Experiments performed on cultured cells are sometimes said to be carried out in vitro (literally, “in glass”) to contrast them with experiments using intact organisms, which are said to be carried out in vivo (literally, “in the living organism”).
Figure 8–1 Microscopic life. A sample of “diverse animalcules” seen by van Leeuwenhoek using his simple microscope.
(A) Bacteria seen in material he excavated from between his teeth. Those in fig. B he described as “swimming first forward and then backwards” (1692). (B) The eukaryotic green alga Volvox (1700). (Courtesy of the John Innes Foundation.)
These terms can be confusing, however, because they are often used in a very different sense by biochemists. In the biochemistry lab, in vitro refers to reactions carried out in a test tube in the absence of living cells, whereas in vivo refers to any reaction taking place inside a living cell, even if that cell is growing in culture.
Tissue culture began in 1907 with an experiment designed to settle a controversy in neurobiology. The hypothesis under examination was known as the neuronal doctrine, which states that each nerve fiber is the outgrowth of a single nerve cell and not the product of the fusion of many cells. To test this contention, small pieces of spinal cord were placed on clotted tissue fluid in a warm, moist chamber and observed at regular intervals under the microscope. After a day or so, individual nerve cells could be seen extending long, thin filaments (axons) into the clot. Thus, the neuronal doctrine received strong support, and the foundation was laid for the cell-culture revolution.
These original experiments on nerve fibers used cultures of small tissue fragments called explants. Today, cultures are more commonly made from suspensions of cells dissociated from tissues. Unlike bacteria, most tissue cells are not adapted to living suspended in fluid and require a solid surface on which to grow and divide. For cell cultures, this support is usually provided by the surface of a plastic culture dish. Cells vary in their requirements, however, and many do not proliferate or differentiate unless the culture dish is coated with materials that cells like to adhere to, such as polylysine or extracellular matrix components.
Cultures prepared directly from the tissues of an organism are called primary cultures. These can be made with or without an initial fractionation step to separate different cell types. In most cases, cells in primary cultures can be removed from the culture dish and recultured repeatedly in so-called secondary cultures; in this way, they can be repeatedly subcultured (passaged) for weeks or months. Such cells often display many of the differentiated properties appropriate to their
Figure 8–2 A fluorescence-activated cell sorter. A cell passing through the laser beam is monitored for fluorescence. Droplets containing single cells are given a negative or positive charge, depending on whether the cell is fluorescent or not. The droplets are then deflected by an electric field into collection tubes according to their charge. Note that the cell concentration must be adjusted so that most droplets contain no cells and flow to a waste container together with any cell clumps.
Figure 8–3 Light micrographs of cells in culture. (A) Mouse fibroblasts.