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Capture and culturing of single microalgae cells, and retrieval of colonies using a perforated hemispherical microwell structure

Authors
Choi, Jong SeobBae, SunwoongKim, Kyung HoonKim, Jaoon Y. H.Sim, Sang JunSeo, Tae Seok
Issue Date
2014
Publisher
ROYAL SOC CHEMISTRY
Citation
RSC ADVANCES, v.4, no.106, pp.61298 - 61304
Indexed
SCIE
SCOPUS
Journal Title
RSC ADVANCES
Volume
4
Number
106
Start Page
61298
End Page
61304
URI
https://scholar.korea.ac.kr/handle/2021.sw.korea/101074
DOI
10.1039/c4ra09730k
ISSN
2046-2069
Abstract
A perforated hemispherical microwell structure is shown to efficiently capture single Chlamydomonas reinhardtii (C. reinhardtii) cells, culture them to form colonies, and retrieve these colonies to serve as seeds for large-scale cultivation. This solution-phase formation and recovery of colonies could overcome the tedious and time-consuming process of selecting colonies from a solid-phase agar plate. The fabricated microdevice was composed of three layers: a top layer consisting of a cell solution for injection and recovery of a microalgal solution, a hemispherical perforated microwell array in the middle, and a bottom layer in which the solution is manipulated by controlling the hydrodynamic force. The microalgal (wild type and hygromycin B-resistant mutant) cells loaded in the top layer rapidly diffused into the microwell holes, and individual such cells were captured with high efficiency (>90%) and within 1 min by applying a withdraw mode in the bottom layer. Single-cell-based cultivation in a medium containing hygromycin B was then performed to generate colonies in the hemispherical microwell. While the wild type cells died, mutant cells resistant to hygromycin B survived well and grew into a colony within 2 days. The produced colonies in the microwells were recovered by applying a release mode in the bottom layer, so that a hydrodynamic force was exerted vertically to push out the colonies through the outlet in 10 s. The recovered cells were cultured on a large scale in medium by using a flask. The recovered C. reinhardtii was confirmed as a hygromycin B-resistant mutant by identifying the hygromycin gene in the polymerase chain reaction (PCR). The microdevice described here could in solution perform single-cell capture, colony formation, and retrieval of colonies for further large-scale cultivation, which could replace tedious and time-consuming solid-phase agar plate processes with a 7-fold reduction in the duration of the process.
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공과대학 (화공생명공학과)
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