Cell Line (Molecular Biology)

When a primary cell culture (see Tissue Culture) is subcultured, it becomes a cell line. This implies that multiple lineages of cells, not necessarily distinct from each other, coexist in the culture (1). Serial subculture, while maintaining these parallel lineages, will tend to show convergence towards whatever common phenotype is best adapted to the culture conditions being employed, as the cell type with the greatest proliferative ability will predominate. The cell line may be finite and die out after a fixed number of population doublings, or become a continuous cell line (see Immortalization) (Fig. 1). Cell lines, particularly continuous cell lines, can become a valuable resource, particularly if preserved in liquid nitrogen after appropriate characterization and validation. Many continuous cell lines are currently available, with wide-ranging properties, including drug resistance markers, inducibility for specific enzymes, estrogen sensitivity, cornification, blood vessel formation,hemoglobin synthesis, myogenesis, and transfection susceptibility (Table 1). Finite cell lines with distinct phenotypic properties are also becoming available through the development of serum-free selective media (2-4). (see Serum Dependence) and can be derived in the laboratory or purchased from commercial suppliers.

Figure 1. Effect of continued culture passage on cumulative cell number, assuming that no cells are discarded. The curve shows the initial decline due to selection, then exponential growth during the replicative phase, and then growth arrest and eventual deterioration following senescence, in a finite cell line, or continued proliferation, often at an enhanced rate, following transformation.

Table 1. Cell Lines in Common Use3

Cell line Morphology	Origin	Age Status	Ploidy	Characteristics	Reference
MRC5 Fibroblast	Human lung	Embryonic Normal	Diploid	Susceptible to human viral infection	Jacobs (1970) Nature 2 168
WI38 Fibroblast	Human lung	Embryonic Normal	Diploid	Susceptible to human viral infection	Hayflick Moorhea (1961)E Cell Res. 585

IMR90	Fibroblast	Human lung	Embryonic	Normal	Diploid	Susceptible to human viral infection	Nichols e (1977) Science 1 60
A2780	Epithelial	Human ovary	Adult	Neoplastic	Aneuploid	Chemosensitive with resistant variants	Tsuruo et (1986) Jp Cancer R 77, 941
A549	Epithelial	Human lung	Adult	Neoplastic	Aneuploid	Synthesizes pulmonary surfactant	Giard et ; (1972) J. Natl.Can Inst. 51, 1
A9	Fibroblast	Mouse subcutaneous	Adult	Neoplastic	Aneuploid	HGPRT-ve: deriv. L929	Littlefiel< (1964) Nature 21 1142
BHK21-C13	Fibroblast	Syrian hamster kidney	NB	Normal	Aneuploid	Transformable by polyoma virus	Macphers and Stoke (1962) Virology 147
BRL3A	Epithelial	Rat liver	NB	Normal		Produce IGF-II	Coon (19 J. Cell Bi 39, 29a
Caco-2	Epithelial	Human colon	Adult	Neoplastic	Aneuploid	Transports ions and amino acids	Fogh (19 J. Natl. Cancer In 58, 209
CHANG liver	Epithelial	Human liver	Embryonic	Normal?	Aneuploid	HeLa contaminated	Chang (1 Proc. Soc Exp.Biol. Med. 87,
CHO-K1	Fibroblast	Chinese hamster ovary	Adult	Normal	Diploid	Simple karyotype	Puck et a (1958) J. Exp. Med 108, 945
EB-3	Lymphocytic Human		Juvenile	Neoplastic	Diploid	EB virus + ve	Epstein a Barr (196 Lancet 1 ,
GH1, GH3	Epithelial	Rat	Adult	Neoplastic	Aneuploid	Produce growth hormone	Yasumur al. (1966) Science 1 1186
HeLa	Epithelial	Human cervix	Adult	Neoplastic	Aneuploid	G6PD type A	Gey et al (1952) Cancer R 12, 364
HeLa-S3	Epithelial	Human cervix	Adult	Neoplastic	Aneuploid	High plating efficiency; will grow well in suspension	Puck and Marcus (1955) Pr Natl. Aca

							Sci. USA 432
Hep-2	Epithelial	Human larynx	Adult	Neoplastic	Aneuploid	HeLa contaminated(su}c	Moore et (1955) CancerR 15, 598
HT-29	Epithelial	Human colon	Adult	Neoplastic	Aneuploid	Differentiation inducible with NaBt	Fogh and Trempe (1975)in Human Tumor C in vitro, J Fogh, ed. Academi Press, Ne York, p.
KB	Epithelial	Human oral	Adult	Neoplastic	Aneuploid	HeLa contaminated	Eagle (19 Proc. Soc Exp. Biol (N.Y.) 89 362
L1210	Lymphocytic	Mouse	Adult	Neoplastic	Aneuploid	Rapidly growing; suspension	Law et al (1949) J. Natl. Can Inst. 10, 1
L5178Y	Lymphocytic	Mouse	Adult	Neoplastic	Aneuploid	Rapidly growing suspension
L929	Fibroblast	Mouse	Adult	Normal	Aneuploid	Clone of L cell	Sanford e (1948) J. Natl. Can Inst. 9, 22
LS	Fibroblast	Mouse	Adult	Neoplastic	Aneuploid	Grow in suspension: deriv. L929	Paul and Struthers (unpublis
MCF7	Epithelial	Human breast pleural effusion	Adult	Neoplastic	Aneuploid	Estrogen recep +ve	Soule et < (1973) J. Natl.Can Inst. 51, 1
P388D	Lymphocytic	Mouse	Adult	Neoplastic	Aneuploid	Grow in suspension	Dawe and Potter (19 Am. J. Pa 33, 603; Koren et (1975) J. Immunol. 114, 894
S180	Fibroblast	Mouse	Adult	Neoplastic	Aneuploid	Cancer chemotherapy screening	Dunham Stewart (1953) J. Natl. Can Inst. 13, 1
STO	Fibroblast	Mouse	Embryonic Normal		Aneuploid	Used as feeder	Bernstein

					layer for embryonal stem cells	(1975) Pr Natl. Aca Sci. USA 1441
3T3-L1 Fibroblast	Mouse, Swiss	Embryonic	Normal	Aneuploid	Adultipose differentiation	Green an Kehinde (1974)C 1, 113
3T3 A31 Fibroblast	Mouse BALB/c	Embryonic	Normal	Aneuploid	Contact inhibited; readily transformed	Aaronson and Toda (1968) J. Physiol. 7 141
NRK49F Fibroblast	Rat kidney	Adult	Normal	Aneuploid	Induction of suspension growth by transforming growth factors	DeLarco Todaro (1978) J. Physiol. 9 335
Vero Fibroblast	Monkey kidney	Adult	Normal	Aneuploid	Viral substrate and assay	Hopps et (1963) J. Immunol. 416
ZR-75-1 Epithelial	Human breast, ascites fluid	Adult	Neoplasti	c Aneuploid	ER-ve, EGFr +ve	Engel (19 Cancer R 38, 3352

1. Origin of Cell Lines

Regenerating tissues in vivo are made up of a small, self-repopulating, stem cell pool, an expandable pool of proliferating progenitor cells, and a nonproliferating differentiated cell pool. On demand, cells leave the stem cell pool and enter the progenitor compartment, where expansion is regulated to meet the current demand in the differentiated cell compartment. When cells enter the differentiated cell compartment, this may be an irreversible process, as seen with erythrocytes, keratinocytes, and neurons; or it may be reversible, as seen with hepatocytes, endothelial cells, and fibrocytes. Cell lines can be derived from any tissue with a proliferating compartment, or from cells that can re-enter a proliferating compartment. It is possible that cell lines will contain stem cells, but, except for hemopoietic cells (see Hematopoiesis), markers are unavailable to determine this. As many cell lines express lineage markers and can, under appropriate conditions, differentiate, it seems most likely that they are derived from the progenitor cells of the tissue.

As cell lines are derived from random outgrowth or from disaggregated cells, multiple lineages of cells will be present. The purity of the culture will be determined by how well these lineages are matched, that is, derived from cells with the same phenotypic fate, or are derived from cells with dissimilar fates. In practice, the selective pressure exerted by the medium will tend to limit the cell population to cells of like phenotype with similar survival capacity, and cells that proliferate less rapidly will gradually be overgrown. It is possible that cells within one phenotypic group have different proliferative capacities but interactions between them produces a uniform proliferative rate in the entire population.

2. Finite Cell Lines

When a cell line is generated at the first subculture of a primary culture, its lifespan is determined, initially, by the environmental conditions. If the conditions are inadequate, the cell line will die out within one or two subcultures, but if the medium, substrate, and other conditions are satisfactory, then the cell line may progress through several serial subcultures. The ultimate limit to the number of subcultures will be determined by the potential regenerative life-span of the cells (see Senescence; Immortalization). Most cell lines from normal tissues will undergo a fixed number of population doublings (Fig. 1), given by the product of the number of passages and the estimated number of doublings per passage (5). These are known as finite cell lines. Normal human skin fibroblasts will usually achieve around 50-60 population doublings and then enter senescence. Senescent cells no longer proliferate, may be partially differentiated, and can remain viable for several months. Cultures of finite cell lines must, therefore, be stored frozen at an early passage level, thawed when required, and used between predetermined passage levels, usually between 10 and 30, to ensure an adequate and consistent supply.

3. Continuous Cell Lines

Some cell lines, such as those derived from mice or from tumors of many species, are not limited by a finite lifespan. They progress either by smooth transition to an immortal cell line (see Immortalization) or undergo neoplastic transformation at some stage and, instead of dying out after a set number of population doublings, continue to proliferate unchecked. Some cultures show evidence of selection and go through a period called crisis, when most cells in the population die out by senescence, but a transformed subpopulation survives, usually with an enhanced growth rate, increased cloning efficiency, loss of contact inhibition, acquisition of anchorage-independent growth, ability to grow to a higher saturation density, and increased tumorigenicity in animals (see: Neoplastic Transformation).

There are a large number of continuous cell lines in existence, many of which are banked in repositories such as the American Type Culture Collection (ATCC) or the European Collection of Animal Cell Cultures (ECACC). They form a valuable resource of vigorously growing cultures, capable of unlimited expansion, but are subject to several caveats.

1. Their origin must be validated before use. Acquisition from a reputable cell bank, or the originator, will usually guarantee this, but there are many instances of continuous cell lines being cross-contaminated by other, more vigorously growing cell lines such as HeLa Cells (6-8). The identity of a cell line needs to be confirmed prior to extensive use. This is done most effectively by performing a DNA fingerprinting, but it is also possible by immunotyping, isoenzyme analysis, or chromosome analysis.

2. The cells must be shown to be free from infection. Continuous cell lines form an ideal substrate for mycoplasma, which can grow undetected in the culture (9). Again, most reputable cell banks will be able to demonstrate that cell lines being distributed are mycoplasma-free, but stocks still need regular checks every 1 or 2 months to ensure that they remain uninfected.

3. Continuous cell lines are genetically unstable. They are usually aneuploid (the chromosomal complement differs in number, and by chromosomal rearrangements, from the donor) and heteroploid (contain subpopulations with differing chromosomal constitutions). This is reflected in their phenotypic diversity, including variations in morphology, enzyme activity, antigenic expression, and growth characteristics. It is normal practice to clone such populations, select a clone with the required properties, and cryopreserve sufficient ampoules (12-100) for future use. Cultures are generally maintained for a limited period, usually about 3 months, and then replaced from frozen stock.

4. Subculture

Subculture, or passage, is the transfer of a culture from one vessel to another, usually using trypsin to dislodge the cells from the substrate and dissociate them from each other. If the cell line is proliferating, then subculture will also imply diluting the cell concentration achieved at the end of the culture period, to a new seeding concentration to initiate a new culture. Growth from seeding until the next subculture is known as a growth cycle, which is repeated each time the culture is passaged. A series of growth cycles constitute serial propagation, and this should not normally proceed beyond about 3 months without replacing stock from the freezer (see above).

Each growth cycle is composed of a lag phase (Fig. 2), where the cells are recovering from trypsinization, synthesizing new extracellular matrix, adhering to the substrate, spreading, repolymerizing the actin microfilament cytoskeleton, and responding to signaling from extracellular matrix adhesions and cytoskeletal rearrangements, which leads to expression of cyclins and, eventually, reentry to the cell cycle. When the cells start to divide, they enter the exponential or "log" phase of the growth cycle and will continue exponential growth until all the available growth surface is occupied or the medium is exhausted. If the medium is limiting, it is customary to replace it half way through the exponential phase of growth. When all the available growth surface is occupied, the culture is said to be confluent and will require to be subcultured again. If the culture is allowed to progress beyond confluence, growth will slow down, as a result of density limitation of growth (see Contact Inhibition), and the culture enters the plateau phase of the growth cycle. If the cells are normal and respond to normal growth control signals, there will be little evidence of cell proliferation or cell loss in plateau, specifically, minimal turnover. If, however, the cells are transformed, they will continue to proliferate for several cell generations after reaching confluence and reach a higher saturation density at plateau, due to deficiencies in contact inhibition of cell motility and density limitation of cell proliferation (10, 11). Even in plateau, there will be continued cell proliferation, although at a lower level than in exponential growth, balanced by increased cell loss from apoptosis, resulting in a greater turnover than occurs in normal cells in plateau.

Figure 2. Phases in the growth cycle of cultured cells following subculture.

The numerical parameters that can be derived from the growth curve are the duration of the lag period, the doubling time in mid-log phase, and the saturation density in plateau. The last will depend on the feeding regimen and, if cell kinetics are being determined, should be measured under nonlimiting medium conditions. Where these parameters are consistent, they can be used to calculate the split ratio, the degree of dilution required to reinitiate a new growth cycle with a short lag period and to achieve the appropriate density for subculture at a convenient time in the future, usually around one week. The split ratio should be a power of 2 when handling finite cell lines. This allows an approximate calculation of the number of generations that have elapsed since the last subculture; for instance, a split ratio of 8 would imply that the cells had undergone 3 population doublings in each growth cycle. Split ratios are less useful with continuous cell lines. They are not needed to determine the stage in the life cycle of the cell line, as it is immortal, and the population doubling time is so much shorter that split ratios of the order of 1-100 are required to produce a week-long growth cycle. At this level of dilution, it is more advisable to determine the cell concentration at each subculture and to dilute to give the seeding concentration that will allow growth to the next subculture in a suitable interval, say, one week.

5. Selective Culture

Alterations in the choice of medium and/or substrate will determine which cells survive in primary and early passage cultures. Most selective media are serum-free (see Serum Dependence), and recipes are available for the culture of many different cell types, including fibroblasts, epithelial cells (epidermal, mammary), glial cells, melanocytes, endothelial cells, and smooth muscle Table 2. Selection of a purified cell culture, with defined characteristics, by cloning, use of a selective medium, or physical separation, allows the resultant culture to be called a cell strain.

Table 2. Examples of Selective Media3

Cells or Cell Line	Medium	Reference
Fibroblasts	MCDB 202	McKeehan et al. (1977) In Vitro 13, 399.
Fibroblasts	MCDB 110	Bettger et al. (1981) Proc. Natl. Acad. Sci. USA 78, 5588.
Keratinocytes	MCDB 153	Tsao et al. (1982) J. Cell Physiol. 110, 219
Bronchial epithelium	LHC	Lechner and LaVeck (1985) J. Tissue Cult. Meth. 9, 43
Mammary epithelium	MCDB 170	Hammond et al. (1984) Proc. Natl. Acad. Sci. USA 81, 5435
Prostate epithelium (rat)	WAJC 401	McKeehan et al. (1982) In Vitro 18, 87.
Prostate epithelium (human)	WAJC 404	McKeehan et al. (1984) Cancer Res. 44, 1998
Glial cells		Michler-Stuke and Bottenstein (1982) J. Neurosci. Res. 7, 215
Melanocytes		Naeyaert et al. (1991) Br. J. Dermatol. 125, 297

Small cell lung cancer	HITES	Carney et al. (1981) Proc. Natl. Acad. Sci. USA 78, 3185
Adenocarcinoma of lung		Brower et al. (1986) Cancer Res. 46, 798
Colon carcinoma		Van der Bosch et al. (1981) Cancer Res. 41, 611
Endothelium	MCDB 130	Knedler and Ham (1987) In Vitro 23, 481

6. Characterization

Cell morphology and growth characteristics are simple criteria for characterization. Spindle-shaped cells are usually considered to be fibroblast-like, without necessarily implying that they are known to be of the fibrocyte lineage. Similarly, cells that grow in patches of polygonal, or pavement-like, cells with distinct boundaries between the cells are said to be epithelial-like, although numerous other cell types can assume a similar morphology. Characterization of a cell strain employs such criteria as (1) identification of the type of intermediate filament protein (eg, cytokeratin subtype in different epithelial cells, desmin in muscle cells, glial fibrillary acidic protein in astrocytes, and neurofilament protein in neurons and some neurendocrine cells) (12); (2) expression of cell-surface antigens, such as epithelial membrane antigen (13); (3) enzymes, such as tyrosine aminotransferase and its inducibility by glucocorticoids in hepatocytes (14); (4) marker chromosomes (15); (5) isoenzymes (16); and (6) the DNA fingerprint (17, 18). The same criteria may be used to characterize a cell line, but would give only an average for the population in a cell strain.

7. Cell Banks

Most of the cell lines in common use are lodged in cell banks containing records of their identifying characteristics. Cell lines or strains that have been characterized and shown to be free of mycoplasma can be submitted to a cell bank for safe-keeping, with or without authority to distribute to other users. Cell lines may be obtained from cell banks for a set charge (Table 3) (19).

Table 3. Cell Banks®

United States and Canada	American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, MD 20852
	National Institute of General Medical Sciences (NIGMS), Human Genetic Mutant Cell and National Institute on Ageing Cell Culture Repositories, Coriell Institute for Medical Research, Copewood Street, Camden, NJ 08103
	Repository for Human Cell Strains, and Cell Repository for Neuromuscular Diseases, Children’s Hospital Research Instsitute, McGillUniversity, 2300 rue Tupper Street, Montreal, Quebec H3H 1P3, Canada

Europe	European Collection for Animal Cell Cultures (ECACC), PHLS/CAMR, Porton, Salisbury, England
	European Collection for Biomedical Research,Dept. Cell Biology and Genetics, Erasmus University, P.O. Box 1783, Rotterdam,Netherlands
	Collection Nationale des Cultures des Microorganismes,Institute Pasteur, 25 rue du Dr. Roux, F-75724 Paris Cedex 15, France
	Human Genetic Cell Repository, Hospices Civilsde Lyon, Hopital Debrousse, 29 rue Soeur Bourier, F-69322 Lyon Cedex05, France
	Tumorbank, Institut fur Experimentelle Pathologie (dkfz), Deutsche Krebsforschungszentrum, in Neuenheimer Feld 280, Postfach 101949, D-6900 Heidelberg 1, Germany
	Centro Substrati Cellulari, Instituto Zooprofilattico Sperimentale della Lombardia, e dell’Emilia, Via A.Biandri 7, I-25100 Brescia, Italy
	National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC), Blvd. Lenin 125 BL 2, V floor, Sofia, Bulgaria
	National Collection of Agricultural and Industrial Microorganisms (NCAIM), Dept. Microbiology, University of Horticulture, Somloiut 14-16, H-1118 Budapest, Hungary
Japan	Japanese Cancer Research Resources Bank (JCRB), National Institute of Hygienic Sciences, Kami-Yoga, Setagaya-Ku, Tokyo
	General Cell Bank, Institute of Physical andChemical Research of RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-01
Australia	Commonwealth Serum Laboratories, 45 Poplar Road,Parkville, Victoria 3052