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 mitochondria^5,6,7,8,9,10 (Figure 1). Cybrid cell lines contain an isogenic nuclear background and mitochondria with a specified percentage of heteroplasmy for the mtDNA variant under study^11,12. In this way, functional assays such as respirometry (by polarography) or electron transport enzyme activity (by spectrophotometry) assays can be performed in cybrid lines ranging between 0% and 100% heteroplasmy levels to determine the potential pathogenic effects of a given mtDNA variant, as well as the relative influence of mitochondrial haplogroups that may differ between cybrid lines¹. Other cell types can be used to study pathogenicity and are best used to complement cybrid generation and functional analysis, as every cell type has its own advantages and disadvantages. Patient fibroblasts directly originate from the patient but are limited in the inherited heteroplasmy and lack an isogenic nuclear control to eliminate nuclear gene effects. mtDNA editing is becoming more popular but suffers from off-target editing and limited editable sites in mtDNA¹³. Induced pluripotent stem cells (iPSCs) can be differentiated to study a mutation's effect on different cell types, such as neurons and retinal ganglion cells, but induction of pluripotency may lead to loss of the mtDNA variant of interest or result in additional de novo variants¹⁴. Cybrid study involves the generation of a more artificial system that is based on a cancer cell line (classically 143B osteosarcoma) nuclear background, but classically grows well, making it amenable to use in high-throughput screening studies of candidate interventions. Limitations to high heteroplasmy level cybrid generation include platelet quality, starting heteroplasmy, and the selective disadvantages of the mutant mitochondria, which are discussed at length in this protocol.

Although cybrid generation is the gold standard for functional determination of mtDNA variant pathogenicity, few labs generate cybrids for the study of mitochondrial disease^{9,10,15,16,17,18,19,20,21,22,23,24,25,26,27}. Rather, more cybrids are generated to study cancer and aging^28,29. Here, we use a suspected mitochondrial disease patient case study to demonstrate optimization strategies developed to facilitate cybrid generation from mtDNA patient platelets, including selection processes and cell preparations needed to establish cybrid cell lines with high versus low heteroplasmy levels for the target mtDNA variant. Specifically, we demonstrate the methodology to generate cybrid cell lines for a complex I (CI) subunit MT-ND1 variant m.3985G>A, in which we perform biochemical studies that demonstrate the pathogenic nature of this variant. Figure 2 depicts the timeline for cybrid generation, where major methodological steps include fusion, selection, and isolation methods to yield distinct colonies.

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. Transfer the supernatant (plasma) to a new centrifuge tube.
5. Record the volume of the plasma.
6. Centrifuge the plasma at 1500 × g for 20 min at 12 °C.
7. Record the estimated platelet pellet volume.
   NOTE: Too many platelets can be cumbersome in this protocol. After fusion, when plating the Rho⁰ cells and platelets, an excess of platelets can stick to the bottom of the flask and prevent Rho⁰ cell adherence. It is recommended to use less than 50 µL of the pellet volume of platelets for the fusion. The excess platelets can be frozen by splitting the platelets in the following steps.
8. Remove the supernatant.
   NOTE: If the pellet is large, removal of the pellet can be performed by aspiration. Patient platelets are sometimes drastically reduced in number and are not very sticky. If this is the case, use a pipette to slowly remove the plasma.
9. If continuing with fresh platelets, resuspend the pellet in 1 mL of Spinner modification of Minimum essential Eagle's medium (SMEM), and transfer to a microcentrifuge tube.
10. If freezing, resuspend the pellet (platelets) in 1.05 mL of SMEM and 450 µL of platelet freezing solution (350 µL of fetal bovine serum [FBS] + 100 µL of dimethyl sulfoxide [DMSO]).
    NOTE: Some patient platelets are not stable during the freezing procedure, and a high mutant cybrid cannot be obtained after frozen. It is recommended to use the fresh preparation.
11. If the Rho⁰ cells are not ready in time, place the platelets at 37 °C with light shaking at 60 rpm.
    NOTE: Ultimately, it is best to time the platelets and Rho⁰ cells to be ready at the same time. It is not suggested to wait for more than 2 h.

2. Preparing Rho⁰ cells

1. Use either freshly thawed 143B osteosarcoma Rho⁰ cells or Rho⁰ cells that were growing to near confluence.
   NOTE: It is recommended to use Rho⁰ cells that were growing in the incubator to near confluence. Freshly thawed Rho⁰ cells can be used, and it is recommended to use a frozen vial of Rho⁰ cells with a high cell density. Allow the cells to attach for two or more hours prior to fusion.
2. Wash the T75 flask with 5 mL of PBS.
3. Add 1 mL of Trypsin-EDTA 0.25% to cover the Rho⁰ cells.
4. Incubate for 5 min at 37 °C.
5. After rocking the flask, wash the bottom of the flask with 5 mL of SMEM.
6. Collect Rho⁰ cells in a 15 mL conical tube.
7. Centrifuge at 200 × g for 5 min to concentrate for cell count measurement.
8. Remove supernatant and resuspend in 1 mL of SMEM.
9. Count the cells.
   NOTE: Ideally, there should be 1.5 million cells for each fusion; however, successful fusions have been done with as few as 30,000 cells.
10. Aliquot 1.5 million cells into 1.5 microcentrifuge tubes and bring the volume to 1 mL.
    NOTE: Be sure to have an extra "mock fusion" tube. This tube will only contain Rho⁰ cells and will be used to help guide the selection process.
11. Centrifuge the platelets at 1700 × g for 15 min at room temperature.
    NOTE: Rho⁰ cells are sensitive to cold temperature.
12. Remove the supernatant from the platelets.
13. Add 1 mL of Rho⁰ cells on top of the platelet pellet.
14. Centrifuge the Rho⁰ cells and the platelets at 200 × g for 5 min at room temperature.

3. Fusion

1. Remove the supernatant from the Rho⁰ cells and the platelet pellet.
2. Add 150 µL of fusion buffer (50% polyethylene glycol [PEG]/10%DMSO) and triturate for 90 s to resuspend.
   NOTE: Some pellets are quite sticky and need to be manually stirred and broken up in addition to pipetting up and down. Record if the pellet was sticky.
3. Add 12 mL of Cybrid media (DMEM) + 10% FBS + 50 µg/mL uridine to a T75 flask.
4. Add the 1 mL of fused platelets and Rho⁰ cells to the flask.
5. Cells are incubated at 37 °C with 5% CO₂ in high humidity.
   NOTE: All cell growth steps are under these conditions.

4. Growth

1. Day 2: Remove Cybrid media and add fresh 12 mL of Cybrid media + 10% FBS + 50 µg/mL uridine.
   NOTE: Rho⁰ cells take about one week to incorporate the mtDNA.
2. Day 5: Remove Cybrid media and add fresh 12 mL of Cybrid media + 10% FBS + 50 µg/mL uridine.
   NOTE: If the Rho⁰ cells are at 80% confluence, split the cells into two flasks.

5. Selection

1. Day 8: Remove media and wash the cells with 5 mL of PBS.
2. Remove PBS and add 1 mL of trypsin.
3. Cover the bottom of the flask with trypsin and incubate for 5 min at 37 °C.
4. Add 5 mL of Cybrid media + 10% FBS + 50 µg/mL uridine.
5. Count the cells.
   NOTE: Selection should start at 10% confluence, which is 125,000 cells in a T75 or tissue culture-treated petri dish. It is recommended to use petri dishes if the colony picking method is preferred. Use a T75 if using the dilution method.
6. Add 125,000 cells to the appropriate container and add the rest of the volume up to 12 mL with Selection media (DMEM).
   NOTE: Selection media is designed to separate fused 143B cells with mitochondria from the Rho⁰ cells. Rho⁰ cells require pyruvate and uridine to survive. Dialyzed FBS does not contain pyruvate and uridine and should only be used for selection. Use two different selection media if possible. One should be 10% dialyzed FBS, and the other should be less restrictive of growth, like 20% dialyzed FBS + 50 µg/mL uridine. A less restrictive selection medium is suggested in parallel for severe mutants.
7. Freeze remaining cells in two cryovials with 1 mL of freezing solution each.
   NOTE: These are preselection cells, and different selections can be tried down the line.
8. Change the media every other day for about 1 week.
   NOTE: The Rho⁰ cells will float when they die and will wash away after the media change. Use the "mock" fusion cells to determine when selection is complete (no more cells in the flask). This flask will be entirely empty because all the Rho⁰ cells will have washed away. It is a good idea to wait another day, even when the mock flask is empty, because fused cells can provide the nutrients in the media to help other Rho⁰ cells persist in the flask. If the cells grow to more than 20% confluence, split and replate. More growth will mask the selection.

6. Colony selection growth method

1. Grow the colonies until they are visible by eye with Cybrid media + 20% FBS + 50 µg/mL uridine.
   NOTE: Tilt the dish and hold it up to the light. The colonies appear as cloudy circles. Under the microscope, there should not be a lot of background cells in between the colonies.
2. Circle independent non-overlapping colonies with a marker at the bottom of the flask while holding them up to the light.
3. Use a P20 to both scrape (mechanically disturb cells attached to the bottom of the flask) and aspirate 20 µL at the same time within the drawn circle to place cells from the selected colony into the tip of the P20.
   NOTE: Scraping manually detaches the cells and pulling up 20 µL is collecting the cells in the pipette tip.
4. Plate each colony in its own well in a 96-well plate.
   NOTE: The advantage of this method is that 143B cells tend to grow well together, so one should not have a problem growing and will get faster results. The disadvantage is that one might have a mixed population.

7. Dilution growth method

1. When the cells reach 75% confluence in Cybrid media + 20% FBS + 50 µg/mL uridine, wash the cells with 5 mL of PBS.
2. Remove PBS and add 1 mL of trypsin.
3. Cover the bottom of the flask with trypsin and incubate for 5 min at 37 °C.
4. Add 5 mL of Cybrid media + 20% FBS + 50 µg/mL uridine.
5. Count the cells.
6. Add 1 × 10⁵ cells in 20 mL of Cybrid media + 20% FBS + 50 µg/mL uridine to 50 mL conical tube 1.
   NOTE: This is 5000 cells/mL, which will be 500 cells in 100 µL when plated in a 96-well plate.
7. Prepare a 50 mL conical tube with a 100-fold dilution (200 µL from Tube 1 in 20 mL of Cybrid media + 20% FBS + 50 µg/mL uridine).
   NOTE: This is about 5 cells per well.
8. Prepare a 50 mL conical tube 3 with a 1:1 dilution (10 mL from Tube 2 in 10 mL of Cybrid media + 20% FBS + 50 µg/mL uridine).
   NOTE: This is about 2.5 cells per well.
9. Prepare a 50 mL conical tube 4 with a 1:1 dilution (10 mL of Tube 3 in 10 mL of Cybrid media + 20% FBS + 50 µg/mL uridine).
   NOTE: This is about 1 cell per well.
10. Plate Tubes 2, 3, and 4 in 96-well plates with 100 µL each.
    NOTE: Be sure to mix the cells before plating. It is hard to check the day of to see if one cell is in each well because the cells need to sink to the bottom and attach. The advantage of this method is that a single cell grows into a colony, ensuring the absence of a mixed background. The disadvantage is that severe mutants may not survive if isolated.
11. The next day, cross out wells that clearly have more than one cell per well.
12. After a few days, check the wells again.
    NOTE: Now there should be colonies, and it is more apparent if there are single cells near the edges of the wells. If there is more than one colony, remove those wells. If there is no cell, remove the well.

8. Growth

1. Start in the 96-well plate and transfer cells to bigger plates step-wise as needed.
   NOTE: These cells like to grow best with high cell density, and not transferring enough cells could cause death. Always keep the previous plate with some cells in it and mark which well came from where so that one can always go back if needed. Importantly, 143B cells die and detach if overconfluent, so check these cells every day.

9. Genetic confirmation

1. Centrifuge the aliquoted cells at 450 ×g for 5 min.
2. Remove media.
3. Add 5 µL of DNA Extraction buffer (50 mM NaOH).
   NOTE: This number can change if there are a lot of cells. Significant cell pellets can be in 10 µL of buffer.
4. Incubate at 95 °C for 30 min.
5. Add an equivalent volume of DNA Stabilization buffer (0.1 M Tris-HCl; pH 8) to the extraction buffer.
6. Perform polymerase chain reaction (PCR) according to the enzyme's guidelines.
   NOTE: Some point mutants can use restriction enzymes to distinguish between control and patient cells. Other point mutations cannot be distinguished and require sequencing. In this instance, it is important to use PCR mixes without dyes for high-quality results. Sanger sequencing and next-generation sequencing can be used. 2.5 µL of DNA is sufficient for a 25 µL reaction.
7. Use PCR amplicons specific to this mutation for Sanger sequencing.
   NOTE: The PCR amplicons were sent for Sanger's sequencing. Mutation heteroplasmy level was confirmed both after clone expansion and after functional analysis.

10. High-resolution respirometry using high-resolution respirometry

1. Perform air calibration as per the respirometer (e.g., Oroboros Instruments) guidelines.
2. Wash cybrids in a T75 flask with PBS.
   NOTE: The cells should not be overconfluent; 70%–80% confluent is ideal.
3. Add 1 mL of trypsin and incubate for 4 min.
4. Add 5 mL of Cybrid media and count cells.
5. Add 500,000 cells to a tube to centrifuge down at 450 × g for 5 min.
   NOTE: The cell number and volumes for the respirometer are with the 0.5 mL chamber.
6. Wash cells by resuspension with PBS and centrifuge again.
7. Resuspend cells in 510 µL of MiR05 buffer and add to an empty chamber.
8. After stabilization, add 0.65 µL of digitonin (50 mg/mL), 0.8 µL of malate (2.5 M), and 2 µL of pyruvate (2.5 M).
   NOTE: Before the addition of digitonin, this is where basal respiration measurement is taken. Every subsequent step should be done after stabilization.
9. Add 12 µL of adenosine diphosphate (ADP) (0.25 M).
10. Add 4 µL of glutamate (2.5 M).
11. Add 12 µL of succinate (1.25 M).
12. Add 1 µL of oligomycin (1 mg/mL).
13. Add 1 µL of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP] (0.2 mM).
    NOTE: Titrate with multiple additions until no further increase is observed (typically 3–5).
14. Add 1 µL of rotenone (2 mM).
15. Add 1 µL of antimycin A (1 mg/mL).
16. Add 0.5 µL of ascorbate (0.8 M) and 0.5 µL of tetramethylphenylenediamine [TMPD] (0.2 M).
17. Open the chamber with a spacer for 20 min.
18. Close the chamber and stabilize for 5 min.
19. Add 22.5 µL of azide (4 M).
20. Collect cells for protein quantification using a bicinchoninic acid (BCA) assay kit.
21. Perform calculations after stabilization of the oxygen consumption rate and normalize by protein. Basal Respiration is the stabilized oxygen consumption rate after the addition of cells to the chamber.
    NOTE: All other measurements are taken after the noted substrate or inhibitor.
22. Calculate OXPHOS_CI: (Glutamate-Antimycin A)/(FCCP-Antimycin A).
23. Calculate OXPHOS_CI+CII: (Succinate-Antimycin A)/(FCCP-Antimycin A).
24. Calculate ETS_CI+CII: (FCCP-Antimycin A).
25. Calculate ETS_CII: (Rotenone-Antimycin A)/(FCCP-Antimycin A).
26. Calculate LEAK_CI+CII: (Oligomycin-Antimycin A)/(FCCP-Antimycin A).
27. Calculate OXPHOS_CIV: (TMPD/ASC-Azide)/(FCCP-Antimycin A).
28. Use at least 4–6 biological replicates for respirometry measurements.

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