6:  6.2 Growth of Animal Cells in Culture

Rich Media Are Required for Culture of Animal Cells

Nine amino acids, referred to as the essential amino acids, cannot be synthesized by adult vertebrate animals and thus must be obtained from their diet. Animal cells grown in culture also must be supplied with these nine amino acids, namely, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. In addition, most cultured cells require cysteine, glutamine, and tyrosine. In the intact animal, these three amino acids are synthesized by specialized cells; for example, liver cells make tyrosine from phenylalanine, and both liver and kidney cells can make glutamine. Animal cells both within the organism and in culture can synthesize the 8 remaining amino acids; thus these amino acids need not be present in the diet or culture medium. The other essential components of a medium for culturing animal cells are vitamins, which the cells cannot make at all or in adequate amounts; various salts; glucose; and serum, the noncellular part of the blood (Table 6-2, top).

Serum, a mixture of hundreds of proteins, contains various factors needed for proliferation of cells in culture. For example, it contains insulin, a hormone required for growth of many cultured vertebrate cells, and transferrin, an iron-transporting protein essential for incorporation of iron by cells in culture. Although many animal cells can grow in a serum-containing medium, such as Eagle’s medium, certain cell types require specific protein growth factors that are not present in serum. For instance, precursors of red blood cells require the hormone erythropoietin, and T lymphocytes of the immune system require interleukin 2 (IL-2). These factors bind to receptor proteins that span the plasma membrane, signaling the cells to increase in size and mass and undergo cell division (Chapter 20). A few mammalian cell types can be grown in a completely defined, serum-free medium supplemented with trace minerals, specific protein growth factors, and other components (Table 6-2, bottom).

Most Cultured Animal Cells Grow Only on Special Solid Surfaces

Within the tissues of intact animals, most cells tightly contact and interact specifically with other cells via various cellular junctions. The cells also contact the extracellular matrix, a complex network of secreted proteins and carbohydrates that fills the spaces between cells (Chapter 22). The matrix, whose constituents are secreted by cells themselves, helps bind the cells in tissues together; it also provides a lattice through which cells can move, particularly during the early stages of animal differentiation.

The extracellular matrices in various animal tissues consist of several common components: fibrous collagen proteins; hyaluronan (or hyaluronic acid), a large mucopolysaccharide; and covalently linked polysaccharides and proteins in the form of proteoglycans (mostly carbohydrate) and glycoproteins (mostly protein). However, the exact composition of the matrix in different tissues varies, reflecting the specialized function of a tissue. In connective tissue, for example, the major protein of the extracellular matrix is a type of collagen that forms insoluble fibers with a very high tensile strength. Fibroblasts, the principal cell type in connective tissue, secrete this type of collagen as well as the other matrix components. Receptor proteins in the plasma membrane of cells bind various matrix elements, imparting strength and rigidity to tissues (see Figure 5-40).

The tendency of animal cells in vivo to interact with one another and with the surrounding extracellular matrix is mimicked in their growth in culture. Unlike bacterial and yeast cells, which can be grown in suspension, most cultured animal cells require a surface to grow on. Many types of cells can adhere to and grow on glass, or on specially treated plastics with negatively charged groups on the surface (e.g., SO₃^2-). The cultured cells secrete collagens and other matrix components; these bind to the culture surface and function as a bridge between it and the cells. Cells cultured from single cells on a glass or a plastic dish form visible colonies in 10 – 14 days (Figure 6-3). Some tumor cells can be grown in suspension, a considerable experimental advantage because equivalent samples are easier to obtain from suspension cultures than from colonies grown in a dish.

Primary Cell Cultures Are Useful, but Have a Finite Life Span

Normal animal tissues (e.g., skin, kidney, liver) or whole embryos commonly are used to establish primary cell cultures. To prepare tissue cells for culture (or to remove adherent cells from a culture dish for biochemical studies), trypsin or another protease is used to destroy the proteins in the junctions that normally interconnect cells. For many years, most cell types were difficult, if not impossible, to culture. But the identification and preparation of various protein growth factors that stimulate the replication of specific cell types, as well as other recent modifications in culture methods, now permit experimenters to grow various types of specialized cells.

Many studies with vertebrate cells, however, still are performed with those few cell types that grow most readily in culture. These are not cells of a defined type; rather, they represent whatever grows when a tissue or an embryo is placed in culture. The cell type that usually predominates in such cultures is called a fibroblast because it secretes the types of proteins associated with fibroblasts in fibrous connective tissue of animals. Cultured fibroblasts have the morphology of tissue fibroblasts, but they retain the ability to differentiate into other cell types; thus they are not as differentiated as tissue fibroblasts.

Some studies are conducted with primary cultures of epithelial cells. In general, external and internal surfaces of tissues and organs are covered by a layer of epithelial cells called an epithelium (Figure 6-4). These highly differentiated cells are said to be polarized because the plasma membrane is organized into at least two discrete regions. For example, the epithelial cells that line the intestine form a simple columnar epithelium (see Figure 6-4b). That portion of the plasma membrane facing the lumen of the intestine, the apical surface, is specialized for absorption; the rest of the plasma membrane, the basolateral surface, mediates transport of nutrients from the cell to the blood and forms junctions with adjacent cells and the underlying extracellular matrix called the basal lamina.

Certain cells cultured from blood, spleen, or bone marrow adhere poorly, if at all, to a culture dish but nonetheless grow well. In the body, such nonadherent cells are held in suspension (in the blood), or they are loosely adherent (in the bone marrow and spleen). Because these cells often come from immature stages in the development of differentiated blood cells, they are very useful for studying normal blood cell differentiation and the abnormal development of leukemias.

When cells are removed from an embryo or an adult animal, most of the adherent ones grow continuously in culture for only a limited time before they spontaneously cease growing. Such a culture eventually dies out after many cell doublings, even if it is provided with fresh supplies of all the known nutrients that cells need to grow, including serum. For instance, when human fetal cells are explanted into cell culture, the majority of cells die within a relatively short time; “fibroblasts,” although also destined to die, proliferate for a while and soon become the predominant cell type. They divide about 50 times before they cease growth. Starting with 10⁶ cells, 50 doublings can produce 10⁶ × 2⁵⁰, or more than 10²⁰ cells, which is equivalent to the weight of about 10⁵ people. Thus, even though its lifetime is limited, a single culture, if carefully maintained, can be studied through many generations. Such a lineage of cells originating from one initial primary culture is called a cell strain (Figure 6-5a).

Transformed Cells Can Grow Indefinitely in Culture

To be able to clone individual cells, modify cell behavior, or select mutants, biologists often want to maintain cell cultures for many more than 100 doublings. This is possible with cells derived from some tumors and with rare cells that arise spontaneously because they have undergone genetic changes that endow them with the ability to grow indefinitely. The genetic changes that allow these cells to grow indefinitely are collectively called oncogenic transformation, and the cells are said to be oncogenically transformed, or simply transformed. A culture of cells with an indefinite life span is considered immortal; such a culture is called a cell line to distinguish it from an impermanent cell strain.

The ability of cultured cells to grow indefinitely or their tendency to be transformed varies depending on the animal species from which the cells originate. Normal chicken cells rarely are transformed and die out after only a few doublings; even tumor cells from chickens almost never exhibit immortality. Among human cells, only tumor cells grow indefinitely. The HeLa cell, the first human cell type to be grown in culture, was originally obtained in 1952 from a malignant tumor (carcinoma) of the uterine cervix. This cell line has been invaluable for research on human cells.

In contrast to human and chicken cells, cultures of embryonic adherent cells from rodents routinely give rise to cell lines. When adherent rodent cells are first explanted, they grow well, but after a number of serial replatings they lose growth potential and the culture goes into crisis (Figure 6-5b). During this period most of the cells die, but often a rapidly dividing variant cell arises spontaneously and takes over the culture. A cell line derived from such a variant will grow forever if it is provided with the necessary nutrients. Cells in spontaneously established rodent cell lines and in cell lines derived from tumors often have abnormal chromosomes. In addition, their chromosome number usually is greater than that of the normal cell from which they arose, and it continually expands and contracts in culture. Such cells are said to be aneuploid (i.e., have an inappropriate number of chromosomes) and are obviously mutants.

Although most cell lines are undifferentiated, some can carry out many of the functions characteristic of the normal differentiated cells from which they are derived. One example is certain hepatoma cell lines (e.g., HepG2) that synthesize most of the serum proteins made by normal hepatocytes (the major cell type in the liver) from which they are derived. These highly differentiated hepatoma cells are often studied as models of normal hepatocytes. Cultured myoblasts (muscle precursor cells) are another example of transformed cells that continue to perform many functions of a specialized, differentiated cell. When grown in culture, transformed myoblasts can be induced to fuse to form myotubes. These resemble differentiated multinucleated muscle cells and synthesize many of, if not all, the specialized proteins associated with contraction (Figure 6-6). Certain lines of epithelial cells also have been cultured successfully. One such line, Madin-Darby canine kidney (MDCK) cells, forms a continuous sheet of polarized epithelial cells one cell thick that exhibits many of the properties of the normal canine kidney epithelium from which it was derived (Figure 6-7). This type of preparation has proved valuable as a model for studying the functions of epithelial cells.

Fusion of Cultured Animal Cells Can Yield Interspecific Hybrids Useful in Somatic-Cell Genetics

Cultured animal cells infrequently undergo cell fusion spontaneously. The fusion rate, however, increases greatly in the presence of certain viruses that have a lipoprotein envelope similar to the plasma membrane of animal cells. A mutant viral glycoprotein in the envelope promotes cell fusion (see the photograph on the first page of this chapter); the mechanism of this effect is discussed at the end of Chapter 17. Cell fusion also is promoted by polyethylene glycol, which causes the plasma membranes of adjacent cells to adhere to each other and to fuse (Figure 6-8). As most fused animal cells undergo cell division, the nuclei eventually fuse, producing viable cells with a single nucleus that contains chromosomes from both “parents.” The fusion of two cells that are genetically different yields a hybrid cell called a heterokaryon.

Because some somatic cells from animals can be cultured from single cells in a well-defined medium, it is possible to select for genetically distinct cultured animal cells, just as is done with bacterial and yeast cells. Moreover, during mitosis the chromosomes in an animal cell are large and highly visible after staining, making it easy to distinguish individual chromosomes (Chapter 9). Genetic studies of cultured animal cells are called somatic-cell genetics to distinguish them from classical genetics, which deals with whole organisms derived from germ cells (sperm and eggs).

Cultured cells from different mammals can be fused to produce interspecific hybrids, which have been widely used in somatic-cell genetics. For instance, hybrids can be prepared from human cells and mutant mouse cells that lack an enzyme required for synthesis of a particular essential metabolite. As the human-mouse hybrid cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. In a medium that can support growth of both the human cells and mutant mouse cells, the hybrids eventually lose all human chromosomes. However, in a medium lacking the essential metabolite that the mouse cells cannot produce, the one human chromosome that contains the gene encoding the needed enzyme will be retained, because any hybrid cells that lose it following mitosis will die. All other human chromosomes eventually are lost.

By using different mutant mouse cells and media in which they cannot grow, researchers have prepared various panels of hybrid cell lines. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes. Because each chromosome can be identified visually under a light microscope, such hybrid cells provide a means for assigning, or “mapping,” individual genes to specific chromosomes. For example, suppose a hybrid cell line is shown microscopically to contain a particular human chromosome. That hybrid cell line can then be tested biochemically for the presence of various human enzymes, exposed to specific antibodies to detect human surface antigens, or subjected to DNA hybridization and cloning techniques (Chapter 7) to locate particular human DNA sequences. The genes encoding a human protein or containing a human DNA sequence detected in such tests must be located on the particular human chromosome carried by the cell line being tested. Panels of hybrids between normal mouse and mutant hamster cells also have been established; in these hybrid cells, the majority of mouse chromosomes are lost, allowing mouse genes to be mapped to specific mouse chromosomes.

Hybrid Cells Often Are Selected on HAT Medium

One metabolic pathway has been particularly useful in cell-fusion experiments. Most animal cells can synthesize the purine and pyrimidine nucleotides de novo from simpler carbon and nitrogen compounds, rather than from already formed purines and pyrimidines (Figure 6-9, top). The folic acid antagonists amethopterin and aminopterin interfere with the donation of methyl and formyl groups by tetrahydrofolic acid in the early stages of de novo synthesis of glycine, purine nucleoside monophosphates, and thymidine monophosphate. These drugs are called antifolates, since they block reactions involving tetrahydrofolate, an active form of folic acid. Many cells, however, contain enzymes that can synthesize the necessary nucleotides from purine bases and thymidine if they are provided in the medium; these salvage pathways bypass the metabolic blocks imposed by antifolates (Figure 6-9, bottom).

A number of mutant cell lines lacking the enzyme needed to catalyze one of the steps in a salvage pathway have been isolated. For example, cell lines lacking thymidine kinase (TK) can be selected because such cells are resistant to the otherwise toxic thymidine analog 5-bromodeoxyuridine. Cells containing TK convert 5-bromodeoxyuridine into 5-bromodeoxyuridine monophosphate. This nucleoside mono- phosphate is then converted into a nucleoside triphosphate by other enzymes and is incorporated by DNA polymerase into DNA, where it exerts its toxic effects. This pathway is blocked in cells with a TK mutation that prevents production of functional TK enzyme. Hence, TK⁻ mutants are resis- tant to the toxic effects of 5-bromodeoxyuridine. Similarly, cells lacking the HGPRT enzyme have been selected because they are resistant to the otherwise toxic guanine analog 6-thioguanine. As we will see next, HGPRT⁻ cells and TK⁻ cells are useful partners in cell fusions with one another or with cells that have salvage-pathway enzymes but that are differentiated and cannot grow in culture by themselves.

The medium most often used to select hybrid cells is called HAT medium, because it contains hypoxanthine (a purine), aminopterin, and thymidine. Normal cells can grow in HAT medium because even though aminopterin blocks de novo synthesis of purines and TMP, the thymidine in the media is transported into the cell and converted to TMP by TK and the hypoxanthine is transported and converted into usable purines by HGPRT. On the other hand, neither TK⁻ nor HGPRT⁻ cells can grow in HAT medium because each lacks an enzyme of the salvage pathway. However, hybrids formed by fusion of these two mutants will carry a normal TK gene from the HGPRT⁻ parent and a normal HGPRT gene from the TK⁻parent. The hybrids thus will produce both functional salvage-pathway enzymes and grow on HAT medium. Likewise, hybrids formed by fusion of mutant cells and normal cells can grow in HAT medium.

Hybridomas Are Used to Produce Monoclonal Antibodies

Each normal B lymphocyte in an animal is capable of producing a single type of antibody directed against a specific determinant, or epitope, on an antigen molecule. If an animal is injected with an antigen, B lymphocytes that make antibody recognizing the antigen are stimulated to grow and proliferate. Each antigen-activated B lymphocyte forms a clone of cells in the spleen or lymph nodes, with each cell of the clone producing identical antibody, termed monoclonal antibody. Because most natural antigens contain multiple epitopes, exposure of an animal to an antigen usually stimulates formation of several different B-lymphocyte clones, each producing a different antibody; a mixture of antibodies that recognize different epitopes on the same antigen is said to be polyclonal.

For many types of studies involving antibodies, monoclonal antibody is preferable to polyclonal antibody. However, biochemical purification of monoclonal antibody from serum is not feasible, in part because the concentration of any given antibody is quite low. For this reason, researchers looked to culture techniques in order to obtain usable quantities of monoclonal antibody. Because primary cultures of normal B lymphocytes do not grow indefinitely, such cultures have limited usefulness for production of monoclonal antibody. This limitation can be avoided by fusing normal B lymphocytes with oncogenically transformed lymphocytes called myeloma cells, which are immortal.

Fusion of a myeloma cell with a normal antibody-producing cell from a rat or mouse spleen yields a hybrid that proliferates into a clone called a hybridoma. Like myeloma cells, hybridoma cells are immortal. Each hybridoma produces the monoclonal antibody encoded by its B-lymphocyte partner. Many different myeloma cell lines from mice and rats have been established; from these, HGPRT⁻ lines have been selected based on their resistance to 6-thioguanine as described above. If such mutant myeloma cells are fused with normal B lymphocytes, any fused cells that result can grow in HAT medium, but the parental cells cannot (Figure 6-10). Each selected hybridoma then is tested for production of the desired antibody; any clone producing that antibody then is grown in large cultures, from which a substantial quantity of pure monoclonal antibody can be obtained.

Such pure antibodies are very valuable research reagents. For example, a monoclonal antibody that interacts with protein X can be used to label, and thus locate, protein X in specific cells of an organ or in specific cell fractions. Once identified, even very scarce proteins can be isolated by affinity chromatography in columns to which the monoclonal antibody is bound (see Figure 3-43c). Monoclonal antibodies also have become important diagnostic and therapeutic tools in medicine. Monoclonal antibodies that bind to and inactivate toxic proteins (toxins) secreted by bacterial pathogens are used to treat diseases caused by these pathogens. Other monoclonal antibodies are specific for cell-surface proteins expressed by certain types of tumor cells; chemical complexes of such monoclonal antibodies with toxic drugs are being developed for cancer chemotherapy.

SUMMARY

• Growth of vertebrate cells in culture requires rich media containing essential amino acids, vitamins, and peptide or protein growth factors, frequently provided by serum. Most cultured vertebrate cells will grow only when attached to a negatively charged substratum that mimics the extracellular matrix in animal tissues.

• Primary cells, which are derived directly from animal tissue, have limited growth potential in culture and may give rise to a cell strain.

• Transformed cells, which are derived from animal tumors or arise spontaneously from primary rodent cells, grow indefinitely in culture (see Figure 6-5b). They usually have an unstable, aneuploid complement of chromosomes, including abnormal chromosomes. Transformed cells derived from a single parental cell are called cell lines.

• Cultured cells can be induced to fuse into heterokaryons (hybrids) by treatment with certain viruses or polyethylene glycol. Heterokaryons between cells of different species tend to lose the chromosomes of one species as they divide.

• Panels of hybrid lines prepared from mutant mouse cells and normal human cells, each containing different human chromosomes, can be used to map the gene encoding a specific human protein to a specific human chromosome.

• Fusion of an HGPRT⁻ myeloma cell and a single B lymphocyte yields a hybrid cell that can grow on HAT medium and proliferate indefinitely, forming a clone called a hybridoma (see Figure 6-10). Since each individual B lymphocyte produces antibodies specific for one antigenic determinant (epitope), a hybridoma produces only the monoclonal antibody synthesized by its original B-lymphocyte parental cell.