DISSOCIATION AND REAGGREGATION OF EMBRYONIC CELLS 



225 



STANDARD CULTURE MEDIUM (MOSCONA) 



Eagle's basal medium* 1% glutamine; with 10% unfiltered and sterile 



Horse serum 2% fresh chick embryo extract, 



(10 day chick embryos and Tyrode's solution 1:1); and 



1% penicillin-streptomycin mixture {50 units each per cc. ) 



Equal portions of the cell suspensions are distributed into sterile, 25 cc. Erlen- 

 mayer flasks each containing 3 cc. of the standard culture medium. The flasks are 

 gassed with the COp-air mixture (5%/95%), tightly stoppered, and placed on a gyra- 

 tory table shaker with a 3/4 inch eccentric rotary motion of 70 r. p. m. at 38 C. 

 constant temperature. Aggregates may be collected after 24-48 hours for analysis, 

 and others may be further cultured on plasma clots or in roller tubes using the cul- 

 ture medium as a liquid phase. 



1. Chop tissue. 2 Trypsinise. 



5 After resuspendinq 

 in medium inoculate 

 vessels. 



r-^^T-] 



3 Count cells. 



4. Spin and remove 

 trypsin. 



6. Add medium 

 to make 

 final volume. 



TRYPSINISATION OF FRESH TISSUE 



Figure reproduced with permission from J. Paul's *'Cell and 

 Tissue Culture" 1960, E. & S. Livingston, Pub., Edinburgh. 



The shape, number, size distribution and internal structure of aggregates formed 

 under a set of specific conditions within 24 hours is known as the aggregation pattern 

 of the cells in testing. Different cell types or cell populations produce different 

 aggregation patterns. Under constant conditions the patterns are highly reproduc- 

 ible. Even slight variations in any of the conditions (i. e. changes in culture raedium, 

 temperature, rotation, shape of flask) may affect them. Cell cohesiveness is prob- 

 ably the major force which aids in the formation of aggregates. According to Mos- 

 cona ('60) "In suspensions of freshly dispersed embryonic cells, a characteristic 

 course of events takes place, as a result of which the cells convene and aggregate 

 into clusters and reestablish tissue-like continuity". 



* Obtainable from The Microbiological Association, Bethesda, Maryland. 



