Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids

Summary

This protocol reports a unique method of using a streaming cytometer and multiple antibodies for simultaneous assessment of multiple mitochondrial functional parameters, including changes in mitochondrial volume, amounts of the mitochondrial respiratory chain (MRC) complex subunits, and mitochondrial DNA (mtDNA) replication.

Abstract

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain's three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

Introduction

Mitochondria are essential cellular organelles and the major site of the adenosine triphosphate (ATP) production. In addition to providing energy for cells, mitochondria also participate in multiple cellular processes, including cell information transmission, cell differentiation, and apoptosis, and have the ability to regulate cell growth and cell cycle. Changes in mitochondrial function have been identified in various neurodegenerative diseases, including Parkinson's disease (PD)^1,2, Alzheimer's disease (AD)³, and amyotrophic lateral sclerosis (ALS)⁴. Mitochondrial dysfunction plays a role in the aging process, accumulating somatic mtDNA mutations and declining respiratory chain function⁵.

Various types of mitochondrial dysfunction occur in neurodegeneration, and the ability to measure such changes is extremely useful when studying disease mechanisms and testing potential treatments. Furthermore, establishing suitable in vitro model systems that recapitulate disease in human brain cells is vital for better understanding disease mechanisms and developing new therapies. iPSCs from patients with neurodegenerative diseases have been used to generate diverse brain cells that manifest mitochondrial damage^6,7,8,9. The development of 3D brain organoids derived from iPSCs is a major step in disease modeling. These iPSC-derived brain organoids provide complexity and contain the patient´s own genetic background, thus providing a disease model that more accurately reflects pathology in the patient's brain.

While some research has been conducted on mitochondrial studies using iPSC-derived brain organoids^10,11,12, convenient and reliable techniques for determining multiple mitochondrial functional parameters in iPSC-derived brain organoids remain limited. Flow cytometry provides a powerful tool to measure mitochondrial parameters at the single-cell level, as we have demonstrated previously¹³. This study provides a detailed protocol for generating cortical organoids from iPSCs, combined with a novel flow cytometry-based approach to simultaneously measure multiple mitochondrial parameters, including mitochondrial mass, respiratory chain complex subunits, and mtDNA copy number (Figure 1). Importantly, by using mitochondrial mass as a denominator, these protocols allow one to measure both the total and specific levels per mitochondrial unit.

Protocol

1. Differentiation of iPSCs into cortical organoids

1. Preparation of matrix-coated plates
   1. Thaw the vial of commercially available basement membrane matrix on ice overnight. Dilute into 1:100 in cold Advanced Dulbecco's Modified Eagle's Medium/Ham's F12 DMEM/F12 (1% final concentration). Make aliquots and store them at -20 °C (see Table of Materials).
   2. Thaw the membrane matrix solution at 4 °C (keep it cold) and coat the required number of wells (1 mL for one well in a 6-well plate). Incubate at 37 °C for 1 h.
   3. Take the plate from the incubator and let it reach room temperature (RT) (leave it for an hour).
      NOTE: The plates can be stored for up to 2 weeks in the refrigerator (remember to take it out and warm the coated wells required for that day at RT until it is no longer cool to the touch before use). It is recommended to use the plates within 3 days. For long time storage, add 1 mL of Essential 8 Medium (see Table of Materials) to the coated plate to avoid the gel drying out.

2. Preparation Essential 8 (E8) medium for iPSC culture

1. In a sterile environment, prepare E8 Medium by combining E8 basal medium with E8 supplement (see Table of Materials). This can be stored at 4 °C for 2 weeks.
   NOTE: Thaw frozen E8 supplement at 4 °C overnight before preparing the complete medium. Thawing the frozen supplement at 37 °C is not recommended; use E8 Medium for 2 weeks. Prior to use, warm E8 Medium required for that day at RT until it is no longer cool to the touch.

3. Subculturing of iPSCs

1. Pre-warm matrix-coated plates in RT or incubator at 37 °C for 20-30 min. Pre-warm the amount of E8 Medium needed at RT.
2. Aspirate the culture medium with a pipette.
3. Rinse iPSCs (see Table of Materials) with Dulbecco's Phosphate-Buffered Saline (Ca²⁺/Mg²⁺ free) (DPBS-/-) (4 mL for one well in a 6-well plate).
4. Add Ethylenediaminetetraacetic acid (EDTA) (0.5 mM) (1 mL for one well in a 6-well plate). Incubate at 37 °C until the edges of the colonies begin to detach from the plate (usually 3-5 min).
5. Aspirate the dissociation solution.
6. Add pre-warmed E8 Medium (4 mL for one well in a 6-well plate) and use high pressure to detach iPSC colonies.
7. Transfer into two different wells in a matrix-coated 6-well plate (2 mL for one well in a 6-well plate) and incubate at 37 °C.
   NOTE: Gently shake before putting the plate into the incubator. The split ratio can be 1:2-1:4.
8. Exchange media daily till the colonies get 80% confluence with good size and connections.
   NOTE: Monitor for colonies of an appropriate size, typically characterized by a well-defined, circular shape and a diameter ranging from 0.5 to 1 mm. The colonies should exhibit dense, compact structures with clearly delineated, smooth borders.

4. Embryoid body (EB) formation and neural induction

1. Prepare neural induction medium (NIM) by combining the components listed in the Table of Materials.
2. Aspirate medium and rinse the cells with DPBS -/- (4 mL per well in a 6-well plate).
3. Add pre-warmed Accutase (1 mL per well in a 6-well plate) (see Table of Materials) in the well and incubate at 37 °C for 10 min.
4. Gently pipette the solution up and down 3 to 4 times using a 1000 µL pipette tip.
5. Neutralize with 2 mL DMEM with 10% fetal bovine serum (FBS) and centrifuge the 15 mL conical tube containing the cells at 600 x g for 5 min at RT. Aspirate the supernatant.
6. Resuspend the cell pellets by gently pipetting up and down till single-cell pallets.
7. Count and calculate live cell concentration using trypan blue¹⁴ in an automated cell counter.
8. Calculate the dilution ratio and dilute cells in NIM to make final cell concentration of 60,000 live cells/mL.
9. Add Rho-associated protein kinase (ROCK) inhibitor Y-27632 (final concentration 50 µM) (see Table of Materials) to the cell suspension and gently mix.
10. Add 150 µL of single cell suspension into each well of ultra-low attachment 96-well plate (see Table of Materials) and place in the incubator. Set up as Day 0.
11. On day 1, check the cells and EB forms in each well.
    NOTE: Evaluate successful EB formation based on their bright, spherical, and translucent appearance under a microscope, their ability to remain free-floating and separate, and the absence of dead cells, which appear as dark spots.
12. On day 2, remove 75 µL medium from each well carefully and add 150 µL NIM along with 50 µM Y-27632 to each well.
13. On days 4, 6 and 8, remove 100 µL medium from each well and add 150 µL NIM to each well.

5. Generation of cortical organoids

1. Prepare neural differentiation medium minus vitamin A (NDM-) by combining the components listed in the Table of Materials.
2. On day 10, transfer EBs from the ultra-low attachment 96-well plate to the ultra-low attachment 6-well plate using a 5 mL pipette. Transfer eight EBs to each well cautiously (Figure 2). Add 5 mL NDM- to each well.
3. Place the plate on the orbital shaker inside the incubator and start spinning at 80 rpm.
4. On days 12, 14, and 16, replace the NDM- with 4 mL after aspirating 3 mL old medium.

6. Maturation and long-term culture of cortical organoids

1. Prepare neural differentiation medium with vitamin A (NDM+) and brain-derived neurotrophic factor (BDNF). For details, see Table of Materials.
2. On day 18, remove the 3 mL medium and add 4 mL NDM+.
3. Return the plate to the spinning culture.
4. Feed every 4 days and maintain the differentiated cortical organoids in NDM+ until use. 25-40-day-old cortical organoids were used for the downstream analysis.
   NOTE: The cortical organoids can be maintained longer (up to 3-4 months); but cell viability will be dropped due to the lack of nutrition exchange.

7. Cell characterization by immunocytochemistry and immunofluorescence staining

1. Use a 1000 µL pipette to gently collect the organoid from the medium and place it gently on a medium with minimal medium on a standard microscope slide.
2. Let it dry completely at RT. Add 4% paraformaldehyde (PFA) (300 µL per sample) for 30 min. Remove PFA and then rinse twice with PBS.
   NOTE: PFA is toxic and suspected to be carcinogenic. Avoid contact with skin and eyes, and handle under a chemical fume hood.
3. Add 30% sucrose solution (300 µL per sample) to the slides and incubate overnight at 4 °C.
4. Block and permeabilize the organoids with blocking buffer (BB) (300 µL per sample) containing PBS (with Ca²⁺/Mg²⁺), 0.3% Triton X-100, and 10% normal goat serum for 2 h at RT (or overnight at 4 °C). Outline the organoids with a hydrophobic barrier pen to keep the buffer on the slide.
5. Overlay samples with primary antibody in blocking buffer (300 µL per sample), anti-SRY (sex determining region Y)-box 2 (SOX2, 1:100), and anti-neural marker tuberin beta III (Tuj1, 1:1000), and incubate overnight at 4 °C in the dark (see Table of Materials).
6. Wash in PBS for 3 h with two to three buffer changes on a gentle rocking platform.
7. Incubate with secondary antibody (300 µL per sample), anti-Alexa Flour 488 (1:800), and anti-Alexa Flour 594 (1:800), overnight at 4 °C in a humidified darkroom. Add Hoechst 33342 (1:5000) nuclear stain at the same time (see Table of Materials).
8. Aspirate the antibody and rinse quickly with PBS, then add PBS containing 0.01% NaAzide for 1-2 days at 4 °C to prevent contamination.
9. Aspirate the PBS and remove the excess solution. Mount the organoids with mounting medium (see Table of Materials), then add a coverslip. Avoid the formation of air bubbles and place in a dark room at RT for at least 12 h to allow the mounting to fully polymerize.
   NOTE: The sample is now ready to be imagined under a fluorescence microscope.

8. Dissociation of cortical organoids

1. Add 30 mL DPBS -/- in a 50 mL centrifuge tube.
2. Gently collect the brain organoids in the tube using a 10 mL pipette. At least 3 organoids are used for one staining, and they should be more than 25 days but less than 40 days old.
3. Sediment organoids for approximately 5 min. Do not centrifuge.
4. Aspirate DPBS and add 2 mL pre-warmed Accutase in the tube. Incubate the sample in the water bath for 5 min at 37 °C.
5. Triturate 15 times gently by using a 1000 µL pipette tip without generating bubbles. Incubate the sample for another 5 min at 37 °C in a water bath.
6. Dissociated gently by using 1000 µL pipette tips 15 times without generating bubbles (Figure 3A).
   NOTE: If there are still some undissociated cells, transfer the undissociated cells into a new 50 mL centrifuge tube and then incubate for another 5 min at 37 °C water bath. Then dissociate gently by using 1000 µL pipette tips 20 times.
7. Neutralize with 4 mL DMEM with 10% FBS and centrifuge the 15 mL conical tube containing the cells at 600 x g for 5 min at RT.
8. Aspirate supernatant and resuspend with 200 µL DPBS using 1000 µL pipette tips 10 times without generating bubbles.
9. Add 200 µL DPBS to make a total volume of 400 µL cell suspensions in DPBS.
10. Filter with the filter or filter tube (use 35 or 40 µm filters and rinse the filter with DPBS before using the filter).
11. Separate half of the solution in a tube and use it as unstained sample.

9. Flow cytometry measurement of MRC complex subunits and TFAM in fixed cells

1. Add Live and Dead (L/D) staining dye (1: 1000) (see Table of Materials) (either with Far Red or Near-infrared, depending on the set-up for the markers for flow cytometry).
   NOTE: If a new vial for L/D dye is opened, add 50 µL DMSO to reconstitute and make a 10 µL aliquot to store them at -20 °C.
2. Keep in RT and dark for 30 min. Add 40 mL DPBS -/-. Centrifuge at 600 x g for 5 min.
3. Resuspend into single-cell suspension after discarding the supernatant.
4. Fix and permeabilize the cells with 1 mL ice-cold 90% methanol at -20 °C for 20 min.
5. Block the samples in 1 mL block buffer containing 0.3 M glycine, 5% goat serum, and 1% bovine serum albumin (BSA) in PBS (with Ca²⁺/Mg²⁺).
6. Wash with 20 mL flow buffer containing PBS (with Ca²⁺/Mg²⁺) with 0.2% BSA once or twice.
7. Add the primary antibodies anti-NDUFB10 conjugated with Alexa Fluor 405 (1:100), anti-VDAC 1 conjugated with Alexa Fluor PE (1:100) and anti-TFAM antibody conjugated with Alexa Fluor 488 (1:200) for 30 min (see Table of Materials).
8. Wash with 20 mL flow buffer.
9. Aspirate and leave approximately 100 µL and reconstitute the cell pellets into 300 µL flow buffer.
10. Transfer the cells to 1.5 mL flow tubes. Keep in the dark and ice.
11. Analyze on the flow cytometer (see Table of Materials). Signals were detected for Anti NDUFB10-Alexa 405 using a 450/50 bandpass filter, TFAM-Alexa 488 using 530/30 bandpass filter, VDAC 1-Alexa 647 using 670/14 bandpass filter and L/D dye in 780/60 bandpass filter (Figure 3B).

10. Flow cytometry acquisition and analysis

1. Use unstained and single stained samples of the control organoid to set the voltage of cytometer settings.
2. Use the unstained control tube to set the forward scatter area (FSC-A) and side scatter area (SSC-A) scatter plots based on the size and granularity of the cell population (Figure 4A).
3. Gate out debris to select live cells from FSC-A and SSC-A plots. Gate out doublets using side scatter height (SSC-H) vs. SSC-A plot (Figure 4B).
4. Select the live cells based on the staining of L/D dye by switching to APC-cy7 vs. FSC-A plot (Figure 4C).
5. Using the unstained samples of each line, draw a gate above the main population of the events in FSC-A vs. fluorochrome channels (FITC, APC, BV 421) plots.
6. Set up compensates for the flow cytometer¹⁵.
7. Use isotype control for negative control to observe background staining.
8. Perform data acquisition.
   1. Select the live cells based on the staining of L/D dye by switching to APC-cy7 vs. the FSC-A plot (Figure 4C).
   2. Utilize the unstained as negative control. Then, while examining the FSC-A vs. fluorochrome channels (FITC, APC, BV 421) plots, establish a gate above the main population of single-cell events (Figure 4D-F).
9. Perform data analysis using FlowJo software (see Table of Materials).
   1. Duplicate the positions of the gates onto the stained cell samples. Document the number of cells that exhibit positive staining.
   2. For each targeted population, assess the median fluorescence intensity (MFI) of different channels (FITC, APC, BV 421) plotted on the x-axis of a histogram to identify the mitochondrial signal. Specific values for the complex I subunit NDUFB10 and TFAM can be determined by dividing the MFI of the complex expression or TFAM by the mitochondrial mass indicator, VDAC 1.

Results

Figure 1 provides a diagrammatic representation of the differentiation process and the strategies used for flow cytometric analysis. Human iPSCs were cultured in non-adherent 96-well plates to form EBs and then transferred to non-adherent 6-well plates to obtain fully grown cortical organoids. The cellular composition of organoids was validated using confocal microscopy after immunostaining with neuronal¹⁶ and glial markers¹⁷. Organoids we...

Discussion

A protocol is presented for generating cortical brain organoids from human iPSCs and for performing the flow cytometric analysis of mitochondrial parameters in single cells isolated from these organoids. The cellular composition of the organoids was verified by confocal microscopy with immunohistochemical staining for neuronal and glial cell markers. The flow cytometry-based strategy co-staining with anti-NDUFB10, VDAC 1, and TFAM has been shown to allow the measurement of specific levels of complex I and mtDNA relative ...

Materials

Name	Company	Catalog Number	Comments
Antibodies using in flow cytometry
anti-DUFB10 Alexa Fluor 405	NOVUS biologicals	NBP2-72915AF405
anti-VDAC1 Alexa Fluor 647	Santa cruz technology	sc-390996
anti-TFAM Alexa Fluor 488	Abcam	ab198308
L/D fixable near-IR dead cell stain kit	Life technologies	L10119
Antibodies using in immunofluorence staining
anti-Tuj1	Abcam	ab78078
anti-SOX2	Abcam	ab97959
anti-Alexa Flour 488	Thermo Fisher Scientific	A28175
anti-Alexa Flour 594	Thermo Fisher Scientific	A-21442
Commercial cells
AG05836 (RRID:CVCL_2B58)			Provided by Gareth John Sullivan from the Institute of Basic Medical Sciences at the University of Oslo, Norway
Essential 8 Medium (iPSC culture medium)
Essential 8 Basal Medium	Thermo Fisher Scientific	A1516901
Essential 8 Supplement (50x) 2% (v/v)	Thermo Fisher Scientific	A1517101
Store at 4 °C and warm up to RT before use.
Instruments
Heracell 150i CO2 Incubators	Fisher Scientific, USA
Orbital shakers - SSM1, SSL1	Stuart Equipment, UK
CCD Microscope Camera Leica DFC3000 G	Leica Microsystems, Germany
Water Bath Jb Academy Basic Jba5 JBA5 Grant Instruments	Grant Instruments, USA
Fluid aspiration system BVC control	Vacuubrand, Germany
Leica TCS SP8 STED confocal microscope	Leica Microsystems, Germany
50 mL falcon tube	Sigma-Aldrich	CLS430828
BD LSR Fortessa	BD Biosciences, USA
Flowjo Sampler Analysis	FlowJo LLC, USA
10 mL pipette	Sigma-Aldrich	SIAL1100
1, 10, 100, 1000 mL pipette	Sigma-Aldrich
40 µm Cell stariner	Sigma-Aldrich	CLS431750
ultra-low attachment 96-well plate	S-BIO	MS-9096UZ
Countess II automated cell counter	Thermo Fisher Scientific
Neural differentiation medium  (NDM+)
DMEM/F12	Life technologies	11330032
Neurobasal medium	Life technologies	2110349
Insulin 0.025% (v/v)	Roche	11376497001
MEM-NEAA 0.5% (v/v)	Life technologies	11140050
Glutamax supplement 1% (v/v)	Life technologies	35050
Penicilin/Streptomycin 1% (v/v)	Life technologies	15140-122
N2 supplement 0.5% (v/v)	Life technologies	17502-048
B27 supplement 1% (v/v)	Life technologies	17504-044
β-Mercaptoethanol 50 µM	Sigma-aldrich	M3148
BDNF 20 ng/mL	Peprotech	450-02
Ascorbic acid 200 µM	Sigma-Aldrich	A92902
Store at 4° C for upto 2 weeks
Neural differentiation medium minus viatmin A (NDM-)
DMEM/F12	Life technologies	11330032
Neurobasal medium	Life technologies	2110349
Insulin 0.025% (v/v)	Roche	11376497001
MEM-NEAA 0.5% (v/v)	Life technologies	11140050
Glutamax supplement 1% (v/v)	Life technologies	35050
Penicilin/Streptomycin 1% (v/v)	Life technologies (recheck)	15140-122
N2 supplement 0.5% (v/v)	Life technologies	17502-048
B27 supplement W/O vit. A 1% (v/v)	Life technologies	12587010
β-Mercaptoethanol 50 µM	Sigma-aldrich	M3148
Store at 4° C for upto 8 days
Neural Induction Medium (NIM)
DMEM/F12	Life technologies	11330032
Knockout serum replacement 15% (v/v)	Life technologies	10828028
MEM-NEAA 1% (v/v)	Life technologies	11140050
Glutamax supplement 1% (v/v)	Life technologies	35050
β-Mercaptoethanol 100 µM	Sigma-Aldrich	M3148
LDN-193189 100 nM	Stemgent/Reprocell	04-0074
SB431542 10 µM	Tocris	1614
XAV939 2 µM	Sigma-Aldrich	X3004
Store at 4° C for upto 10 days
Neutralisation medium
IMDM	Life technologies	21980032
FBS 10%	Sigma-Aldrich	12103C
Other reagents
DPBS (Ca2+/Mg2+ free)	Thermo Fisher Scientific	14190250
Bovine Serum Albumin	Europa Bioproducts	EQBAH62-1000
Accutase	Life technologies	A11105-01
Geltrex	Life technologies	A1413302
EDTA	Life technologies	15575038
Advanced DMEM / F12	Life technologies	12634010
Neural tissue dissociation kit	Miltenyi biotec	130-092-628
Y-27632 dihydrochloride Rock Inhibitor	Biotechne Tocris	1254
Fluoromount-G™ Mounting Medium	SouthernBiotech	0100-20
PFA	Thermo Fisher Scientific	28908