TISSUE CULTURE 



246 



TISSUE CULTURE 



bryos removed from the uteri by aseptic 

 methods. Whatever the source of tis- 

 sue, the extract must be prepared with 

 rigid asepsis because no means of steri- 

 lization has proved satisfactory. Fil- 

 tration through a bacteriological candle 

 results in great reduction in potency. 



Extracts of malignant tissues are very 

 effective in stimulating growth of some 

 types of cells. Earle, W. R., Arch. f. 

 exp. Zellforsch., 1937, 20, 140 for ex- 

 ample, using horse serum and extracts 

 of Walker 256 rat mammary carcinoma, 

 in certain instances obtained media 

 which had no stimulative action on rat 

 subcutaneous fibroblasts and on the epi- 

 thelium of normal rat mammary, but 

 which had a very great stimulative 

 effect on the growth of cells of car- 

 cinoma from the rat mammary epi- 

 thelium. 



In exploring the possibilities of grow- 

 ing any cell type various percentage 

 combinations of embryonic extract and 

 serum are among the first media to be 

 tried; but, in attempting to make syn- 

 thetic culture media for particular kinds 

 of cells, tissue and protein hydrolysates, 

 amino acids, vitamin and hormone 

 preparations and nucleic acids, have 

 been used (Fischer and Parker). 



Because of limited knowledge of the 

 nutritional requirements of cells it is 

 not possible to obtain survival or 

 growth for any considerable period. 

 As knowledge increases we can reason- 

 ably expect greater facility in the 

 growth of many kinds of cells, and an 

 increased ease in obtaining an optimal 

 selective growth of any one cell type in 

 a mixed tissue with the suppression of 

 other unwanted types. 



Slide Cultures. The tissue clump is 

 planted in a drop of plasma and nutrient 

 culture medium on a round coverslip 

 of 24 mm. diameter. This coverslip is 

 laid, culture side up on a coverslip 48 

 mm. square, and is attached to the 

 larger coverslip through capillarity by 

 allowing a small drop of culture medium 

 to run between them. 



A hollow ground slide, charged with 

 a vaseline ring, is then lowered onto the 

 large coverslip until contact of the 

 coverslip with the vaseline ring on the 

 slide seals the preparation: For cover- 

 slips of the size cited a rectangular hol- 

 low ground slide 55 x 80 nun. by 6 nmi. 

 thick and with a polished concavity 

 40 nxm. in diameter and about 4.5 mm. 

 deep at its deepest point is convenient 

 (these Pj're.x slides may be obtained on 

 special order from Bausch and Lomb 

 Optical Company). 



Such preparation can then be given 

 an outer edge -seal of paraffin. By us- 



ing very thin coverslips, and if neces 

 sary, by even omitting the small innei 

 slip, the cells can be critically studiec 

 with high numerical aperture lenses. 

 In fact this type of preparation is prob- 

 ably the best for work with short work- 

 ing distance high resolution objectives. 



Since the total amount of culture 

 medium is only 1-3 drops, a tissue clump 

 of very limited size must be used and 

 the reasonably healthy life of the prepa- 

 ration is onlj' a few days. At the end 

 of that time however, the culture may 

 be opened, the inner coverslip with the 

 actual culture lifted out, rinsed in iso- 

 tonic saline, fresh nutrient fluid added 

 and the whole resealed onto a new outer 

 coverslip and hollow ground slide. By 

 this partial renewal of the culture 

 medium everj^ 2 or 3 days such a culture 

 may be carried for a long time. Pogo- 

 geff, I. A., and Murray, M. R., Anat. 

 Rec, 1946, 94, 321-335, report carrying 

 such cultures of muscle cells for over 

 a year. When the cell clump gets too 

 large a small fragment of it may be re- 

 explanted to a new culture. 



Such slide cultures may be killed and 

 fi.xed and stained in tolo. For even 

 more e.xacting visual or photographic 

 work the plasma may be omitted and 

 the cells grown or allowed to migrate 

 out directly on the glass coverslip. In 

 migrations under these conditions the 

 cells spread on the glass in extremely 

 thin sheets. These are suitable for 

 critical microscopic study of chromo- 

 somes, mitochondria, Golgi apparatus 

 and other cellular components. If 

 grown on thin plastic sheets they can 

 even be fixed and examined with the 

 electron microscope (Porter, K. R., 

 Anat. Rec, 1946, 94, 490). 



Slide culture technique is recom- 

 mended for beginners; but, when it is 

 necessary to carry slide cultures 

 through consecutive changes of media, 

 it is difficult to maintain sterility. 

 When dangerous infectious agents are 

 employed, slide cultures should be 

 handled with great care to avoid hazard 

 to the operator, as the preparations 

 frequently develop leaky seals, and be- 

 cause the coverslip used for very high 

 resolution microscopic study is ex- 

 tremely fragile. Accurate control of 

 conditions over long periods of time is 

 more difficult in slide culture than in 

 Carrel flask cultures containing more 

 media which can be changed with less 

 disturbance of cells embedded in the 

 fibrin clot. 



Roller Tube Cultures introduced by 

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

 1936, 27, 45. The culture vessel is a 

 round bottle, or a pyrex test tube about 



