548 GROWTH IN TISSUE CULTURE 6 



motion, endocrine secretion became evident only later, when methods became 

 available which were sufficiently refined to make quantitative studies in some of 

 these fields possible. So, while interest in proliferative growth has produced a 

 large literature, the reader is invited to keep in mind that proliferation is only one 

 facet of the concept of "growth" in tissue cultures. Survival without overall 

 increase in mass, in volume or in the number of the cell population (zero growth), 

 or even decrease in population size (negative growth) may occur in cultures 

 maintained over long periods of time. Their value as experimental material is 

 not necessarily lessened because they are not increasing in size. 



Tissue cultures have been made in many ways since the first simple beginning 

 in a study of a fragment of frog nervous tissue in clotted lymph(Harrison, 1906-07; 

 1906-08; 1908). The embryonic chick was found to be a most convenient source 

 of tissues, having the advantages that cells can be readily obtained aseptically 

 from the egg, and that much activity was quickly evident. The use of clotted 

 chicken blood plasma as a nutrient and support for the cells was introduced by 

 Burrows (191 1); and Carrel (1913) produced a nutrient mvich superior for proli- 

 feration and active migration of chick embryonic fibroblasts by adding to the 

 plasma an extract of chicken embryonic tissues. 



Many variants of the now classical plasma-embryonic extract mixture have 

 since been introduced, e.g. the use of heterologous plasma, serum and tissue 

 extracts; of extracts from embryos of different ages, from adult tissues and from 

 different organs and cells ; of ascitic fluid ; of ultrafiltrates and other fractions from 

 both plasma and tissue extracts {cf. review by Stewart and Kirk, 1954). In addi- 

 tion, a variety of artificial media, some fully defined in chemical terms {e.g. White, 

 1946, 1949; Morgan, Morton and Parker, 1950; Healy, Fisher and Parker, 1954b, 

 1955; Trowell, 1955; Evans, Bryant, McQiiilkin, Fioramonti, Sanford, Westfall 

 and Earle, 1956; Waymouth, 1955), some serum-free [e.g. Baker and Ebeling, 

 1939; Waymouth, 1956b), and some still supplemented with serum or other 

 protein-containing fluids [e.g. Wilson, Jackson and Brues, 1942; Fischer, 1948; 

 Eagle, 1955a, b), have been contrived. The use of a plasma clot, long thought es- 

 sential for the success of cultures of both organized and unorganized tissues, has 

 recently become less common. Fluid nutrient media can now be used even for 

 organized cultures, when the cultures are supported upon paper (Chen, 1954; 

 Trowell, 1954), agar (Wilde, 1948; Trowell, 1952) or other inert material. Plasma 

 clots still have an important place in tissue culture technique, but the possibility 

 of growing many types of culture without them broadens the scope of possible 

 experiments which can be made with the cells. For example, separation of the cells 

 from the nutrient is difficult when the cells are embedded in a coagulum. Cells 

 grown in a fluid medium can readily be separated from the nutrient and vised for 

 quantitative studies. The large scale cultivations of cells as hosts for viruses are 

 now almost exclusively made in fluid culture media. 



Tissue cultures can be made in a number of ways, and special techniques select- 

 ed for different types of experiment. Very simple preparations made by placing 

 a fragment of tissue in a drop of plasma on a coverslip sealed over a depression 

 slide constitute the classical "hanging drop" cultures (Carrel, 1929). Variation 

 and elaborations on this method include the Maximow double coverslip technique 



