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% CO2. Mycoplasma tests should be conducted weekly to ensure mycoplasma-free cultures. 143BTK– rho0 cells can be generated as previously described5.
1. Culture of cells
2. Enucleation of fibroblasts
3. Fusion of the enucleated fibroblasts with rho 0 cells
4. Cybrid selection and expansion
Generating cybrids requires 3 days of laboratory work plus a selection period (~2 weeks) and additional 1-2 weeks for the growth of clones. The critical steps are the quality of cytoplasts and the selection period. The morphology of cybrids resembles that of the rho0 donor cells. Assignment of the correct mtDNA and nDNA in the cybrids is mandatory to confirm the identity of the cells. An example is given in Figure 2. In this case, we generated cybrids starting from fibroblasts derived from a patient carrying the heteroplasmic m.3243A>G, one of the most common mtDNA mutations associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Analysis of VNTR showed that cybrid nDNA is identical to that of the rho0 cells (Figure 2A), confirming the replacement of the patient's nDNA with the 143B genome. RFLP and/or sequencing analyses can be used to assess the presence of the mtDNA mutation and the heteroplasmy percentage (Figure 2B,C).
Figure 1: Schematic diagram of the cybrid generation protocol. Patient-derived fibroblasts are treated with cytochalasin B and centrifuged to obtain cytoplasts (enucleated cells). Cytoplasmic fusion of cytoplasts and mtDNA-depleted cells (143BTk– rho0) allows the generation of cybrids that can be isolated after selection in the appropriate medium. Picking up and amplification of single clones yields different heteroplasmy percentages, which in theory can vary from 100% wild-type to 100% mutated. Abbreviation: mtDNA = mitochondrial DNA. Please click here to view a larger version of this figure.
Figure 2: Molecular characterization of cybrids. (A) Analysis of Apo-B microsatellites shows that the nDNA extracted from the generated cybrids is identical to the nuclear DNA of the rho0 cell line. (B) HaeIII restriction maps of the PCR product spanning the MELAS-associated m.3243A>G, used to quantify the amount of WT) and Mut mtDNA species. (C) Representative results of RFLP analysis showing m.3243A>G heteroplasmy levels in different cybrid clones (c1, c2, c3). Abbreviations: M = marker; bp = base pairs; WT = wild-type; Mut = mutant; RFLP = restriction fragment length polymorphism; MELAS = mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. Please click here to view a larger version of this figure.
Media Composition | |
Complete culture medium | Final conc. |
DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate) | [1:1] |
FetalClone III (Bovine Serum Product) | 10% |
Sodium Pyruvate 100 mM | 1 mM |
Penicillin-Streptomycin (solution 100x) | 1% |
L-Glutamine 200 mM (100x) | 2 mM |
Supplemented culture medium | Final conc. |
DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate) | [1:1] |
FetalClone III (Bovine Serum Product) | 10% |
Sodium Pyruvate 100 mM | 1 mM |
Penicillin-Streptomycin (solution 100x) | 1% |
L-Glutamine 200 mM (100x) | 4 mM |
Uridine | 50 µg/mL |
Enucleation medium | Final conc. |
DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate) | [1:1] |
FetalClone III (Bovine Serum Product) | 5% |
Sodium Pyruvate 100 mM | 1 mM |
Penicillin-Streptomycin (solution 100x) | 1% |
L-Glutamine 200 mM (100x) | 2 mM |
Cytochalasin B from Drechslera dematioidea | 10 µg/mL |
Fusion medium | Final conc. |
DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate) | [1:1] |
FetalClone III (Bovine Serum Product) | 5% |
Sodium Pyruvate 100 mM | 1 mM |
Penicillin-Streptomycin (solution 100x) | 1% |
L-Glutamine 200 mM (100x) | 2 mM |
Selection medium | Final conc. |
DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate) | [1:1] |
Dialyzed FBS | 5% |
Sodium Pyruvate 100 mM | 1 mM |
Penicillin-Streptomycin (solution 100x) | 1% |
L-Glutamine 200 mM (100x) | 2 mM |
5-Bromo-2'-Deoxyuridine | 100 µg/mL |
Table 1: Details of media used for cybrid generation.
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 |
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.
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.
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.