Key Concepts Of Section

Plant Tissues

■ The integration of cells into tissues in plants is fundamentally different from the assembly of animal tissues, primarily because each plant cell is surrounded by a relatively rigid cell wall.

■ The plant cell wall comprises layers of cellulose mi-crofibrils embedded within a matrix of hemicellulose, pectin, extensin, and other less abundant molecules.

■ Cellulose, a large, linear glucose polymer, assembles spontaneously into microfibrils stabilized by hydrogen bonding.

■ The cell wall defines the shapes of plant cells and restricts their elongation. Auxin-induced loosening of the cell wall permits elongation.

■ Adjacent plant cells can communicate through plas-modesmata, which allow small molecules to pass between the cells (see Figure 6-34).

■ Plants do not produce homologs of the common adhesive molecules found in animals. Only a few adhesive molecules unique to plants have been well documented to date.

6.7| Growth and Use of Cultured Cells

Many technical constraints hamper studies on specific cells or subsets of cells in intact animals and plants. One alternative is the use of intact organs that are removed from animals and perfused with an appropriately buffered solution to maintain their physiologic integrity and function. Such organ perfusion systems have been widely used by physiologists. However, the organization of organs, even isolated ones, is sufficiently complex to pose numerous problems for research on many fundamental aspects of cell biology. Thus molecular cell biologists often conduct experimental studies on cells isolated from an organism and maintained in conditions that permit their survival and growth, a procedure known as culturing.

Cultured cells have several advantages over intact organisms for cell biology research. First, most animal and plant tissues consist of a variety of different types of cells, whereas cells of a single specific type with homogeneous properties can be grown in culture. Second, experimental conditions (e.g., composition of the extracellular environment) can be controlled far better in culture than in an intact organism. Third, in many cases a single cell can be readily grown into a colony of many identical cells, a process called cell cloning, or simply cloning (Figure 6-36). The resulting strain of cells, which is genetically homogeneous, is called a clone. This simple technique, which is commonly used with many bacteria, yeasts, and mammalian cell types, makes it easy to isolate genetically distinct clones of cells.

▲ FIGURE 6-36 Cultured mammalian cells viewed at three magnifications. (a) A single mouse cell attached to a plastic petri dish, viewed through a scanning electron microscope. (b) A single colony of human HeLa cells about 1 mm in diameter, produced from a single cell after growth for 2 weeks. (c) After

A major disadvantage of cultured cells is that they are not in their normal environment and hence their activities are not regulated by the other cells and tissues as they are in an intact organism. For example, insulin produced by the pancreas has an enormous effect on liver glucose metabolism; however, this normal regulatory mechanism does not operate in a purified population of liver cells (called hepa-tocytes) grown in culture. In addition, as already described, the three-dimensional distribution of cells and extracellular matrix around a cell influences its shape and behavior. Because the immediate environment of cultured cells differs radically from this "normal" environment, their properties may be affected in various ways. Thus care must always be exercised in drawing conclusions about the normal properties of cells in complex tissues and organisms only on the basis of experiments with isolated, cultured cells.

Culture of Animal Cells Requires Nutrient-Rich Media and Special Solid Surfaces

In contrast with most bacterial cells, which can be cultured quite easily, animal cells require many specialized nutrients and often specially coated dishes for successful culturing. To permit the survival and normal function of cultured tissues or cells, the temperature (37 °C for mammalian cells), pH, ionic strength, and access to essential nutrients must simulate as closely as possible the conditions within an intact organism. Isolated animal cells are typically placed in a nutrient-rich liquid, the culture medium, within specially treated plastic dishes or flasks. The cultures are kept in incubators in which the temperature, atmosphere, and humidity can be controlled. To reduce the chances of bacterial or fungal contamination, antibiotics are often added to the culture medium. To further guard against contamination, investigators usually transfer cells between dishes, add reagents to the culture medium, and otherwise manipulate the specimens within special cabinets cells initially introduced into a 6-cm-diameter petri dish have grown for several days and then been stained, individual colonies can easily be seen and counted. All the cells in a colony are progeny of a single precursor cell and thus genetically identical. [Part (a) courtesy of N. K. Weller. Parts (b) and (c) courtesy of T T Puck.]

containing circulating air that is filtered to remove microorganisms and other airborne contaminants.

Media for culturing animal cells must supply histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threo-nine, tryptophan, and valine; no cells in adult vertebrate animals can synthesize these nine essential amino acids. In addition, most cultured cells require three other amino acids (cysteine, glutamine, and tyrosine) that are synthesized only by specialized cells in intact animals. The other necessary components of a medium for culturing animal cells are vitamins, various salts, fatty acids, glucose, and serum—the fluid remaining after the noncellular part of blood (plasma) has been allowed to clot. Serum contains various protein factors that are needed for the proliferation of mammalian cells in culture. These factors include the polypeptide hormone insulin; transferrin, which supplies iron in a bioaccessible form; and numerous growth factors. In addition, certain cell types require specialized protein growth factors not present in serum. For instance, hematopoietic cells require erythro-poietin, and T lymphocytes require interleukin 2 (Chapter 14). A few mammalian cell types can be grown in a chemically defined, serum-free medium containing amino acids, glucose, vitamins, and salts plus certain trace minerals, specific protein growth factors, and other components.

Unlike bacterial and yeast cells, which can be grown in suspension, most animal cells will grow only on a solid surface. This highlights the importance of cell adhesion molecules. Many types of cells can grow on glass or on specially treated plastics with negatively charged groups on the surface (e.g., SO32~). The cells secrete ECM components, which adhere to these surfaces, and then attach and grow on the secreted matrix. A single cell cultured on a glass or a plastic dish proliferates to form a visible mass, or colony, containing thousands of genetically identical cells in 4-14 days, depending on the growth rate (see Figure 6-36c). Some specialized blood cells and tumor cells can be maintained or grown in suspension as single cells.

Primary Cell Cultures and Cell Strains Have a Finite Life Span

Normal animal tissues (e.g., skin, kidney, liver) or whole embryos are commonly used to establish primary cell cultures. To prepare tissue cells for a primary culture, the cell-cell and cell-matrix interactions must be broken. To do so, tissue fragments are treated with a combination of a protease (e.g., trypsin or the collagen-hydrolyzing enzyme collagenase or both) and a divalent cation chelator (e.g., EDTA) that depletes the medium of usable Ca2+ or Mg2 + . The released cells are then placed in dishes in a nutrient-rich, serum-supplemented medium, where they can adhere to the surface and one another. The same protease/chelator solution is used to remove adherent cells from a culture dish for biochemical studies or subculturing (transfer to another dish).

Often connective tissue fibroblasts divide in culture more rapidly than other cells in a tissue, eventually becoming the predominant type of cells in the primary culture, unless special precautions are taken to remove them when isolating other types of cells. Certain cells 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 removed from an embryo or an adult animal are cultured, most of the adherent ones will divide a finite number of times and then cease growing (cell senescence, Figure 6-38a). For instance, human fetal fibroblasts divide about 50 times before they cease growth. Starting with 106 cells, 50 doublings can produce 106 X 250, or more than 1020 cells, which is equivalent to the weight of about 10 people. Normally, only a very small fraction of these cells are used in any one experiment. 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.

Cell strains can be frozen in a state of suspended animation and stored for extended periods at liquid nitrogen temperature, provided that a preservative that prevents the formation of damaging ice crystals is used. Although some cells do not survive thawing, many do survive and resume growth. Research with cell strains is simplified by the ability to freeze and successfully thaw them at a later time for experimental analysis.

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. Such prolonged growth is exhibited by cells derived from some tumors. In addition, rare cells in a population of primary cells that undergo certain spontaneous genetic changes, called oncogenic transformation, are able to grow indefinitely. These cells are said to be oncogenically transformed or simply transformed. A culture of cells with an indefinite life span is considered immortal and is called a cell line.

The HeLa cell line, the first human cell line, was originally obtained in 1952 from a malignant tumor (carcinoma) of the uterine cervix. Although primary cell cultures of normal human cells rarely undergo transformation into a cell line, rodent cells commonly do. After rodent cells are grown in culture for several generations, the culture goes into senescence (Figure 6-37b). During this period, most of the cells stop growing, but often a rapidly dividing transformed cell arises spontaneously and takes over, or overgrows, the culture. A cell line derived from such a transformed variant will grow indefinitely if provided with the necessary nutrients.

Regardless of the source, cells in immortalized lines often have chromosomes with abnormal structures. In addition, the number of chromosomes in such cells is usually greater than that in the normal cell from which they arose, and the chromosome number expands and contracts as the cells continue to divide in culture. A noteworthy exception is the Chinese hamster ovary (CHO) line and its derivatives, which have fewer chromosomes than their hamster progenitors. Cells with an abnormal number of chromosomes are said to be aneuploid.

(a) Human cells

Phase Phase

(a) Human cells

Phase Phase

Cell generations

(b) Mouse cells

(b) Mouse cells

Days after initiation of culture

▲ FIGURE 6-37 Stages in the establishment of a cell culture. (a) When cells isolated from human tissue are initially cultured, some cells die and others (mainly fibroblasts) start to grow; overall, the growth rate increases (phase I). If the remaining cells are harvested, diluted, and replated into dishes again and again, the cell strain continues to divide at a constant rate for about 50 cell generations (phase II), after which the growth rate falls rapidly. In the ensuing period (phase III), all the cells in the culture stop growing (senescence). (b) In a culture prepared from mouse or other rodent cells, initial cell death (not shown) is coupled with the emergence of healthy growing cells. As these dividing cells are diluted and allowed to continue growth, they soon begin to lose growth potential, and most stop growing (i.e., the culture goes into senescence). Very rare cells survive and continue dividing until their progeny overgrow the culture. These cells constitute a cell line, which will grow indefinitely if it is appropriately diluted and fed with nutrients: such cells are said to be immortal.

Most cell lines have lost some or many of the functions characteristic of the differentiated cells from which they were derived. Such relatively undifferentiated cells are poor models for investigating the normal functions of specific cell types. Better in this regard are several more-differentiated cell lines that exhibit many properties of normal nontransformed cells. These lines include the liver tumor (hepatoma) HepG2 line, which synthesizes most of the serum proteins made by normal liver cells (hepatocytes). Another example consists of cells from a certain cultured fibroblast line, which under certain experimental conditions behave as muscle precursor cells, or myoblasts. These cells can be induced to fuse to form myotubes, which resemble differentiated multinucleated muscle cells and synthesize many of the specialized proteins associated with contraction. The results of studies with this cell line have provided valuable information about the differentiation of muscle (Chapter 22). Finally, as discussed previously, the MDCK cell line retains many properties of highly differentiated epithelial cells and forms well-defined epithelial sheets in culture (see Figure 6-6).

Hybrid Cells Called Hybridomas Produce Abundant Monoclonal Antibodies

In addition to serving as research models for studies on cell function, cultured cells can be converted into "factories" for producing specific proteins. In Chapter 9, we describe how it is done by introducing genes encoding insulin, growth factors, and other therapeutically useful proteins into bacterial or eukaryotic cells. Here we consider the use of special cultured cells to generate monoclonal antibodies, which are widely used experimental tools and increasingly are being used for diagnostic and therapeutic purposes in medicine.

To understand the challenge of generating monoclonal antibodies, we need to briefly review how antibodies are produced by mammals. Each normal B lymphocyte in a mammal is capable of producing a single type of antibody directed against (can bind to) a specific chemical structure (called a determinant or epitope) on an antigen molecule. If an animal is injected with an antigen, B lymphocytes that make antibodies recognizing the antigen are stimulated to grow and secrete the antibodies. Each antigen-activated B lymphocyte forms a clone of cells in the spleen or lymph nodes, with each cell of the clone producing the identical antibody—that is, a monoclonal antibody. Because most natural antigens contain multiple epitopes, exposure of an animal to an antigen usually stimulates the formation of multiple different B-lymphocyte clones, each producing a different antibody. The resulting mixture of antibodies that recognize different epitopes on the same antigen is said to be polyclonal. Such polyclonal antibodies circulate in the blood and can be isolated as a group and used for a variety of experiments. However, monoclonal antibodies are required for many types of experiments or medical applications. Unfortunately, the biochemical purification of any

Inject mouse with antigen X

Inject mouse with antigen X


Mutant mouse myeloma cells unable to grow in HAT medium

Mouse spleen cells; some cells (red) make antibody to antigen X

Mix and fuse cells

Mix and fuse cells

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