PREPARATION OF ES CELLS FOR AGGREGATION CHIMERAS 

TRANSGENIC FACILITY

Samuel Lunenfeld Research Institute, Mount Sinai Hospital

600 University Ave., Toronto, Ontario, M5G 1X5

416-586-8382

ES cell lines to be used to form aggregation chimeras should be maintained under the optimal conditions  
(see  /1/ Wurst W., A.L. Joyner. 1993: Production of targeted embryonic stem cell clones. In Gene targeting: A Practical Approach. (ed. A. Joyner) IRL Press at Oxford University Press).  

Cells should be passed at least once after thawing, before using them for aggregations.  
The protocol given here was developed for R1.  
The goal is to produce clumps of 8-15 loosely connected cells just before aggregations.  

1. Three or four days prior to aggregation (Day -4 or -3):  

• thaw ES cells (passage a confluent plate of cells) on feeders as described /1/ 

2. Change the medium the next day (Day -3 or -2).  

3. One or two days prior to aggregation (Day -2 or -1):  

• trypsinize a plate of ES cells to give a single cell suspension as described /1/; 
• let any large clumps settle after spinning and resuspending (optional); 
• plate cell suspension on gelatinized tissue culture plates instead of the usual 1:5 ratio pass them 1:50 or even more dilute. 

4.  24 hours are usually enough to produce clumps of right size (8-15 cells), but cells can also be cultured  one more day (in this case medium should be changed);  
  
5.  Day of aggregation (Day 0):  
 (after preparation of the embryos)  
  

• add the necessary volume of trypsin (i.e. 0.5 ml into 6 cm plate) to the plates of ES cells; 
• trypsinize for 3-5 minutes at room temperature or 37 C or until they form clumps of loosely connected cells then add ES cell medium to normal volume of plate; 
• keep the cell suspension in the plate and use it to assemble aggregates within next hour or two.