The Translational Path of Mitochondrial Transplantation for Pulmonary Fibrosis

1. Introduction: The Need for New Therapeutic Strategies in IPF

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disease that affects approximately five million people worldwide and is associated with a poor prognosis, with a median survival of only 2-5 years following diagnosis [1,2] . IPF is one of the most common and severe forms of interstitial lung disease (ILD), a diverse group of more than 200 disorders characterized by varying degrees of inflammation and fibrosis within the lung parenchyma [3] . Although the cause of IPF remains unknown, both genetic and environmental factors are thought to contribute to abnormal healing and increased susceptibility of the alveolar epithelium to injury [4] . Current models of IPF pathogenesis propose that repeated epithelial injury and aberrant activation of alveolar epithelial cells promote the migration and proliferation of fibroblasts and their differentiation into myofibroblasts. The persistence of these activated myofibroblasts leads to the formation of fibroblastic foci and excessive deposition of extracellular matrix (ECM) [5] . Over time, the accumulation of ECM remodels normal lung architecture, leading to irreversible fibrosis, honeycomb cyst formation, loss of pulmonary function and, ultimately, respiratory failure [6] .

Despite advances in research, there is currently no cure for IPF. Pharmacological management primarily consists of antifibrotic agents, including pirfenidone, nintedanib, and, more recently, the FDA-approved phosphodiesterase 4B inhibitor—nerandomilast [6] . These therapies can slow down the decline in lung function but cannot reverse established fibrosis or recover lung function. Furthermore, treatment-associated adverse effects often limit long-term tolerability and adherence to these drugs.

For patients with advanced IPF, lung transplantation (LTx) remains the only intervention capable of significantly extending survival [7] . However, LTx is available to only a proportion of patients because of the limited supply of donor organs, strict eligibility criteria, and the risk of post-transplant complications [7,8] . Outcomes also remain a concern, as patients transplanted for IPF have been reported to experience poorer post-transplant outcomes than recipients transplanted for other chronic respiratory diseases, including chronic obstructive pulmonary disease and cystic fibrosis [9] .

Together, these limitations highlight the urgent need for innovative therapeutic approaches that go beyond slowing disease progression to target the fundamental mechanisms driving fibrosis. One emerging approach is mitochondrial transplantation (MTx), which has been investigated for its potential to restore cellular bioenergetics, enhance tissue repair, and improve regenerative capacity in the injured lung [10] .

2. Mitochondrial Dysfunction in the Pathogenesis of IPF

IPF develops through the dysregulation of multiple interconnected cellular and molecular pathways, many of which overlap with biological processes associated with aging. Aging is one of the strongest risk factors for IPF. Many of the hallmarks of aging including cellular senescence, impaired proteostasis, and mitochondrial dysfunction, are prominent features of the disease. Among these, mitochondrial dysfunction has emerged as a contributor to IPF pathogenesis and progression [11] . Increasing evidence suggests that disruptions in mitochondrial quality control mechanisms, including mitochondrial biogenesis, mitochondrial dynamics (fusion and fission), and mitophagy, have roles in the development of fibrotic lung remodeling [11,12,13,14] .

Mitochondria are often referred to as the “energy powerhouse of the cell”; however, these organelles have further functions beyond producing about 90% of cellular ATP through oxidative phosphorylation. Mitochondria regulate several processes, including ionic homeostasis, innate immune signalling, and apoptosis, many of which are relevant to pulmonary fibrosis [15,16,17] . When mitochondria are unable to effectively carry out their essential functions, defects in oxidative phosphorylation, loss of mitochondrial membrane potential, defective respiratory chain activity, and excessive generation of mitochondrial reactive oxygen species (mtROS) can promote cellular stress, senescence, and apoptosis [11] . Dysfunction of these processes contributes to abnormal tissue repair and the persistence of fibrogenic signalling within the injured lung.

Evidence of mitochondrial dysfunction has been identified in several cell populations involved in IPF. Alveolar epithelial type II (AECII) cells, which play an important role in maintaining and repairing the alveolar epithelium, contain enlarged and structurally abnormal mitochondria in IPF lungs and show reduced mitochondrial abundance compared with healthy controls [12] . Reduced expression of PTEN-induced kinase 1 (PINK1), a key regulator of mitophagy, has been associated with defective clearance of damaged mitochondria and the accumulation of mitochondrial stress in AECII cells [12] . Similarly, dysregulation of mitofusins, which regulate mitochondrial fusion, have been linked to disrupted mitochondrial dynamics and the development of pulmonary fibrosis [14] . In lung fibroblasts, reduced expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a master regulator of mitochondrial biogenesis, has been associated with persistent fibroblast activation and enhanced ECM deposition [13] . Together, these findings suggest that defects in mitochondrial quality control may contribute to IPF through effects on both epithelial dysfunction and fibroblast-driven fibrosis (Figure 1).

Taken together, this evidence supports the concept that mitochondrial dysfunction is not only a consequence of fibrosis, but also an active driver of disease initiation and progression [16,18] . Importantly, mitochondrial dysfunction in IPF involves several interconnected processes, raising the possibility that therapeutic approaches targeting a single signalling pathway may have limited efficacy. This has generated considerable interest in mitochondrial replenishment strategies, including mitochondrial transplantation, which aim to restore functional mitochondria and re-establish cellular bioenergetic homeostasis. By simultaneously improving energy production, reducing oxidative stress, and potentially correcting several interconnected mitochondrial defects, mitochondrial transplantation has emerged as a promising therapeutic approach to mitigate fibrotic remodelling [19] .

3. Mitochondrial Transplantation (MTx)

Mitochondrial transplantation therapy has recently emerged as a promising regenerative medicine strategy for diseases associated with mitochondrial dysfunction [19] . It has shown therapeutic benefits in preclinical models of several human diseases, including ischemia-reperfusion injury in multiple organs [10,20,21,22] , neurodegenerative disorders [23] , and pulmonary diseases [10] .

The approach relies on the isolation and delivery of functional mitochondria from autologous, allogeneic, or xenogeneic cell or tissue sources [24,25] . The isolated mitochondria can be administered through a variety of routes, including co-incubation in cell culture systems [26] , direct tissue injection [27] , intravenous infusion [28] , intratracheal delivery [29] , and nebulization [21] . Following administration, recipient cells rapidly internalize exogenous mitochondria through endocytic mechanisms, allowing their integration into the recipient’s mitochondrial network [19,30,31] .

Advances in mitochondrial labelling techniques such as mitochondrial probes and fluorescently tagged mitochondria have improved the ability to track mitochondrial uptake and intracellular fate [32] . Biomaterial-based approaches involving nanoparticles and biopolymers have also been developed to enhance mitochondrial stability and delivery efficiency [26,29,33] . Collectively, these findings support MTx as a therapeutic strategy capable of targeting multiple aspects of mitochondrial dysfunction simultaneously.

4. Current Evidence Supporting MTx in Pulmonary Fibrosis

Growing evidence supports the therapeutic potential of MTx in pulmonary fibrosis. Preclinical studies have demonstrated that the delivery of healthy mitochondria can improve mitochondrial function, reduce fibrotic responses, and restore cellular homeostasis in experimental models of lung injury and fibrosis [10] .

However, the available evidence is largely limited to in vitro and animal (non-human) studies, each of which captures only selected features of IPF and fails to fully reproduce the complexity of the human disease [34,35] . Consequently, the translation of these promising findings into clinical therapies remains challenging.

To better understand the opportunities and limitations of MTx in IPF, the following sections examine the experimental platforms currently used in the field, with particular emphasis on their physiological relevance, translational value, and ability to bridge the gap between mechanistic discovery and clinical application.

5. Experimental Platforms for Studying MTx in IPF

5.1. Animal Models

Animal models of pulmonary fibrosis have played an important role in the preclinical evaluation of novel therapeutic strategies [34] , including MTx [28,29] . Among these, the bleomycin-induced mouse model remains the most widely used and extensively characterized. These models have been invaluable for investigating mechanisms of fibrogenesis and assessing the biodistribution, pharmacodynamics, safety, and therapeutic efficacy of emerging interventions in vivo. However, despite decades of promising preclinical findings, relatively few therapies that demonstrate efficacy in animal models have successfully translated into effective treatments for patients with IPF [34] .

This limited translation reflects the inherent challenges of modelling IPF in animals. Unlike human IPF, which is a heterogeneous, progressive disease of unknown etiology, bleomycin-induced fibrosis results from a defined acute injury and does not fully recapitulate the histopathological features of usual interstitial pneumonia or the chronic disease course observed in patients. Nevertheless, animal models remain essential for evaluating therapeutic efficacy within the context of systemic physiology and for investigating treatment-related effects that cannot be assessed in vitro. Adherence to the American Thoracic Society’s recommendations for preclinical pulmonary fibrosis studies may improve the translational value and rigour of these investigations [34] .

At least two studies have demonstrated the feasibility of MTx in animal models of pulmonary fibrosis using different delivery routes. Lee et al. showed that mitochondria isolated from human umbilical cord mesenchymal stem cells attenuated bleomycin-induced lung fibrosis and accelerated post-injury weight recovery following intravenous administration [28] . Similarly, Wang et al. reported that intratracheally delivered mitophagy-enhanced engineered mitochondria (Mito-MEN) restored mitochondrial function and reduced fibrosis-associated phenotypes in bleomycin-treated mice [29] . These experimental findings in mice support the therapeutic potential of MTx in vivo and provide important proof-of-concept evidence for its application in pulmonary fibrosis.

Despite these encouraging results, whether the benefits observed in animal models will translate to patients remains uncertain. Consequently, animal studies should be viewed as a step in the preclinical development pipeline rather than definitive predictors of clinical efficacy given their limited ability to recapitulate the complexity and progression of human IPF.

5.2. 2D Cell Culture

Two-dimensional (2D) cell culture systems are the most widely used in vitro models for studying IPF and typically employ cell types involved in disease pathogenesis, including fibroblasts, alveolar epithelial cells, endothelial cells, and macrophages derived from healthy or diseased human or animal tissues [35] . These models provide a highly controlled platform for investigating disease mechanisms and evaluating novel therapeutic strategies. Their simplicity, reproducibility, and scalability make them particularly attractive for studying MTx.

Fibroblast-based models are commonly used because fibroblast activation, myofibroblast differentiation, and excessive ECM deposition are central features of IPF. Primary fibroblasts isolated from healthy donors or IPF lungs, as well as immortalized cell lines, are frequently stimulated with transforming growth factor-β (TGF-β) to induce a profibrotic phenotype and to model key aspects of fibrosis in vitro [26,35] .

Baudo et al. treated TGF-β-stimulated fibroblasts with a functionalized mitochondrion for 24 hours. MTx partially restored cellular bioenergetics and reduced myofibroblast differentiation and ECM production, providing proof of concept for the antifibrotic potential of MTx [26] . However, these findings require validation in primary IPF cells and more physiologically relevant models.

Epithelial cell models are also widely used because epithelial injury and dysfunction are recognized drivers of fibrosis [35] . Lee et al. investigated MTx in TGF-β-treated fibroblasts and bronchial epithelial cells using mitochondria isolated from human umbilical cord mesenchymal stem cells. Mitochondrial treatment reduced mitochondrial injury, attenuated epithelial-to-mesenchymal transition, and decreased ECM accumulation following co-incubation [28] .

More complex 2D systems have also been developed to model epithelial-fibroblast interactions. These include conditioned media approaches and transwell co-culture systems, in which epithelial cells and fibroblasts communicate via soluble factors while remaining physically separated [35,36,37,38,39] . Such models provide additional insight into intercellular signalling and may represent useful platforms for evaluating mitochondrial-based therapies.

Overall, 2D cell culture models are valuable for defining cell-specific mechanisms, confirming mitochondrial uptake, and assessing the bioenergetic and antifibrotic effects of MTx. However, their major limitation is the inability to reproduce the multicellular complexity, ECM architecture, and mechanical properties of the fibrotic lung. Consequently, findings obtained in 2D systems should be considered as an important first step that requires validation in more complex experimental platforms [35] .

5.3. Organoid Models

Three-dimensional (3D) in vitro models provide a more physiologically relevant platform for studying IPF than conventional 2D cultures [35] . These models include hydrogel-based constructs, spheroids, and organoids that better recapitulate the architecture and cellular interactions of lung tissue. Spheroids are self-assembled 3D structures composed of lung fibroblasts, epithelial cells, macrophages, or combinations thereof, derived from either cell lines or primary healthy and diseased tissues [35,39,40,41] .

Among these platforms, lung organoids represent a major advance in disease modelling. Derived from stem cells, organoids contain multiple lung cell populations organized into complex 3D structures that mimic key features of human lung development and tissue organization [35] . As a result, they provide a valuable system for investigating fibrotic mechanisms and evaluating MTx in a controlled yet biologically relevant environment. Compared with 2D cultures, organoids better capture cell-cell interactions and tissue-level responses, making them particularly attractive for studying therapeutic efficacy [35,42] .

However, several challenges limit their application in MTx studies. The dense ECM and increased structural complexity of organoids may impair the penetration, distribution, and uptake of exogenous mitochondria. In addition, the absence of a functional vascular network can result in oxygen and nutrient gradients, leading to hypoxia and reduced viability in the organoid core [24,42,43] . These limitations should be carefully considered when designing and interpreting MTx experiments.

5.4. Lung-on-a-Chip Models

Lung-on-a-chip devices are microfluidic platforms that recreate key structural and functional features of the lung by co-culturing multiple lung-relevant cell types on opposite sides of a thin, porous membrane that mimics the alveolar-capillary interface [35] . Unlike conventional 2D and 3D culture systems, these platforms incorporate controlled fluid flow and/or mechanical stimuli, such as cyclic stretch to simulate breathing, thereby providing a more physiologically relevant environment.

The integration of microfluidics represents a major advantage of lung-on-a-chip technology, as it enables the study of cell-cell interactions, vascular transport, and tissue responses under dynamic conditions that more closely resemble the alveolar units. Several lung fibrosis-on-a-chip models have been successfully developed and applied to disease modelling and drug discovery [35,44] . These features make lung-on-a-chip systems particularly attractive for investigating mitochondrial-based therapies, as they may permit the evaluation of mitochondrial delivery, uptake, biodistribution, and therapeutic efficacy within a biomimetic setting [46] .

Despite these advantages, lung-on-a-chip platforms remain technically demanding, costly, and relatively low throughput compared with conventional cell culture systems. Furthermore, most current models do not fully reproduce the cellular diversity and long-term remodelling observed in IPF. Nevertheless, by incorporating physiological architecture, mechanical forces, and vascular-like flow, lung-on-a-chip technology represents a promising intermediate platform that may help bridge the gap between simplified in vitro studies and in vivo models [35,45] .

5.5. Precision-Cut Lung Slices (PCLS)

Precision-cut lung slices are a valuable ex vivo platform for studying pulmonary diseases and accelerating drug discovery. PCLS are generated from human or animal lung tissue, including donor lungs, explanted lungs, or surgical specimens, which are inflated with low-melting-point agarose and sectioned into thin slices that can be maintained in culture for several days to weeks [46,47] .

A major advantage of PCLS is the preservation of the native three-dimensional lung architecture, including resident cell populations, ECM composition, and endogenous cell-cell interactions. To model pulmonary fibrosis, PCLS can be generated directly from fibrotic lungs or derived from healthy tissue and stimulated with profibrotic mediators to induce fibrosis-like features [47] . This flexibility enables the evaluation of therapeutic strategies in human disease within a complex tissue environment that more closely resembles the diseased human lung than conventional cell culture systems [35] .

PCLS may be particularly well suited for studying MTx, as they allow simultaneous assessment of mitochondrial uptake, tissue bioenergetics, inflammatory responses, and fibrotic remodelling within an intact multicellular microenvironment. However, several limitations should be considered. Similarly to organoids, PCLS lack a functional vascular circulation, which may influence mitochondrial distribution and long-term tissue viability. Tissue thickness, agarose content, and culture duration may also affect mitochondrial penetration, uptake efficiency, and experimental outcomes. Furthermore, access to human lung tissue is limited to specialized centers with donor or explanted lungs [35,47,48] .

Overall, PCLS may represent one of the most powerful pre-clinical platforms to evaluate mitochondrial related treatment. By preserving native lung architecture while maintaining experimental control, they offer a highly translational system for evaluating mitochondrial-based therapies in pulmonary fibrosis [47,48] .

5.6. Explanted Human IPF Lungs and Ex Vivo Lung Perfusion

Ex vivo lung perfusion (EVLP) is an advanced platform that enables the assessment, preservation, and treatment of intact lungs outside the body under normothermic ventilated and perfused conditions [49] . While EVLP was originally developed to evaluate and optimize donor lungs for transplantation, it has also emerged as a powerful tool for translational lung research and development of therapeutics. Donor lungs deemed unsuitable for transplantation can be maintained on EVLP and utilized for experimental studies, offering a unique opportunity to evaluate novel therapies for human lung disease in actual human lungs.

In addition to donor lungs, explanted diseased lungs obtained from patients undergoing lung transplantation can be studied ex vivo, allowing the investigation of therapies directly within human tissue with the disease. By preserving physiological ventilation, perfusion, and tissue architecture, EVLP provides a highly relevant platform for assessing therapeutic efficacy, biodistribution, and safety. For mitochondrial transplantation studies, both intravenous and intratracheal routes of delivery can be explored in a controlled and clinically representative environment.

Although EVLP has already been used to investigate MTx in models of ischemia-reperfusion injury [50] , its application to pulmonary fibrosis remains largely unexplored. Nevertheless, the ability to evaluate therapeutic interventions in intact human lungs makes EVLP one of the most important translational platforms available for preclinical testing of mitochondrial-based therapies.

The major limitations of EVLP include high cost, technical complexity, relatively short experimental duration and limited availability; as specialized equipment, expertise, and access to donor or explanted lungs are typically restricted to lung transplant centers. Despite these challenges, EVLP represents a critical bridge between conventional preclinical models and clinical implementation (Figure 2).

6. Conclusions

Mitochondrial transplantation is an emerging therapeutic strategy for IPF that aims to address a driver of disease progression: mitochondrial dysfunction. While promising results have been reported in preclinical studies, significant challenges remain before this approach can be translated into clinical practice.

The selection of appropriate experimental models is critical for advancing the field. Each platform offers distinct advantages and limitations, ranging from the mechanistic simplicity and high throughput of 2D cultures to the high translational relevance of EVLP and human lung tissue models. Combining complementary platforms may provide the most effective strategy for evaluating mitochondrial-based therapies and accelerating clinical translation. Finally, greater emphasis on methodological studies and on reporting both positive and negative findings will improve reproducibility, refine experimental approaches, and help move mitochondrial transplantation closer to becoming a viable therapy for pulmonary fibrosis.