# Protocol for generation of transmitochondrial cybrids under pyruvate/uridine-supplemented conditions using a microfluidic device

## TL;DR

This paper introduces a new protocol using a microfluidic device to generate transmitochondrial cybrids even when pyruvate and uridine are present in the medium.

## Contribution

A novel microfluidic-based method for generating transmitochondrial cybrids under PU-supplemented conditions.

## Key findings

- The microfluidic device enables mitochondrial transfer between single cells.
- mtDNA repopulation and typing in cybrids can be confirmed using RFLP analysis.
- The protocol works effectively even with pyruvate/uridine in the medium.

## 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

•Protocol for the generation of transmitochondrial cybrids using a microfluidic device•Instructions for the fabrication of a microfluidic device•Steps for mitochondrial transfer between single cells using a microfluidic device•Guidance on harvesting cybrids under pyruvate/uridine-supplemented conditions

Protocol for the generation of transmitochondrial cybrids using a microfluidic device

Instructions for the fabrication of a microfluidic device

Steps for mitochondrial transfer between single cells using a microfluidic device

Guidance on harvesting cybrids under pyruvate/uridine-supplemented conditions

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.