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Selection of viable cardiomyocytes for cell transplantation using three-dimensional tissue culture
S B Watzka1, J Lucien, M Shimada
1Division of Cardiovascular Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada.
Background:
We developed a three-dimensional cardiac tissue culture system to select viable and contractile cells for the purpose of cardiomyocyte transplantation. In this study we will show that reaggregation of cardiomyocytes in culture is an active process indicative of cardiomyocyte viability and functionality.
Methods:
Myocardial tissue from newborn mice has been enzymatically digested, incubated in culture inserts, and studied by phase contrast microscopy, conventional histology, immunohistochemistry, electron microscopy, and TUNEL assay.
Results:
Cells that are plated on nonadhesive surfaces, reaggregate to spontaneously contracting cell aggregates. The capacity to reaggregate was significantly dependent from the age of the tissue donor (P<0.0001) and on the method of enzymatic dissociation (P<0.0001). The majority of cells within the aggregates consisted of cardiomyocytes. After 24 hr incubation, significant amounts of laminin and fibronectin had been deposited between the cells. Ultrastructural analysis revealed viable cardiomyocytes attached to each other by tight junctions. The apoptotic rate within the aggregates was 11.4+/-4.6 vs. 44.5+/-10.5% immediately after dissociation (P<0.05).
Conclusions:
The capacity to form spontaneously contracting aggregates is an inherent characteristic of viable cardiomyocytes in 3-dimensional cultures, which could be successfully exploited for cellular cardiomyocyte transplantation.