A novel electron emission-based cell culture device promotes cell proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells.

Fumiaki Sugimori,Kenichi Morita,Hiroyuki Hirakawa,Mai Takasaki,Ai Tsutsui,Takashi Tsumura,Hiroyuki Yamaji,Shohei Komaru,Yo Uemura,Toshimasa Uemura,Tadashi Iwamatsu,Satoshi Fujita

doi:10.1371/journal.pone.0213579

Abstract

In this report we demonstrate the effect of a novel electron emission-based cell culture device on the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells. Our device has an electron emission element that allows, for the first time, stable emission of electrons into an atmosphere. Atmospheric electrons react with gas molecules to generate radicals and negative ions, which induce a variety of biochemical reactions in the attached cell culture system. In this study, we demonstrated the effect of this new electron emission-based cell culture device on cell proliferation and differentiation using pre-osteoblastic MC3T3-E1 cells. Electron emission stimulation (EES) was applied directly to culture medium containing plated cells, after which the number of living cells, the mRNA levels of osteogenesis-related genes, and the alkaline phosphatase (ALP) activity were evaluated. The growth rate of EES-exposed cells increased by approximately 20% in comparison with unexposed control cells. We also found the mRNA levels of osteogenic specific genes such as collagen type I α-1, core-binding factor α-1, and osteocalcin to be up-regulated following EES. ALP activity, a marker for osteogenic activity, was significantly enhanced in EES-treated cells. Furthermore, reactive oxygen species generated by EES were measured to determine their effect on MC3T3-E1 cells. These results suggest that our new electron emission-based cell culture device, while providing a relatively weak stimulus in comparison with atmospheric plasma systems, promotes cell proliferation and differentiation. This system is expected to find application in regenerative medicine, specifically in relation to bone regeneration.

Highlights

Electron emission devices typically operate only in a vacuum and have long been used in vacuum tubes, CRTs, electron microscopes, and similar instruments
Cells in the neighboring region under the electron emission element more strongly expressed hydroxyl radical (HO) and hypochlorous acid (HClO) compared with other areas when the medium depth was shortened (0.5 ml medium/well) compared with the normal experimental condition (1.5 ml medium/well). These results suggested that the reactive oxygen species (ROS) including HO or HClO generated by Electron emission stimulation (EES) might be key factors that promote the differentiation of MC3T3-E1 cells
Our study demonstrated the effect of an electron emission-based cell culture device on the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells

Summary

Introduction

Electron emission devices typically operate only in a vacuum and have long been used in vacuum tubes, CRTs, electron microscopes, and similar instruments. Few studies have been reported in which the operation of electron emission in an atmosphere was attempted. The MIS (Metal-Insulator-Semiconductor)-type electron emission device has been reported to operate from low vacuum up to atmospheric pressure [1,2]. In the process of developing a charger for an MFP (Multifunction Printer), our group at SHARP CORPORATION recently succeeded in developing the first electron emission device capable of stable operation in atmosphere [3]. Our previous electron spin resonance (ESR) study using spin trap reagent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) showed that products generated from an electron emission device in the aqueous phase contained the hydroxyl radical (HO ), hydrogen radical (H ), and superoxide (O2-) [4]. Hydrogen peroxide (H2O2) was detected, and it is considered that these reactive species are derived from O2 and H2O dissociated by ionized O2

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