Isolation and cryopreservation of neonatal rat cardiomyocytes.
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.
Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North Carolina State University.
Adam C
AC
Vandergriff
Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North Carolina State University.
Michael Taylor
MT
Hensley
Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North Carolina State University; The Cyrus Tang Hematology Center, Soochow University; kcheng3@ncsu.edu.
Ke
K
Cheng
D000185:Actinin / Q000032:analysis / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005615:Freezing; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D012518:Sarcomeres / Q000737:chemistry / Q000378:metabolism
D003451:Cryoprotective Agents; D000185:Actinin; D004121:Dimethyl Sulfoxide