TISSUE CULTURE 



343 



TISSUE CULTURE 



find it convenient to employ beef em- 

 bryos removed from the uteri by asep- 

 tic methods (Gey and Gey, cited 

 above). 



At present, whatever the source of 

 tissue, the extract is routinely pre- 

 pared with rigid asepsis because no 

 means of sterilization has yet proven 

 satisfactory. Filtration through a bac- 

 teriological filter results in rapid clog- 

 ging of the filter and in great reduction 

 in potency. However, it has been 

 recently reported (Bryant, Jay C, 

 Earle, "W. R. and Peppers, E. V., at 

 the 1951 Annual Meeting of the Tissue 

 Culture Association, Detroit) that by 

 treating the extract first with hyalu- 

 ronidase, and then by centrifuging at 

 approximately 40,000 G, extract from 

 9-day chicks may be rapidly filtered 

 by low pressure through a ^03 porosity, 

 4 mm. wall thickness Selas filter (Selas 

 Filter Co., Philadelphia, Penn.). The 

 resulting extract, used with serum, 

 caused rapid proliferation of mouse 

 strain L fibroblasts for the 17 day 

 interval studied. If these results are 

 confirmed for other cell types and for 

 longer intervals of culture, this method 

 of preparation of embryo extract should 

 substantially facilitate tissue culture 

 work. 



Extracts of malignant tissues have 

 often been extremely effective in 

 stimulating growth of some types of 

 cells. A balanced saline extract of 

 Walker 256 rat mammary carcinoma, 

 used with horse serum, has caused 

 rapid proliferation of the Walker 

 256 carcinoma cells in culture, but 

 no stimulative action was noted on 

 either rat normal mammary epithelium 

 or subcutaneous fibroblasts (Earle, 

 W. R., Arch. f. Exp. Zellf., 1937, 20, 

 140). 



Horse serum has been routinely used 

 in this laboratory over a period of 

 years as a serum component of nutrient 

 fluid. The mixture consisted of 40% 

 horse serum, 20% 1 : 1 extract from 9-day 

 chick embryos and 40% balanced saline 

 solution. This has given luxuriant 

 long-term growth with such different 

 cells as Walker 256 rat mammary car- 

 cinoma (Earle, W. R., Arch, of Path., 

 1939, 27, 80), fibroblasts from rats, 

 mice, and humans, and a number of 

 mouse fibrosarcoma strains (Earle, 

 W. R., J. Nat. Cancer Inst., 1943, 4, 

 165). Not all cells do well in this me- 

 dium. A high concentration of chicken 

 serum with low embryo extract gave 

 superior growth of chicken monocvtes. 

 Parker, R. C. (J. Exp. Med., 1932, 

 55, 713; 1933, 58, 97; 1933, 58, 401) has 

 found that various strains of fibroblasts 



required very different concentrations 

 of embryo extract and serum to attain 

 their optimal growth. 



Where it can be used horse serum 

 offers certain technical advantages. 

 Eight to 10 liters of horse blood can 

 be obtained from one bleeding without 

 injury to the horse, and after clotting 

 and separation of the blood cells by 

 centrifugation the serum may be 

 sterilized by pressure filtration through 

 a #03 Selas filter at 5 p.s.i., and stored 

 under refrigeration for a year or longer. 

 Before filtration extreme care should be 

 taken to prevent any fungus or bac- 

 terial growth in the serum; to.xic sul)- 

 stances produced can pass the filter 

 and injure or kill the culture. 



Numerous other types of sera have 

 been used in tissue culture. Beef 

 serum would probably be as satisfac- 

 tory as horse. If local facilities make 

 it available sheep serum could be 

 tried. In hospital centers human cord 

 serum has been available and has been 

 found extremely satisfactory bj^ many 

 workers (Gey, G. O. and M. K. cited 

 above). Dr. Margaret Murray finds 

 that for human tumor material it is 

 superior to horse serum (personal 

 communication) . 



In exploring the possibilities of grow- 

 ing any cell type, various percentage 

 combinations of embryo extract and 

 serum are among the first media to be 

 tried, after which these combinations 

 may be supplemented by addition of 

 other physiologically active substances. 



There has been a great deal of work 

 toward preparing a chemically defined 

 or synthetic culture medium for cells 

 growing in culture (Vogelaar, J. P. 

 M., and Erlichman, E., Am. J. Cancer, 

 1933, 18, 28; Baker, L. E., Science, 

 1936, 83, 605; Fischer, A., Astrup, T., 

 Ehrensvard, G. and Oehlenschlager, 

 v., Proc. Soc. Exp. Biol. & Med., 1948, 

 67, 40; White, P. R., Growth, 1946, 

 10, 231; Davidson, J. N., Leslie, I. 

 and Waymouth, C, Biochem. J., 1949, 

 44, 5; Morgan, J. F., Morton, H. J. 

 and Parker, R. C, Proc. Soc. Exp. 

 Biol. & Med., 1950, 73, 1). Substan- 

 tial progress has been and is being 

 made in defining the nutritional re- 

 quirements of cells in culture, but at the 

 present time no chemically defined 

 medium now available appears to be 

 satisfactory for the continued pro- 

 liferation of any type of tissue cell 

 in vitro. This study of cell nutrition 

 of both the normal and the malignant 

 cell will undoubtedly continue as one of 

 the most interesting and active fields 

 of tissue culture research. 



At present our knowledge of the 



