552 GROWTH IN TISSUE CULTURE 5 



were above the size which others have shown to be hmiting for proHferation and 

 moreover most of the expansion which he observed could have been due to mi- 

 gration rather than proHferation. 



Earle, Sanford, Evans, Waltz and Shannon (1951) made a study of capacity 

 for proliferation and survival in relation to inoculum size (using strain L mouse 

 connective tissue cells), and to volume of nutrient (using a medium containing 

 chick embryonic extract, horse serum and a balanced salt solution). These cells 

 are capable of rapid and continuous proliferation in this medium, if large enough 

 inocula are used. A minimum of 100,000-500,000 cells/2 ml of this nutrient 

 was found to be necessary to ensure 100% survival of the cultures; with inocula 

 of 10,000-50,000 cells, only 64% of the cultures proliferated, and with much smaller 

 inocula (600 cells), no cultures survived. Hollomon and Fisher (1950) have con- 

 sidered the limiting factors in such systems in terms of the geometry of the con- 

 tainer, critical colony size and the concentration of a hypothetical substance pro- 

 duced by the cells themselves. 



The first successful production of clones from single, isolated cells was achieved 

 by Sanford, Earle and Likely in 1948. They based their method on the old hypo- 

 thesis that heavily populated cultures can modify or "condition" the medium, to 

 make it adequate for growth. The same strain of mouse cells (strain L), which had 

 been cultivated in liquid medium under perforated cellophane, was selected for 

 the isolations. Cells from this strain were taken up in capillary tubes, 100 y. 

 internal diameter, containing a film of plasma. Plasma and embryonic extract 

 were drawn in, the capillary tubes sealed at both ends and incubated at 37.5 °C for 

 15-20 h. Portions of capillary containing a single cell near the middle of a 4-5 mm 

 length were selected, the ends cut open, and each tube placed in a D3.5 Carrel 

 flask containing 0.5 ml plasma and a total of 1.5 ml of a "conditioned" medium 

 (fortified with 100 mg/ioo ml glucose) from an actively growing culture. The 

 proportion of cells which survived and grew into colonies was only about 4%. This, 

 however, was sufficient to enable several lines of cells, which have become im- 

 portant laboratory tools, to become established. For example, progeny from a 

 single mouse subcutaneous tissue cell have given rise to several cell lines with 

 different capacities to induce sarcomas on injection into animals of the parent 

 strain (C3H) (Sanford, Likely and Earle, 1954). 



Another study of growth in relation to number of cells and amount of medium 

 was made by Harris (1955), who used freshly isolated rat fibroblasts brought into 

 suspension by controlled treatment with trypsin. For his experiments he used a 

 culture chamber enclosing a space for medium between two coverslips. The cells 

 grew on one coverslip, and serial counts were made in selected, marked areas, to 

 determine changes in cell number. Over short periods of time, he found that growth 

 rates were independent of inoculum size (above a minimum size), though the total 

 amount of growth was proportional to inoculum size. Inocula of 4500-5000 cells 

 in 0.43 ml medium, corresponding to 16-18 cells/mm2, evenly distributed on the 

 coverslip, usually failed to survive. Use of medium (diluted serum) taken from 

 cultures which had been multiplying for some hours did not permit growth of single 

 cells or of small inocula. The concept of "adaptation" is therefore regarded by 

 Harris as, if not untenable, at least an incomplete explanation of the facts. It is 



