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Generation of transmitochondrial cybrids using a microfluidic device.

Ken-Ichi Wada1, Kazuo Hosokawa2, Yoshihiro Ito3

  • 1R&D Laboratory for Innovative Biotherapeutics, Graduate School of Pharmaceutical Sciences, Kyushu Univ., 3-1-1 Maidasi, Higashi, Fukuoka, 8112-8582, Japan; Bioengineering Laboratory, Cluster for Pioneering Research, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan; Nano Medical Engineering Laboratory, Cluster for Pioneering Research, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.

Experimental Cell Research
|June 6, 2022
PubMed
Summary
This summary is machine-generated.

This study presents a microfluidic device for efficient mitochondrial DNA (mtDNA) transfer, successfully creating transmitochondrial cybrids. This method enables precise control over cellular mtDNA for research and therapeutic applications.

Keywords:
Cell fusionCybridHomoplasmyMicrofluidic deviceMicrotunnelMitochondrial transfer

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Area of Science:

  • Cell Biology
  • Genetics
  • Bioengineering

Background:

  • Mitochondrial DNA (mtDNA) mutations are linked to various diseases.
  • Achieving homoplasmic mtDNA mutations is crucial for studying mitochondrial function and disease.
  • Previous methods for mtDNA manipulation were often inefficient or lacked precision.

Purpose of the Study:

  • To develop and validate a microfluidic device for generating transmitochondrial cybrids.
  • To demonstrate the efficiency and specificity of single mitochondrion transfer using microfluidics.
  • To establish a method for selective culture and harvest of generated cybrid cells.

Main Methods:

  • Utilized a microfluidic device to transfer mitochondria from HeLa cells to thymidine kinase-deficient ρ0 143B cells.
  • Employed selective culture conditions (lacking pyruvate and uridine, supplemented with 5-bromo-2'-deoxyuridine) to isolate cybrid cells.
  • Implemented puromycin-based selection within the microfluidic device for harvesting cybrids.

Main Results:

  • Successfully generated transmitochondrial cybrid cells containing HeLa mtDNA and 143B nuclei.
  • Confirmed the absence of parental mtDNA (143B) and nuclei (HeLa) in the resulting cybrids.
  • Observed reduced lactate production and loss of pyruvate/uridine auxotrophy in cybrid cells, indicating successful mitochondrial integration.
  • Demonstrated selective harvest of cybrids using puromycin selection.

Conclusions:

  • The microfluidic device provides an effective platform for generating transmitochondrial cybrids.
  • This technique allows for precise control over cellular mtDNA composition.
  • The developed method holds potential for disease modeling and gene therapy research involving mitochondrial disorders.