34 



Methods and Techniques 



tain a sublethal state of cytolysis, as a result 

 of reaction to the medium, which may exert 

 hitherto unsuspected effects on other cells 

 nearby in the cultures. 



Even in cases, however, where such effects 

 may be discounted in the interpretation of 

 results, many other difficulties arise in at- 

 tempts to prepare a suitable medium. Cells 

 can never be independent of mechanical 

 factors in their environment, and indeed are 

 notoriously susceptible to their influence, 

 as was so clearly demonstrated by Harrison 

 ('14). In a liquid environment they take 

 quite different shape than when they have 

 access to a solid substrate, and the physical 

 framework of the matrix in which they de- 

 velop is of paramoimt importance in deter- 

 mining their form (cf. Weiss, '49). Some- 

 times hidden mechanical influences quite 

 prejudice the interpretation of investigations 

 designed to analyze the effects of quite dif- 

 ferent factors; for an example, the reader is 

 referred to Weiss' ('50) critique of Marsh 

 and Beams' ('46) experiments where develop- 

 ing nerve cells were subjected in vitro to 

 apparent modification by the passage of elec- 

 trical currents. Indeed the demonstration of 

 the degree to which cells are susceptible to 

 influences of external mechanical factors has 

 been one of the most fruitful contributions 

 of the isolation method. 



From the point of view of nutritive and 

 chemical effects of the milieu on isolates, the 

 analysis is more highly complex. If a part 

 is isolated in inorganic media of easily re- 

 producible composition, many components of 

 the normal environment are lacking which 

 may be essential to foster the processes of 

 normal differentiation; and even such simple 

 factors as a change in pH (though in view of 

 the widely divergent systems in the cell 

 which this might affect its simplicity is only 

 apparent) can alter the accomplishment of 

 cells in such media (Holtfreter, '45). If the 

 isolate is explanted to parts of another or- 

 ganism, as in implants to the eye cavity, the 

 anterior chamber of the eye, the chorio- 

 allantois, or to the varioTis sites employed for 

 the window techniques, or even to culture 

 fluids containing embryonic extract, plasma 

 or other body fluids, it is impossible in our 

 present state of knowledge to ascertain what 

 components are present. While to some the 

 ultimate aim may seem the perfection of 

 synthetic media — and the embryologists 

 proper lag far behind the tissue culture ex- 

 perts and the microbiologists in their progress 

 towards this goal — it is a little soon to divine 

 what all the ingredients of such media might 



be, since embryologists have hardly yet ex- 

 hausted the knowledge of all the biochemical 

 requirements of their material. 



A fundamental problem arises as to the 

 criteria by which a neutral or indifferent 

 medium could be recognized as such, granted 

 the validity of the assumption that it exists 

 and granted that it would be capable of 

 preparation. Just as there are various con- 

 ditions luider which cells removed from an 

 embryo behave differently than in the nor- 

 mal embryo, so there are various sets of 

 conditions imder which cells removed from 

 the embryo might carry out the same per- 

 formance as in the embryo; the most striking 

 manifestations of embryonic organization 

 are those regulatory phenomena whereby 

 processes resembling the normal are carried 

 out under a great variety of abnormal con- 

 ditions. 



Devillers ('50), for instance, has found the 

 trout blastoderm incapable of differentiation 

 in triple-strength Holtfreter's solution but 

 able to differentiate in modified White's solu- 

 tion. It is unthinkable that this is the single 

 solution capable of supporting differentiation 

 in this form. Devillers' result demonstrates 

 the fact that triple-strength Holtfreter's solu- 

 tion is unsatisfactory for his particular ex- 

 periment, but provides no essential informa- 

 tion about the blastoderm; the fact that 

 modified White's solution is more favorable 

 furnishes little information about differen- 

 tiation as such, but signifies primarily that 

 the medium used permits certain embryonic 

 processes to occur. The absence of differentia- 

 tion of cultures in solutions of particular com- 

 position does not necessarily demonstrate that 

 the cells are characterized by the presence 

 or absence of particular potencies, but rather 

 may indicate that the media lack certain 

 factors required as stimuli for the realization 

 of normal potencies, or even that they include 

 agents which may actively inhibit such real- 

 ization. 



Whether or not the cells will differentiate, 

 furthermore, is not the only test of the suit- 

 ability of the medium; the direction of dif- 

 ferentiation and what the factors are which 

 determine it are as important considerations. 

 Using the prospective nervous system of the 

 yoiuig urodele gastrula as an example, when 

 isolated in salt solution it will under some 

 conditions form only simple epidermis, under 

 others nervous tissue (Holtfreter, '45). Im- 

 planted in vivo, where it is subjected to a 

 wider variety of influences, it is capable of 

 differentiating widely divergent structures 

 (Holtfreter, '29; Bautzmann, '29; Kusche, '29) 



