Cell lysis is a crucial step to conduct functional mitochondrial assays, where the primary objective is to provide access to the enzymes in the mitochondrial membranes and matrix while preserving their native activities. Mitochondria are organelles frequently referred to as the powerhouse of the cell, due to their vital function of generating ATP through oxidative phosphorylation (OXPHOS) (Glover et al., 2024, Mitchell, 1961, Monzel et al., 2023). OXPHOS dysfunction leads to mitochondrial diseases (MDs) or mitochondriopathies. Mitochondria exist in most tissues and play a critical role in cellular metabolism so their disfunction affects most functions in the body (Gropman, 2001). Organs and tissues with high energy demand, such as the brain, eyes, and cardiac and skeletal muscles, are particularly susceptible to energy failure due to OXPHOS defects. This leads to phenotypes often manifesting in ophthalmological, neurological, and cardiological systems (Klopstock et al., 2021).

MDs results from deleterious mutations of either mitochondrial DNA (mtDNA) or of nuclear DNA encoding for mitochondrial proteins related to organelle maintenance and translation, such as mitochondrial myophaty, Leigh syndrome, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and chronic progressive external ophthalmoplegia (CPEO) (Goto et al., 1990, Lake et al., 2016, Van Goethem et al., 2001).

Currently, MDs are being labeled as pathway-base diseases rather than merely energy-deficit sicknesses (McCormick et al., 2013, Wen et al., 2025). This is due to the fact that mitochondria are involved in multiple cellular metabolic processes, performing intricate functions networks and information processing, which are pivotal in both health regulation and diseases progression (Zong et al., 2024). Hence, many common pathologies cause secondary mitochondrial dysfunction (SMD), such as neurodegeneration, heart failure, chronic degenerative manifestations, metabolic syndrome, inflammation, autoimmune diseases, and cancer (Chouchani et al., 2014, Eldeeb et al., 2022, Prasun, 2020, Russell et al., 2020, Xu et al., 2025). Furthermore, dysfunctional proteostasis, abnormal dynamics and quality control, inhibited ATP production, calcium dyshomeostasis, and metabolic reprogramming often are concurrent and interact within pathological conditions, thereby impacting mitochondria's function as biosynthetic and signaling hubs (Murphy and Hartley, 2018, Picard and Shirihai, 2022).

Several diagnostic methodologies for MDs have been developed. Some of them involve respiratory chain and Krebs cycle enzyme analysis, requiring the use of tissue biopsies. This is critical in cases where genetic testing has failed to provide a definitive diagnosis (Parikh et al., 2019). Muscle tissue biopsies are often preferred due to the tissue´s high energy demands, which make it highly susceptible to OXPHOS dysfunction, thus establishing it as the gold standard method for MDs studies (Gill et al., 2023, Wen et al., 2025). However, the invasive nature of the technique limits tissue sampling, thus restricting the number of experimental assays that can be conducted. Besides, the obtention of a single sample precludes the performance of comparative, longitudinal, and prospective clinical studies. This problem has been resolved using primary fibroblasts cultures that provide numerous cells from patients, that can be expanded and stored (van den Heuvel et al., 2004).

Fibroblast cultures have emerged as a promising alternative to evaluate mitochondrial function (Saada, 2014), and can be applied to chronic degenerative diseases, cosmetology, and cell toxicity, among others (Law and Ren, 2023, Letsiou, 2021, Li et al., 2024, Olesen et al., 2022, Park et al., 2025, Sreedhar et al., 2020, Teixeira et al., 2021). Also, skin biopsy is less invasive than muscle biopsy, and fibroblasts can be easily cultured, expanded, and preserved from a small sample, increasing the possibility to perform further studies.

Despite comprehensive mitochondrial enzymatic assays in fibroblasts, comparative studies on cell disruption methods and their impact on enzymatic activities are not available, leading to significant variability in the results from control samples disrupted using different techniques (Bujan et al., 2022, Gellerich et al., 2004, Medja et al., 2009). Thus, it is necessary to establish which cell-disruption method should be used to analyze mitochondrial enzymes without compromising their integrity and activity. To approach this problem, we here performed a systematic study, focusing on the most representative techniques among the wide range of cell lysis methods (Gomes et al., 2020, Hopkins, 1991, Islam et al., 2017).

The results show that it is possible to standardize conditions for measuring the activity of all OXPHOS complexes using a fibroblast disruption method that significantly reduces experimental variability. We believe that following a universal disruption method will increase reproducibility of the assays carried out in pharmacological comparative studies, mitochondria-targeted therapies, oxidation and antioxidation assays, mitochondrial dynamics studies (Murphy and Hartley, 2018, Xu et al., 2025), and contribute to more accurate interpretations in longitudinal and prospective studies.