**1. Introduction**

Mitochondrial function is essential to meet energy demand and coordinate cellular function. Mitochondria are often referred to as the powerhouse of the cell. However, in addition to converting nutrients into energy that can be used by the cell, mitochondria also play an important role in intercellular signaling, calcium buffering, biosynthesis, and apoptosis [1]. Consumed nutrients get broken down into small molecules that can be used either to enter catabolic or anabolic processes. In the catabolic processes, nutrients break down into small molecules that are used to fuel the mitochondrial electron transport chain (ETC) to either produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) or generate heat. On the other hand, the anabolic processes involve building up molecules that can be used by the cells as storage or for structural purposes. The balance between catabolism and anabolism defines the metabolic signature in every tissue [2–5]. Therefore, mitochondrial metabolism is critical for maintaining energetic homeostasis and

**Citation:** Acin-Perez, R.; Benincá, C.; Shabane, B.; Shirihai, O.S.; Stiles, L. Utilization of Human Samples for Assessment of Mitochondrial Bioenergetics: Gold Standards, Limitations, and Future Perspectives. *Life* **2021**, *11*, 949. https://doi.org/ 10.3390/life11090949

Academic Editors: Giorgio Lenaz and Salvatore Nesci

Received: 28 July 2021 Accepted: 23 August 2021 Published: 10 September 2021

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mitochondrial dysfunction promotes the development and progression of metabolism- and aging-related disorders.

Mitochondrial diseases are genetically inherited, clinically heterogeneous disorders mainly affecting post-mitotic tissues with high energy demand, such as the brain, heart, and skeletal muscle. Since symptoms are varied, affecting multiple organs in the body, diagnosing mitochondrial disease can be challenging. Genetic testing (Figure 1) is the most reliable way to diagnose and categorize a mitochondrial disorder but requires the right choice of starting material and sequencing technique [6]. Specifically, for mitochondrial deoxyribonucleic acid (mtDNA) encoded diseases, the variability observed by mutational heteroplasmy and copy number in different tissues needs to be considered. A negative genetic result does not exclude a mtDNA-related disorder and sampling of tissues such as the urinary tract and buccal epithelial cells or skeletal muscle is often needed. Moreover, some mtDNA variants, specifically deletions and depletion-causing mutations, are detectable only in the high-energy demand affected tissue [7]. Therefore, even with genetic testing available, molecular diagnosis often requires functional characterization. For that, muscle biopsy is the gold standard sample to perform enzymatic activities and/or histology in clinical settings. In research, besides the use of muscle biopsies, skin biopsy-derived fibroblasts are also used for functional characterization of mitochondrial disorders with newly discovered mutations and genes. In this case, assessing the ETC function is required for analysis of mitochondrial-dependent cell metabolism with measuring oxygen consumption, namely respirometry, as the gold standard measurement of mitochondrial function. Respirometry provides an integrated measurement of oxidative mitochondrial metabolism. Platforms and applications for measuring respiration in fresh samples are well established and have been extensively described and reviewed [8–24].

However, measuring respiration in primary tissue samples from humans can be challenging given that a muscle biopsy may not be accessible, sample amount may be limiting, and, when measuring respiration using the traditionally established protocols, there is the limitation that fresh tissue and extensive processing is required. This is due to the fact that freeze-thawing samples damages membranes resulting in cytochrome c release from the mitochondrial intermembrane space, which makes the electron transport chain inefficient as cytochrome c is an essential electron carrier. In addition, damage to mitochondrial membranes causes respiration to uncouple energy production. For these reasons, respirometry must be performed quickly and is done primarily on-site, which limits the use of respirometry as a diagnostic approach, for use in clinical trials, and in previously frozen samples stored in biobanks. These limitations make translational mitochondrial research challenging and largely unfeasible on a large scale. To address these challenges, new approaches are emerging to make use of existing samples for respirometry and use less invasive samples, which will be discussed later in this review.

**Figure 1.** Overview of human samples and bioenergetic assessment used for clinical diagnosis and research. The primary approach for medical diagnosis of mitochondrial disorders (green boxes) is genetic testing of non-invasive or minimally invasive samples. Follow-up testing may be required in skeletal muscle biopsies when additional molecular characterizations are needed for diagnosis or to determine the extent of mitochondrial dysfunction. These include histology and biochemical analysis. For mitochondrial function evaluation in research (purple box), skeletal muscle, primary fibroblasts, and circulating blood samples are routinely used for molecular and biochemical analysis, histology, and bioenergetic testing. Further optimization and development of non-invasive samples may provide a future path for expanding bioenergetics research in human subjects to include large-scale population studies and clinical trials. Created with BioRender.com on 10 August 2021.
