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248 results on '"Ohnuma, Kiyoshi"'

4. Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves

Author

Kurashina, Yuta, Imashiro, Chikahiro, Hirano, Makoto, Kuribara, Taiki, Totani, Kiichiro, Ohnuma, Kiyoshi, Friend, James, and Takemura, Kenjiro

Subjects
Biochemistry and Cell Biology, Biological Sciences, Biotechnology, Animals, CHO Cells, Cell Adhesion, Cell Proliferation, Cell Separation, Cricetulus, Culture Media, Serum-Free, DNA Damage, Equipment Design, Membrane Proteins, Microscopy, Electron, Scanning, Trypsin, Ultrasonic Waves, Assay systems, Tissue engineering, Biological sciences, Biomedical and clinical sciences
Abstract

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.

Published
2019

6. Generation of human induced pluripotent stem cell lines derived from four Rett syndrome patients with MECP2 mutations

Author

Mori, Miyu, primary, Yoshii, Shoko, additional, Noguchi, Michiya, additional, Takagi, Daigo, additional, Shimizu, Tomoya, additional, Ito, Hidenori, additional, Matsuo-Takasaki, Mami, additional, Nakamura, Yukio, additional, Takahashi, Satoru, additional, Hamada, Hiromichi, additional, Ohnuma, Kiyoshi, additional, Shiohama, Tadashi, additional, and Hayashi, Yohei, additional

Published
2024
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8. Pluripotent Stem Cell Heterogeneity

Author

Hayashi, Yohei, Ohnuma, Kiyoshi, Furue, Miho K., COHEN, IRUN R., Editorial Board Member, LAJTHA, ABEL, Editorial Board Member, LAMBRIS, JOHN D., Editorial Board Member, PAOLETTI, RODOLFO, Editorial Board Member, REZAEI, NIMA, Editorial Board Member, and Birbrair, Alexander, editor

Published
2019
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44. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

Author

Yomo Tetsuya, Ohnuma Kiyoshi, Asashima Makoto, and Kaneko Kunihiko

Subjects
Cytology, QH573-671
Abstract

Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea) and side scatter (SSheight and SSarea). The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases cell viability but decreases the proliferative ability of the PC12 cells. Conclusion We demonstrated a pretreatment method to collect well-characterized, viable, single cells without using fluorescent labels and without significant damage to the cells. This method is quantitative, rapid, single-step, and yields cells of high purity, making it applicable for a variety of single-cell level analyses.

Published
2006
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