Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Summary

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Abstract

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

Introduction

Cell culture of cardiomyocytes is a critical tool in modern cardiac research. Neonatal rat cardiomyocytes (NRCMs) are commonly used since the isolation and culture is easier than that of adult rat cardiomyocytes¹. The NRCM method still has several limitations including a long isolation procedure and limited cell proliferation in the dish. There are numerous protocols for the isolation of NRCMs with most generally requiring 4-48 hr of work^2–6. In addition, the cells are frequently isolated from 1 to 2-day old rat pups^2,4–7; the timing of the birth can be unpredictable and conflict with other work in the lab. The isolations c....

Protocol

The following protocol is designed for the isolation of cardiomyocytes from one litter of neonatal rat pups (10-14 pups). If litter size is significantly different, the procedure may have to be scaled to compensate. The pups should be around 48±6 hr old. All procedures herein have been approved by the North Carolina State University Institutional Animal Care and Usage Committee (IACUC).

1. Cell Isolation Preparation

NOTE: Perform the following steps the day before isolation.

1. Autoclave the following: large scissors, small scissors, small sharp forceps, large forceps. Use a gravity cycle for 60 min....

Results

Following the isolation of the neonatal rat cardiomyocytes, the cells are frozen down to liquid nitrogen, and can be stored for at least several months. Typically upon thawing, the viability determined by Trypan Blue analysis will be between 40-60%. Though this is lower than other cell types, with proper seeding and culture the cells will proliferate (Figure 1). In order to verify contractility, the cells can be imaged using a phase contrast microscopy. The cells should begin to spontaneously contract wi.......

Discussion

This protocol allows for the NRCMs to be isolated, cryopreserved, and thawed. Thawing the cells is a crucial portion of the procedure. A series of DMSO dilutions is used to slowly remove DMSO from the cells¹⁴. It is important that the extraction of DMSO be performed quickly as the cells are particularly sensitive to dying immediately after thawing. If more or less cells are required for thawing, the volumes of the DMSO solutions can be scaled as needed.

One challenge to NRCM isolati.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IMDM (+25 mM HEPES +L-glutamine)	Gibco	12440-046	With added 25 mM HEPES and L-glutamine
L-glutamine	Gibco	25030-081
FBS	Hyclone	SH30070.03
Gentamicin	Gibco	15710-064
2-Mercaptoethanol	Gibco	21985-023
HBSS (+Ca +Mg)	Corning	21-020-CV	With added calcium and magnesium, pH 7.1-7.4
Trypsin 0.25%	Gibco	25300-056
Trypsin 0.05%	Gibco	25300-054
Cryostor CS5	BioLife Solutions	205102	Freezing media
Cryogenic Vial	Corning	430659
Collagenase	Sigma	C1889-50MG
40 μm Cell Strainer	Greiner Bio-One	542040
Sterilizing Vacuum Filter (0.22μm)	Corning	431118
50 ml Conical	Corning	430828
15 ml Conical	Corning	430790
trypan blue	Cellgro	25-900-CI
Mr Frosty Freezing Container	ThermoScientific	5100-0001
Millicell EZ SLIDES	Millipore	PEZGS0416
α-sarcomeric actinin antibody	Sigma	A7811
Fibronectin	Corning	356008
Bromodeoxyuridine	BD Biosciences	51-7581KZ