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A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination

BACKGROUND: Budding yeast, Saccharomyces cerevisiae, has been extensively favored as a model organism in aging and age-related studies, thanks to versatile microfluidic chips for cell dynamics assay and replicative lifespan (RLS) determination at single-cell resolution. However, previous microfluidi...

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Autores principales: Wang, Yingying, Zhu, Zhen, Liu, Ke, Xiao, Qin, Geng, Yangye, Xu, Feng, Ouyang, Shuiping, Zheng, Ke, Fan, Yimin, Jin, Nan, Zhao, Xiangwei, Marchisio, Mario A., Pan, Dejing, Huang, Qing-an
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8973578/
https://www.ncbi.nlm.nih.gov/pubmed/35361237
http://dx.doi.org/10.1186/s12951-022-01379-9
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author Wang, Yingying
Zhu, Zhen
Liu, Ke
Xiao, Qin
Geng, Yangye
Xu, Feng
Ouyang, Shuiping
Zheng, Ke
Fan, Yimin
Jin, Nan
Zhao, Xiangwei
Marchisio, Mario A.
Pan, Dejing
Huang, Qing-an
author_facet Wang, Yingying
Zhu, Zhen
Liu, Ke
Xiao, Qin
Geng, Yangye
Xu, Feng
Ouyang, Shuiping
Zheng, Ke
Fan, Yimin
Jin, Nan
Zhao, Xiangwei
Marchisio, Mario A.
Pan, Dejing
Huang, Qing-an
author_sort Wang, Yingying
collection PubMed
description BACKGROUND: Budding yeast, Saccharomyces cerevisiae, has been extensively favored as a model organism in aging and age-related studies, thanks to versatile microfluidic chips for cell dynamics assay and replicative lifespan (RLS) determination at single-cell resolution. However, previous microfluidic structures aiming to immobilize haploid yeast may impose excessive spatial constraint and mechanical stress on cells, especially for larger diploid cells that sprout in a bipolar pattern. RESULTS: We developed a high-throughput microfluidic chip for diploid yeast long-term culturing (DYLC), optical inspection and cell-aging analysis. The DYLC chip features 1100 “leaky bowl”-shaped traps formatted in an array to dock single cells under laminar-perfused medium and effectively remove daughter cells by hydraulic shear forces. The delicate microstructures of cell traps enable hydrodynamic rotation of newborn buds, so as to ensure bud reorientation towards downstream and concerted daughter dissection thereafter. The traps provide sufficient space for cell-volume enlargement during aging, and thus properly alleviate structural compression and external stress on budding yeast. Trapping efficiency and long-term maintenance of single cells were optimized according to computational fluid dynamics simulations and experimental characterization in terms of critical parameters of the trap and array geometries. Owing to the self-filling of daughter cells dissected from traps upstream, an initial trapping efficiency of about 70% can rapidly reach a high value of over 92% after 4-hour cell culturing. During yeast proliferation and aging, cellular processes of growth, budding and daughter dissection were continuously tracked for over 60 h by time-lapse imaging. Yeast RLS and budding time interval (BTI) were directly calculated by the sequential two-digit codes indicating the budding status in images. With the employed diploid yeast strain, we obtained an RLS of 24.29 ± 3.65 generations, and verified the extension of BTI in the first couple of generations after birth and the last several generations approaching death, as well as cell de-synchronization along diploid yeast aging. CONCLUSIONS: The DYLC chip offers a promising platform for reliable capture and culturing of diploid yeast cells and for life-long tracking of cell dynamics and replicative aging processes so that grasping comprehensive insights of aging mechanism in complex eukaryotic cells. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12951-022-01379-9.
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spelling pubmed-89735782022-04-02 A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination Wang, Yingying Zhu, Zhen Liu, Ke Xiao, Qin Geng, Yangye Xu, Feng Ouyang, Shuiping Zheng, Ke Fan, Yimin Jin, Nan Zhao, Xiangwei Marchisio, Mario A. Pan, Dejing Huang, Qing-an J Nanobiotechnology Research BACKGROUND: Budding yeast, Saccharomyces cerevisiae, has been extensively favored as a model organism in aging and age-related studies, thanks to versatile microfluidic chips for cell dynamics assay and replicative lifespan (RLS) determination at single-cell resolution. However, previous microfluidic structures aiming to immobilize haploid yeast may impose excessive spatial constraint and mechanical stress on cells, especially for larger diploid cells that sprout in a bipolar pattern. RESULTS: We developed a high-throughput microfluidic chip for diploid yeast long-term culturing (DYLC), optical inspection and cell-aging analysis. The DYLC chip features 1100 “leaky bowl”-shaped traps formatted in an array to dock single cells under laminar-perfused medium and effectively remove daughter cells by hydraulic shear forces. The delicate microstructures of cell traps enable hydrodynamic rotation of newborn buds, so as to ensure bud reorientation towards downstream and concerted daughter dissection thereafter. The traps provide sufficient space for cell-volume enlargement during aging, and thus properly alleviate structural compression and external stress on budding yeast. Trapping efficiency and long-term maintenance of single cells were optimized according to computational fluid dynamics simulations and experimental characterization in terms of critical parameters of the trap and array geometries. Owing to the self-filling of daughter cells dissected from traps upstream, an initial trapping efficiency of about 70% can rapidly reach a high value of over 92% after 4-hour cell culturing. During yeast proliferation and aging, cellular processes of growth, budding and daughter dissection were continuously tracked for over 60 h by time-lapse imaging. Yeast RLS and budding time interval (BTI) were directly calculated by the sequential two-digit codes indicating the budding status in images. With the employed diploid yeast strain, we obtained an RLS of 24.29 ± 3.65 generations, and verified the extension of BTI in the first couple of generations after birth and the last several generations approaching death, as well as cell de-synchronization along diploid yeast aging. CONCLUSIONS: The DYLC chip offers a promising platform for reliable capture and culturing of diploid yeast cells and for life-long tracking of cell dynamics and replicative aging processes so that grasping comprehensive insights of aging mechanism in complex eukaryotic cells. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12951-022-01379-9. BioMed Central 2022-03-31 /pmc/articles/PMC8973578/ /pubmed/35361237 http://dx.doi.org/10.1186/s12951-022-01379-9 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Wang, Yingying
Zhu, Zhen
Liu, Ke
Xiao, Qin
Geng, Yangye
Xu, Feng
Ouyang, Shuiping
Zheng, Ke
Fan, Yimin
Jin, Nan
Zhao, Xiangwei
Marchisio, Mario A.
Pan, Dejing
Huang, Qing-an
A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination
title A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination
title_full A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination
title_fullStr A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination
title_full_unstemmed A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination
title_short A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination
title_sort high-throughput microfluidic diploid yeast long-term culturing (dylc) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8973578/
https://www.ncbi.nlm.nih.gov/pubmed/35361237
http://dx.doi.org/10.1186/s12951-022-01379-9
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