560 GROWTH IN TISSUE CULTURE 6 



principal reason for the employment, especially in cultures of small volume, of 

 hypotonic media is that in such cultures some evaporation is apt to take place, 

 which, if the medium was isotonic at the start, would leave the cells in a hyper- 

 tonic environment. Hypotonicity is in general less traumatic to cells than hyper- 

 tonicity. Brues and Masters (1936) observed reversible swelling of fibroblasts in 

 media down to 0.2 and 0.3% NaCl (34-51 m.eq./l), with maximum migration 

 at 0.86% NaCl ( 1 50 m.eq./l) and irreversible damage at 2.00% (342 m.eq./l) . 



i§) Effects of the physical nature of the substrate 



For a long time the idea was firmly entrenched that all cells, but particularly 

 connective tissue fibroblasts and various kinds of epithelium, required a particular 

 type of supporting substrate. A coagulum of lymph or plasma appeared to be 

 ideal, because cells could migrate throughout the fibrous meshwork or on the 

 surface of the clot. Such a substrate does indeed effectively support the migration 

 of many types of cells, though others are quite unable to grow in such an environ- 

 ment. Strains of cells have been isolated which will actually not tolerate a plasma 

 clot, but which grow luxuriantly in fluid media (Evans, Earle, Wilson, Waltz and 

 Mackey, 1952). The use of a clot has the additional disadvantages that it is com- 

 posed of materials which the cells may metabolize, and that its composition is 

 variable and indeterminate. Many efforts have been made to find truly inert sub- 

 strates for various kinds of cells. Agar is rather better in this respect than plasma, 

 though it may carry nutrient substances adsorbed upon it, and has been used for 

 diverse types of tissue cultures by Smyth (1914-15), Wolff' and Haffen (1952), 

 Trowell (1952) and Puck, Marcus and Cieciura (1956). Hotchin (1955) has 

 successfully substituted methylcellulose for agar. Fibrous supports of several kinds 

 have been tried, e.g. spider's web (Harrison, 19 14), silk and cotton fibres, lens 

 paper (Beard and Rous, 1934; Trowell, 1954; Chen, 1954), cellulose fibres and 

 films (Pearce and Paterson, 1952; Moscona and Moscona, 1953), nylon cloth 

 (Goossens, 1953, 1954), and glass fibres (Warner, Hanawalt and Bischoff", 1949; 

 Frisch, 1952). Many, but not all, cells can spread upon a glass surface and are 

 able to proliferate there. A few cell types have been grown in fluid suspension, 

 being maintained so by methods of agitation. Among inert substrates other than 

 glass which permit cell attachment and migration is cellophane, first used by 

 Fischer (1930), and widely adopted in the form of perforated sheets since the 

 introduction of these by Evans and Earle (1947). Other plastic surfaces, e.g. 

 Formvar (Wirth and Barski, 1947) and Araldite (Barski, 1954) are satisfactory. 



All these substrates can be used in conjvmction with a fluid nutrient. This is not 

 to claim that all types of cultures can best be grown in fluid media, though it is 

 often a great advantage to be able to separate the cells from the medium, and the 

 possibilities of doing so have been widened to include many types of unorganized 

 and organized tissues, among the latter being organ rudiments (Chen, 1954), 

 whole lymph nodes (Trowell, 1952, 1954) and tumours (Leighton, 1951, 1954). 

 A three dimensional type of growth is made possible by the use of pieces of cellu- 

 lose or gelatin sponge (Leighton, 1951, 1954; Leighton and Kline, 1954; Leighton, 

 Kline, Belkin and Tetenbaum, 1956), in the interstices of which a high degree of 

 tissue organization is possible, so that the growth characteristics of certain tumours 



