation, systems are used to replace or complement cleaning of glassware, sterilization, and quality animal models. Genetically engineered cells control. This approach is particularly advanta- are used successfully for high-throughput geous for small laboratories. screening. The practice of standard culture methods is best acquired in a laboratory where culturing is Cell-Derived Products routinely performed. Also, in the beginning, it Cell culture systems of different complexity is best to use only proven protocols from well- can also be used for the synthesis of biological established laboratories. Once the basic condi- products. Various cell lines synthesize and re- tions are working, new protocol variants may lease cytokines and other trophic molecules be attempted. Much time is saved if a particular that can be used for research or as media addi- culture technique is mastered in a laboratory tives for other culture systems. The hybridoma where it is used. Many published protocols lack technology is employed for monoclonal anti- small but critical technical details, and there- body production. Cultured human epidermal fore do not allow reliable reproduction. In any keratocytes serve as autografts for burn victims. case, sterile techniques and standard good labo- Cell cultures may also be used for virus propa- ratory practice are essential for successful gation and purification, and as host cell lines work. Besides the basic cell culture equipment, for the infection of primary cells with viral such as a laminar flow hood and a CO2 incuba- vectors. Transfection of various types of eu- tor, a special facility may be needed to meet the karyotic cells is also used for the production of safety standards for working with pathogenic certain eukaryotic proteins. Embryonic stem viruses, human cell cultures, or transformed cells are most suitable for gene targeting, and cell lines. Overview of Cell are employed in transgenic technology. Fur- and Tissue Culture thermore, genetically engineered cell lines that Techniques 12.1.10

Supplement 4 Current Protocols in Pharmacology LITERATURE CITED Handler, J.S. and Kreisberg, J.I. 1991. Biology of Baba, H., Nakahira, K., Morita, N., Tanaka, F., renal cells in culture. In The Kidney, 4th ed. Akita, H., and Ikenaka, K. 1997. GFAP gene (B.M. Brenner and F.C. Rector, eds.) pp. 110- expression during development of astrocytes. 131. W.B. Saunders, Philadelphia. Dev. Neurosci. 19:49-57. Hay, R.J., Park, J.-G., and Gazdar, A. 1994. Atlas of Banker, G. and Goslin, K. 1991. Culturing Nerve Human Tumor Cell Lines. Academic Press, San Cells. MIT Press, Cambridge, Mass. Diego. Bornstein, M.B. 1995. Organotypic cultures of Jakoby, W.B. and Pastan, I.H. (eds.) 1979. Cell mammalian nerve tissues: A model system for Culture. Methods Enzymol. vol. 58. neurotoxicological investigations. In Neurotoxi- Jat, P.S., Noble, M.D., Ataliotis, P., Tanaka, Y., Yan- cology. Approaches and Methods (L.W. Chang noutsos, N., Larsen, L., and Kioussis, D. 1991. and W. Slikker, eds.) pp. 573-579. 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Current Protocols in Pharmacology Supplement 4 Verity, M.A. 1995. Cell suspension techniques in neurotoxicology. In Neurotoxicology. Ap- proaches and Methods (L.W. Chang, and W. Slikker, Jr., eds.) pp. 573-579. Academic Press, San Diego. Wiesinger, H., Schuricht, B., and Hamprecht, B. 1991. Replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from newborn mouse brain. Brain Res. 550:69-76.

Contributed by Paul Honegger Universite de Lausanne Lausanne, Switzerland

Overview of Cell and Tissue Culture Techniques 12.1.12

Supplement 4 Current Protocols in Pharmacology