Dimethyl sulfoxide (DMSO) has been used for several decades as the most efficient cryoprotective agent (CPA) for many types of cells in spite of its cytotoxicity and its effect on differentiation. Recently we showed that carboxylated poly-L-lysine, which is classified as a polyampholyte, has a cryoprotective effect on cells in solution without any other cryoprotectant. Here we developed high molecular weight polyampholytes with an appropriate ratio of amino and carboxyl groups and evaluated their cryopreservation efficiency. A novel polyampholyte based on naturally available polymer dextran, in which we introduced both amino and carboxyl groups shows an excellent post thaw-survival efficiency of more than 90% of murine L929 cells. It can serve as the sole high molecular weight CPA for tissue engineering applications without animal derived materials.