Abstract
A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES) cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.
Highlights
Embryonic stem (ES) or induced Pluripotent stem cells are widely expected to be used in clinical therapeutics for transplantation [1,2,3,4] or as drug screening tools [5,6,7,8]
Fabrication of a collection dish for cardiomyocytes First, sodium alginate was transferred to a culture dish that was coated with a spin-coater and dried in air
PIPAAm exhibits hydrophilic/hydrophobic alterations with external temperature changes, and cells on a PIPAAm surface can be collected by lowering the culture temperature from 37 to 20uC, while avoiding the use of digestive enzymes
Summary
Embryonic stem (ES) or induced Pluripotent stem (iPS) cells are widely expected to be used in clinical therapeutics for transplantation [1,2,3,4] or as drug screening tools [5,6,7,8]. For each passaging of cells, cell lines are usually detached from the culture dish using collagenase or trypsin, which degrades the extracellular matrix or proteins. This is harmful to cells, so fragile cells, such as cultured primary neurons, cannot be recultured. PIPAAm is hydrophobic at 37uC and hydrophilic at 20uC, so cells on a PIPAAm-coated culture dish can be detached without destroying the extracellular matrix and intercellular connections, such as tight junctions. These methods yield cell sheets that maintain their intercellular connections. Cell sheets made from corneal epithelial stem cells have been investigated for cornea therapeutics [11]
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