TISSUE CULTURE 



248 



TISSUE CULTURE 



and growth from this explant will often 

 cover the floor of the flask at 28 days. 



There are several advantages over 

 the roller tube culture. The plasma 

 clot is usually thicker (though a thin 

 clot can be used) so that there is less 

 trouble from clot erosion. "Patching" 

 of the clot, with fresh plasma is rarely 

 necessary. The clot is of such thick- 

 ness and texture that it can be slipped 

 loose from the flask as a sheet, and 

 poured out onto a sterile glass plate, 

 where the culture can be easily and 

 accurately cut up for subinoculation 

 by means of a Von Graefe No. 5 cataract 

 knife. Because a single very thin strip- 

 shaped explant is placed in each flask, 

 actual sub-planting of cultures is much 

 more rapid than with the roller tube prep- 

 arations in general use and the actual 

 amount of tissue probably greater. 



If desired these cultures may be in- 

 cubated on slowly rocking shelves but 

 this is required only in more exacting 

 studies in which it is necessary to have 

 the whole surface of the culture verj' 

 uniformly washed with the fluid. Such 

 cultures can be routinely photographed 

 at magnifications in excess of 200 to 400 

 diameters and can be examined regu- 

 larly with up to a 4 mm. .65 KA. achro- 

 matic objective. A satisfactory lens 

 is the Bausch and Lomb 5.5 mm. .65 NA. 

 objective. For higher numerical aper- 

 ture photographs, subinoculation must 

 be made to slide cultures. 



Among the objections to employing 

 Carrel flasks is their initial cost. (D 

 3.5 flasks are now quoted at about $1.75 

 each). They must be made accurately 

 and can only be obtained from a few 

 manufacturers. (Satisfactory flasks of 

 3.5 and 5.0 cm. diameter can be pur- 

 chased on specification from Otto Hopf , 

 Glassblower, Upper Black Eddy, Pa. 

 and from E. Machlett and Son, 220 E. 

 23rd St., New York.) When the re- 

 search is such that a rocking shelf unit 

 for the cultures is necessary this device 

 is mechanically slightly more complex 

 than is the mechanism of the roller 

 tube unit. 



Limitations of Tissue Culture. 1. 

 All tissue cultures more of than just a 

 few hours duration must be planned so 

 as to establish and maintain the steril- 

 ity of the cultures and of the media used 

 on them. This often greatly increases 

 the difficulty of otherwise simple tech- 

 nical operations. Recent introduction 

 of antibiotics offers help to the worker 

 to obtain sterile cultures from infected 

 tissues such as skin. The new "Selas" 

 porcelain filters (Selas Corporation of 

 America, Philadelphia 34, Pa.) on pre- 



liminary study, give promise of being 

 useful in sterilization. 



2. It is not possible at present to es- 

 tablish a tissue cell strain from a single 

 cell. Instances have been described in 

 which a single tissue cell grown in a 

 culture dish with other cells has pro- 

 liferated and developed into a colony, 

 but such cultures are always suspect as 

 having possibly received cells from the 

 other colonies in the dish. 



3. Culture conditions cannot be con- 

 sidered as "normal" to any cell or group 

 of cells, nor can they be made so. Any 

 extrapolation of cell or tissue behavior 

 from in vitro to normal in vivo condi- 

 tions must therefore be made with 

 reserve. 



4. While practically any tissue cell 

 can probably be kept viable in culture 

 for a limited time, our present knowl- 

 edge of the metabolic and other cultural 

 requirements of various cell types is so 

 incomplete that only a limited number 

 of cell types have as yet been kept 

 growing in culture for a year. Cells 

 from normal adult tissue are generally 

 more difficult to grow than embryonic 

 cells, particularly when the adult cells 

 are highly specialized and are under 

 complex endocrine control, as in the 

 case of normal mammary epithelium. 

 Even in the adult animal, however, the 

 relatively undifferentiated mesoderma,! 

 cells, loosely termed "fibroblasts" can 

 be readily grown from many species and 

 are well suited to experiments. In any 

 study when an easily grown cell tj'pe 

 (the "fibroblast") can be used with 

 equal value to a cell type which has not 

 been satisfactorily grown, the more 

 easily grown cell type is obviously the 

 one of choice leaving the more difficult 

 cell types until special studies can de- 

 fine and remove difficulties complicating 

 their gi'owth in culture. Incomplete 

 cells, such as adult erythrocytes, which 

 lack a nucleus, or certain nerve cells 

 which lack a centriole, of course cannot 

 be expected to proliferate in vitro. 

 Malignant cells can frequently be grown 

 for indefinite intervals in cases where 

 attempts to grow normal tissues of the 

 same sort have not yet succeeded. For 

 instance, growth of the normal mam- 

 mary gland epithelium has been possible 

 as yet for only limited periods, but cer- 

 tain mammary carcinomas have been 

 grown for a year or longer (Earle, W. R., 

 Am. J. Cancer, 1935, 24, 566). 



5. The media now used for growing 

 cultures for extended intervals of time 

 are made up of such complex sub- 

 stances as serum, chick embryo extract, 

 liver digest, etc., which canuot be de- 

 fined chemically. There s at 3 sent 



