An Enhanced Method for Transmitochondrial Cybrid Generation

Transmitochondrial cybrid technology is a key approach for elucidating the effects of mitochondrial DNA (mtDNA) mutations in defined nuclear genetic backgrounds and for studying nuclear-mitochondrial interactions. However, its application is limited by the availability of suitable recipient cell lines and by technically demanding enucleation procedures. We report three advances in cybrid technology: (1) enucleation using mitomycin C, a widely used agent for generating feeder layers in stem cell culture, which does not depend on cell attachment and provides a gentler alternative to actinomycin D; (2) selection of cybrids using mitochondrial uncouplers, which can reduce background survival of non-cybrid cells; and (3) cryopreservation of enucleated donor cells in liquid nitrogen, preserving fusion competence and increasing experimental flexibility. Additionally, we validate newly developed mtDNA-free (ρ⁰) derivatives of HCT116, HT1080, and U2OS cell lines as recipients for cybrid generation. These advances facilitated donor cell preparation, improved cybrid selection, and enhanced experimental flexibility, including the demonstration of preserved fusion competence of enucleated HeLa cells after 10 years of cryostorage. The ρ⁰ derivatives of HCT116, HT1080, and U2OS cells were confirmed as effective recipients. Together, these improvements enhance the efficiency and accessibility of transmitochondrial cybrid technology and are expected to facilitate its broader application.