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J Biosci Bioeng. 2014 Nov;118(5):588-92. doi: 10.1016/j.jbiosc.2014.04.008. Epub 2014 May 21.

Effective Rho-associated protein kinase inhibitor treatment to dissociate human iPS cells for suspension culture to form embryoid body-like cell aggregates.

Author information

1
Division of Medicine and Engineering Science, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan.
2
Division of Medicine and Engineering Science, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan. Electronic address: kurohiro@yamanashi.ac.jp.

Abstract

Treatment conditions using Y-27632 in the preparation of cell suspension of dissociated human pluripotent stem cells (hiPSCs) were investigated in the context of embryoid body (EB)-like cell aggregates. The effectiveness of a pretreatment with Y-27632 before cell dissociation and that of a Y-27632 treatment during cell dissociation were investigated from the viewpoint of simplicity and robustness. The duration of Y-27632 treatment in the preparation process affected the circularity and agglomeration of dissociated hiPSCs. A single application of pretreatment failed to prevent the onset of blebbing. However, a pretreatment promoted the agglomeration of dissociated hiPSCs when combined with the addition of Y-27632 to cell suspension. Our results indicate that pretreatment enhances the agglomeration potential of dissociated hiPSCs. When cell dissociation was performed in the presence of Y-27632, dissociated hiPSCs possessed the highest circularity and significant agglomerating property. It was shown that treatment with Y-27632 during cell dissociation is a simple and robust method to prepare dissociated hiPSCs for suspension culture to form EB-like cell aggregates.

KEYWORDS:

Aggregate; Blebbing; Dissociation; Embryoid body; Rho-associated protein kinase inhibitor; Suspension; iPS cells

PMID:
24856590
DOI:
10.1016/j.jbiosc.2014.04.008
[Indexed for MEDLINE]
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