Isolation and Purification of Adult Mouse Cardiomyocytes by Langendorff Perfusion and Gravity Sedimentation

Summary

This study aims to optimize and streamline the extraction procedure for adult mouse cardiomyocytes, with the ultimate goal of improving research methodologies in cardiovascular diseases. This protocol efficiently obtains cardiomyocytes with high viability and purity within a reduced processing time, enabling more reliable experimentation and accelerating therapeutic advancements.

Abstract

Cardiovascular diseases remain a major global health challenge, creating a critical need for reliable in vitro models. This study presents an optimized protocol for the isolation and culture of adult mouse cardiomyocytes to address limitations of low viability and non-cardiomyocyte contamination in existing methods. The protocol is based on retrograde Langendorff perfusion, utilizing heparin sodium for anticoagulation and employing collagenase type II alone (1.2 mg/mL) for digestion. Cardiomyocytes were purified by gravity sedimentation and cultured on laminin-coated surfaces. This method consistently yielded 8.75 ± 0.36 × 10⁵ cardiomyocytes per heart with a high proportion of rod-shaped cells (91.2% ± 2.11%), and a high purity (the percentage of cTNT⁺DAPI⁺ cells relative to DAPI⁺ cells was approximately 97.47% ± 1.365%), which exhibited clear sarcomeric structures. By providing a standardized and reproducible method for obtaining high-quality adult cardiomyocytes, this protocol effectively bridges the gap between in vitro studies and pathophysiological relevance, thereby supporting advanced cardiovascular research.

Introduction

Cardiovascular diseases remain the leading cause of mortality worldwide¹. Cardiomyocytes, as a central focus in cardiac research, have long served as essential tools for investigating cardiac physiology and pathology². While commercially available cardiomyocyte cell lines and the recently popularized human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer greater advantages in terms of accessibility and ease of manipulation^3,4,5, primary cardiomyocytes isolated ex vivo demonstrate superior structural and fun....

Protocol

All animal procedures in this study were strictly performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines approved by the Ethics Committee of Deqing People's Hospital. The study used 8-12-week-old male C57BL/6 mice purchased from GemPharmatech Co., Ltd. All animals were housed in SPF-grade barrier facilities with environmental parameters meeting national standards (room temperature 22 ± 2 °C, relative humidity 50 ± 10%, 12/12-h light/dark cycle). Throughout the experiments, this study rigorously adhered to the "3R" principles (Replacement, Reduction, Refinement) and implemented standard procedures, inc....

Results

The isolation and culture procedure of adult mouse cardiomyocytes is demonstrated in the protocol, with the required instrumentation and tubing setup illustrated in Figure 1. Morphological assessment revealed that freshly isolated cardiomyocytes, as well as those cultured for 1 h and 24 h, all exhibited characteristic rod-shaped or fusiform morphology with clearly visible striations (Figures 2A-C). Immunofluorescence staining for cardiac tro.......

Discussion

This study presents a significant methodological breakthrough in cardiovascular research by establishing an optimized protocol for adult mouse cardiomyocyte isolation and culture. Through systematic refinement of buffer composition and enzymatic digestion parameters, this approach markedly enhances the reliability of the culture system while improving both cardiomyocyte purity and viability. These technical advancements provide a robust platform for modeling cardiovascular diseases using primary adult cardiomyocytes.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BDM	Aladdin Scientific Corporation, Shanghai, China	D111028
BSA	Aladdin Scientific Corporation, Shanghai, China	B265994
C57BL/6 mice	GemPharmatech Co., Ltd		8–12-week-old male
CaCl2	Aladdin Scientific Corporation, Shanghai, China	C431202
Collagenase type II	Worthington Biochemical Corporation (USA)	LS004176	LOT#:43D23502   Enzyme activity units: 270u/mg dw
FBS	Beyotime Biotechnology,China	C0226
Glucose	Aladdin Scientific Corporation, Shanghai, China	D639737
HEPES	Sigma-Aldrich	H7006
KCl	Aladdin Scientific Corporation, Shanghai, China	P112134
KH2PO4	Aladdin Scientific Corporation, Shanghai, China	P104075
L-glutamine	Thermo Fisher Scientific	25030081
MEM	Thermo Fisher Scientific	11095080
MgSO4	Aladdin Scientific Corporation, Shanghai, China	M116444
Na2HPO4	Aladdin Scientific Corporation, Shanghai, China	S274390
NaCl	Aladdin Scientific Corporation, Shanghai, China	C111547
NaH2PO4	Aladdin Scientific Corporation, Shanghai, China	S108339
NaHCO3	Aladdin Scientific Corporation, Shanghai, China	S112338
Penicillin-streptomycin (100x)	Beyotime Biotechnology,China	C0222
Primovert Inverted Cell Culture Microscope	Carl Zeiss
Taurine	Aladdin Scientific Corporation, Shanghai, China	T103829