M E T H O D S I N M O L E C U L A R M E D I C I N E TM Cancer Cell Culture Methods and Protocols Edited by Simon P Langdon Basic Principles of Cancer Cell Culture Basic Principles of Cancer Cell Culture Simon P Langdon Introduction Cell culture is practiced extensively throughout the world today The techniques required to allow cells to grow and be maintained outside the body have been developed throughout the 20th century In the 50 years since the publication of the first human cancer cell line, HeLa (1), thousands of cell lines representing most of the spectrum of human cancer have been derived These have provided tools to study in depth the biochemistry and molecular biology associated with individual cancer types and have helped enormously in our understanding of normal as well as cancer cell physiology Although some caution is required in interpreting data obtained by studying cells in vitro, it has allowed investigation of a complex disease such as cancer to be simplified to its component parts The aim of this chapter is to introduce some of the basic concepts involved in the practice of cell culture Evolution of Cancer Cell Culture The science of cell and tissue culture has evolved steadily throughout the last century and its origins can be traced back to 1885 (see Table 1) In that year, Wilhelm Roux reported that the medullary plate of a chick embryo could be maintained in saline solution for several days Many of the early experiments used material derived from amphibians as it was cold blooded and often demonstrated tissue regeneration In 1887, Arnold demonstrated that frog lymphocytes could migrate and survive in saline Soon after, in 1898, the first experiment using human tissue was reported when Ljunggren showed that human skin could survive in vitro if placed in ascitic fluid With the turn of the century, longer culture experiments were attempted and in 1903, Jolly was able to maintain salamander leukocytes in vitro for a month However, despite these early experiments, it is Ross Harrison who is generally regarded as the “father” of tissue culture Harrison explanted tissue from frog embryos into frog lymph clots and the fragments of tissues not only survived but nerve fibres grew from the cells (2) These experiments were fundamental in showing continuation of function in vitro and also in establishing a general technique of tissue culture This technique From: Methods in Molecular Medicine, vol 88: Cancer Cell Culture: Methods and Protocols Edited by: S P Langdon © Humana Press Inc., Totowa, NJ Langdon Table Early Milestones in Cancer Cell Culture Date Event Investigator 1885 First tissue (chicken embryo) maintained in vitro (for several days) Wilhem Roux 1898 First human tissue (skin) maintained in vitro (in ascitic fluid) Ljunggren 1903 First tissue (salamander leucocytes) to be maintained for mo Jolly 1907 First functional experiment (frog nerve fibre growth) and first general technique (use of lymph clot) Ross Harrison 1911 First investigations of factors in medium required for growth and survival Warren Lewis 1922 First culture of epithelial cells Albert Ebeling 1943 First continuous rodent cell line Wilton Earle, George Gey 1951 First continuous human cancer cell line (HeLa) George Gey 1955 Systematic definition of nutritional needs of animal cells in culture Harry Eagle 1961 Normal cells (fibroblasts) have a finite lifespan in culture Hayflick/Moorhead 1965 First defined serum-free medium Ham 1965–present Development and use of large numbers of cell lines Multiple was developed further by Montrose Burrows, who replaced lymph clot with plasma clot, and by Alexis Carrell who showed that embryo extracts had useful growth promoting activities and could aid growth within culture (3,4) In 1911, Warren Lewis began studies to identify factors required for growth in culture, and by 1914 Losee and Ebeling were culturing cancer cells The first continuous rodent line was generated by Wilton Earle in 1943 at the National Cancer Institute and this investigator is credited with being the first to grow cells on glass and from single cells (5) In 1951, George Gey developed the first human cancer continuous cell line, HeLa, and this cell line is still used extensively today (1,6) The 1950s and 1960s were marked by detailed studies by a host of investigators, including Eagle, Fischer, Parker, Healy, Morgan, White, and Waymouth, defining the nutritional requirements of cells in culture leading to the development of the media in current use In the 1960s, Ham designed a fully defined serum-free medium (7,8), and in the 1970s, Sato and his colleagues optimized the addition of hormones and growth factors to serum-free media (9) Since the 1970s there has been the continuous development of thousands of cancer cell lines providing large numbers of models for most forms of cancer Basic Principles of Cancer Cell Culture Table Definition of Cell Culture Terms Term Definition Cell culture Maintenance of dissociated cells in culture Tissue culture Maintenance of tissue explants in culture Cell line A culture that is subcultured beyond the initial primary culture phase Finite cell line A cell line with a limited lifespan that eventually undergoes senescence Continuous cell line A cell line that is essentially immortal and continues indefinitely Primary culture The initial culture derived from in vivo material Clone The progeny isolated from a single cell Immortalization Enabling of cells to extend their life in culture Lag phase of growth Initial phase of growth when cells are subcultured Log phase of growth Most rapid growth phase when culture shows exponential growth Plateau phase of growth Phase when cell become confluent Population doubling time Time for cell number to double Cell banks Repositories of cancer cell lines and related materials Substrate The matrix on which a culture is grown Passage Subculture of cells from one container to another Confluent Situation wherein cells completely cover the substrate Cell Culture Definitions and General Germs Cell culture, like many other areas of technology, has developed its own language Some of the more commonly used definitions are listed in Table The term “cell culture” refers to the culture of disaggregated cells while “organ culture” describes the use of nondispersed tissue, both encompassed by the description “tissue culture” The initial culture taken directly from an individual is referred to as the “primary culture” and when diluted and transferred into further containers (a process referred to as “subculture” or “passage”), it becomes a “cell line.” Cell lines may be categorized as either “continuous” lines, which have the potential for indefinite population expansion, or “finite,” lines, which undergo a limited number of population doublings before “senescence.” Becoming a continuous cell line requires “transformation” and this necessitates either the presence of cells that are already transformed at the initiation of the culture or undergoing transformation in the early generations Cell lines may exist either as adherent cultures or they may grow in “suspension.” Most cell types will adhere to a “substrate” such as plastic or glass and proliferate as a monolayer, while suspension cultures not attach to a substrate and will grow floating in medium Langdon Table Components of Media Components of Eagle’s basal medium (BME) Amino acids Vitamins Inorganic salts Other Arginine Cystine Glutamine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine Biotin D-Ca pantothenate Choline Folic acid i-Inositol Nicotinamide Pyridoxal HCl Riboflavine Thiamine HCl CaCl2 KCl MgSO4 NaCl NaHCO3 NaH2PO4 D-Glucose Phenol red Additional components added in other media Amino acids Vitamins Inorganic salts Other Alanine Asparagine Aspartic acid Cysteine Glutamic acid Glycine Hydroxyproline Proline Serine Ascorbic acid Biotin Cholesterol Niacin p-aminobenzoic acid Nicotinic acid Pyridoxine Fe(NO3)3 KH2PO4 MgCl2 Na2SeO3 CuSO4 FeSO4 ZnSO4 Ca(NO3)2 KNO3 HEPES Hypoxanthine Linoleic acid Putrescine Pyruvate Basic Requirements of Cells in Culture For cells to thrive in culture, a variety of conditions must be met As for the in vivo setting, nutritional and environmental conditions are essential for cell health Nutrition is provided by media with or without the addition of serum For adherent cells, attachment to a substrate (now generally plastic) is important while carbon dioxide and oxygen levels have to be maintained within certain limits 4.1 Media The synthetic media currently in use today were developed in the 1950s These basal media contain amino acids, carbohydrates, vitamins, and salts (see Table and Appendix 1) Serum or further components, such as growth factors and hormones, are added to the basal media to provide the complete mix necessary for growth The composition of Basic Principles of Cancer Cell Culture media range from those containing the most essential ingredients, for example, Eagle’s basal medium (BME) to those containing a much broader range of components, such as Medium 199 The medium must also be buffered to allow a stable pH, ideally around pH 7.4 One of the first media to be developed was BME This emerged from Harry Eagle’s studies that sought to identify a medium that would support a wide variety of both normal and malignant cell types (10,11) This medium was subsequently modified to produce a number of popular media Increasing the amounts of individual amino acids gave Eagle’s minimum essential medium (MEM) (11), while Dulbecco’s modified Eagle’s medium (DMEM) contained a fourfold increased concentration of amino acids and vitamins (12) with a further change increasing the glucose content 4.5-fold Iscove’s modified Dulbecco medium (IMDM) was developed to support hemopoietic precursors and is a modification of DMEM, containing selenium, additional amino acids and vitamins, sodium pyruvate, and HEPES buffer (13) Glasgow minimum essential medium (GMEM) was a variation of Eagle’s medium that was used to study factors affecting cell competence This contains a twofold increased concentration of individual amino acids and vitamins (14) In 1950, Morgan and colleagues described a medium that could support the growth of explanted tissue (15) This became known as Medium 199 and contained many more components than found in Eagle’s medium and had broad applicability for the culture of many cell types A less complex version of Medium 199 is CMRL 1066 developed at the Connaught Medical Research Institute (16) Initially designed as a serum-free medium, it can be supplemented with serum to support the growth of many cell types Another medium developed to address serum-free growth was Waymouth’s medium, designed as a totally serum-free medium for cultivation of mouse L929 cells but proven useful for other cell lines also (17) Ham’s Nutrient Mixtures F10 and F12 were originally developed for the growth of Chinese Hamster Ovary (CHO) cells either with or without serum supplementation (7,8) The combination of F12 and DMEM as a 1/1 mix has found widespread use in serum-free formulations combining the richness of F12 with its trace elements and increased vitamins with the nutrient potency of DMEM Ham and coworkers went on to develop the MCDB series of media which were developed for serum-free growth of individual cell lines using supplements or low levels of fetal bovine serum protein In 1959, McCoy described a basic formulation that was subsequently modified to create a medium supporting the growth of a wide variety of primary cultures (18) RPMI 1640, developed by Moore and colleagues at Roswell Park Memorial Institute (19), is a medium extensively used for supporting the growth of many types of cell culture This has become the medium of choice for many tissue culture laboratories Media is available from commercial sources in several formats—normal strength medium (shelf life, 9–12 mo), concentrates that are diluted down (generally 10X) (shelf life, 12–24 mo) and powdered medium that has a long shelf life (2–3 yr) and can be made when needed Concentrates and powdered medium require the addition of sterile water Langdon 4.2 Serum Although media contains many of the essential nutrients required for growth, additional key elements are provided by serum Serum supports the survival and growth of cells in culture to the extent that it is capable of replacing many of the in vivo hormonal, nutritional, and stromal elements present in the in vivo cell environment Serum proteins include hormones, growth factors, lipids, transport (binding) proteins, enzyme cofactors, and attachment factors (9) The concentrations of individual components of serum will vary with the age and health status of the animals of origin and for this reason fetal and newborn calf sera are extensively employed for most cancer culture studies though human and equine sera are also used Typically, concentrations of 5–20% serum in media are considered optimal depending on cell type Higher percentages generally add little benefit for increased cost The levels of serum proteins will vary to some degree from batch to batch of sera and individual cell cultures will have differing requirements for these components It is generally recommended that a batch of serum is bought that can last from 1–2 yr stored at –20°C Since batches of sera from individual suppliers differ to some degree in their composition, several small amounts should be obtained from a range of suppliers and then a number of cell lines used by the laboratory should be tested The parameters to be assessed will generally include growth rates and attachment efficiency Generally, some information on the biochemical analysis together with certain growth and microbiological tests is available on the Certificate of Analysis 4.3 Serum-Free Media The use of serum has permitted the growth of many cell types in culture, however there are a number of drawbacks associated with its use First, although the ingredients of media are clearly defined, sera will vary in its composition dependent on its batch These differences in composition may produce some changes in a number of parameters including growth rate, attachment, and other functional endpoints Some components in serum are in fact growth inhibitory although further supplementation may still be required where some components are present at insufficient levels For certain purposes, the presence of proteins at undefined levels may also prove a complication, for instance where measurements of a cell secreted protein are being made or the effects of media conditioned by cells in a bioassay necessitate a fully defined background Finally, although sera are now routinely checked for the presence of contaminants, viruses in particular have frequently been present in the past For these reasons, together with considerations of cost and greater reproducibility, there has been a move to the use of totally defined media In the 1960s and 1970s, two strategies were developed to define media that did not require the addition of serum Sato and colleagues pioneered the addition of specific supplements to existing basal media while Ham and coworkers increased the concentrations of components of the basal medium until they could support growth The key categories of additives include hormones, binding proteins, lipids, trace elements, and attachment factors Sato’s experiments in supplementing medium identified several factors that appeared to have widespread value in maintaining the growth of many cell types in Basic Principles of Cancer Cell Culture media alone (9,20) These included insulin, transferrin, and selenite (ITS) and often epidermal growth factor and hydrocortisone (HITES) Serum albumin and fibronectin are also widely used The media selected is frequently a 1/1 mixture of DMEM and Ham’s F12 though other media such as RPMI 1640, IMDM, and the various MCDB media are also commonly used Other additives that can have great value for specific cell lines include fibroblast growth factor, estrogen, glucagon, prostaglandins, and triiodothyronine (9) Ham’s laboratory developed the MCDB series of media to provide a defined and optimally balanced environment to promote growth of specific cell types For example, fibroblasts grow in MCDB 110, keratinocytes in MCDB 201, and 1551 and CHO cells in MCDB 302 media (21) When transferring from serum-containing medium to serum-free it is advisable to reduce the serum content gradually to aid adaptation to a reduced nutritional environment Transferring to a serum-free medium requires more care when trypsinization is undertaken Generally, serum will rapidly inactivate trypsin, however if serum is not being used, then a trypsin inhibitor can be added to neutralize the trypsin If attachment to the substrate is poor, precoating with a collagen or fibronectin solution may help 4.4 The Substrate For growth and differentiation in culture almost all types of cancer cells require interaction with a substrate Most cancer cell types will grow as monolayers on plastic or glass The major exceptions are hematopoietic cell lines and most small cell lung cancer cell lines that prefer to grow in suspension as single cells and as clusters of cells respectively Polystyrene is the most widely used plastic, though other substrates such as polycarbonate, polytetrafluoroethylene and polyvinyl are also available These plastics require treatment with irradiation or chemicals to produce a charged surface Cell adhesion can be greatly increased by coating the substrate with extracellular matrix (ECM) components Widely used ECM proteins include collagen, fibronectin, and laminin Alternatively, substrates can be precoated with serum or with medium conditioned by cells which produce ECM molecules Although monolayer culture is often the simplest and most convenient mode of culture, more complex systems can provide a greater level of information Growth within an ECM such as collagen or Matrigel in three dimensions, rather than on two, can provide more complete morphological and biochemical differentiation (22,23) The use of dual cultures wherein two populations of cells, e.g., carcinoma cells and fibroblasts, are separated by a filter, allows study of paracrine interactions and the roles of diffusible factors (see Chapters 28 and 29) 4.5 Physical Environment Maintenance of the physical environment conditions within certain limits is required for optimal cell growth For most mammalian cultures, the preferred temperature is 36.5° ± 1°C and although cells can still grow at lower temperatures, they will die rapidly at temperatures above 40°C Culture media contain buffering systems that generally require a CO2 atmosphere and frequently 5% CO2 is preferred Most cancer cells 10 Langdon require a pH value that is around 7.2–7.4 and media should be changed regularly as cell growth produces respiratory byproducts that acidify the media The pH indicator commonly added to medium is phenol red which is yellow at pH 6.5, orange at pH 7.0, red at pH 7.4, and purple at pH 7.8, thus providing a simple visual indication of the pH status of the culture Finally, humidity levels are important as evaporation of water from the medium will concentrate salt levels which could eventually cause cell lysis For optimal growth, the osmolality of the media should be kept within relatively narrow limits Primary Cell Culture Primary culture, i.e., the initial culture established from an individual, represents the situation most closely related to the original tissue The primary material used may be either a fragment, for example an explant, that can be made to attach to the substrate wherein cells can migrate and grow directly from the fragment, or tumor material that can be broken up by mechanical or enzymatic means into single cells or clusters of cells The source of the material can have an impact on the efficiency of this process, with cultures being more easily established from primary ascitic or pleural effusions already containing cells in suspension than from solid tumors Enzymes routinely used for disaggregation include trypsin and collagenase Many of the cell types within the initial cell mix may not adhere to a substrate readily or grow under the culture conditions, and the balance of cell types in culture may change rapidly with time as the fast-growing cells outgrow the slower or nonproliferating cell types This loss of heterogeneity has both advantages and disadvantages With selection to produce a more homogeneous cell population, if the predominant emerging cell type obtained is the one of interest then this might be considered helpful and desirable For the development of cell lines, this is necessary to allow a pure population to emerge The disadvantage of selective growth is that the heterogeneity and diversity of the multicellular tumor is lost with the subsequent absence of key intracellular interactions Cancer Cell Lines The development of a culture beyond the primary culture results in a “cell line.” The importance of a cell line lies in its ability to provide a renewable source of cell material for repeat studies Cell line models should reflect the properties of their original cancers, e.g., maintenance of histopathology when transplanted into immunodeficient mice, genotypic and phenotypic characteristics, gene expression and drug sensitivity (24) However, as it is frequently fast growing cell lines from poorly differentiated tumors that are generally selected for growth in vitro, the cell lines in widespread use may not necessarily always reflect those found in the majority of the clinical disease (24) Virtually all types of cancer cells can now be grown in culture The classical studies of Hayflick and Moorhead in the early 1960s demonstrated that diploid human fibroblasts could undergo a limited number of divisions (approx 50) in culture before enter- Basic Principles of Cancer Cell Culture 11 ing “crisis” and senescence (25) Most cancer cell lines will undergo indefinite numbers of divisions, and immortal cell lines can extend to thousands of divisions For a cell line to become “continuous” (rather than “finite”) cells must be present at low levels in the initial culture that have the ability to divide indefinitely or cells have to undergo “transformation.” This transformation can be produced via chemical or viral means (see Chapter 26) Once a new cell line has been established, it should be characterized and confirmed to be free of contamination These aspects are covered in Chapters and and 31 As cell lines undergo increasing numbers of passages, they may lose certain features such as differentiation characteristics; however they may also demonstrate greater homogeneity as the most rapidly growing subclones will emerge Stocks of the cell lines should periodically be frozen at a variety of passage numbers to provide a renewable resource As model systems, cell lines possess a number of advantages over primary cultures As mentioned above, their predominant strength is the ability to repeat studies with a well characterized culture system that can be used in multiple laboratories With continued culturing a relatively homogeneous cell population will arise unlike the primary culture that may contain many types of stromal and infiltrating cell types potentially complicating the interpretation of data 6.1 General Growth Characteristics of Cell Lines When cell lines are subcultured, they will pass through several well-defined stages of growth Monolayer cells when initially subcultured will take time to adhere to the substrate and, if they have been disaggregated by proteolytic enzymes (like trypsin), need time to repair the damage caused by these enzymes At this point the culture will grow relatively slowly and this stage is referred to as the “lag phase.” As the culture starts growing, paracrine exchange of growth factors will help accelerate the growth rate and an increasing percentage of cells will undergo cell division The initial cell density is a factor here and the higher the cell density, the more rapidly the culture will grow The culture will demonstrate its most rapid phase of growth, often exponential and therefore called the “log phase.” Finally, as the monolayer fills the available substrate area with cells in close contact with each other (confluency) and relatively high use of sera and media components, the growth rate will slow down to a “plateau” at a particular “saturation density” that is characteristic for a cell line (for a particular set of conditions) and this is referred to as the “plateau phase.” It is generally recognized that the longer the cells are in the plateau phase before subculture, the longer they remain in the lag phase after subculture 6.2 Cancer Cell Collections Cancer cell lines are widely available through a number of large cell banks The largest of these are listed in Table and in addition there are many other national collections that are often government sponsored and nonprofit making The World Federation for Culture Collection has 469 culture collections in 62 countries (http:// www.wfcc.info), although not all hold cancer cell cultures The number of more spe- 334 Uphoff and Drexler antibiotics Mycoplasmas trapped within clumps of eukaryotic cells or even in cavities formed by the cell membrane of a single cell might be protected from the antibiotic This is also the reason for the advice to keep the concentration of the antibiotic constantly high by frequently exchanging the medium Depending on the growth rate of the cell line, which might be severely altered by the antibiotic, the cell density should be diluted, kept constant, or even concentrated If no data are available at all for a given cell culture, or if the cell culture is in a very bad condition, the cell density, growth rate, and viability should be recorded frequently to improve the condition of the culture The concentration of the antibiotics should be kept at a constant level throughout the treatment period Low antibiotic concentrations attributable to degradation in culture or dilution by passaging the cell culture may lead to the development of resistant mycoplasma strains Thus, exchange of the complete medium and washing the cells with PBS is recommended References Barile, M F and Rottem, S (1993) Mycoplasmas in cell culture In: Rapid Diagnosis of Mycoplasmas (Kahane, I and Adoni, A., eds.), Plenum Press, New York, pp 155–193 Drexler, H G and Uphoff, C C (2000) Contamination of cell cultures, mycoplasma In: The Encyclopedia of Cell Technology (Spier, E., Griffiths, B., Scragg, A H., eds.), Wiley, New York, pp 609–627 Uphoff, C C and Drexler, H G (2001) Prevention of mycoplasma contamination in leukemia-lymphoma cell lines Human Cell 14, 244–247 Uphoff, C C., Meyer, C., and Drexler, H G (2002) Elimination of mycoplasma from leukemia-lymphoma cell lines using antibiotics Leukemia 16, 284–288 Schmidt, J and Erfle, V (1984) Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines Exp Cell Res 152, 565–570 Cell Culture Media APPENDIX Formulations of Commonly Used Cell Culture Media 335 336 Formulations of Commonly Used Cell Culture Media 336 Appendix Cell Culture Media Formulations of Commonly Used Cell Culture Media (Cont.) 337 337 338 Formulations of Commonly Used Cell Culture Media (Cont.) 338 Appendix Cell Culture Media Formulations of Commonly Used Cell Culture Media (Cont.) 339 339 Lines from ATCC and DSMZ APPENDIX Human Cancer Cell Lines Available from the ATCC and DSMZ Cell Banks (Combined List) 341 342 Appendix Human Cancer Cell Lines Available from the ATCC and DSMZ Lines from ATCC and DSMZ Human Cancer Cell Lines Available from the ATCC and DSMZ (Cont.) 343 344 Human Cancer Cell Lines Available from the ATCC and DSMZ (Cont.) Appendix Lines from ATCC and DSMZ Human Cancer Cell Lines Available from the ATCC and DSMZ (Cont.) 345 346 Appendix Human Cancer Cell Lines Available from the ATCC and DSMZ (Cont.) Lines from ATCC and DSMZ Human Cancer Cell Lines Available from the ATCC and DSMZ (Cont.) 347 ... From: Methods in Molecular Medicine, vol 88: Cancer Cell Culture: Methods and Protocols Edited by: S P Langdon © Humana Press Inc., Totowa, NJ Langdon Table Early Milestones in Cancer Cell Culture... major exceptions are hematopoietic cell lines and most small cell lung cancer cell lines that prefer to grow in suspension as single cells and as clusters of cells respectively Polystyrene is... changed reguFrom: Methods in Molecular Medicine, vol 88: Cancer Cell Culture: Methods and Protocols Edited by: S P Langdon © Humana Press Inc., Totowa, NJ 17 18 Macleod and Langdon larly and not allowed