Cellular engineering of ventricular adult rat cardiomyocytes.
OBJECTIVE
Preparation of viable cultured adult cardiomyocytes (vARCs) is a prerequisite for cell-based transplantation and tissue engineering. Ectopic gene expression is important in this context. Here, we present an in vitro cell replating strategy using Accutase for cultured vARCs, allowing ectopic gene expression.


METHODS
Cultured vARCs from 6- to 8-week-old rats were used. Transfections with EGFP (enhanced green fluorescent protein) constructs, Mlc-3f-EGFP or alpha-actinin-EGFP were performed using adenovirus-enhanced transferrin-mediated infection (AVET). Accutase (PAA Laboratories, Linz, Austria) was used for the detachment of cultured cells. Immunohistochemical analysis, together with confocal laser microscopy was used for structural analysis of the cells.


RESULTS
Cultured vARCs could be detached with a high yield (40 to 60%) from primary cultures using Accutase. The cultivation period plays an important role in the yield of viable cells. Resultant replated vARCs (rep-vARCs) rapidly (1-2 h) acquired a rounded up shape without degradation of their contractile apparatus, which is in contrast to the rod-shaped freshly isolated vARCs (fi-vARCs). The detached cells survived passage through a narrow syringe needle. After seeding, detached cells rapidly attached to various substrates, increased their content of the contractile apparatus, and formed cell-cell contacts within 3 days after reseeding. The detached cells survived passage through a narrow syringe needle. The high recovery of cells after replating enabled the use of the AVET system for gene delivery. AVET is free of infectious particles and does not lead to expression of viral proteins. Transfection of vARCs prior to detachment had a small effect on cell recovery and ectopically synthesized proteins were properly localized after replating.


CONCLUSIONS
Detachment of cultured vARCs using Accutase is well compatible with ectopic gene expression and yields a viable transgenic population of vARCs that eventually may be suitable as transgenic cardiomyocyte grafts.
Weikert
Christian
C
Institute of Cell Biology, ETH-Hoenggerberg Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland.
Eppenberger-Eberhardt
Monika
M
Eppenberger
Hans M
HM
D000185:Actinin / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media; D010450:Endopeptidases; D005260:Female; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins; D006352:Heart Ventricles; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D032383:Myocytes, Cardiac / Q000637:transplantation; D051381:Rats; D011919:Rats, Inbred Strains; D014162:Transfection / Q000379:methods
D003470:Culture Media; D008164:Luminescent Proteins; D000185:Actinin; D049452:Green Fluorescent Proteins; D010450:Endopeptidases