An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Summary

Transmitochondrial cybrids are hybrid cells obtained by fusing mitochondrial DNA (mtDNA)-depleted cells (rho⁰ cells) with cytoplasts (enucleated cells) derived from patients affected by mitochondrial disorders. They allow the determination of the nuclear or mitochondrial origin of the disease, evaluation of biochemical activity, and confirmation of the pathogenetic role of mtDNA-related variants.

Abstract

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes.

The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho⁰) could be repopulated by exogenous mitochondria to obtain the so-called "transmitochondrial cybrids." Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho⁰ cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

Introduction

Mitochondrial disorders (MDs) are a group of multisystem syndromes caused by an impairment in mitochondrial functions due to mutations in either nuclear (nDNA) or mitochondrial (mtDNA) DNA¹. They are among the most common inherited metabolic diseases, with a prevalence of 1:5,000. mtDNA-associated diseases follow the rules of mitochondrial genetics: maternal inheritance, heteroplasmy and threshold effect, and mitotic segregation². Human mtDNA is a double-stranded DNA circle of 16.6 kb, which contains a short control region with sequences needed for replication and transcription, 13 protein-coding genes (all subunits of t....

Protocol

NOTE: The use of human fibroblasts may require ethical approval. Fibroblasts used in this study were derived from MD patients and stored in the Institutional biobank in compliance with ethical requirements. Informed consent was provided for the use of the cells. Perform all cell culture procedures under a sterile laminar flow cabinet at room temperature (RT, 22-25 °C). Use sterile-filtered solutions suitable for cell culture and sterile equipment. Grow all cell lines in a humidified incubator at 37 °C with 5% C.......

Discussion

The mtDNA has a very high mutation rate compared to nDNA because of the lack of protective histones and its location close to the respiratory chain, which exposes the molecule to damaging oxidative effects not efficiently counteracted by the repair systems¹⁶. The first pathogenic mtDNA mutations were identified in 1988^17,18, and since then, a large number of mutations have been described. NGS technology is a relevant approach to screen the.......

Materials

Name	Company	Catalog Number	Comments
5-Bromo-2'-Deoxyuridine	Sigma-Aldrich (Merck)	B5002-500MG
6 well Plates	Corning	3516
96 well Plates	Corning	3596
Blood and Cell Culture DNA extraction kit	QIAGEN	13323
Centrifuge	Beckman Coulter	Avanti J-25	7,200 rcf, 37 °C
Centrifuge bottles, 250 mL	Beckman Coulter	356011
Cytochalasin B from Drechslera dematioidea	Sigma-Aldrich (Merck)	C2743-200UL
Dialyzed FBS	Gibco	26400-036 100mL
DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate)	EuroClone	ECB7501L
Dulbecco's Phosphate Buffered Saline - PBS (w/o Calcium w/o Magnesium)	EuroClone	ECB4004L
Ethanol Absolute Anhydrous	Carlo Erba	414601
FetalClone III (Bovine Serum Product)	Cytiva - HyClone Laboratories	SH30109.03
Glass pasteur pipettes	VWR	M4150NO250SP4
Inverted Research Microscope For Live Cell Microscopy	Nikon	ECLIPSE TE200
JA-14 Fixed-Angle Aluminum Rotor	Beckman Coulter	339247
Laboratory autoclave Vapormatic 770	Labotech	29960014
L-Glutamine 200 mM (100x)	EuroClone	ECB 3000D
Minimum Essential Medium MEM	Euroclone	ECB2071L
MycoAlert Mycoplasma Detection Kit	Lonza	LT07-318
PEG (Polyethylene glicol solution)	Sigma-Aldrich (Merck)	P7181-5X5ML
Penicillin-Streptomycin (solution 100x)	EuroClone	ECB3001D
Primo TC Dishes 100 mm	EuroClone	ET2100
Primo TC Dishes 35 mm	EuroClone	ET2035
Sodium Pyruvate 100 mM	EuroClone	ECM0542D
Stereomicroscope	Nikon	SMZ1000
Trypsin 2.5% in HBSS	EuroClone	ECB3051D
Uridine	Sigma-Aldrich (Merck)	U3003-5G