IEICE TRANS. ELECTRON., VOL.E96–C, NO.3 MARCH 2013 353 PAPER Special Section on Recent Progress in Molecular and Organic Devices Label-Free and Noninvasive Monitoring of Cell Differentiation on Spheroid Microarray Hidenori OTSUKA † a) , Member, Masako NAGAMURA † , Akie KANEKO † , Koichi KUTSUZAWA † , and Toshiya SAKATA †† , Nonmembers SUMMARY A two-dimensional microarray of ten thousand (100 × 100) chondrocyte-spheroids was successfully constructed with a 100- μm spacing on a micropatterned gold electrodes that were coated with poly(ethylene glycol) (PEG) hydrogels. The PEGylated surface as a cy- tophobic region was regulated by controlling the gel structure through pho- tolithography. In this way, a PEG hydrogel was modulated enough to in- hibit outgrowth of chondrocytes from cell adhering region in the horizontal direction. These structural control of PEG hydrogel was critical for induc- ing formation of three-dimensional chondrocyte condensations (spheroids) within 24 hours. We report noninvasive monitoring of the cellular func- tional change at the cell membrane using a chondrocyte-based field ef- fect transistor (FET), which is based on detection of extracellular potential change induced as a result of the interaction between extracellular matrix (ECM) protein secreted from spheroid and substrate at the cell membrane. The interface potential change at the cell membrane/gate insulator inter- face can be monitored during the uptake of substrate without any labeling materials. Our findings on the time course of the interface potential would provide important information to understand the uptake kinetics for cellular differentiation. key words: 3D cell culture, spheroid, bovine articular cartilage, gly- cosaminoglycan (GAG), field effect transistor (FET) 1. Introduction Micro-patterning and surface engineering techniques are emerging as important tool to clarify the effects of a mi- croenvironment on cellular behaviour by coordinating cell- surface, cell-cell, and cell-culture medium interactions in a controlled manner [1]–[4], since the microenvironment is a defining factor in a wide range of cellular processes including proliferation, differentiation, and expression of phenotype-specific functions. Particularly, in cell-based biosensors (CBBs) and tissue engineering, in vitro mainte- nance of well-differentiated primary cells as a tissue is nec- essary for long term culture and cell monitoring with spe- cific function. Since isolated primary cells are known to readily lose many cell-specific functions during culture, the most crucial issues in CBBs are long-term viability and re- tention of cell-specific functions of cultured cells. In this regards, the formation of multicellular spheroids is of partic- Manuscript received July 14, 2012. Manuscript revised November 19, 2012. † The authors are with the Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, 162-8601 Japan. †† The author is with the Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, 113-8656 Japan. a) E-mail: h.otsuka@rs.kagu.tus.ac.jp (Corresponding author) DOI: 10.1587/transele.E96.C.353 ular interest, since spheroids show not only morphological but also functional similarities to tissues and organs [5], [6]. In this study, we achieved to construct microarray of cell- spheroid by controlling cyto-phobic surface precisely using poly(ethylene glycol) (PEG) hydrogels which are useful in biomedical and pharmaceutical applications such as drug delivery and tissue engineering. Because PEG is a nontoxic, water-soluble polymer that resists recognition by immune system, and has been approved biocompatible [7], we se- lected hydrogels prepared from PEG with multi arm for non- fouling and cyto-phobic coating. Although there are many reports investigating a relation between the hydrogel’s bulk property [8], [9], viscoelasticity [10], [11], molecular mobil- ity and permeation and biocompatibility, the interfacial phe- nomenon between cells and substrate surface is complicated and difficult to understand. The primary aim of the present study is to control the surface properties of PEG hydrogel for the array formation of spheroid on the gold surface. In particular, we report the formation of primary chondrocyte spheroid on the gold elec- trode for the potential of long term monitoring the cell be- havior, which can be expected to estimate a cellular differ- entiation in response to the signaling molecules. 2. Experimental Section A branched poly(ethylene glycol) derivative used in this study, and a substrate using the photosensitive composition containing the poly(ethylene glycol) are described specifi- cally. 2.1 PEG Synthesis Synthesis of branched poly(ethylene glycol) derivative (4PA20K). 12 g (93.6 mmol) of 4-azide-benzoic acid was dissolved in 40 mL of thionyl chloride, and the mixture was heated under reflux for 1.5 hours. The reaction was concen- trated under reduced pressure, and a small amount of hexane was added thereto. The mixture was concentrated again un- der reduced pressure, and dried under vacuum, as a result of which 9.3 g (51.2 mmol, yield 70%) of 4-azide-benzoic acid chloride, which is a target product, as a white solid was ob- tained. 1 H-NMR (CDCl3) δ 8.11–8.15 (2H, m), 7.11–7.16 (2H, m). Next, 81 mg (0.8 mmol) of triethylamine and 147 mg (1.2 mol) of 4-dimethylaminopyridine were added to 3 mL Copyright c  2013 The Institute of Electronics, Information and Communication Engineers