The cell in culture.

It is now more than 50 years since Ross Harrison (1907) inaugurated the technique of tissue culture in vitro, and at the same time settled for ever the controversy as to the origin of nerve fibres; he explanted fragments of a tadpole's spinal cord in a drop of lymph spread on a coverslip sealed to a hollow-ground slide and observed the outgrowth of naked nerve fibres into the surrounding fluid. During the next few years the work of Carrel and Burrows enabled many other cell types to be grown in culture for long periods; since then tissue culture has rapidly developed until now it ramifies far and wide into cell biology, cancer research, physiology, biochemistry, pharmacology, virology, and many other branches of science. To-day I am going to talk about the contribution that the technique has made to our knowledge of the structure and behaviour of individual living cells, as this aspect of the subject seemed the most appropriate to the present occasion. The hanging-drop method used by Ross Harrison in his classical experiments on nerve outgrowth remains the standard technique for studying individual cells in culture. There are many different forms of hanging-drop preparation, but typically it consists of a coverslip sealed over a hollow-ground slide; the tissue fragment or explant is contained in a drop of nutritive medium, usually a mixture of plasma and embryo extract, spread on the under surface of the coverslip. When the culture is incubated at body temperature, amoeboid cells crawl out of the explant which soon becomes surrounded by a broad zone of new growth in which mitotic division is active. Every few days the tissue has to be cut out of the clot and transferred to a fresh medium or the cells degenerate. Methods have been devised to avoid this periodic disturbance of the tissue. One is Maximow's double coverslip technique to which I shall refer later. Another is some form of continuous perfusion of the cells with an oxygenated fluid nutrient; there are many types of perfusion chamber, but in our laboratory we use the Buchsbaum apparatus (Buchsbaum and Kuntz, 1954) in which cells can be observed with phase contrast under the highest magnifications and remain in good condition for many days. During the early period of tissue culture only two optical methods were available for the study of living cultures: ordinary direct illumination and the dark-ground microscope. Direct illumination was the more commonly used, and with its aid many accurate and beautiful observations were made by such pioneers of tissue culture cytology as Margaret and Warren Lewis in the United States, Giuseppe Levi in Italy, and by my old chief, Dr. T. S. P. Strangeways, in this country. Let us consider how tissue culture cells appear when viewed on the warm stage of the microscope with direct lighting. In his book published in 1924, Dr. Strangeways describes the emergence of a fibroblast from an explant of the choroid and sclerotic of a chick embryo in the following words: "A slender process of cytoplasm is seen to protrude from the edge of the explant; this process enlarges, the nucleus passes into it, and gradually the whole mass of cytoplasm detaches itself and passes into the medium. . . . The cytoplasm of the outwandering cell appears as a clear homogeneous, jelly-like substance with no obvious cell wall, although the limits can be readily discerned.... The outline is constantly changing, and larger or smaller processes are thrown out and withdrawn in amoeboid movement." He mentions rod-like mitochondria, fine granules, and fat droplets moving in the cytoplasm. There is little to be said about the nucleus of which he writes: