Embryoid body methods

Hopfl, G., Gassmann, M., and Desbaillets, 1.2004. Differentiating embryonic stem cells into embryoid bodies. Methods Mol Biol 254 79-98. 

The ES cells were encapsulated in the PMBV/PVA hydrogel by the same method used for the L929 cells. In general, ES cells form a cell aggregate called an embryoid body in suspension culture. However, it was observed that the encapsulated ES cells in the hydrogel did not form any embryoid body for 72 h. 

The embryonic stem cell test is an animal-free alternative test method for developmental toxicity. Mouse embryonic stem cells are cultured in a hanging drop method to form embryoid bodies. These embryoid bodies, when plated on tissue culture dishes, differentiate to form contracting myocardial cell foci within 10 days. Inhibition of cardiomyocyte differentiation by test compounds serves as the end point of the assay, as monitored by cormting contracting muscle foci under the microscope. 

Fig. 2. The embryonic stem cell test. Droplets with 750 ES cells per 20 l complete medium are pipetted on the inner side of a cover lid of a 94 x 16 mm bacterial Petri dish (a). 56 droplets are cultured in a Flanging drop method.The lid is placed on a Petri dish, containing 5 ml of PBS (b).The cells will form Embryoid bodies (EB) (c) by proliferation and clustering. After 3 days the EB are transferred to a Petri dish containing 5 ml suspension medium (d). The EB are cultured for 2 days and then transferred to a 24-well plate (e) where they will further differentiate into contracting cardiomyocytes. The scoring is pertormed at day 10 by examining the EB under a light microscope (f).

All of these methods follow the typical protocol for embryoid body generation as described below. 

Kurosawa H (2007) Methods for inducing embryoid body formation in vitro differentiation system of embryonic stem cells. J Biosci Bioeng 103(5) 389-398... 

The EST has been developed with the aim to exploit the characteristics and differentiation potential of mouse embryonic stem cells (ES cells), established from the early embryo in 1981 [4, 5], ES cells are cultured in suspension to induce the formation of embryoid bodies, and afterwards they are transferred in 24-well dishes to allow attachment and differentiation in contracting cardiomyocytes. The toxicological endpoint is the inhibition of cardiac differentiation. In parallel a cytotoxicity test is performed on undifferentiated ES cells and a control somatic (fibroblast) cell line (3T3). The concentrations of testing chemicals that induce 50 % of differentiation inhibition (ID50) and 50 % cytotoxicity (IC50) in ES cells and 3T3 cells are inserted in a validated prediction model to classify the test chemical as non-embryotoxic, moderate, or strong embryotoxic [2, 6, 7], The validation of the method has been... 

Kurosawa, H., Imamura, T., Koike, M., Sasaki, K. and Amano, Y. (2003) A simple method for forming embryoid body from mouse embryonic stem cells. J. Biosci. Bioeng., 96, 409-411. 

Come, J., Nissan, X., Aubry, L. et al. 2008. Improvement of culture conditions of human embryoid bodies using a controlled perfused and dialyzed bioreactor system. Tissue Eng C, Methods 14(4) 289-98. 

C6me, J., Nissan, X., Aubry, L. et al. 2008. Improvement of culture conditions of human embryoid bodies using a controlled perfused and dialyzed bioreactor system. Tissue Eng C, Methods 14(4) 289-98. Cormier, J. T., Zur Nieden, N. I., Rancourt, D. E., and KaUos, M. S. 2006. Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors. Tissue Eng 12(11) ... 

Fu, X., Toh, W.S., Liu, H., Lu, K., Li, M., Hande, M.R, et al. Autologous feeder cells from embryoid body outgrowth support the long-term growth of human embryonic stem cells more effectively than those from direct differentiation. Tissue Eng., Part C. Methods 16, 719-733 (2009)... 

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