TISSUE CULTURE 



342 



TISSUE CULTURE 



3. Consequently, with some cell types 

 at least the fibrillar structure of the 

 clot probably facilitates the final 

 stages of cell division, since final 

 separation of the cells is accomplished 

 by the daughter cells migrating in 

 opposite directions. 4. The freshly 

 prepared fibrin clot contains some 

 serum and possibly other factors which 

 enhance the growth of many cell types. 



Chicken plasma is most often used 

 for the preparation of the clot because 

 it is less likely to clot spontaneously 

 and since a gel of good consistency can 

 be more routinely prepared from it; 

 but plasma homologous with the cells 

 is frequently employed, as is also 

 plasma from other animals. Premature 

 clotting of the plasma is usually pre- 

 vented by addition of a small amount 

 of purified heparin. 



Although the plasma or fibrin clot is 

 widely used, it is in many ways un- 

 satisfactory. 1. With certain combina- 

 tions of cells and media the clot rapidly 

 dissolves and ruins the culture. 2. 

 In the case of very slow-growing cul- 

 tures the clot may gradually become so 

 opaque as to make optical examina- 

 tion impossible. 3. The chemically un- 

 defined nature of the plasma or fibrin 

 clot and the variability of its physical 

 structure interfere with many types of 

 work, such as studies in cell nutrition. 



4. Since the cells are embedded in the 

 clot and cannot be separated from it, 

 differential staining, weighing of the 

 cells, or chemical analyses of the cells 

 alone in the absence of the clot, are 

 difficult or impossible. 



Perforated cellophane (Evans, V. J., 

 and Earle, W. R., J. Nat. Cancer Inst., 

 1947, 8, 103) offers a substrate superior 

 to plasma for many kinds of work. In 

 the cellophane substrate culture the 

 cell clump is placed on the floor of the 

 culture container, is inamersed in a 

 nutrient fluid medium, and is covered 

 with a perforated cellophane sheet which 

 holds it in position against the glass 

 floor of the flask. This type substrate 

 is cheap, easily handled, may be steri- 

 lized by autoclaving, is relatively inert, 

 and is chemically defined (regenerated 

 cellulose, Earle, 'W. R., Evans, V. J., 

 Edward, M. F., and Duchesne, E., J. 

 Nat. Cancer Inst., 1949, 10, 291). 

 Experience to date indicates that when 

 grown on this substrate in an adequate 

 nutrient culture fluid, many cell types, 

 both normal and malignant, continue 

 to proliferate with far greater luxuri- 

 ance than when embedded in the 

 plasma clot. Loose cells from the 

 implant float loose, reattach to the 

 cellophane or the glass floor of the cul- 



ture vessel, and rapidly extend the 

 area covered by the cells (Earle, W. R., 

 Evans, V. J. and Schilling, E. L., J. 

 Nat. Cancer Inst., 1950, 10, 943). The 

 luxuriance of growth is such that cul- 

 ture flasks of 60 square cm. floor area 

 may often be used for established stock 

 cultures. Frequently this area is cov- 

 ered bj^ cells so rapidly that trans- 

 planting is necessary in as short time 

 as 5-10 days. Transplanting of cello- 

 phane substrate cultures is most easily 

 accomplished by scraping or shaking 

 the culture. Many cells come loose 

 from the substrate and are trans- 

 ferred as a cell suspension to a new flask. 

 Once a luxuriant cell growth is ob- 

 tained on cellophane and a heavy 

 cell suspension is prepared from it, the 

 cellophane sheet may in instances be 

 discarded and the cells may be allowed 

 to settle on the glass floor of the flask 

 on which they attach and proliferate 

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

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

 H. K., J. Nat. Cancer Inst., in press; 

 Shannon, J. E., Jr. and Earle, W. R., 

 J. Nat. Cancer Inst., in press). 



When survival or growth is desired 

 for longer than a few hours, nutrients 

 must be included in the culture medium. 

 In successful instances of cell prolif- 

 eration over an extended period of time 

 the nutrient materials used have been 

 a tissue extract, most often from em- 

 bryonic tissue, a serum, or usually a 

 combination of the two. In many in- 

 stances, particularly in the older litera- 

 ture, where a tissue extract alone has 

 been found satisfactory as a nutrient, 

 serum was actually also present in sub- 

 stantial amounts owing to use of a 

 plasma substrate for the culture. 



The tissue extract now commonly 

 employed for routine tissue cultures of 

 cells from many species is made by 

 briefly extracting minced embryonic 

 tissue with an equal volume of balanced 

 salt solution, and by decanting the 

 supernatant fluid after centrifuging. 

 This concentration is known as 1:1 

 embryo extract. An attempt is gener- 

 ally made to eliminate living tissue 

 cells from the solution by freezing in 

 CO2 snow and recentrifuging. The 

 extract loses potency rapidly and 

 should therefore be used within a few 

 days after preparation, but if lyophil- 

 ized it can be kept for much longer 

 periods (Hetherington, D. C, and 

 Craig, J. S., Proc. Soc. Exp. Biol, and 

 Med., 1939, 42, 831). 



One current source of embryo extract 

 is chick embryos of 9-11 days incuba- 

 tion. Where facilities of a local slaugh- 

 ter house are available some workers 



