Isolation, Purification, and Characterization of Functional Mitochondria Derived from Mouse Skeletal Muscle for Mitochondrial Transplantation

Summary

A standardized protocol combining trypsin digestion and differential centrifugation was established for the isolation, purification, and functional characterization of mitochondria from mouse skeletal muscle, as well as for evaluating their application in mitochondrial transplantation.

Abstract

Mitochondria are essential organelles that regulate energy metabolism, signal transduction, and cellular homeostasis in eukaryotic cells. Mitochondrial dysfunction contributes to the pathogenesis of numerous diseases and has prompted the development of mitochondrial transplantation as a regenerative therapeutic strategy. The successful application of mitochondrial transplantation depends on the availability of highly purified and functionally intact mitochondria. Skeletal muscle is a suitable donor source due to its high mitochondrial content, metabolic activity, and accessibility. This study established a standardized, reproducible protocol for the isolation, purification, and characterization of functional mitochondria from mouse skeletal muscle and evaluated their use in mitochondrial transplantation. The procedure consisted of two major stages. First, mitochondria were isolated from the skeletal muscle of C57BL/6 mice using trypsin digestion followed by differential centrifugation. Second, the isolated mitochondria were characterized to evaluate purity, ultrastructure, and functional activity. Mitochondrial purity was assessed by bicinchoninic acid (BCA) protein quantification and Western blot analysis. Ultrastructural integrity was examined by transmission electron microscopy. Functional activity was evaluated using JC-1 and mitochondrial fluorescent labeling together with measurements of oxygen consumption, ATP production capacity, and respiratory control ratio using a high-resolution respirometry system. The isolated mitochondria exhibited preserved membrane potential, intact ultrastructure, and stable respiratory activity, indicating suitability for downstream functional studies and mitochondrial transplantation applications.

Introduction

Mitochondria are the primary sites of adenosine triphosphate (ATP) production and play central roles in apoptosis, calcium homeostasis, and the metabolism of reactive oxygen species (ROS). The functional integrity of mitochondria is therefore critical for maintaining cellular homeostasis and determining cell fate^1,2. Mitochondrial dysfunction, including mitochondrial DNA (mtDNA) mutations and electron transport chain (ETC) abnormalities, leads to impaired energy metabolism, oxidative stress, and dysregulated apoptosis^3,4. These abnormalities are associ....

Protocol

All animal experiments were performed in a specific pathogen-free (SPF) facility and approved by the Animal Ethics and Use Committee of the Third Affiliated Hospital of Air Force Medical University (Approval No.: IACUC-2024kq-065).

Male C57BL/6 mice (6-8 weeks old, 18-20 g) were purchased from the Laboratory Animal Center of Air Force Medical University (Production License: SCXK (Shaanxi) 2024-002). The mice were housed in individually ventilated cages (IVCs) under positive pressure and clean conditions, with a temperature of 20-26 °C, relative humidity 40%-70%, a 12 h light-dark cycle, and at least 15 air changes per hour. Sterilized ....

Results

A schematic overview of the mitochondrial isolation and purification workflow is shown in Figure 1. All procedures were performed at 4 °C to preserve mitochondrial integrity and functional activity. Freshly isolated skeletal muscle tissue was minced and digested with trypsin to facilitate tissue dissociation, then homogenized in isotonic isolation buffer using a glass homogenizer. Differential centrifugation was subsequently performed to separate mitochondria from tissue debris and cytosolic.......

Discussion

Mitochondrial transplantation is an emerging strategy in cell therapy and regenerative medicine that restores physiological function in cells with impaired energy metabolism through the delivery of functionally intact mitochondria. In addition to regulating cellular energy metabolism, this technique can modulate key biological processes such as apoptosis and oxidative stress, thereby providing novel therapeutic opportunities for a variety of diseases^4,17,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-Cytochrome C antibody	Abcam	EPR1327	Mitochondrial Characterization
Anti-fade mounting medium	PlantChemMed	PC-90016	Reduce photobleaching of fluorescent dyes
BCA Protein Assay Kit	Zhonghui Hecai Biotechnology Co., Ltd.	PQ003	Protein concentration assay
Beta-actin antibody	Cell Signaling technology	3700s	Beta-actin antibody was used to detect the purity of isolated mitochondria
BSA	Sigma	A6003	During isolation, it binds free fatty acids to protect mitochondrial activity.
Centrifuge	Eppendorf	5418R	4 °C Centrifugation
CCCP	Solarbio	C6700	to uncouple mitochondrial oxidative phosphorylation and induce maximal proton leakage
Confocal Laser Scanning Microscope	Olympus Corporation	FV4000	Immunofluorescence Staining Observation
COX IV polyclonal antibody	Proteintech	11242-1-AP	Mitochondrial Characterization
DAPI	Plant Chem Medicine	PC-90004	For nuclear counterstaining
D-mannitol	Solarbio	IM00402	maintain osmotic balance and prevent mitochondrial swelling during isolation
EGTA	Sigma	E4378	Commonly used in mitochondrial isolation buffers to protect mitochondrial function by specifically binding Ca2+
Fetal bovine serum	Every Green	11011-08611	Culture of L929 cells
Forceps	RWD Life Science Co., Ltd.	F11003-11	Used to grasp, hold and lift tissues
Glass homogenizer	Beyotime	FGH005	Used for tissue homogenization and isolation of mitochondria
Glutaraldehyde	LEAGENE	DF0151	It rapidly fixes and stabilizes cellular ultrastructure by cross-linking proteins, preventing autolysis to preserve the natural morphology of organelles.
HEPES	Thermo Fisher	J67485	Provides a stable pH environment.
Incubator	Thermo Fisher	51032874	Culture of L929 cells
JC-1	Solarbio	M8650	Detecting changes in the membrane potential of purified mitochondria.
L929 cell line	Procell	CL-0137	For mitochondrial endocytosis assay
Lead citrate	Sigma	15326	to stain ultrathin sections for fine subcellular structure observation.
MEM Alpha basic (1x)	Gibco	6125510	Culture of L929 cells
Methanol	Fuyu Chemical (China)	67-56-1	As a component of the transfer buffer, it is used to remove SDS and facilitate the transfer of proteins from the gel to the solid-phase membrane.
Microplate Reader	Thermo Fisher	Varioskan LUX	Measure the absorbance of JC-1-stained mitochondria
Mitochondrial Membrane Potential Detection Kit	Solarbio	M8650	Detection of Mitochondrial Membrane Potential in Skeletal Muscle
Mitochondrial isolation Buffer	Solarbio	SM0020	Kits for Extraction Based on Differential Centrifugation
Mitochondria Storage Buffer	Solarbio	SM0020	For mitochondrial preservation
Mito Tracker  Red CMXRos	Yeasen Biotechnology	40741ES50	Mitochondrial Characterization
MOPS	MedChemExpress	HY-D0859	AS a buffer in solution preparation
Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (H+L)	Proteintech	RGAM001	RGAM001 targets Mouse IgG (H+L) in Western blot
Multi-rAb HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (H+L)	Proteintech	RGAR001	RGAR001 targets to Rabbit IgG (H+L) in ELISA and Western blot
Non-fat dry milk	NCM Biotech	WB6503	It is mainly used as a blocking agent to reduce background signal by occupying non-specific binding sites on the membrane.
O2k-FluoRespirometer	Oroboros Instruments	O2K	Determine mitochondrial respiratory function and oxygen metabolism.
Osmium tetroxide	Sigma	75633	or postfixation to preserve lipids and enhance membrane contrast in TEM samples.
PBS (1x)	Plant Chem Medicine	PC-00003	Clean Muscle Tissue
Penicillin-Streptomycin	Procell	PB180120	Culture of L929 cells
Phalloidin-488	Beyotime	C1035	Label cytoskeletal F-actin for fluorescence microscopic imaging
Polyvinylidene fluoride (PVDF) membrane	Millipore (Merck)	IPVH00010	Western blotting
PowerPac Basic	BIO-RAD	164-5050	For protein separation by SDS-PAGE electrophoresis
Precast gradient polyacrylamide gel (4–12%)	ACE Bioscience	ET12412LGel	Used for SDS-PAGE separation of mitochondrial and cellular total proteins
Protease inhibitor	Shaanxi Zhonghui Hecai Biomedical Technology	PL032	Inhibits the activity of endogenous and exogenous proteases, preventing the degradation of target proteins during sample processing.
RIPA lysis buffer	Zhonghui Hecai Biotechnology Co., Ltd.	PL001-2A	Used for total protein extraction of animal and cellular tissues
Sodium Chloride Injection (0.9%)	Xi’an Jingshuang Shuanghe Pharmaceutical Co., Ltd.	Approval No. H61020014	Cell/tissue experimental buffering, rinsing and osmotic pressure maintenance
Sterile gloves	Jiangsu Yangzi Lide Medical Devices Co., Ltd.	YMZ000-1	Isolate contamination and protect operators and experimental samples
Sucrose	Sucrose	S818048	Provides osmotic support and maintains mitochondrial morphology and functional integrity.
TBST	NCM Biotech	WB20500	Washing to reduce background, serving as a diluent for antibodies and blocking solutions, while protecting the stability of antigen-antibody binding by maintaining a stable pH and ionic environment.
Tissue Homogenate Assay Kit(contains Adenosine diphosphate (ADP) ,Succinate,Rotenone,Antimycin A ,Oligomycin,Pyruvate,Malate, Glutamate,Ascorbate,N,N,N',N'- tetramethyl-p-phenylenediamine (TMPD))	Beijing Huawei Zhongyi Technology Co., Ltd.	023012	Detection of mitochondrial OCR in tissue homogenate adapted for Oroboros O2k respirometer
Tissue scissors	RWD Life Science Co., Ltd.	S12003-09	For cutting and dissecting soft tissues, separating organs and fascia in animal dissection
Transmission electron microscope	HITACHI	HT7800	Analyze the ultrastructural morphology of mitochondria
Trypsin	Plant Chem Medicine	PC-90001	Organ pre-digestion
Universal Antibody Diluent	NCM Biotech	WB100D	Antibody Diluent
Ultramicrotome	Leica	EMUC7	Used to prepare ultrathin sections for transmission electron microscopy (TEM)
Uranyl acetate	Electron Microscopy Sciences	22400	to stain ultrathin sections and improve intracellular structural contrast.
Vertical electrophoresis cell	BIO-RAD	165-8001	For protein separation by SDS-PAGE electrophoresis
Wash Buffer	Solarbio	SM0020	Wash and purify isolated mitochondria, maintaining their structural and functional integrity
75% ethanol	Shanghai Macklin Biochemical Co., Ltd.	E885996	For surface disinfection of laboratory apparatus and experimental materials