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

Wahid, Maheen; Mackenzie, Graeme; Rooney, Liam M.; Greig, Justin C.; McConnell, Gail; Combet, Emilie; Gray, Stuart; Murray, James; Currie, Susan; Gould, Gwyn W.; Cunningham, Margaret R.

doi:10.1186/s44330-024-00017-0

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

Maheen Wahid

, Graeme Mackenzie, Liam M. Rooney

, Justin C. Greig

, Gail McConnell

, Emilie Combet

, Stuart Gray

, James Murray

, Susan Currie, Gwyn W. Gould

, Margaret R. Cunningham

BMC Methods, Volume: 1, Start page: 16

Swansea University Author: James Murray

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DOI (Published version): 10.1186/s44330-024-00017-0

Abstract

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 misinterp...

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Published in:	BMC Methods
ISSN:	3004-8729
Published:	Springer Nature 2024
Online Access:	Check full text
URI:	https://cronfa.swan.ac.uk/Record/cronfa68448

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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. 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spelling	2025-02-04T14:12:35.6070009 v2 68448 2024-12-03 From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies 12d0a585fcfe66f83c4c21c6bca3197b 0000-0002-6928-2347 James Murray James Murray true false 2024-12-03 MEDS 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. Journal Article BMC Methods 1 16 Springer Nature 3004-8729 Skeletal muscle biopsy, sample handling, processing, storage, imaging, mitochondria, analysis 2 12 2024 2024-12-02 10.1186/s44330-024-00017-0 Methodology COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University Another institution paid the OA fee MW is funded as a PhD student through the University of Strathclyde Faculty of Science Global Research Scholarship Programme (awarded 2023) Reference number 229057368. JM is funded by Medical Research Scotland PhD Reference PhD-50007–2-19. LMR is funded by Leverhulme Trust. The EVOS FL Auto microscope was purchased through a gift to the University of Strathclyde provided by Mr. Ken Wye Saw. 2025-02-04T14:12:35.6070009 2024-12-03T13:33:55.8996470 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science Maheen Wahid 0000-0003-1500-0671 1 Graeme Mackenzie 2 Liam M. Rooney 0000-0002-2237-501X 3 Justin C. Greig 0009-0004-5624-2030 4 Gail McConnell 0000-0002-7213-0686 5 Emilie Combet 0000-0002-9302-8971 6 Stuart Gray 0000-0001-8969-9636 7 James Murray 0000-0002-6928-2347 8 Susan Currie 9 Gwyn W. Gould 0000-0001-6571-2875 10 Margaret R. Cunningham 0000-0001-6454-8671 11 68448__33038__fc7b3e2559bf411ab8580125ce66a18d.pdf 44330_2024_Article_17.pdf 2024-12-03T13:33:55.8995961 Output 5573258 application/pdf Version of Record true © The Author(s) 2024. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License (CC-BY 4.0). true eng http://creativecommons.org/licenses/by/4.0/
title	From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies
spellingShingle	From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies James Murray
title_short	From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies
title_full	From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies
title_fullStr	From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies
title_full_unstemmed	From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies
title_sort	From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies
author_id_str_mv	12d0a585fcfe66f83c4c21c6bca3197b
author_id_fullname_str_mv	12d0a585fcfe66f83c4c21c6bca3197b_***_James Murray
author	James Murray
author2	Maheen Wahid Graeme Mackenzie Liam M. Rooney Justin C. Greig Gail McConnell Emilie Combet Stuart Gray James Murray Susan Currie Gwyn W. Gould Margaret R. Cunningham
format	Journal article
container_title	BMC Methods
container_volume	1
container_start_page	16
publishDate	2024
institution	Swansea University
issn	3004-8729
doi_str_mv	10.1186/s44330-024-00017-0
publisher	Springer Nature
college_str	Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id	facultyofmedicinehealthandlifesciences
hierarchy_top_title	Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id	facultyofmedicinehealthandlifesciences
hierarchy_parent_title	Faculty of Medicine, Health and Life Sciences
department_str	Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
document_store_str	1
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description	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.
published_date	2024-12-02T05:33:19Z
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From freeze to function: optimised cryopreservation and mitochondrial analysis workflow for skeletal muscle biopsies

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