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In Vitro Differentiation Protocols

For the development of ES cells into differentiated phenotypes, cells are cultivated in three-dimensional aggregates or EBs. The differentiation of cardiac muscle, neuronal, pancreatic and hepatic cells requires different conditions. In this chapter, differentiation protocols utilizing the hanging drop method are described (see Note 7, Fig. 1). 1. Prepare a single cell suspension containing a defined ES cell number of 200, 400, or 600 (see Subheading 3.2.2.) cells in 20 L of differentiation...

Media Reagents and Stock Solutions for Cell Culture

Phosphate-buffered saline (PBS) containing 10 g of NaCl, 0.25 g of KCl, 1.44 g of Na2HPO4, and 0.25 g of KH2PO42H2O L, filter-sterilized through a 0.22- im filter. 2. Trypsin solution 0.2 trypsin 1 250 (Serva, Heidelberg, Germany) in PBS for routine passaging, 0.1 trypsin 1 500 in PBS for replating of EB outgrowths, filter-sterilized through a 0.22- im filter. 3. Ethylenediamine tetraacetic acid (EDTA) solution 0.02 EDTA (Sigma, St. Louis, MO) in PBS for routine passaging, 0.08 EDTA in PBS for...