From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies

Background: Skeletal muscle biopsies are valuable in clinical and research settings, contributing to advancements in diagnosing, understanding, and treating muscle-related conditions. Traditional freezing methods often cause artefacts mistaken for disease, leading to incorrect diagnoses or misinterpretation of research findings. Proper handling of muscle biopsies is critical for accurate histopathological and mitochondrial analysis. It is essential to preserve the entire tissue, especially for small needle biopsies. While most research focuses on mitochondrial analysis in cells, there are few studies on whole tissue samples. This study aimed to provide an effective methodological workflow to improve cryopreservation techniques for human and rodent muscle biopsies and create a reliable method for mitochondrial analysis in muscle tissues. Methods: Human muscle samples were preserved with different concentrations of formaldehyde after freezing with liquid nitrogen to study the effects of freeze–thaw cycles. We compared the edge and belly of muscle samples embedded in Optimal Cutting Temperature compound (OCT) to see how OCT affects ice crystal formation. Rat muscle biopsies were frozen using six different methods, using liquid nitrogen and precooled isopentane as freezing media. Each medium involved direct immersion, OCT dip before immersion, and placement in histocassettes before immersion. Effectiveness of these methods was evaluated using histological and immunohistochemical staining. Mitochondrial analysis in type I and II myofibres was attempted by employing the Trainable Weka Segmentation plugin using Fiji. Results: Histologically stained human tissue sections showed that freeze–thaw and formaldehyde fixation led to freezing artefacts, disrupted endomysium, and widely spaced cells. Quantitative differences in ice crystal artefacts between edge and belly of rat whole muscle samples demonstrated effects of OCT in crystal formation. Histological and immunohistochemical staining of sections from rat muscle biopsies frozen in six different cryopreservation techniques revealed that only isopentane/histocassette combination preserved tissue integrity in both core and periphery of tissue sections. Moreover, an optimised Fiji workflow enabled accurate quantification and mapping of mitochondrial networks. Discussion: The isopentane/histocassette combination is an effective cryopreservation method, ensuring artefact-free preservation of both core and periphery of tissue sections. Our workflow utilising Trainable Weka Segmentation plugin provides a reliable method for mitochondrial analysis in skeletal muscle tissues, facilitating future studies in muscle research.