28. THE DISSOCIATION AND 

 REAGGREGATION OF EMBRYONIC CELLS* 



PURPOSE: To separate differentiating embryonic cells from each other by disrupting the 

 intercellular matrix, and observing the reaggregation of similar and dissimilar cells. 



MATERIALS: Early organ rudiments and tissues of embryos such as the chick and mouse. 



Technical materials are the same as for tissue culture procedures, in addition to 

 which the solutions listed below. 



METHOD: Since Moscona has shown that tissues from various warm-blooded vertebrates 

 can be dissociated by trypsinization and the cells may then reaggregate along lines of 

 prospective differentiation rather than generic source, the general method of pre- 

 paring the separated cell masses will be given so that the investigator may try the 

 process on other forms as well as repeat those reported. It is obvious that those 

 with prior experience in tissue culture methods and cell physiology will, most likely, 

 be the most successful. While Moscona and others report their researches in a way 

 which might convey the impression that the technique is simple, one should not be 

 misled. However, the results are so rewarding and so significant that anyone so in- 

 clined to work should be encouraged. 



Freshly dissected fragments of early differentiating embryonic tissue, measuring no 

 more than 1 mm in diameter, are collected in small centrifuge tubes and incubated 

 (warm-blooded animals) for 10 minutes in calcium and magnesium free solution 

 (CMF) at 37°C. and at a pH. of 7. 2 under a mixture of 5% CO^ and 95% air. 



CMF Solution 

 NaCl - 8. gr. KH2PO4 - 0. 025 gr. 



KCl - 0. 30 gr. NaHCOj - 1. 00 gr. 



NaH2PO^- H^O - 0. 05 gr. Glucose - 2 gr. 



Glass Dist. Water - 1,000 cc. 



The fragments are then transferred to trypsin in CMF at 37°C. for 15-20 minutes, 

 also under the COn-air mixture. The trypsin is a crystallized, lyophilized prepar- 

 ation (Worthington Co. , Freehold, N. J. ) made up as a 1% solution in CMF. Also use 

 crystalline soybean trypsin inhibitor which is non-toxic in concentrations of 1 mgm 

 per 5 ml of culture medium. 



The fragments are then gently rinsed several times with CMF, avoiding disruption 

 or disaggregation at this time by careful handling. The purpose of the rinsing is to 

 free the fragments of adherent and excess trypsin. It is the tryptic degradation of 

 the intercellular mucoproteins which brings about the separation of the cells without 

 their destruction. But the tryptic action must not be allowed to go far enough to des- 

 troy the cells. To each fragment add 1 cc of the culture medium kept at room tem- 

 perature, and, with tapered pipette, disperse the cell masses into single cells by 

 flushing the fragments in and out briskly through the tip of the pipette without froth- 

 ing. This could be done under the dissecting microscope with the pipette containing 

 no air, only the medium used. 



* This exercise has been organized with the (generous cooperation of Dr. A. Moscona. 



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