556 GROWTH IN TISSUE CULTURE 6 



there is some evidence that peptides, and a suggestion that whole protein mole- 

 cules (Winnick, 1952) may be assimilated by the cells. In brief, the requirements 

 for growth are probably relatively simple, and consist chiefly of ( i ) substances which 

 act as energy sources; (2) those which act as building blocks for the synthesis of new 

 protoplasm (and some substances, e.g. glucose and the amino acids may fall into 

 both categories) ; and (3) components or activators of varous biocatalysts, e.g. the 

 vitamins and some ions (see Chapter i). 



II. PHYSICAL FACTORS TEMPERATURE, IRRADIATION, pH, TONICITY, 



PHYSICAL NATURE OF SUBSTRATE 



{a) Effects of temperature on growth in tissue culture 



It is generally assumed that the optimum temperature for survival and growth 

 of cells from a warm-blooded animal is the body-temperature of the donor animal. 

 Quite early in the history of the tissue culture technique (Lambert, 191 2), the 

 tolerance of tissue from the chick embryo heart to a wide range of temperatures 

 was established. At 44 °C diffuse outgrowth occurred for several days, though less 

 than at 38 °C: at 46 °C no growth took place, though subjection of the cells to 

 46 °C for 45 min. did not prevent subsequent growth at 38". Very severe inactiva- 

 tion took place at 50°, and complete suppression of all activity at 55°. These 

 results were corroborated and extended by Pincus and Fischer (1931), who found 

 that the maximum duration of exposure to a supranormal temperature consistent 

 with survival of chick osteoblasts fell with increasing temperature; e.g., even a 

 prolonged sojourn at 42° or 44° was not lethal to these cells, but they were killed 

 by 1 05 min. at 47°, 6 min. at 50° and 3.5 min. at 52°. Lambert ( 1 9 1 2) also studied the 

 sub- phvsiological range of temperatures. Chick heart fragments in hanging-drop 

 beat more regularly and for longer at 29° than at 38°; outgrowth was slower at 

 2i°-27° than at 38° and not so extensive, though it continued for a longer time. 

 A culture kept at 26° for 3 days and then cooled to — 1° for I2h. resumed beating 

 45 min. after return to 26°. Lambert's cultures survived — 4° for 48 h., — 10° for 

 10 min. and — 18° for 2 min., though 15 min. at — 18° was lethal. In a further study, 

 Lambert (191 3) stored fragments of chick or rat embryonic tissue, embedded in a 

 plasma clot in hanging drop preparations, at various temperatures ( — 7° to +21°) 

 for different periods of time (2-6 days). The cultures were then returned to 37° or 

 37.5° and the effects upon subsequent outgrowth of cells observed. The freezing 

 point of chicken plasma is — 7°, and those of the chick cell cultures kept in the 

 range — 7° to — 6° which froze did not recover, though unfrozen cultures showed 

 occasional outgrowth up to 8 days. Viable cells were found after maintenance 

 at — 1° to 0° for 10 days; at +6° to +7° for 16 to 18 days; at +12° to + 14° for 

 12 days; and at + 18° to +21° for 1 1 days. Rat tissues were more sensitive to freez- 

 ing than those of the chick. Lake (191 6, 191 7) made a study similar to Lambert's, 

 with similar results. Later studies on storage of cells at very low temperatures 

 have shown the importance for viability of (i) the nature of the suspension medium 

 (Scherer and Hoogasian, 1954; Taylor and Gerstner, 1955; Parshley and Deter- 

 ling, 1956) and (ii) the rates of freezing and thawing (Smith, 1952; Smith and 



