Biotechnology and Bioprocess Engineering 2019; 24(3): 536-543  
Effective and Intact Cell Detachment from a Clinically Ubiquitous Culture Flask by Combining Ultrasonic Wave Exposure and Diluted Trypsin
Hanako Tauchi1,†, Chikahiro Imashiro2,†, Taiki Kuribara3, Genichiro Fujii2, Yuta Kurashina4,5, Kiichiro Totani3, and Kenjiro Takemura5,*
1Futaba High School, Tokyo 102-0085, Japan
2School of Science for Open and Environmental Systems, Graduate School of Science and Technology, Keio University, Kanagawa 223-8522, Japan
3Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, Tokyo 180-8633, Japan
4Department of Materials Science and Engineering, Tokyo Institute of Technology, Kanagawa 226-8503, Japan
5Department of Mechanical Engineering, Keio University, Kanagawa 223-8522, Japan
Correspondence to: Kenjiro Takemura
Tel: +81455661826; Fax: +81455661826
E-mail: takemura@mech.keio.ac.jp

These authors contributed equally to this work.
Received: December 22, 2018; Revised: March 27, 2019; Accepted: April 15, 2019; Published online: June 30, 2019.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Bioengineering research and applications are supported by cell culture technologies that produce a large number of homogeneous cells. However, trypsin used in the general culture procedure for cell detachment decreases cell activity and culture efficiency. Furthermore, manually conducted culture procedures, especially pipetting after trypsin treatment, can induce inhomogeneous mechanical stress in cells, which may influence cellular functions. Alternate detachment methods using specialized culture devices without trypsin and/or manual pipetting have been reported. However, conventional trypsinization is still widely used. Diluted trypsin increases culture efficiency. Therefore, we developed a cell-detaching method using diluted trypsin and ultrasonic vibration for cell detachment from ubiquitous culture vessels. To demonstrate our concept, we used a T25 flask. Vibration of the culture surface was excited by ultrasonic waves propagated from an ultrasonic transducer placed under the flask. Using the proposed method, cells were completely detached by diluted trypsin, whereas 8.6% of cells remained on the flask with manual pipetting. The viability and proliferation of cells detached by the proposed method were higher than those of cells detached by the conventional method, owing to the low concentration of trypsin. Furthermore, glucose consumption after detachment showed no abnormality, eliminating possible oncogenesis. Two membrane proteins were quantified immediately after detachment and at 24 h of culture, and there were no differences between the detachment methods. Thus, we conclude that our proposed method improves culture efficiency without any adverse effects and ensures homogeneous mechanical stress on cells.


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