Enzyme-free passage of human pluripotent stem cells by controlling divalent cations.

Kiyoshi Ohnuma,Saoko Tachikawa,Tatsuo Michiue,Yuzuru Ito,Miho K Furue,Yohei Hayashi,Techuan Chan,Kana Yanagihara,Yasuko Onuma,Ayaka Fujiki,Makoto Asashima

doi:10.1038/srep04646

Abstract

Enzymes used for passaging human pluripotent stem cells (hPSCs) digest cell surface proteins, resulting in cell damage. Moreover, cell dissociation using divalent cation-free solutions causes apoptosis. Here we report that Mg2+ and Ca2+ control cell-fibronectin and cell-cell binding of hPSCs, respectively, under feeder- and serum-free culture conditions without enzyme. The hPSCs were detached from fibronectin-, vitronectin- or laminin-coated dishes in low concentrations of Mg2+ and remained as large colonies in high concentrations of Ca2+. Using enzyme-free solutions containing Ca2+ without Mg2+, we successfully passaged hPSCs as large cell clumps that showed less damage than cells passaged using a divalent cation-free solution or dispase. Under the same conditions, the undifferentiated and early-differentiated cells could also be harvested as a cell sheet without being split off. Our enzyme-free passage of hPSCs under a serum- and feeder-free culture condition reduces cell damage and facilitates easier and safer cultures of hPSCs.

Highlights

Enzymes used for passaging human pluripotent stem cells digest cell surface proteins, resulting in cell damage
To achieve enzyme-free and less damaging passage of human pluripotent stem cells (hPSCs), we focused on the roles of Ca21 and Mg21 in cellcell and cell-fibronectin binding
We hypothesized that solution containing physiological concentration of Ca21, but no Mg21, could be used to passage hPSCs cultured on fibronectin-coated dishes as large cell clumps without the need for enzyme-based cell dissociation

Summary

Introduction

Enzymes used for passaging human pluripotent stem cells (hPSCs) digest cell surface proteins, resulting in cell damage. We hypothesized that solution containing physiological concentration of Ca21, but no Mg21, could be used to passage hPSCs cultured on fibronectin-coated dishes as large cell clumps without the need for enzyme-based cell dissociation. We tested this hypothesis using our serum- and feeder-free culture medium (ESF9a) in fibronectincoated dishes[14,16,17], allowing us to examine hPSC attachments without masking by undefined adherent factors derived from the serum and feeder cells

Methods

Results

Conclusion