DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS IN TISSUE CULTURES 



OF CHICK EMBRYOS. 



BY MARGARET REED LEWIS. 



Up to the present time the study of fixed and stained preparations of embryos 

 has failed to decide the question of the origin of the connective-tissue fibers. This 

 is partly because the methods used necessarily coagulate and distort the delicate 

 cell processes and also the intercellular substances, and such results readily lend 

 themselves to various interpretations by different investigators. 



In the following observations on tissue culture an attempt has been made 

 to study the formation of the connective-tissue fibers directly within the living 

 tissue, and not only within living tissue, but within tissue which has developed 

 in an environment entirely free from fibrin or any substances other than those 

 within the cell which coagulates upon fixation. 



The pieces of tissue to be explanted for the tissue cultures were washed through 

 several changes of Locke's solution until such fibrogen as was present had become 

 coagulated within the pieces themselves before they were explanted. The medium 

 thus remained free from fibrin, for no fibrin network was observed in the medium 

 of any preparation. In order to see whether the substance forming fibrin would 

 be dissolved out from the explanted piece and deposited as a fibrin network in the 

 medium of the culture, a few cultures were made in which the explanted pieces 

 of tissue were not carefully washed. No fibrin network was found in these cul- 

 tures, even after many days. However, in such preparations a delicate network 

 formed over the growth (not within the growth) upon fixation, showing that some 

 substance had been dissolved out from the explanted piece, which coagulated 

 upon fixation. Although this network did not resemble fibrin network, or the 

 delicate processes from the cells, or even the fibrous tissue itself, all such prepara- 

 tions were discarded in order to avoid any possible chance of error. 



By this method it is taken for granted that such connective-tissue fibrils as 

 form in the tissue-culture growths arise from the cells, either as a secretion formed 

 by the cells and deposited in the form of fibrils and fibers, or from the transforma- 

 tion of the cytoplasm of the cell itself. As will be seen later, while there is evi- 

 dence of a possible secretory activity of these mesenchyme cells, as Renaut (1904) 

 and Renaut and Dubreuil (1906) have claimed, due to the presence of the "grains 

 de segregation," or the so-called vacuoles of Lewis and Lewis (1915), nevertheless, in 

 these tissue cultures, the connective-tissue fibrils formed by a transformation of 

 the cytoplasm of the cells. 



Tissue culture is not an entirely satisfactory method for the study of any 

 highly differentiated tissue, owing to the fact that the cells which migrate out 

 from the explanted piece and later increase by division attach themselves so 

 closely to the cover-slip and become spread out in such a thin layer that the differ- 



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