TISSUE CULTURE 



348 



TISSUE CULTURE 



normal and malignant, and should 

 ultimately allow the growth of large 

 cultures from them. To date, however, 

 (March 1951) technical difficulties have 

 interfered with carrying the proliferat- 

 ing isolated cell of most of these other 

 types through to large cultures. 



For accurate cell descriptions which 

 are to be compared with descriptions 

 from other cultures at other times, 

 culture conditions and media should 

 be standardized as accurately as pos- 

 sible. Cultures should be well estab- 

 lished yet relatively young, and of 

 comparable size (Earle, W. R. and 

 Thompson, J. W., U. S. Pub. Health 

 Rep., 1930, 45, 2672). Chilling of the 

 cultures, inadequate frequency of 

 change of culture media, severe vibra- 

 tion, all tend to produce aberrant cell 

 shapes. Prolonged exposure to light 

 in the presence of erythrocytes can 

 injure or kill otherwise relatively in- 

 sensitive cells (Earle, W. R., J. Exp. 

 Med., 1928, 48, 457; 48, 683). The cen- 

 tral part of a dense culture is often 

 degenerating or necrotic. Cells which 

 have migrated out from an explant 

 frequently exhibit aberrant and "giant 

 cell" forms at the extreme periphery of 

 the culture. Cells located in a plasma 

 substrate at its interface with the 

 supernatant fluid often show different 

 structural features from cells at the 

 interface of the plasma with the glass 

 surface of the culture dish (Earle, 

 W. R., Schilling, E. L. and Shelton, 

 E., J. Nat. Cancer Inst., 1950, 10, 

 865 and 1067). 



These variations should be recognized 

 by the worker and every effort made to 

 standardize conditions to eliminate them 

 as complicating factors in the records. 

 Photographic records of the culture 

 and of its living cells are extremely sat- 

 isfactory as records if correctly made. 

 Phase contrast optics are often useful 

 or essential. For following changes 

 in cell activities the time-lapse cinemat- 

 ograph is extremely useful. 



4. Tissue Culture and Quantitative 

 Research. When an attempt is made 

 to prepare substantial numbers of repli- 

 cate cultures for quantitative studies 

 involving changes of cell proliferation 

 rate in culture, numerous difficulties are 

 encountered. If fresh tissue is used, 

 the amount of residual tissue material 

 brought over from the host, and the 

 variability among the explants of an 

 extensive series of cultures often raise 

 serious questions as to the significance 

 of results obtained. By using plasma 

 substrate tissue colonies of a well estab- 

 lished cell strain, and by bisecting 

 each colony, one half of each colony 



may be used as a control on the other, 

 but even this type preparation contains 

 the residual materials of the plasma 

 matrix, while the number of control 

 cultures necessary to achieve reason- 

 ably accurate conclusions is often pro- 

 hibitive. 



If the cell type can be grown satis- 

 factorily on a surface substrate (e.g. 

 cellophane or glass), and if a cell sus- 

 pension can be prepared from it, this 

 suspension may be handled by special 

 burettes and other accessory equip- 

 ment, and from the suspension large 

 numbers of replicate cultures having a 

 high degree of accuracy may be rapidly 

 planted (Evans, V. J., Earle, W. R., 

 Sanford, K. K., Shannon, J. E. Jr. and 

 Waltz, H. K., J. Nat. Cancer Inst., 

 1951, 11, 907). 



The term growth has been so loosely 

 used in the tissue culture literature, 

 and particularly in the early literature, 

 as to be confusing and often mislead- 

 ing (Essentials of Tissue Culture, 

 Parker, R. C, Cunningham, B. and 

 Kirk, P. L., J. Cell, and Comp. Physiol., 

 1942, 20, 343). Estimation of change 

 in culture area or diameter has prob- 

 ably been the most widely used and 

 most easily applied index of culture 

 "Growth". Substantial cell prolifera- 

 tion for an extended period of time can 

 be easily recognized, but in the early 

 stages of the life of the culture the 

 method may be grossly inaccurate due 

 to increase in the culture area resultant 

 from cell migration rather than from 

 cell proliferation. Even with older 

 cultures the method is accurate only 

 where cell density per uuit area is 

 relatively constant and if necrosis has 

 not supervened. 



Actual observation of cell division is 

 the ultimate evidence of cell prolifera- 

 tion. By determining the number of 

 cells which undergo mitosis in a cul- 

 ture area per unit time relative to the 

 total number of cells in that area, an 

 estimate may be made of the relative 

 frequency of cell proliferation. But 

 such observations are arduous and are 

 subject to a number of possible errors. 

 Cell proliferation rate may be very 

 different in different parts of the same 

 culture, so that the method is valid 

 only for a constant zone or for com- 

 parable areas of cultures. If the 

 enumerations are made visually on 

 living cells, the accuracy of enumera- 

 tion is often very poor while the con- 

 tinuous exposure to light may be in- 

 jurious. When fixed preparations are 

 counted the method is valid only if 

 the average interval of duration of 

 mitosis is known and if the experi- 



