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 the respiratory chain), 22 tRNA, and 2 rRNA genes³.

In healthy individuals, there is one single mtDNA genotype (homoplasmy), whereas more than one genotype coexists (heteroplasmy) in pathological conditions. Deleterious heteroplasmic mutations must overcome a critical threshold to disrupt OXPHOS and cause diseases that can affect any organ at any age⁴. The dual genetics of OXPHOS dictates inheritance: autosomal recessive or dominant and X-linked for nDNA mutations, maternal for mtDNA mutations, plus sporadic cases both for nDNA and mtDNA.

At the beginning of the mitochondrial medicine era, a landmark experiment by King and Attardi⁵ established the basis to understand the origin of a mutation responsible for an MD by creating hybrid cells containing nuclei from tumor cell lines in which mtDNA was entirely depleted (rho⁰ cells) and mitochondria from patients with MDs. Next-generation sequencing (NGS) techniques were not available at that time, and it was not easy to determine whether a mutation was present in the nuclear or mitochondrial genome. The method, described in 1989, was then used by several researchers working in the field of mitochondrial medicine^6,7,8,9; a detailed protocol has been recently published¹⁰, but no video has been made yet. Why should such a protocol be relevant nowadays when NGS could precisely and rapidly identify where a mutation is located? The answer is that cybrid generation is still the state-of-the-art protocol to understand the pathogenic role of any novel mtDNA mutation, correlate the percentage of heteroplasmy with the severity of the disease, and perform a biochemical investigation in a homogeneous nuclear system in which the contribution of the autochthonous nuclear background of the patient is absent^11,12,13.

This protocol describes how to obtain cytoplasts from confluent, patient-derived fibroblasts grown in 35 mm Petri dishes. Centrifugation of the dishes in the presence of cytochalasin B allows the isolation of enucleated cytoplasts, which are then fused with rho⁰ cells in the presence of polyethylene glycol (PEG). The resulting cybrids are then cultivated in selective medium until clones arise. The representative results section shows an example of molecular characterization of the resulting cybrids to prove that the mtDNA is identical to that of the donor patients' fibroblasts and that the nDNA is identical to the nuclear DNA of the tumoral rho⁰ cell line.

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% 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

1. Before starting any procedure, verify the presence of the mutation in fibroblasts derived from MD patient(s) and quantify the percentage of heteroplasmy or homoplasmy by Restriction Fragment Length Polymorphism (RFLP) and/or whole mtDNA sequencing analyses¹⁴.
2. Seed fibroblasts in four 35 mm Petri dishes, each containing 2 mL of Complete Culture Medium (Table 1). Let the cells grow until 80% confluent (48 h).
3. Grow 143BTK^- rho⁰ cells in 8 mL of Supplemented Culture Medium (Table 1) in a 100 mm Petri dish.
4. Maintain the cells in an incubator at 37 °C with 5% CO₂.
5. Check the absence of mtDNA in rho⁰ cells by sequencing techniques¹⁴.

2. Enucleation of fibroblasts

1. Sterilize four 250 mL centrifuge-suitable bottles by autoclave sterilization at 121 °C for a 20 min cycle. Dry them in a laboratory oven or at RT.
2. Prewarm the centrifuge at 37 °C.
3. Wash the 35 mm dishes containing the fibroblasts twice, using 2 mL of 1x phosphate-buffered saline (PBS) without (w/o) calcium and magnesium.
4. Clean the outer surface of the dishes with 70% ethanol and wait until the alcohol evaporates.
5. Remove the lids from the dishes and the screw caps from the bottles. Place each dish, without the lid, upside down on the bottom of each 250 mL centrifuge bottle.
6. Slowly add 32 mL of Enucleation Medium to each bottle (Table 1), allowing the medium to enter the dish and come into contact with the cells. Remove any bubbles from the dishes using a long glass Pasteur pipette, curving the tip in a Bunsen flame.
   NOTE: It is important to remove the bubbles to allow the medium to enter the dish and come in contact with the cells.
7. Close each bottle with the screw cap and transfer them to the centrifuge.
8. Centrifuge for 20 min at 37 °C and 8,000 × g, acceleration max, deceleration slow. Pay attention to balance the centrifuge: counterweight each bottle. If necessary, adjust the weight by adding a suitable volume of Enucleation Medium.
9. During centrifugation, use vacuum or a 10 mL serological pipette to aspirate and discard the medium from the 143BTK^- rho⁰ culture plates and wash them twice using 4 mL of 1x PBS w/o calcium and magnesium.
10. Add 2 mL of trypsin to cover the cell monolayer completely.
11. Place the dishes in a 37 °C incubator for ~2 min.
12. Remove the dishes from the incubator, observe cell detachment using an inverted microscope for live cells (objectives 4x or 10x), and inhibit the enzyme activity by adding 2 mL of Supplemented Culture Medium.
13. Aspirate the 4 mL of the cell suspension in the dish with a 10 mL pipette and transfer it to a 15 mL conical tube.
14. Count the cells using a Burker hemocytometer chamber or an automated counter.
15. At the end of centrifugation (step 2.8), aspirate the medium from the bottles and discard it.
16. Remove the dishes by inverting the bottles on a sterile gauze previously sprayed with 70% ethanol. Clean the outer surface of the dishes and their lids with 70% ethanol. Wait until the alcohol evaporates, and then close the dishes.
17. Before proceeding, check for cytoplast (ghost) formation using an inverted microscope for live cells (objective 4x or 10x). Look for extremely elongated fibroblasts due to the extrusion of their nuclei induced by cytochalasin B.
18. To each 35 mm dish, add 1 × 10⁶ of 143BTK^- rho⁰ cells resuspended in 2 mL of 143BTK^- rho⁰ culture medium supplemented with 5% fetal bovine serum (FBS).
19. Leave the dishes for 3 h in a humidified incubator at 37 °C and 5% CO₂ and let the 143BTK^- rho⁰ cells settle on the ghosts. Do not disturb the dishes.

3. Fusion of the enucleated fibroblasts with rho ⁰ cells

1. After 3 h of incubation, aspirate and discard the medium from the dishes.
2. Wash the adherent cells twice with 2 mL of Dulbecco's Modified Eagle Medium (DMEM) high glucose w/o serum or with Minimum Essential Medium (MEM).
3. Aspirate and discard the medium.
4. Add 500 µL of PEG solution (see the Table of Materials) to the cells and incubate for exactly 1 min.
5. Aspirate and discard the PEG solution.
6. Wash the cells three times using 2 mL of DMEM high glucose w/o serum or with MEM.
7. Add 2 mL of Fusion Medium (Table 1) and incubate overnight in the incubator at 37 °C with 5% CO₂.

4. Cybrid selection and expansion

1. After overnight incubation, remove the plates from the incubator, trypsinize the cells as described above (steps 2.9-2.13), and transfer the content of each 35 mm dish into a 100 mm dish.
2. Add 8 mL of Selection Medium (Table 1) and place the plates in the incubator at 37 °C with 5% CO₂.
3. Change the medium every 2-3 days.
4. Wait for ~10-15 days of selection until colonies of cells appear.
5. Freeze one of the four Petri dishes by collecting all the clones and generating a "massive" culture as a backup of the cybrids, which can be eventually recloned and used for further investigations.
6. Trypsinize the cells in the remaining culture dishes, count, and seed into one or more Petri dishes at 50-100 cells/dish in the Supplemented Culture Medium (Table 1) until clones appear. Let them grow for some days.
7. Pellet the remaining cells by centrifugation at 1,200 × g for 3 min at RT and discard the supernatant.
8. Extract DNA from the pellet (see the Table of Materials).
9. Perform genotyping by variable number of tandemly repeated (VNTR) analysis as previously reported¹⁵.
10. Pick up clones from the Petri dish with cloning cylinders or a pipette tip, using a stereomicroscope to avoid pooling of different clones, and transfer them to a 96-well plate, each well containing 200 µL of Supplemented Culture Medium (Table 1).
11. Expand every clone until there are enough cells for freezing and extracting DNA.
12. Verify the mutation percentage of each clone by RFLP or other sequencing methods. Ideally, try to obtain both clones with wild-type mtDNA (0% mutation) and clones with different mutation percentages, both adding up to homoplasmic mutant mtDNA (100% mutation). See Figure 1 for a schematic diagram of the cybrid generation protocol.

Results

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 der...

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

List of materials used in this article

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