TISSUE CULTURE 



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TISSUE CULTURE 



media and other conditions prevailing 

 in culture, and of the nutrients and 

 other materials influencing the cells 

 in vitro are all too inadequate. Con- 

 sequently any extrapolation of cell or 

 tissue behavior from in vitro to normal 

 in vivo conditions must be made with 

 great reserve. Great caution should 

 be observed in undertaking any tissue 

 culture study the significance of which 

 is dependent on in vitro conditions 

 being identical or closely comparable 

 with those in vivo. 



2. Types of Cultures. In cover slip 

 preparations the tissue clump is planted 

 in a drop of plasma and nutrient cul- 

 ture 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 

 the large coverslip until contact of 

 the coverslip with the vaseline ring on 

 the slide seals the preparation: For 

 coverslips of the size cited a rectangular 

 hollow ground slide 55 x 80 mm. by 6 

 mim. thick and with a polished concav- 

 ity 40 mm. in diameter and about 4.5 

 mm. deep at its deepest point is excel- 

 lent. (The more usual Maximow slide is 

 75 X 45 X 8 mm., with a cavity 36 mm. 

 in diameter, and requires a 40 x 40 mm 

 coverslip.) 



Coverslip preparations can be given 

 a final outer edge-seal of paraffin. By 

 using very thin coverslips, and if 

 necessary, by even omitting the small 

 inner slip, the cells can be critically 

 studied with high numerical aperture 

 lenses. This type of preparation is 

 probably the best for routine work with 

 short working 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 

 preparation is only 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 balanced saline; fresh nutrient 

 fluid is added and the whole resealed 

 onto a new outer coverslip and hollow 

 ground slide. By this partial renewal 

 of the culture medium every 2 or 3 

 days the culture may be carried for ex- 

 tended periods. Pogogeff" and Murray 

 (Anat. Rec, 1946, 95, 321) report carry- 

 ing such cultures of muscle cells for 

 more than a year and a half. When the 

 cell clump gets too large a small frag- 



ment of it may be re-explanted to a new 

 culture. 



Instead of using a plasma substrate 

 for the culture, the cell clump may be 

 placed on the coverslip, a few drops of 

 nutrient fluid added, and a disc of 

 perforated cellophane dropped on the 

 cell clump (Schilling, E. L., Earle, 

 W. R. and Evans, V. J., J. Nat. Cancer 

 Inst., 1950, 10, 883). Or the cell clump 

 may be placed on an inner coverslip 

 in nutrient fluid, and covered with a 

 disc of perforated cellophane. Trans- 

 fer of the culture to a fresh slide is 

 similar to transfer of the usual double 

 coverslip culture preparation. 



Coverslip cultures may be killed and 

 fixed and stained in toto. For even 

 more exacting 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 ex- 

 tremely thin sheets. These are suit- 

 able for critical microscopic study of 

 chromosomes, mitochondria, Golgi ap- 

 paratus and other cellular components. 

 If grown on thin plastic sheets they 

 can even be fixed and examined with 

 the electron microscope. 



Coverslip cultures for short periods 

 of time are recommended in beginning 

 tissue culture work, but when it is 

 necessary to carry them through con- 

 secutive changes of media, sterility is 

 difficult to maintain. When dangerous 

 infectious agents are employed, cover- 

 slip preparations should be handled 

 with great care to avoid hazard to the 

 operator, as they frequently develop 

 leaky seals and because the thin cover- 

 slips are easily broken. Accurate con- 

 trol of culture conditions over long 

 periods of time is more difficult in 

 coverslip preparations than in tube or 

 Carrel flask cultures. 



For preparations of high optical per- 

 fection, such as are necessary in high 

 resolution microcinematography, the 

 culture is often prepared on a large 

 coverslip as described, and over this a 

 thin glass or metal slide, having a hole 

 through it the diameter of the usual 

 hollow-slide concavity, is placed and 

 sealed. The open top of the prepara- 

 tion is then sealed by means of another 

 coverslip. In this type of mount the 

 nutrient fluid of the culture can form 

 a continuous film joining a small 

 central area of the upper and lower 

 coverslips. While the optical perfec- 

 tion of such a preparation is high, the 

 cell clump used must be small and its 

 life is short due to limited volume of 



