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

Abstract

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. Since the 1970s, there have been a number of attempts to freeze isolated mitochondria for storage, with various degrees of success. In most of these studies, the functionality of frozen-thawed mitochondria was assessed with regard to bioenergetic parameters. In a recent and relatively successful attempt, frozen mitochondria were reported to have roughly 50% of normal respiratory function, and the mitochondria were well coupled.

Improved tolerance to salt and water stress in Drosophila melanogaster cells conferred by late embryogenesis abundant protein

“…The observed functional integrity after freezing is quite remarkable. In contrast, mouse liver mitochondria that naturally lack LEA proteins show a dramatic reduction in the respiratory control ratio after freeze-thawing in trehalose containing medium (Yamaguchi et al, 2007). Viability for non-permeabilized cells expressing AfLEA1.3 was higher in the presence of increasing concentrations of sucrose compared to control cells (Fig.…”

Heightened mitochondrial priming is the basis for apoptotic hypersensitivity of CD4+CD8+thymocytes

“…Final digitonin concentrations of 0.001-0.005% were effective in gently permeabilizing the plasma membrane without compromising mitochondrial potential for up to 3 h in cell lines and primary samples. To stabilize mitochondria in permeabilized cells we also changed our buffer to a trehalose-based buffer described by the Newmeyer laboratory, referred to here as T-EB (23).…”

Light-harvesting chlorophyll pigments enable mammalian mitochondria to capture photonic energy and produce ATP

“…Mitochondrial isolation started within 1 hour of the death of the animal and the hearts were transported to the laboratory in a bath of 0.25 M sucrose, 0.1 M tris(hydroxymethyl)aminomethane (Tris) at pH 7.5, which was surrounded by ice. Mitochondria were isolated and stored in 250 ml aliquots at a concentration of ,60 mg of protein/ml in 300 mM trehalose, 10 mM HEPES-KOH pH 7.7, 10 mM KCl, 1 mM EGTA, 1 mM EDTA and 0.1% BSA at 280˚C (Yamaguchi et al, 2007) until use. The thawed mitochondria exhibited a respiratory control ratio of ,1, indicating mitochondrial fragmentation.…”