Mitochondria frozen with trehalose retain a number of biological functions and preserve outer membrane integrity.

In apoptosis, Bcl-2-family proteins regulate the barrier function of the mitochondrial outer membrane (MOM), controlling the release of proapoptotic proteins from the intermembrane space into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria. Unfortunately, mitochondria frozen/thawed in standard sucrose-mannitol buffers become leaky and useless for apoptosis research. However, here we show that mitochondria frozen in buffer containing the sugar, trehalose, maintained MOM integrity and responsiveness to Bcl-2-family proteins, much like fresh mitochondria. Trehalose also preserved ultrastructure, as well as biological functions such as ATP synthesis, calcium-induced swelling, transmembrane potential, and the import and processing of protein precursors. However, bioenergetic function was somewhat reduced. Thus, trehalose-frozen mitochondria retained most of the biological features of mitochondria including MOM integrity. Although not ideal for studies involving bioenergetics, this method will facilitate research on apoptosis and other mitochondrial functions that rely on an intact MOM.