570 GROWTH IN TISSUE CULTURE 6 



(i) centrifugal migration of cells 



(ii) mitotic cell division 



(iii) increase in cell size 



(iv) centripetal migration 



(v) death of cells 



(vi) decrease in cell size 



The contribution of each to the "growth" of an individual culture is variable and 

 the relative contributions of each are extremely unequal. Recently Ehrmann and 

 Gey (1953) have vised area measurements for studying the growth of an established 

 strain of rat fibroblasts which grows in a monocellular layer on the glass of a roller 

 tube. Though they are here still dealing with the "balance of growth" resulting 

 from these six factors, in this case the objections raised by differences in thickness 

 are absent. 



Several studies have been made on the eflfect of initial size upon the growth 

 (area) of fibroblast cultures. Earle and Thompson (1930), using hanging drop 

 cultures, studied variations in culture area with size of explant. With fresh explants, 

 division of a fragment I X i X i mm into 4-24 fragments made little difference to 

 the final size. Fragments divided into two halves gave greater final areas; divided 

 into 36-64 fragments, the growth of each was severely reduced; and when each 

 culture was less than i/64th of the original, only a few cells (1-40) migrated out 

 in 48 h. Passage cultures gave a larger final area than fresh explants when subdivi- 

 ded below I /36th of the colony size, but division into 64 or more pieces also in this 

 case greatly limited the outgrowth area. Earle and Thompson's conclusion was 

 that the absolute increase in the area of a culture varies more or less directly to 

 the size of the explant. More recently, Firket (1954) has analyzed the growth of 

 fresh explants and of established cultures of chick fibroblasts in relation to size of 

 explant. He showed that, whereas with fresh explants the relative growth (in 

 terms of superficial area, S) log S^^/S^ was well correlated with initial area, 

 for cultures established for several passages in vitro, there appeared to be complete 

 independence between initial and final size. 



Other factors were taken into consideration in a study by Ephrussi and Teissier 

 (1932-33). According to these findings, the final sizes of colonies of fibroblasts, 

 grown in Carrel flasks in media without embryonic extract, are proportional to the 

 initial size, and small colonies stop growing sooner than large. By contrast, with 

 colonies in media containing embryonic extract, those of smaller size catch up 

 the larger during the first day in vitro, and the final sizes are nearly independent 

 of the initial size. The final size was found to be greater, the higher the concen- 

 tration of embryonic extract in the medium. Fischer (1946), using ample em- 

 bryonic extract in the medium, found that the growth of small fragments ceased 

 earlier than that of large fragments. However, Hull and Kirk (1950b) recalculated 

 Fischer's data on the basis of increase in area per unit initial area, and failed to 

 agree that the final area was proportional to the surface area at the beginning. 

 Migration being so important a contributor to final area, it is pertinent to recall 

 that Willmer (1927) found the amount of migration of cells from the eleven day 



