Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation.


Type
Article
Change log
Authors
Kleine-Brüggeney, Hans  ORCID logo  https://orcid.org/0000-0002-1179-0509
Gielen, Fabrice 
Agley, Chibeza C 
Abstract

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.

Description
Keywords
hydrogels, microdroplets, pluripotency, single cell analysis, stem cells, Animals, Cell Culture Techniques, Cell Differentiation, Cell Line, Cell Proliferation, Hydrogels, Mice, Microspheres, Mouse Embryonic Stem Cells, Optics and Photonics, Perfusion, Phenotype, Rheology, Time Factors
Journal Title
Small
Conference Name
Journal ISSN
1613-6810
1613-6829
Volume Title
15
Publisher
Wiley
Sponsorship
European Commission Horizon 2020 (H2020) ERC (695664)
Wellcome Trust (108438/C/15/Z)
Wellcome Trust (101861/Z/13/Z)
Medical Research Council (MR/R017735/1)
European Research Council (772798)
Biotechnology and Biological Sciences Research Council (BB/R018588/1)
Wellcome Trust WT108438/C/15/Z