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

Jong Seob Choi, Sunwoong Bae, Kyung Hoon Kim, Jaoon Y H Kim, Sang Jun Sim, Tae Seok Seo

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)61298-61304
Number of pages7
JournalRSC Advances
Volume4
Issue number106
DOIs
Publication statusPublished - 2014

Fingerprint

Hygromycin B
Agar
Hydrodynamics
Recovery
Polymerase chain reaction
Cell culture
Seed
Genes

ASJC Scopus subject areas

  • Chemical Engineering(all)
  • Chemistry(all)

Cite this

Capture and culturing of single microalgae cells, and retrieval of colonies using a perforated hemispherical microwell structure. / Choi, Jong Seob; Bae, Sunwoong; Kim, Kyung Hoon; Kim, Jaoon Y H; Sim, Sang Jun; Seo, Tae Seok.

In: RSC Advances, Vol. 4, No. 106, 2014, p. 61298-61304.

Research output: Contribution to journalArticle

Choi, Jong Seob ; Bae, Sunwoong ; Kim, Kyung Hoon ; Kim, Jaoon Y H ; Sim, Sang Jun ; Seo, Tae Seok. / Capture and culturing of single microalgae cells, and retrieval of colonies using a perforated hemispherical microwell structure. In: RSC Advances. 2014 ; Vol. 4, No. 106. pp. 61298-61304.
@article{4b3c9294f4414b5a8591e6af2fa65e0f,
title = "Capture and culturing of single microalgae cells, and retrieval of colonies using a perforated hemispherical microwell structure",
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.",
author = "Choi, {Jong Seob} and Sunwoong Bae and Kim, {Kyung Hoon} and Kim, {Jaoon Y H} and Sim, {Sang Jun} and Seo, {Tae Seok}",
year = "2014",
doi = "10.1039/c4ra09730k",
language = "English",
volume = "4",
pages = "61298--61304",
journal = "RSC Advances",
issn = "2046-2069",
publisher = "Royal Society of Chemistry",
number = "106",

}

TY - JOUR

T1 - Capture and culturing of single microalgae cells, and retrieval of colonies using a perforated hemispherical microwell structure

AU - Choi, Jong Seob

AU - Bae, Sunwoong

AU - Kim, Kyung Hoon

AU - Kim, Jaoon Y H

AU - Sim, Sang Jun

AU - Seo, Tae Seok

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84913559533&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84913559533&partnerID=8YFLogxK

U2 - 10.1039/c4ra09730k

DO - 10.1039/c4ra09730k

M3 - Article

AN - SCOPUS:84913559533

VL - 4

SP - 61298

EP - 61304

JO - RSC Advances

JF - RSC Advances

SN - 2046-2069

IS - 106

ER -