TISSUE CULTURE 



245 



TISSUE CULTURE 



that the fibrin clot contributes other 

 chemical or physical factors favorable 

 to cell growth and migration. 



Chicken plasma is usually employed 

 for the preparation of the clot since it 

 is less likely than others to clot spon- 

 taneously and since a gel of satisfactory 

 consistency can be more routinely pre- 

 pared from it. Plasma homologous 

 with the cells is often used, however, as 

 is also plasma from other convenient 

 animals. Premature clotting of the 

 plasma can be prevented by addition of 

 a small amount of purified heparin. 



While the fibrin clot seems the best 

 available culture matrix for general 

 work, it is far from perfect. For in- 

 stance, with certain combinations of 

 cells and media the solid matrix may 

 completely dissolve in the area of the 

 cells and ruin the culture ; or, in the case 

 of very slow-growing cultures, the clot 

 may gradually become so opaque as to 

 handicap optical examination. In 

 studies of cell nutrition the chemically 

 undefined nature of the plasma clot and 

 the difficulties of making a routine 

 quantitative separation of cells and clot 

 interfere with chemical analysis. 



Isotonic Saline. A satisfactory iso- 

 tonic saline solution is necessary for 

 washing cultures and for suitable dilu- 

 tion of plasma and nutrient media. 

 Often only minor differences exist in 

 their formulae. Mammalian Ringer, 

 Drew, Locke, Tyrode, or Earle's Solu- 

 tion are all satisfactory for mammalian 

 cells, while such solutions as amphibian 

 Ringer are advised for amphibian cells. 

 Any solution simulating roughly the in- 

 organic salt content of serum, and hav- 

 ing a comparable osmotic pressure can 

 be used for routine tissue cultures. 

 About 1% of glucose is usually included 

 as a source of carbohydrate. For much 

 tissue culture work the solution used 

 by Earle, W. B., J. Nat. Cancer Inst., 

 1943, 4, 165 has the advantage of an 

 alkali reserve, in the form of sodium 

 bicarbonate comparable to that of 

 serum. 



All physiological solutions, such as 

 serum, depending chiefly on sodium 

 bicarbonate for their alkali reserve, can 

 be maintained at a stable pH within 

 workable physiological limits only when 

 kept in a sealed container with an ade- 

 quate tension of CO2 in the air over- 

 lying the fluid. The heat of steriliza- 

 tion changes the bicarbonate to car- 

 bonate, the pH rises to a value in excess 

 of 8.0 and this frequently causes sec- 

 ondary changes like precipitation of the 

 calcium or magnesium. Even steri- 

 lization by filtration through a bac- 

 teriological filter under vacuum can 



cause such an alkaline shift, and there 

 is a slight shift even when filtered under 

 pressure. Probably the most satisfac- 

 tory procedure for sterilizing such a 

 solution is to bring it to a pH somewhat 

 acid to that desired (to compensate for 

 loss of CO2 during handling), to filter 

 by pressure and to store in sealed con- 

 tainers. For routine culture work an 

 initial pH of about 7.8 in the culture is 

 desirable because elaboration of acid 

 by the cells of the culture will carry the 

 pH to somewhat more acid levels. 

 For further control of the pH of the 

 media see Parker, R. C, Methods of 

 Tissue Culture. New York: Hoeber, 

 1938. 



Nutrient Media. When survival or 

 growth is desired for longer than a very 

 limited time adequate nutrient factors 

 (embryonic extracts and sera) must be 

 incorporated into the culture medium. 

 Optimum proportions of extract and 

 serum must be determined for different 

 types of cells. 20% embryo extract, 

 40% horse serum and 40% saline solu- 

 tion is satisfactory for growing mouse, 

 rat and human fibroblasts, rat mam- 

 mary carcinoma and mouse sarcomas, 

 but Carrel and Ebeling obtained their 

 best results with chicken macrophages 

 by using a medium composed chiefly of 

 serum. 



Numerous sera have been mixed with 

 tissue culture media. In some hospital 

 centers human cord serum has been 

 available and has proved satisfactory 

 (Gey, G. O. and M. K., Am. J. Cancer, 

 1936, 27, 45). Horse serum has been 

 found extremely satisfactory in my 

 laboratory for it can be obtained in 

 large amounts with little trouble from 

 hemolj^sis, and it is relatively stable 

 over periods of at least a year. Such 

 sera can be sterilized by pressure filtra- 

 tions but before sterilization by filtra- 

 tion care should be taken to prevent 

 bacterial growth and resultant produc- 

 tion of toxic substance in the serum. 



The tissue extract now , commonly 

 used for routine tissue cultures of cells 

 from many species is made by extract- 

 ing briefly minced embryonic tissue 

 with an equal volume of isotonic saline 

 and by decanting the supernatant solu- 

 tion after centrifuging. To eliminate 

 living tissue cells freeze in CO2 snow 

 and recentrifuge. The extract loses 

 potency rapidly and should therefore be 

 used within a few days after prepara- 

 tion. 



A current source of embryo extract is 

 chick embryos of 9 days incubation. 

 Where facilities of a local slaughter 

 house are available some workers (Gey), 

 find it convenient to employ beef em- 



