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

Hans Kleine-Brüggeney; Liisa D. van Vliet; Carla Mulas; Fabrice Gielen; Chibeza C. Agley; José C.R. Silva; Austin Smith; Kevin Chalut; Florian Hollfelder

doi:10.1002/smll.201804576

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

Hans Kleine-Brüggeney, Liisa D. van Vliet, Carla Mulas, Fabrice Gielen, Chibeza C. Agley, José C.R. Silva, Austin Smith, Kevin Chalut, Florian Hollfelder^*

^*Corresponding author for this work

Centre for Human & Applied Physiological Sciences

Research output: Contribution to journal › Article › peer-review

30 Citations (Scopus)

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.

Original language	English
Article number	1804576
Journal	Small
Volume	15
Issue number	5
DOIs	https://doi.org/10.1002/smll.201804576
Publication status	Published - 1 Feb 2019

Keywords

hydrogels
microdroplets
pluripotency
single cell analysis
stem cells

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

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 ({\O}: 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.",

keywords = "hydrogels, microdroplets, pluripotency, single cell analysis, stem cells",

author = "Hans Kleine-Br{\"u}ggeney and {van Vliet}, {Liisa D.} and Carla Mulas and Fabrice Gielen and Agley, {Chibeza C.} and Silva, {Jos{\'e} C.R.} and Austin Smith and Kevin Chalut and Florian Hollfelder",

note = "Funding Information: H.K.-B. and L.D.v.V. contributed equally to this work. The authors thank S. Kuhaudomlarp for help with preliminary experiments and T. N. Kohler for helpful advice and comments on the manuscript. This research described was funded by the Wellcome Trust (WT108438/C/15/Z), the Engineering and Physical Sciences Research Council (EPSRC), the EU via its Horizon2020 programme, and the Medical Research Council (MRC). The Cambridge Stem Cell Institute receives core funding from the Wellcome Trust and the MRC. A.S. is an MRC Professor; K.C. and F.H. are ERC Investigators, J. C. R. S. is a Senior Wellcome Trust Fellow (WT101861). H.K.-B. held fellowships by the Studienstiftung des deutschen Volkes and the German Academic Exchange Service (DAAD). Publisher Copyright: {\textcopyright} 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim",

year = "2019",

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doi = "10.1002/smll.201804576",

language = "English",

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T1 - Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation

AU - Kleine-Brüggeney, Hans

AU - van Vliet, Liisa D.

AU - Mulas, Carla

AU - Gielen, Fabrice

AU - Agley, Chibeza C.

AU - Silva, José C.R.

AU - Smith, Austin

AU - Chalut, Kevin

AU - Hollfelder, Florian

N1 - Funding Information: H.K.-B. and L.D.v.V. contributed equally to this work. The authors thank S. Kuhaudomlarp for help with preliminary experiments and T. N. Kohler for helpful advice and comments on the manuscript. This research described was funded by the Wellcome Trust (WT108438/C/15/Z), the Engineering and Physical Sciences Research Council (EPSRC), the EU via its Horizon2020 programme, and the Medical Research Council (MRC). The Cambridge Stem Cell Institute receives core funding from the Wellcome Trust and the MRC. A.S. is an MRC Professor; K.C. and F.H. are ERC Investigators, J. C. R. S. is a Senior Wellcome Trust Fellow (WT101861). H.K.-B. held fellowships by the Studienstiftung des deutschen Volkes and the German Academic Exchange Service (DAAD). Publisher Copyright: © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

PY - 2019/2/1

Y1 - 2019/2/1

N2 - 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.

AB - 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.

KW - hydrogels

KW - microdroplets

KW - pluripotency

KW - single cell analysis

KW - stem cells

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U2 - 10.1002/smll.201804576

DO - 10.1002/smll.201804576

M3 - Article

C2 - 30570812

AN - SCOPUS:85058837171

SN - 1613-6810

VL - 15

JO - Small

JF - Small

IS - 5

M1 - 1804576

ER -

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

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Keywords

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