A simplified protocol for the isolation and culture of cardiomyocytes and progenitor cells from neonatal XXXX ventricles.

@entity16899 @entity3111 @entity19 @entity17777 @entity36664 @entity7006 @entity11404 @entity3512 @entity701 @entity3844
UNASSIGNED: The neonatal heart is a very useful tool for the study of biochemical pathways and properties of cardiomyocytes and as it has the potential to regenerate for a brief period of time from birth; it is also useful to study cardiac regeneration. However, as the heart matures, this proficiency for regeneration is reduced. This regenerative potential may be influenced by the microenvironment of the heart in the early stages of postnatal development and therefore, cell cultures derived at this stage may contain functional cardiomyocytes and progenitor cells. The aim of this study was to identify key steps in the isolation and culture of such early stage-neonatal @entity19 hearts to allow maximum migration of cardiomyocytes from the explant and their maintenance as functional, long term cultures. Explant cultures of @entity19 ventricles preserved 3-dimensional structure and generated migrating layers of cardiomyocytes that expressed alpha sarcomeric actin which could be further sub-cultured by enzymatic dissociation. Western blotting demonstrated expression of @entity17777 , @entity7006 , alpha sarcomeric actin and @entity16899 proteins after 20 days of explant culture. @entity36664 , @entity7006 , and @entity11404 continued to express in five weeks old explant cultures while the @entity17777 protein was expressed up to two passages during sub-culture. Real time PCR and SQRT PCR also demonstrated gene expression of cardiomyocyte markers in long term cultures. Migrating cells from the explants assembled into contracting spheroids after subculture and expressed the @entity17777 protein. Progenitor markers @entity3844 , @entity3512 , and extracellular proteins, periostin and @entity3111 demonstrated the preservation of cellular heterogeneity in such cultures. Supplementation with @entity701 maintained a cardioprotective environment and reduced the non-myocyte population. This is an optimized and efficient method for the generation of neonatal heart cultures that is not labor intensive and does not require supplementation with cytokines.
