The Thermo Scientific Pierce Primary Cardiomyocyte Isolation Kit provides isolation and culturing reagents for the optimal yield and viability of primary cardiomyocytes from neonatal heart from mouse and rat.
The Primary Cardiomyocyte Isolation Kit has been designed to obtain high viability and purity of primary cardiomyocytes using validated reagents and an optimized procedure for enzymatic digestion and culturing. The kit contains two tissue-specific dissociation enzymes, media formulated for primary cell culture, and a cardiomyocyte cell culture growth supplement that promotes the isolation, enrichment and growth of functional cardiomyocytes. The validated protocol ensures optimal isolation and significantly reduces the processing time of isolation when compared to other methods. Addition of the cardiomyocyte growth supplement to culture cells further enriches for cardiomycytes, and cultures have been maintained for up to 7 days using the kit components.
- Optimized – procedure and reagents optimized for viability, yield, purity, and ease-of-use
- Time-saving – isolation protocol requires less than 2 hours compared to up to 21 hours by other methods
- Yield – provides a 1.5- to 4-fold increase in yield compared to do-it-yourself methods and other commercial kits
- Viability – provides higher viability than do-it-yourself methods and other commercial kits
- Functional – cultured cardiomyocytes express the appropriate biochemical markers with validated beating activity
- Complete kit includes Cardiomyocyte Isolation Enzyme 1, Cardiomyocyte Isolation Enzyme 2, Hanks' Balanced Salt Solution (HBSS), DMEM for Primary Cell Isolation, and Cardiomyocyte Growth Supplement
- Cardiomyocyte cell differentiation
- Immunohistochemistry (IHC)
- Functional and biochemical assays
- Biologically relevant system for preclinical drug discovery and predictive disease modeling
Current methods for dissociation of cardiac cells from heart tissue require repeated (5 to 8 incubations) or lengthy (>16 hours) enzyme digestion, resulting in reduced yield and viability of isolated cells. The Pierce Primary Cardiomyocyte Isolation Kit provides fully optimized reagents and a protocol that prevents overdigestion for the isolation and culture of cardiomyocytes. The complete process from handling primary tissues to seeding cells in culture vessels can be completed within 2 hours.
Because it uses optimized tissue-specific dissociation enzymes, this kit isolates cardiomyocytes with greater yield and cell viability than cells isolated by existing methods. The isolated primary cardiomyocytes express cardiomyocyte protein markers and exhibit contractile function (beating activity, see video), thereby providing a model system for studies of contraction, ischaemia, hypoxia and the toxicity of various compounds.
|Cell yield and viability after primary cardiomyocyte isolation with different methods. Mouse neonatal hearts at Day 1 were minced, incubated with Thermo Scientific Pierce Cardiomyocyte isolation enzyme 1 (with papain) and 2 (with thermolysin) for 35 minutes, and disrupted by pipetting up and down with a pipette and 1000µL tip 25 times to generate single cell suspension. Cell yield and cell viability were acquired from cell suspension isolated from one mouse neonatal heart at Day 1 in total volume of 1.5mL. Cell viability was determined by trypan blue exclusion assay and total cell yield was determined using an Invitrogen™ Countess™ Automated Cell Counter.
|Viable cardiomyocytes are isolated using the Pierce Primary Cardiomyocyte Isolation Kit. Mouse cardiomyocytes from postnatal mouse hearts were isolated and cultured using the Pierce Cardiomyocyte Isolation Kit. Isolated cells were plated on a 24-well plate at a density of 5 x 10^5 cells per well, and cell contractions were recorded at Day 1 (left panel) and 6 (right panel) after plating. At Day 1, spontaneous contractions from individual cells were recorded at 20X magnification. At Day 6 post-plating, synchronous and spontaneous contractions from the monolayer were recorded at 40X magnification.
|Cardiomyocyte differentiation after cell isolation. Mouse Neonatal cardiomycoytes were isolated and cultured using the Pierce Primary Cardiomyocyte Isolation Kit. Cardiomyocytes were grown on 24 well at the density 5 x 10^5 cells or a 35mm glass bottom plate at the density of 2.5 x 10^6 cells. At Day 1 and Day 7, cells were fixed with 4% paraformaldehyde, permeabilized, and stained with propidium iodide (pink), and Troponin T Cardio Isoform (green, Part No. MA5-12960). Nuclei were visualized using Hoechst 33342 (blue)(Part No. 62249). Images were taken at 20X and 60X. The magnified image indicates a striated pattern, indicative of differentiated and functional cardiomyocytes.
- Louch, W.E., (2011) Methods in cardiomyocyte isolation, culture, and Gene Transfer. J Mol Cell Cardiol. 51(3):288-98.
- Chlopcikova, S., et al. (2001) Neonatal rat cardiomyocytes – a model for the study of morphological, biochemical and electrophysiological characteristics of the heart. Biomed. Papers 145(2):49-55.
- Gorelik, J., et al. (2004) Comparison of arrhytmogenic effects of tauro- and glycoconjugates of cholic acid in an in vitro study of rat cardiomyocytes. Int. J. Obstet. Gynaecol., 111:867-70.
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