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Updated: May 12, 2026
An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model
Published on: March 9, 2022
Protocol for generation of transmitochondrial cybrids under pyruvate/uridine-supplemented conditions using a
Ken-Ichi Wada1, Kazuo Hosokawa2, Yoshihiro Ito2
1Institute for Materials Chemistry and Engineering, Kyushu University, 744 Motooka, Nishi, Fukuoka 819-0395, Japan; Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi, Fukuoka 812-8582, Japan; RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Abstract:
Under conventional approaches, selective culture with a pyruvate/uridine (PU)-free medium is essential for generating transmitochondrial cybrids. Here, we present a protocol for generating transmitochondrial cybrids using a microfluidic device, which works even under PU-supplemented conditions. We describe steps for preparation of the microfluidic device, partial cell fusion (mitochondrial transfer), and harvest of transmitochondrial cybrids. We then detail procedures for confirmation of mtDNA repopulation in cybrids and mtDNA typing by restriction fragment length polymorphism (RFLP) analysis. For complete details on the use and execution of this protocol, please refer to Wada et al.1.
