TISSUE^CULTURE 



341 



TISSUE CULTURE 



on the cells may be studied. The cul- 

 ture medium may be removed and 

 tested to determine what changes the 

 cells have induced in it (Brues, A. M., 

 Rathbun, E. N., Cohn, W. E., J. Cell, 

 and Comp. Physiol., 194-i, 24, 155). 

 The tissue growing in culture may be 

 used as a host for the growth and 

 study of bacteria or viruses (Fo.x, J. 

 P., Amer. J. Hygiene, 1947, 46, 1). 

 Rapidly increasing facility in the han- 

 dling of manj^ types of cells in culture 

 suggests a constantly increasing useful- 

 ness of tissue culture methods in many 

 fields of future scientific work. 



1. The Culture Medium and Sub- 

 strate. A satisfactory isotonic balanced 

 saline solution is necessary for washing 

 cultures and for dilution of plasma and 

 nutrient media. This balanced salt 

 solution can be prepared best from 

 glass distilled water free from fungus 

 or bacterial growth which might give 

 rise to toxic substances. Reagent 

 quality chemicals should be used and 

 heavy metal contaminants in partic- 

 ular should be avoided. Only minor 

 differences exist in the formulae of 

 many of the solutions in current use: 

 Mammalian Ringer (Ringer, S., J. 

 Physiol., 1895, 18, 425), Tyrode (Arch. 

 Intern, de Pharmacodyn. et de Thdr., 

 1910, 20, 205; Gey, G. O. and Gey, M. 

 K., Am. J. Cancer, 1936, 27, 45). Hanks' 

 (Hanks, J. H., J. Cell, and Comp. 

 Physiol., 1948, 31, 235), Simms' (Simms, 

 H. S., and Sanders, M., Arch. Pathol., 

 1942, 33, 619), Earle's solution (J. Nat. 

 Cancer Inst., 1943, 4, 165). Solutions 

 as amphibian Ringer are used with 

 amphibian cells. Any solution ap- 

 proximating the inorganic salt content 

 of serum, and having a comparable 

 osmotic pressure, can be employed as a 

 diluting and wash solution for routine 

 tissue cultures. For skin epithelium 

 Parshley and Simms (Am. J. Anat., 

 1950, 86, 163) have used a diluting 

 solution containing no calcium or bi- 

 carbonate, but with increased phos- 

 phate, and with aspartic acid added. 

 This, however, apparently has not been 

 tested for longer culture intervals than 

 about a week. About 0.1% glucose is 

 usually included in balanced saline 

 solutions as a source of carbohydrate. 

 For much tissue culture work the solu- 

 tion used by Earle has the advantage 

 of an alkali reserve, in the form of so- 

 dium 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 sealed containers with an 

 adequate tension of CO2 in the air over- 

 lying the fluid. Sterilization by heat, 

 or by vacuum filtration through a bac- 

 teriological filter, causes an alkaline 

 shift, due to loss of CO2, and this 

 causes secondary changes in the solu- 

 tion such as precipitation of the cal- 

 cium and magnesium salts. Probably 

 the most satisfactory procedure for 

 sterilizing such a solution is to filter by 

 pressure (2-10 p.s.i.) and to store in 

 sealed containers. Before the filtra- 

 tion CO2 should be passed through the 

 solution to bring it to a pH somewhat 

 more acid than that desired. For 

 routine culture work an initial pH of 

 about 7.6 in the culture is desirable 

 because elaboration of acid by cells in 

 the culture will carry the pH to some- 

 what more acid levels. 



While the balanced salt solution may 

 be used for rinsing cultures, and while 

 tissue fragments may be left in it for 

 short periods, these times should be 

 limited as continued action of the bal- 

 anced saline on the cells is injurious. 

 It is probably wiser to consider the 

 balanced salt solution as having its 

 major use as a diluent for other media 

 such as serum. 



For the satisfactory routine main- 

 tenance of cultures of most tissue 

 cells a solid support or substrate for 

 their growth and migration is neces- 

 sary. Various types of substrates have 

 been employed, such as silk thread, 

 spider web, glass wool, lens tissue, 

 cellophane, gelatin and agar, and for 

 simple cultures, the glass surface of 

 the culture dish. 



By far the most widely used sub- 

 strate, and the one which has proven 

 most satisfactory until recently has 

 been a thin layer of plasma or fibrin 

 clot. To prepare the culture with 

 this substrate, the clump of cells to be 

 planted (the implant) is placed in a 

 thin layer (0.1 to 1.0 mm. thick) of 

 plasma or fibrinogen solution, and this 

 is clotted into a solid gel by addition 

 of a little tissue extract or thrombin 

 solution. When of correct consistency 

 the clot is a solid, somewhat elastic, 

 optically clear gel. Its fibrillar struc- 

 ture enables the cells to migrate 

 through it, although they tend to col- 

 lect at its surfaces. 



This type of culture substrate has 

 certain advantages: 1. The fibrin ma- 

 trix anchors the cells so that as long as 

 the matrix is intact few cells wash 

 loose and are lost when the culture fluid 

 is renewed. 2. The fibrils of the sub- 

 strate present effective surfaces for 

 adhesion and migration of the cells. 



