Isolation, Quantitation and Viability Analysis of Neonatal Cardiomyocytes using Cellometer
The neonatal rat cardiomyocyte model has been used for many years by researchers studying the heart. In addition to increasing understanding of the morphological, biochemical, and electrophysiological characteristics of the normal heart, neonatal cardiomyocytes have been used to study contraction, ischemia, hypoxia, and the toxicity of different compounds. This model has been used to determine the optimal dosage for certain drugs and to develop and evaluate the efficacy of potential therapeutic agents. Apoptosis, or programmed cell death, has been observed in infarcted and re-perfused myocardium, end stages of heart failure, post-infarction left ventricular remodeling, and diabetes, making apoptosis a key focus in the neonatal rat cardiomyocyte model1. In recent years, research into cellular therapy and regenerative medicine has increased. Studies involving the transplant of neonatal rat cardiomyocytes into adult rats following cardiac infarction have generated positive results, yielding improvement in local tissue and overall cardiac function2.
Increased yield and accurate assessment of cell viability of isolated cardiomyocytes are critical to ongoing cardiac research studies involving the neonatal rat cardiomyocyte model. Nexcelom Bioscience and Worthington Biochemical have demonstrated a simple, consistent procedure for isolation and assessment of viable neonatal cardiomyocytes.
1. “Neonatal Rat Cardiomyocytes – A Model for the Study of Morphological, Biochemical, and Electrophysiological Characteristics of the Heart”, S. Chlopčiková, J. Psotová, P. Miketová, Biomed. Papers, Volume 145, Number 2, pages 49-55. 2001
2. “Cell Therapy Enhances Function of Remote Non-Infarcted Myocardium”, A. Moreno-Gonzalez, F. S. Korte, J. Dai, K. Chen, B. Ho, H. Reinecke, C. E. Murry, M Regnier, J. Mol. Cell Cardiol., Volume 47, Number 5, pages 603-613. 2009
Isolation of Neonatal Cardiomyocytes
The isolation of neonatal cardiomyocytes involves enzyme digestion to dissociate cells from the heart tissue followed by purification steps to remove non-muscle cells and tissue debris. The procedure is designed for maximum dissociation with minimal harm to the cardiomyocyte cells. The Worthington Neonatal Cardiomyocyte Isolation System utilizes purified trypsin and collagenase enzyme preparations to maximize cell dissociation and viability and reduce lot-to-lot variability. Each kit lot undergoes strict functional testing to ensure a consistent yield of viable cells.
Determination of Cell Concentration and Viability
The Cellometer Vision Cell Analyzer and Cellometer Auto 2000 Cell Viability Counter offer pre-set viability assays for a wide variety of primary cell types, including PBMCs, stem cells, nucleated cells, and splenocytes. The viability of freshly-isolated neonatal cardiomyocytes was determined using a traditional trypan blue method as well as a dual-fluorescent AO/PI method. The AO/PI method is highly recommended for samples containing debris.
In the AO/PI method, the acridine orange (AO) dye stains DNA in the cell nucleus of both live and dead cells. Propidium iodide (PI) DNA-binding dye is used to determine cell viability. Healthy cells are impermeable to the PI dye. Only dead (non-viable) nucleated cells with compromised membranes are stained. Cells stained with both AO and PI fluoresce red due to Förster resonance energy transfer (FRET), so live nucleated cells fluoresce green and dead nucleated cells fluoresce red. There is no interference from cellular debris or non-nucleated cells, as all Cellometer AO/ PI live and dead cell counts are conducted in the fluorescent channels.
The Cellometer Vision Cell Analyzer and Cellometer Auto 2000 Cell Viability Counter reduce viability analysis time, ensuring the timely transfer of isolated neonatal cardiomyocytes to culture. Using only 20µl of sample, dual-fluorescence staining with Cellometer ViaStain AO/PI Staining Solution offers improved accuracy of viability results with no interference from debris or non-nucleated cells, ensuring a correct cell concentration for in vitro experiments and cell-based assays.
View the Cellometer Neonatal Cardiomyocyte Application Note to view the materials, procedure, and results from the isolation and analysis of neonatal cardiomyocytes.