Molecular Biology of the Cell - Volume 1 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

Introduction to Cell Biology
How Cells Are Studied
Cell Separation and Culture

Organelle Structure and large molecules can be studied under a Microscope; effective staining Methods have been developed to localize specific molecules within The Cell. However, a detailed biochemical analysis is required to understand the Molecular Basis of Cellular Organization. Unfortunately, Biochemical Methods require a significant number of Cells, and the cells are destroyed during the analysis. If a piece of tissue is used as a sample for biochemical analysis, its disruption will yield a mixture of fragments from various cells. And if the tissue is composed of different cell types, which is the rule rather than the exception, resolving this mixture becomes virtually impossible. In an effort to extract maximum information about all the cells that make up Tissues, cell biologists have developed methods to dissociate tissues into cells and isolate individual cell types. The resulting relatively homogeneous cell population can be analyzed directly or after expanding them through cultivation.

4.3.1. Cells can be isolated from tissues and separated into different types [19]

The first step in isolating cells of a single type from a tissue containing various cell types is to disperse the tissue into a suspension of individual cells. This is achieved by disrupting the Extracellular matrix and Intercellular junctions that hold the cells together. Typically, the highest yield of viable cells is obtained from embryonic or neonatal tissues. In this case, the cell Separation Procedure involves treating the tissue with Proteolytic Enzymes (such as Trypsin and collagenase) and Ca²⁺-binding (or chelating) agents (such as ethylenediaminetetraacetic acid - EDTA) that mediate Cell Adhesion. The tissue is then subjected to gentle mechanical disruption to separate it into individual cells.

Several approaches are used to fractionate a mixed cell suspension into individual cell types. One approach is based on differences in the Physical Properties of cells. For example, centrifugation can separate large cells from small ones, and heavy cells from light ones; these methods will be discussed when we address Cell Fractionation (for which these methods were originally developed). Another approach is based on the ability of certain cells to adhere tightly to Glass or plastic, allowing them to be separated from other, less adherent cells.

An important refinement of this method involves The Use of Antibodies. Antibodies that bind specifically to one cell type (among those present in the tissue) can be coupled to various matrices, such as Collagen, polysaccharide beads, or plastic. Only the cells recognized by the antibodies will bind to such a surface. The bound cells are then released either by gentle shaking or by digesting the matrix (e.g., collagen) with enzymes (e.g., collagenase).

The most sophisticated cell separation method involves labeling with antibodies coupled to fluorescent Dyes. An electronic fluorescence-activated cell sorter (FACS) can separate labeled cells from unlabeled ones. In this method, individual cells travel one by one in a narrow stream and pass through a laser beam, where their fluorescence is measured. A vibrating nozzle then forms tiny droplets, most of which contain either a single cell or no cells at all. At the moment of formation, each droplet is automatically given a positive or negative charge depending on whether it contains a fluorescent cell. A strong electric field then deflects the droplets into appropriate containers. Occasional cell clumps are detected by increased light scattering and are discarded into a waste container (Fig. 4-38). The cell sorter can select a single cell from among thousands; it sorts about 5000 cells per second. Once a population of identical cells is obtained by any of these methods, the researcher can use them for biochemical analysis. Alternatively, these cells can be placed in culture, allowing their behavior and properties to be studied under culture conditions.

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Fig. 4-38. Schematic diagram of a fluorescence-activated cell sorter. A laser beam analyzes the fluorescence of cells passing through it. Droplets containing individual cells are charged positively or negatively depending on whether the cell fluoresces. The droplets are then deflected into collection tubes according to their charge. Note that the cell concentration must be adjusted so that most droplets contain no cells. Consequently, most droplets, along with any cell clumps, are directed to the waste container.

4.3.2. Cells can be grown in a culture vessel [20]

Most PLANT AND ANIMAL cell types can survive, multiply, and even differentiate under favorable conditions. Using tissue culture techniques, cells can be studied under a microscope or analyzed biochemically. Furthermore, by adding specific molecules, such as Hormones or growth factors, to the culture vessel and removing them, we can assess their effects on cells. The use of mixed cultures allows The Study of interactions between different cell types. In scientific literature, any experiments with cell cultures are often referred to as experiments performed in vitro, which literally means "in glass"; in contrast, experiments on Living organisms are said to be performed in vivo. Biochemists and cell biologists use these terms in a slightly different sense. For them, in vitro refers to biochemical reactions occurring outside living cells, while in vivo refers to all reactions taking place within living cells. The birth of the tissue culture method dates back to 1907. At that time, an experiment was designed to resolve a debate among neurobiologists. The hypothesis to be tested (known as the "neuron doctrine") was that each nerve fiber is formed by growing out of a single nerve cell, rather than by the fusion of multiple cells. To test this hypothesis, small pieces of Spinal Cord were placed in a warm, moist chamber and observed under a microscope at regular intervals. After about a day, long, thin processes could be seen growing out from individual Nerve Cells. This provided evidence in favor of the neuron doctrine and laid the foundation for the revolution brought about by cell culture techniques.

The pioneering experiments conducted in 1907 involved the use of small tissue fragments, or explants. Today, cultures are typically prepared from a cell suspension obtained by tissue dissociation. Unlike bacterial cells, most cells making up the tissues of Multicellular Organisms are unable to grow in suspension. They require a solid surface to grow and divide. Initially, when the cultivation method was first introduced, a plasma clot was used as a mechanical support, but today it is usually replaced by The surface of a plastic culture dish (Fig. 4-39). Cells vary greatly in their requirements; some can grow or differentiate only if the culture dish is coated with Components of the extracellular matrix, such as collagen.

Fig. 4-39. Scanning electron micrograph of rat fibroblasts growing in tissue culture. (Courtesy of Gunther Albrecht-Buchler.)

Cultures prepared directly from the tissues of an Organism, with or without an initial cell fractionation step, are called primary cultures. In most cases, cells from a primary culture can be removed from the culture dish and used to produce A large number of secondary cultures, which can be repeatedly subcultured for weeks or months. Often, these cells retain the differentiated CHARACTERISTICS OF THE tissues from which they were derived. For example, fibroblasts continue to secrete collagen; embryonic Skeletal Muscle cells fuse to form giant muscle fibers that contract spontaneously in tissue culture dishes; nerve cells extend axons that are electrically excitable and capable of forming synapses with other nerve cells; and epithelial cells form extensive sheets that retain many properties of intact epithelium. Because all these events can be observed as cells grow in culture, they can be studied using many techniques that are not feasible with intact tissues.

4.3.3. Chemically defined media can be used to identify specific growth factors [21]

Until the early 1970s, tissue culture was something of a mixture of science and witchcraft. Although plasma clots were replaced by plastic dishes and liquid media containing a precise mixture of salts, Amino Acids, and Vitamins, most media still contained small amounts of poorly characterized biological Materials, such as horse serum, crude extracts from chick embryos, or fetal bovine serum. For most routine tissue cultures, such media are still used today (Table 4-4), but they are not suitable for studying the specific requirements for cell growth and differentiation.

This led to The Development of chemically defined media for culturing various cell types. In these media, every component is known. Along with low-molecular-weight nutrients, they typically contain one or more specific protein growth factors that cells require to survive and proliferate in culture: for example, certain nerve cells, both in culture and in the animal, require trace amounts of nerve growth factor. Other factors of this type have been discovered that are vital for the development and maintenance of specific cell types. The advent of chemically defined media has greatly facilitated the discovery of new factors.

Table 4-4. Composition of a standard medium for culturing mammalian cells ¹⁾

¹⁾ The glucose concentration should be 5-10 mM. All Amino acids are used in the L-form; with one or two exceptions, they are used at a concentration of 1 or 2 mM. The concentration of vitamins should be 100-fold lower, i.e., about 1 μM. The concentration of serum (horse or calf) should be 10% of the total volume. Penicillin and streptomycin are Antibiotics added to prevent bacterial growth. Phenol red is a pH indicator used to monitor a pH of 7.4.

For cultivation, plastic or glass vessels are typically used, with surfaces treated to allow cell adhesion. The vessels are placed in an incubator at 37°C in an atmosphere of 5% CO₂ and 95% air.

4.3.4. Introduction/5.html">Eukaryotic Cell lines are commonly used to obtain homogeneous cells [18]

Most mammalian cells in culture die after a certain number of divisions; human Skin cells, for example, divide 50-100 times before dying. It is hypothesized that the limited lifespan of cells in culture reflects the limited lifespan of the organism from which they were obtained. Occasionally, mutant cells arise in culture that are practically immortal. They can multiply indefinitely, forming a cell line (Table 4-5). These cells grow best on a solid surface, and their growth typically ceases once they form a confluent monolayer.

Usually, mutant cells capable of indefinite division still differ from Cancer cells, which can divide indefinitely both in vitro and in vivo. Unlike other cell lines, cancer cells can grow without adhering to a solid surface and form much denser populations in culture dishes than normal cells. A similar property can be induced experimentally in normal cells by transforming them with tumor Viruses or chemical compounds. The resulting neoplastically transformed cell lines are capable of causing tumors when injected into animals. Both transformed and untransformed cell lines provide a source of large numbers of cells of a single type and are therefore of great value to researchers. Such cell lines have the additional advantage that they can be stored indefinitely at -70°C, retaining The ability to yield viable cells upon thawing. However, It is important to realize that cells of both types of cell lines almost always differ significantly from their normal progenitors in the tissues from which they were derived.

Table 4-5. Some of the most widely used cell lines

¹⁾ Many of these cell lines are of tumor origin. All of them are capable of multiplying indefinitely in tissue culture and exhibit (at least partially) properties characteristic of the tissues from which they originate. BHK21, SP2, and HeLa cell lines are capable of growing in suspension, whereas other cells require a solid support to proliferate.

The genetic homogeneity of cell lines can be further enhanced by cloning, i.e., by isolating a single cell and allowing it to proliferate to form a large colony. A clone is a population of cells derived from a single progenitor cell. Cell cloning is primarily used to obtain cell lines in which a mutation has affected specific genes. The study of such mutant cells, which are defective in a specific protein, provides valuable insights into the function of the protein in normal cells.

4.3.5. Cell Fusion leads to The formation of cell hybrids [22]

When two cells fuse, they form a heterokaryon—a single combined cell with two nuclei. Typically, to induce cell fusion, the cell suspension is treated with inactivated viruses or polyethylene glycol. Both of these agents alter the cell's Plasma Membrane, which leads to cell fusion. The formation of heterokaryons makes it possible to mix the components of two distinct cells to study their interaction. For example, if an inactive chicken erythrocyte Nucleus is introduced by fusion into the Cytoplasm of a cell growing in tissue culture, this nucleus is reactivated: RNA Synthesis begins, followed by METABOLISM/36.html">DNA Replication. It was in mouse-human cell Hybridization experiments that evidence was first obtained showing that human and mouse cell-surface Proteins, initially restricted to their respective halves of the heterokaryon, rapidly diffuse and mix over its entire surface.

After a certain period, the heterokaryon undergoes mitosis, resulting in a hybrid cell. The nuclear envelopes of this cell break down, and all Chromosomes are integrated into a single large nucleus (Fig. 4-40). Although such hybrid cells can be cloned to establish a hybrid cell line, the primary hybrid cells are unstable and lose chromosomes. For unknown reasons, mouse-human hybrid cells predominantly lose Human chromosomes. This results in a variety of mouse-human hybrid lines, each containing only one or a few human chromosomes. This phenomenon has proven useful for mapping and localizing genes within The Human Genome. For example, human Insulin is synthesized only by those hybrid cells that contain human chromosome 11; therefore, the Gene encoding insulin is located on this specific chromosome.

Fig. 4-40. Diagram illustrating the fusion of human and mouse cells, leading to the formation of heterokaryons containing one or more nuclei. In some cases, heterokaryons give rise to hybrid cells with a single fused nucleus. Such hybrid cells are used for mapping individual genes to specific human chromosomes. This mapping is possible because hybridization is accompanied by the rapid, random loss of most human chromosomes. Only one or a few human chromosomes are retained in the resulting clones. In hybrid cells formed by the fusion of other cell types, most of the original chromosomes are often retained.

Table 4-6. Major Milestones in the development of tissue culture techniques

Fig. 4-41. Preparative ultracentrifuge. The sample under study is contained in tubes placed in circular cylindrical slots within a metal rotor. As the rotor spins rapidly, a significant centrifugal force is generated, causing the particles in the sample to sediment. Under vacuum conditions, friction is reduced; As a result, the rotor does not heat up, and a built-in refrigeration system maintains the sample Temperature at 4°C.

Some Key milestones in the development of tissue culture methods are listed in Table 4-6.

Summary

Cells from embryonic and neonatal tissues are used as starting material to isolate specific cell types, which can then be analyzed biochemically or used to establish cell cultures. Many plant and animal cells survive and are often capable of proliferating in a culture dish when provided with an appropriate nutrient medium. Different cell types require distinct nutrients, including one or more protein growth factors. Most animal cells die after a finite number of divisions, but occasionally, rare variants spontaneously arise in culture that can be maintained indefinitely as cell lines. Cell lines can be used to obtain clones derived from a single progenitor cell. In this way, mutant cells defective in a single protein can be isolated. Different cell types can be fused to form heterokaryons (cells with two nuclei), which eventually give rise to hybrid cells (whose nuclei have fused into one). Hybrid cells can be used to study the interactions between components of two different cells. Furthermore, this method helps determine which specific chromosomes harbor particular genes.