Cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol have been used for the cryopreservation of cells and tissues. DMSO is the most effective CPA but shows high cytotoxicity and can effect differentiation. ɛ-poly-L-lysine (PLL) derivatives show higher cryopreservation efficiency than the conventional CPAs. Culture medium solutions with 7.5 w/w% of PLL whose amino groups of more than 50 mol% were converted to carboxyl groups by succinic anhydride showed higher postthaw survival efficiency of L929 cells than those of current CPAs without the addition of any proteins. In addition, rat mesenchymal stem cells were cryopreserved more effectively than with DMSO and fully retained the potential for proliferation and differentiation. Furthermore, many kinds of cells could be cryopreserved with PLL having the appropriate ratio of COOH groups, regardless of the cell types, including adhesive and floating cells, human- and mouse-derived cells, primary cells, and established cell lines. The properties might be associated with the antifreeze protein properties. These results indicate that these polymeric extracellular CPAs may replace current CPAs and the high viability after thawing and nonnecessity of serum ensure that these CPAs may be used in various preservation fields.