An optimized method to isolate human fibroblasts from tissue for ex vivo analysis
An optimized method to isolate human fibroblasts from tissue for ex vivo analysis
Despite their involvement in many physiological and pathological processes, fibroblasts remain a poorly-characterized cell type. Analysis of primary fibroblasts while maintaining their in vivo phenotype is challenging: standard methods for fibroblast isolation require cell culture in vitro, which is known to alter phenotypes. Previously-described protocols for the dissociation of primary tissues fail to extract sufficient numbers of fibroblasts, instead largely yielding immune cells. Here, we describe an optimized method for generating a fibroblast-enriched single-cell suspension from human tissues using combined mechanical and enzymatic dissociation. This allows analysis of ex vivo fibroblasts without the need for culture in vitro.
Fibroblasts, Lung, Stroma, Tissue disaggregation
Waise, Sara
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Parker, Rachel
ca68b9e6-b3d4-4246-845f-4636068d479f
Rose-Zerilli, Matthew J.J.
08b3afa4-dbc2-4c0d-a852-2a9f33431199
Layfield, David M.
3f5ffdd6-6ec0-43dd-95a8-5752bcefe7f5
Wood, Oliver
dad2f90c-70a5-44a0-914a-52809b75d1c6
West, Jonathan
f1c2e060-16c3-44c0-af70-242a1c58b968
Ottensmeier, Christian H.
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Thomas, Gareth J.
2ff54aa9-a766-416b-91ee-cf1c5be74106
Hanley, Christopher J.
7e2d840d-e724-4389-a362-83741ccdf241
5 December 2019
Waise, Sara
13f667c5-853e-4120-a2c6-bbc8324e3b2a
Parker, Rachel
ca68b9e6-b3d4-4246-845f-4636068d479f
Rose-Zerilli, Matthew J.J.
08b3afa4-dbc2-4c0d-a852-2a9f33431199
Layfield, David M.
3f5ffdd6-6ec0-43dd-95a8-5752bcefe7f5
Wood, Oliver
dad2f90c-70a5-44a0-914a-52809b75d1c6
West, Jonathan
f1c2e060-16c3-44c0-af70-242a1c58b968
Ottensmeier, Christian H.
42b8a398-baac-4843-a3d6-056225675797
Thomas, Gareth J.
2ff54aa9-a766-416b-91ee-cf1c5be74106
Hanley, Christopher J.
7e2d840d-e724-4389-a362-83741ccdf241
Waise, Sara, Parker, Rachel, Rose-Zerilli, Matthew J.J., Layfield, David M., Wood, Oliver, West, Jonathan, Ottensmeier, Christian H., Thomas, Gareth J. and Hanley, Christopher J.
(2019)
An optimized method to isolate human fibroblasts from tissue for ex vivo analysis.
Bio-protocol, 9 (23), [e3440].
(doi:10.21769/BioProtoc.3440).
Abstract
Despite their involvement in many physiological and pathological processes, fibroblasts remain a poorly-characterized cell type. Analysis of primary fibroblasts while maintaining their in vivo phenotype is challenging: standard methods for fibroblast isolation require cell culture in vitro, which is known to alter phenotypes. Previously-described protocols for the dissociation of primary tissues fail to extract sufficient numbers of fibroblasts, instead largely yielding immune cells. Here, we describe an optimized method for generating a fibroblast-enriched single-cell suspension from human tissues using combined mechanical and enzymatic dissociation. This allows analysis of ex vivo fibroblasts without the need for culture in vitro.
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Published date: 5 December 2019
Additional Information:
Funding Information: this protocol was derived from previously-published data 28. This work was supported by Cancer Research UK and Medical Research Council Clinical Research Training Fellowships and a Pathological Society Trainee’s Small Grant to SW. Implementation of Drop-seq was supported by a Medical Research Council Discovery award (MC_PC_15078) and a Southampton Cancer Research UK Centre Development Fund Award to MJJRZ, CHO, JW, CJH & GJT. RP was supported by a John Goldman Fellowship for Future Science (2016/JGF/0003; Leuka Charity) awarded to MJJRZ. The authors thank Evan Macosko, Melissa Goldman and Steve McCarroll for their helpful advice, Dr. Serena Chee (University Hospital Southampton), Benjamin Johnson, Carine Fixmer and Maria Lane (TargetLung Clinical Trials Associates) for enabling access to clinical samples, and the patients involved in this study.
Keywords:
Fibroblasts, Lung, Stroma, Tissue disaggregation
Identifiers
Local EPrints ID: 483994
URI: http://eprints.soton.ac.uk/id/eprint/483994
ISSN: 2331-8325
PURE UUID: 86b80bd6-9c97-45cd-be3f-e0e1e82acdcb
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Date deposited: 08 Nov 2023 18:16
Last modified: 06 Jun 2024 01:54
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Contributors
Author:
Sara Waise
Author:
Rachel Parker
Author:
David M. Layfield
Author:
Oliver Wood
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