V T'joen1, H Declercq, M Cornelissen. 1. Department of Basic Medical Science - Tissue Engineering Group, Faculty of Medicine and Health Science, Ghent University - UGent, Gent, Belgium. veronique.tjoen@ugent.be
Abstract
OBJECTIVES: Human embryonic stem cells (hESC) are promising for tissue engineering (TE) purposes due to their unique properties. However, current standard mechanical passaging techniques limit rates of possible TE experiments, as it is difficult to obtain high enough numbers of the cells for experimentation. In this study, several dissociative solutions and application methods are tested for their applicability to, and influence on, hESC culture and expansion. MATERIALS AND METHODS: Expansion of two hESC lines, H1 and VUB01, subjected to different passaging techniques, was evaluated. Four dissociative solutions - TrypLE™ Express, Trypsin-EDTA, Cell Dissociation Solution and Accutase™- were combined with two application protocols. As reference conditions, manual and bead-based passaging techniques were used. RESULTS: Results showed that use of Cell Dissociation Solution in combination with a slow adaptation protocol, generated the best expansion profile for both cell lines. The hESC single cell lines remained pluripotent, had good expansion profiles and were capable of differentiation into representatives of all three germ layers. Reproducibility of the results was confirmed by adaptation for three other hESC lines. CONCLUSION: Use of Cell Dissociation Solution, combined with slow adaptation protocol, allows a fast switch from the mechanical passaging technique to a single-cell split technique, generating stable and robust hESC cell lines, which allow for large scale expansion of hESC for TE purposes.
OBJECTIVES:Human embryonic stem cells (hESC) are promising for tissue engineering (TE) purposes due to their unique properties. However, current standard mechanical passaging techniques limit rates of possible TE experiments, as it is difficult to obtain high enough numbers of the cells for experimentation. In this study, several dissociative solutions and application methods are tested for their applicability to, and influence on, hESC culture and expansion. MATERIALS AND METHODS: Expansion of two hESC lines, H1 and VUB01, subjected to different passaging techniques, was evaluated. Four dissociative solutions - TrypLE™ Express, Trypsin-EDTA, Cell Dissociation Solution and Accutase™- were combined with two application protocols. As reference conditions, manual and bead-based passaging techniques were used. RESULTS: Results showed that use of Cell Dissociation Solution in combination with a slow adaptation protocol, generated the best expansion profile for both cell lines. The hESC single cell lines remained pluripotent, had good expansion profiles and were capable of differentiation into representatives of all three germ layers. Reproducibility of the results was confirmed by adaptation for three other hESC lines. CONCLUSION: Use of Cell Dissociation Solution, combined with slow adaptation protocol, allows a fast switch from the mechanical passaging technique to a single-cell split technique, generating stable and robust hESC cell lines, which allow for large scale expansion of hESC for TE purposes.
Authors: Tenneille E Ludwig; Veit Bergendahl; Mark E Levenstein; Junying Yu; Mitchell D Probasco; James A Thomson Journal: Nat Methods Date: 2006-08 Impact factor: 28.547
Authors: J A Thomson; J Itskovitz-Eldor; S S Shapiro; M A Waknitz; J J Swiergiel; V S Marshall; J M Jones Journal: Science Date: 1998-11-06 Impact factor: 47.728