In this chapter, we present some of the principal methods used to study cells and their molecular components. We consider how to separate cells of different types from tissues, how to grow cells outside the body, and how to disrupt cells and isolate their organelles and constituent macromolecules in pure form. We also present the techniques used to determine protein structure, function, and interactions, and we discuss the breakthroughs in DNA technology that continue to revolutionize our understanding of cell function. We end the chapter with an overview of some of the mathematical approaches that are helping us deal with the enormous complexity of cells. By considering cells as dynamic systems with many moving parts, mathematical approaches can reveal hidden insights into how the many components of cells work together to produce the special qualities of life.
Although the organelles and large molecules in a cell can be visualized with microscopes, understanding how these components function requires a detailed biochemical analysis. Most biochemical procedures require that large numbers of cells be physically disrupted to gain access to their components. If the sample is a piece of tissue, composed of different types of cells, heterogeneous cell populations will be mixed together. To obtain as much information as possible about the cells in a tissue, biologists have developed ways of dissociating cells from tissues and separating them according to type. These manipulations result in a relatively homogeneous population of cells that can then be analyzed—either directly or after their number has been greatly increased by allowing the cells to proliferate in culture.
Intact tissues provide the most realistic source of material, as they represent the actual cells found within the body. The first step in isolating individual cells is to disrupt the extracellular matrix and cell–cell junctions that hold the cells together. For this purpose, a tissue sample is typically treated with proteolytic enzymes (such as trypsin and collagenase) to digest proteins in the extracellular matrix and with agents (such as ethylenediaminetetraacetic acid, or EDTA) that bind, or chelate, the Ca2+ on which cell–cell adhesion depends. The tissue can then be teased apart into single cells by gentle agitation.
For some biochemical preparations, the protein of interest can be obtained in sufficient quantity without having to separate the tissue or organ into cell types. Examples include the preparation of histones from calf thymus, actin from rabbit muscle, or tubulin from cow brain. In other cases, obtaining the desired protein requires enrichment for a specific cell type of interest. Several approaches are used to separate the different cell types from a mixed cell suspension. One of the most sophisticated cell-separation techniques uses an antibody coupled to a fluorescent dye to label specific cells. An antibody is chosen that specifically binds to the surface of only one cell type in the tissue. The labeled cells can then be separated from the unlabeled ones in a fluorescence-activated cell sorter. In this remarkable machine, individual cells traveling single file in a fine stream pass through a laser beam, and the fluorescence of each cell is rapidly measured. A vibrating nozzle generates tiny droplets, most containing either one cell or no cells. The droplets containing a single cell are automatically given a positive or a negative charge at the moment of formation, depending on whether the cell they contain is fluorescent; they are then deflected by a strong electric field into an appropriate container. Occasional clumps of cells, detected by their increased light scattering, are left uncharged and are discarded into a waste container. Such machines can accurately select 1 fluorescent cell from a pool of 1000 unlabeled cells and sort several thousand cells each second (Figure 8–2).
Although molecules can be extracted from whole tissues, this is often not the most convenient or useful source of material. The complexity of intact tissues and organs is an inherent disadvantage when trying to purify particular molecules. Cells grown in culture provide a more homogeneous population of cells from which to extract material, and they are also much more convenient to work with in the laboratory. Given appropriate surroundings, most plant and animal cells can live, multiply, and even express differentiated properties in a culture dish. The cells can be watched continuously under the microscope or analyzed biochemically, and the effects of adding or removing specific molecules, such as hormones or growth factors, can be systematically explored.