title: Improved Method for Isolation of Neonatal Rat Cardiomyocytes with Increased Yield of C-Kit+ Cardiac Progenitor Cells.
abstract: Cell therapy represents a promising new paradigm for treatment of heart disease, a major cause of death in the industrialized world. The recent discovery of tissue resident c-Kit+ cardiac progenitor cells (CPCs) has fueled scientific efforts to exploit these cells therapeutically for regenerative interventions, and primary culture of cardiomyocytes is a commonin-vitromodel to investigate basic molecular mechanisms underlying cardiac degeneration and regeneration. Current protocols for cardiomyocyte isolation frequently result in low cell yield and insufficient depletion of fibroblasts, which then overgrow the cardiomyocytes in culture. In this protocol we describe an improved method for the isolation of neonatal rat cardiomyocytes that also enables enhanced yields of CPCs. Gentle techniques of enzymatic and mechanical tissue processing ensure high cell numbers and viability, while subsequent Percoll density gradient centrifugation minimizes fibroblasts. We compared the advantages of different enzymes and found that Collagenase 2 alone leads to very high yields of cardiomyocytes, whereas the application of Matrase™ enzyme blend increases the relative yield of c-Kit+ CPCs to up to 35%. Cardiomyocytes and CPCs isolated with this protocol may constitute an important cell source for investigating heart disease as well as cell based therapeutic approaches.
authors.affiliation: Department of Translational Molecular Pathology, Unit 2951, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Rutering
authors.affiliation: Department of Translational Molecular Pathology, Unit 2951, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
authors.forename: Matthias
authors.initials: M
authors.lastname: Ilmer
authors.affiliation: Department of Translational Molecular Pathology, Unit 2951, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Recio
authors.affiliation: InGeneron Incorporated, 8205 El Rio Street, Houston, TX 77054, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Coleman
authors.affiliation: Department of Translational Molecular Pathology, Unit 2951, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
authors.forename: Jody
authors.initials: J
authors.lastname: Vykoukal
authors.affiliation: Applied Stem Cell Laboratory, Heart and Vascular Institute, Department of Medicine, Tulane University Health Science Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Alt
keywords: Cardiac progenitor cells; Cardiomyocytes; Cell isolation; Density gradient centrifugation; Stem cells; c-Kit