Trans-Mitochondrial Cybrid Generation from mtDNA Patient Platelets: An Efficient Protocol Optimizing Colony Selection and Functional Validation

Summary

We describe an optimized trans-mitochondrial cybrid generation protocol from mtDNA patient platelets, emphasizing growth parameters and colony isolation. This approach enables the study of mitochondrial DNA variants at variable heteroplasmy levels in a common nuclear background to quantify their functional significance on electron transport chain enzymatic activities and integrated respiratory capacity.

Abstract

Trans-mitochondrial cybrid cell line generation represents the gold-standard method for determining pathogenicity by enabling biochemical analyses of a specific mitochondrial DNA (mtDNA) variant of interest at high and low percentages (heteroplasmy levels) within an otherwise identical mtDNA and nuclear genome background. Historically, the cybrid generation process has been tedious and poorly efficient. Here, we describe a highly efficient and effective protocol for generating trans-mitochondrial cybrid cell lines by fusing human platelets with a standard osteosarcoma 143B cell line to provide an isogenic nuclear background depleted of mtDNA (Rho⁰ cells). Cell isolates capture a given mtDNA genome of interest to establish stable cell lines harboring different degrees of heteroplasmy, or to compare divergent effects of distinct mitochondrial haplogroups. Because cybrids from mitochondrial patients may be more difficult to establish with standard protocols, this current methodology focuses on isolating mtDNA variants where the electron transport chain activity is affected. We here demonstrate that colony selection techniques reduce time and improve the yield of generating high-level heteroplasmy mtDNA mutant cybrid lines. A case study is provided of cybrid generation for a variant of unknown significance in MT-ND1, m.3985G>A (p.E227K). We analyze the efficiency of the cybrid generation process using this protocol and run functional studies performed by high-resolution respirometry. High-level heteroplasmy MT-ND1 m.3985G>A cybrid mutants generated by this protocol are shown to have impaired complex I-dependent mitochondrial respiration relative to wild-type control, demonstrating m.3985G>A is likely pathogenic.

Introduction

Establishing pathogenicity of mitochondrial DNA (mtDNA) mutations is challenging due to their rarity, heteroplasmy involving variable levels of mutated and wild-type mtDNA genomes in different tissues, diverse pathophysiologic mechanisms, and complex interactions with nuclear genome backgrounds^1,2,3,4. For these reasons, trans-mitochondrial cybrid cell lines remain the gold standard method by which to establish pathogenicity of mtDNA variants, where "cybrids" represent the fusion of one cell's nucleus with another cell's mito....

Protocol

Blood platelets were obtained per the Children's Hospital of Philadelphia's (CHOP) IRB study 08-6177.

1. Preparation of platelets

1. Day 1: Draw blood into acid-citrate-dextrose tubes when drawn.
   NOTE: Blood should be placed on the rotator immediately. Platelets are ideally harvested a maximum of 4 h after blood draw.
2. Record the volume of the blood. A volume of 5–6 mL of blood is recommended.
3. Centrifuge blood at 200 × g for 20 min at 12 °C.
   NOTE: Centrifugation at 12 °C preserves sample integrity and prevents platelet activation.
4. Tran....

Results

To demonstrate methodologic differences and the path to protocol optimization, we present the results of cybrid generation from one patient with suspected mitochondrial disease who harbors a variant of unknown significance (VUS) in MT-ND1 of complex I (CI) in the electron transport chain. After clinical mtDNA genome next-generation sequencing in multiple tissues at two different CLIA-approved clinical diagnostic labs, the only variant suspicious of pathogenicity was MT-ND1 m.3985G>A. The heteroplasmy.......

Discussion

Here, we present a highly efficient protocol for the generation of trans-mitochondrial cybrid cell lines from mtDNA patient platelets. We demonstrate the applicability with a case study of an MT-ND1 variant of uncertain significance, the importance of carefully considering growth and selection parameters. Once these major issues are optimized, the successful selection of cybrids harboring mtDNA variants that may impair respiratory chain function. This protocol can be used for the generation of cybrids with mtDNA.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ADP	Sigma Aldrich	A5285-1G
Antimycin A	Sigma Aldrich	A8674-25MG
Ascorbate	Sigma Aldrich	11140-250G
Azide	Sigma Aldrich	S2002-100G
Dialyzed FBS	Fisher Scientific	35071CV
Digitonin	Sigma Aldrich	D141-100MG
DMEM	Corning	10-013-CV	Cybrid media
DMEM	Corning	10-017-CM	Selection media
FBS	Cytiva	SH30910.03
FCCP	Apex Bio	B5004
Glutamate	Sigma Aldrich	G5889-100G
Graphpad Prism	Graphpad
Hybri-Max	Sigma Aldrich	P7306	fusion buffer
Malate	Sigma Aldrich	M1000-100G
MiR05 buffer	Oroboros Instruments	60101-01
O2K respirometor	Oroboros Instruments	C-0050
Oligomycin	Sigma Aldrich	O4876-5MG
Pyruvate	Sigma Aldrich	P2256-100G
Rotenone	Sigma Aldrich	R8875-1G
SMEM	Sigma	M8167
Succinate	Sigma Aldrich	S2378-100G
TMPD	Sigma Aldrich	T3134-5G
Trypsin-EDTA 0.25%	Thermo Scientific	25200056
Uridine	Sigma Aldrich	U3750-25G