Summary

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

Published: August 04, 2023
doi:

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 damage6,7,8,9. 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 organoids10,11,12, 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 Preparation of matrix-coated plates 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). Thaw the membrane matrix solution at 4 °C (keep it cold) and coat the required numbe…

Representative 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 …

Divulgations

The authors have nothing to disclose.

Acknowledgements

We extend our sincere gratitude to Gareth John Sullivan from the Institute of Basic Medical Sciences at the University of Oslo, Norway, for generously providing us with the AG05836 (RRID:CVCL_2B58) cell line. We kindly thank the Molecular Imaging Centre, Flow Cytometry Core Facility at the University of Bergen in Norway. This work was supported by the following funding: K.L was partly supported by the University of Bergen Meltzers Høyskolefonds (project number:103517133) and Gerda Meyer Nyquist Guldbrandson og Gerdt Meyer Nyquists legat (project number: 103816102). L.A.B was supported by the Norwegian Research Council (project number: 229652), Rakel og Otto Kr.Bruuns legat and Gerda Meyer Nyquist Guldbrandson og Gerdt Meyer Nyquists legat.

Materials

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

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Citer Cet Article
Marjan, S., Yangzom, T., Kristiansen, C. K., Chen, A., Bindoff, L. A., Liang, K. X. Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids. J. Vis. Exp. (198), e65621, doi:10.3791/65621 (2023).

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