II PHYSICAL FACTORS 561 



in vitro are recognizably similar to the histological sections made from the tumours 

 as they grow in vivo. 



Particularly promising substrates for growing cells appear to be the bacterial 

 cellulose membranes introduced by Moscona and Moscona (1953) and the sheets 

 of reconstituted collagen of Ehrmann and Gey (1956). The use of collagen, a nor- 

 mal substrate for many cells in vivo, enables cells to be studied on a ''natural" 

 surface. 



Under certain conditions, some cells will proliferate in suspension, unattached 

 to each other or to a solid substrate. Owens, Gey and Gey (1954) have achieved 

 rapid growth (four-fold increase in five days) of the MB III lymphoblasts with 

 the cells and medium kept in a state of constant agitation, Idv end-over-end rotation 

 of the culture tube at 38 rpm (2280 rph). Measurements have been made by 

 Earle, Schilling and Bryant (1954) of the rates of proliferation of strain L mouse cells 

 in conventional roller tubes (rotating round the long axis of the tube) at speeds 

 from less than 10 rph up to 1200 rph. Using the regular medium of horse serum 

 and embryo extract diluted with balanced salt silution, and estimating growth 

 by nuclear counts, it was found that the cell populations at the end of three 

 weeks in cultures rotated at speeds between 150 and 1200 rph were 50%-7o% 

 greater than in tubes rotated at the more usual rate of 6-9 rph. But even at the 

 higher velocities, most of the cells remained adherent to the glass of the tubes. To 

 maintain the cells in suspension, Earle, Schilling, Bryant and Evans (1954) 

 slightly increased the viscosity of the medium by adding methylcellulose. In this 

 type of suspending fluid, and at a velocity of 2400 rph, none of the strain L cells 

 remained attached to the glass. Most of them were also unattached to each other, 

 though groups of up to about 10 cells could be found. At lower rates of rotation, 

 though most of the cells were detached from the tube, they formed free floating 

 aggregates, the size of which appeared to be approximately proportional to the 

 velocity of rotation. Clumps 0.1-0.5 "^"^ were formed at 300 rph. Still more effec- 

 tive cell suspensions have been made by shaking the cultures on a rotary shaker 

 at 210 or 230 rpm in the methylcellulose-containing medium (Earle, Bryant, 

 Schilling and Evans, 1956). Under these conditions, strain L mouse fibroblasts, 

 HeLa human cervical carcinoma and clone 1469 mouse liver epithelium proliferate 

 very actively. Slower rates permit cell aggregation, impede oxygenation and give 

 poorer growth. This type of system allows efficient utilization of nutrients, e.g. 

 2 g of new cells could be produced in 4 days with the expenditure of 325 ml of 

 medium. 



III. RELATIONSHIP OF GROW^TH I X VITRO TO MAINTENANCE, 



DIFFERENTIATION AND FUNCTION 

 CONDITIONS FOR GROW^TH, SURVIVAL AND DIFFERENTIATION 



It is found empirically that certain conditions can be created, of which the prin- 

 cipal ones are adequate nutrition and appropriate temperature, in which cells can 

 proliferate rapidly over an indefinite period. In general, the more vigorous the 

 proliferative growth, the less readily will the cells in a particular population mani- 



Literaiure p. §8l 



