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

• Published in: Journal of bioscience and bioengineering 2014-11, Vol.118 (5), p.588-592
• Main Authors: Horiguchi, Ayumi, Yazaki, Koyuki, Aoyagi, Mami, Ohnuki, Yoshitsugu, Kurosawa, Hiroshi
• Format: Article
• Language: English
• Subjects: Aggregate; Amides - pharmacology; Biological and medical sciences; Biotechnology; Blebbing; Cell Aggregation - drug effects; Cell Culture Techniques - methods; Dissociation; Embryoid Bodies - cytology; Embryoid Bodies - drug effects; Embryoid body; Fundamental and applied biological sciences. Psychology; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - drug effects; Induced Pluripotent Stem Cells - enzymology; iPS cells; Protein Kinase Inhibitors - pharmacology; Pyridines - pharmacology; rho-Associated Kinases - antagonists & inhibitors; Rho-associated protein kinase inhibitor; Suspension
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2014.04.008

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.