CYTOGENETIC ANALYSIS 493 



Steps to insure genetic continuity 



A number of crucial reports have appeared in the literature regarding the sudden 

 transformation of a particular cell line from its usual fibroblast-like appearance to one 

 of epithelial features. The extent to which such alterations have been witnessed is 

 reviewed in part by Rothfels et a/. 1076 During the initial studies on Chinese-hamster 

 cultures, Ford and Yerganian 391 noted a shift in the appearance of the six-month-old 

 A strain toward polyploidy (70 + chromosomes) after seven months. The 70 + 

 chromosomes suggested cells of this culture to have hyperoctoploid complements, if 

 derived from Chinese-hamster cells. However, the chromosomes lacked any similarity 

 to Chinese-hamster types or their immediate alterations, as noted previously among 

 transplantable tumors. The only plausible explanation was a technical error made 

 when pipetting media and failing to use a separate pipette for each operation. This 

 measure of caution had not been fully incorporated into the routine management of 

 the stock. Direct cellular contamination by a line of human cells was substantiated 

 further by employing serologic tests, using antihamster and antihuman rabbit 

 serum. On another occasion, a culture isolated from a normal adult Chinese hamster 

 was sent to us for a report on the chromosomal situation on the first anniversary of its 

 existence in vitro. Immediately upon viewing the first few metaphases, it was obvious 

 that the entire population of cells was human in origin! Further inquiry disclosed 

 that the culture had changed recently from a fibroblast-like form to one with epithelial 

 features. This, too, was another example of direct cellular contamination. The 

 implications of such accidents are far too complicated to suggest any remedy other 

 than to discard the majority of the suspected forms and to replace them with equally 

 available, and probably more serviceable cell lines of known sex and species. Thus, 

 adequate control and routine guarding against such events in the future would entail 

 the rule that a pipette be used only once. The beginner in tissue culture should either 

 start his or her own cultures or obtain them from a reliable source that has conducted 

 chromosomal and, if possible, immunologic comparisons. Direct cellular contamin- 

 ation is far more widespread than is usually realized. 206, 1076 



Activities that help guard against accidental loss of lines of cells 



1. Freeze representative samples of parental line and variant clones. 534, 1301 



2. Test filtered media for sterility with surplus cultures and by placing 1-ml. 

 samples into sterile Bactothioglycolate while bottling media. 



3. Reduce serum toxicities, particularly for trials of plating efficiency by: (a) 

 purchasing serum from a reliable source that is aware of the problem; (b) preheat at 

 56° C. for 30 minutes prior to filtration (D. K. Ford, personal communication) ; (c) 

 combine serum from two species, such as fetal calf serum and horse serum (Yerganian, 

 unpublished data); (d) use dialyzed serum wherever applicable, and (e) substitute 

 fetuin and albumin in lieu of whole or dialyzed serum, as prescribed by Fisher, Ham, 

 and Puck. 367 The addition of NCTC 109 (4-6 per cent) and 0.5 per cent lactalbumin 



