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1 近畿大学次世代基盤技術研究所報告 Vol. 7(2016)59-63 PEG *1 *2 *3, *3, *4 Immobilization of PEG onto cell microarrays as a substrate for cell fusion and evaluation of their surface properties Akinori IMAJO *1 Shiho NAGAOKA *2 Wonhwan CHOI *3 Masatsugu NAGASHIMA *3, and Kohei SHIRAISHI *4 To prepare a surface-modified microarray(μay) on both PEG segments and specific protein binding sites as a substrate for high-efficiency cell fusion, PEG macromonomer(macpeg) [P(macPEG)] or/and N-succinimidyl arylate(nas) and macpeg copolymer [P(macPEG-co- NAS)] with different compositions were selectively immobilized on the glass spots of the μay by using surface-initiated radical copolymerization. Two kinds of selective immobilization methods of 3-aminopropyltrimethoxysilane were attempted on a glass substrate as a model of spots of μay by using 1)vapor deposition and 2)immersion in the solution, and evaluated their surfaces from the scanning probe microscope(spm) and water contact angle measurements. Poly(macPEG) and poly- (macpeg-co-nas) chains as brushes were observed on the glass(g-glass). As a result, the SPM images and wettability based on the immobilized polymers on the g-glasses were changed by changing treated methods of alkoxysilane. Keywords: PEG immobilization, Suface-initiated radical polymerization, Cell microarray, Cell fusion, Vapor deposition method (ips) (1) (2),(3) DNA (4) 21 (1) ips (5) (6) (HAC) (7) ips (8) (9),(10) HAC MC * * * * siraisi@hiro.kindai.ac.jp

2 MMCT MC (11) 0.001% (12) (PEG) PEG (13)~(17) (18)~(22) HAC MC 30%50% (PEG) MC PEG HAC 1 PEG (μay)μay MC PEG PEG μay μay (23) PEG PEG PEG NH2 N-succinimidyl acrylate(nas)peg Poly(ethylene glycol)methyl ether methacrylate(mn: 1,100)[macPEG] P(macPEG) P(macPEG-co-NAS)μAy g-ay (SPM) () 3-aminopropyltrimethoxysilane[APMSi] N,N-dimethylformamide[DMF] triethylamine[tea] NAS macpeg (Mn: 1,100) 80 (μay). Milli-Q synthesis 4,4 -azobis(4-cyanovaleric acid chloride)[v501-cl] 4.4 -azobis(4-cyanovaleric acid)[v501] [PCl5] [30 /18 M =1/3(v/v)] 8 ml (Fisher Blan)1 h [APMSi ] g (1 mmol) APMSi DMF 2 ml 603 h DMF 15 min (Fig.1-A) - APMSi 2 h (Fig.1-B) - 3 ml APMSi 30 2 h (Fig.1-C) - 3 ml APMSi 80 2 h (Fig

6 L TEA APMSi 3020 h [] macpeg 1.65 g(1.

g-glass 0.0315 g(0.1 mmol)v501-cl 1 ml 30.

- μay APMSi (μay:) SImage Entry (25) 10 μl 1 min

3 1-D) min [] g(0.1 mmol) V501-Cl 1 ml 30.6 L TEA APMSi 3020 h [] macpeg 1.65 g(1.5 mmol) 1 ml, macpeg 0.99 g(0.9 mmol)nas g(0.6 mmol)0.5 ml 0.5 ml DMF 11 1 ml V501-Cl 8020 h 3 45 min PEG g-glass g(0.1 mmol)v501-cl 1 ml 30.6 LTEA APMSi 3020 h macpeg 1.65 g(1.5 mmol) 1 ml V501-Cl 8020 h 3 45 min g-μay Fig.2. - μay APMSi (μay:) SImage Entry (25) 10 μl 1 min 5 (SPM) Shimadzu SPM-9500J3 Innovation Solutions Bulgaria Ltd Budget sensors Tap300AI-G30 μm 30 μm0.5 Hz Fig.1. APMSi [30 /18 M =1/3(v/v)]8 ml 1 h - 3 ml APMSi μay 30 2 h (Fig.2) min APMSi APMSi APMSi

㸬ࡋ ࡋ㸪ᾐₕἲ ࡇ ࢩ ศᏊ ኈࡀ㔜 ࡗ 㔜 ࡍ ᛶࡀ㧗ࡃ 㸪㔜 ᮎ➃ ᇶࡀᇙ 㸪ᇶᯈ 㠃 ᆒ ᅛᐃ ࡋ ࡎ㸪 ᯝ ࡋ ࢫ 㠃 APMSi ࡀከ ᅛ ᐃ ࡁ 㸪㛤ጞ ᅛᐃ ᇶ ࡀῶᑡ ࡋ㸪ᆒ ᅛᐃࡀ㞴ࡋ 㸬 ᪉㸪 APMSi ࡉ ࡏ 㸪 ᇶ ᯈ 㠃 SAM (SelfAssemble Monolayer㸧㓄 ᛶ ᣢࡘ ᕫಟ ࡀᙧᡂࡉ 㸬 ἲ 㸪 ፹ ࡀ ࡃᗫᾮ ῶ㔞㸪ᇶᯈస〇ࢥࢫ ๐ῶ

4 㸬ࡋ ࡋ㸪ᾐₕἲ ࡇ ࢩ ศᏊ ኈࡀ㔜 ࡗ 㔜 ࡍ ᛶࡀ㧗ࡃ 㸪㔜 ᮎ➃ ᇶࡀᇙ 㸪ᇶᯈ 㠃 ᆒ ᅛᐃ ࡋ ࡎ㸪 ᯝ ࡋ ࢫ 㠃 APMSi ࡀከ ᅛ ᐃ ࡁ 㸪㛤ጞ ᅛᐃ ᇶ ࡀῶᑡ ࡋ㸪ᆒ ᅛᐃࡀ㞴ࡋ 㸬 ᪉㸪 APMSi ࡉ ࡏ 㸪 ᇶ ᯈ 㠃 SAM (SelfAssemble Monolayer㸧㓄 ᛶ ᣢࡘ ᕫಟ ࡀᙧᡂࡉ 㸬 ἲ 㸪 ፹ ࡀ ࡃᗫᾮ ῶ㔞㸪ᇶᯈస〇ࢥࢫ ๐ῶ ᇶᯈస〇㛫 ᕤ ࡉ 㸬 ࡓ㸪ᾐₕἲ ᶵࢩ ศᏊ 㔜 Ꮚ ሁ ῶ㔞 ࡁ Ⅼ ࡘ㸬 ἲ ࡋ㸪 ࢫᇶᯈཬ μay ࢫࢫ 㧗ᐦᗘ ᅛᐃἲ ウࡋ㸪᪂つ㛤 ࡋࡓ ἲ ᾐₕἲ ẚ ࡗࡓ㸬 స〇ࡋࡓ ࢫᇶᯈ ಟ㣭ㄆࡍ ࡓ 㸪SPM ᐃ Ỉ ᥋ゐゅ ᐃ ࡗࡓ㸬Ỉ ᥋ゐゅ ᐃ ᮍಟ㣭 ࢫ ǉ=63 ࡗࡓ㸬 P(macPEG-co-NAS)-g-glass㸪P(macPEG)-g-glass APMSi ᅛᐃ ἲ ࡅ Ỉ ᥋ゐゅ Table 1 ࡍ㸬APMSi ᅛᐃ ἲ ኚ ࡉࡏࡓࡑ ࡒ g-glass ᮍฎ ࢫ ẚ ᇶᯈ 㠃 ᛶࡀኚ ࡋ ࡓ㸬P(macPEG) P(macPEG-co-NAS) ẚ ࡍ 㸪P(macPEG) ࡀ ࡎ ᥋ゐゅࡀ㧗ࡃ㸪 Ỉᛶ ࡇ ࡀ ㄆ ࡁࡓ㸬ࡇ 㸪 P(macPEG-co-NAS) ẚ 㸪NAS ඹ㔜 ࡗ 㸪㙐㛫 య㞀ᐖࡀప ࡋ㸪 ᇶᯈ 㠃 ᅛᐃ ࡉ PEG 㙐㔞 ᐦᗘ ࡀ㧗 ࡃ ࡗࡓ ᥎ᐃࡋ 㸬㠃㛤ጞ㔜 ᚋ ࢫ 㠃 poly(macpeg) poly(macpeg-co- NAS) ಟ㣭ㄆࡍ ࡓ 㸪 Ẽ㞺ᅖẼ 㠃 SPM ᐃ(30 μm 30 μm) ᯝ Fig.3,4 ࡍ㸬ᮍฎ ࢫ พฝࡀ ࡃ ࢫࢣ ᖹ ࡇ ᑐࡋ㸪 APMSi ᅛᐃ ᪉ἲ ኚ ࡋࡓ ᯝ㸪 ࡆἲ㸪 ࡅἲ㸪㟼 ἲ poly(macpeg) poly(macpeg-co- NAS) ࢢ 㔜 ࡋࡓ g-glass 㸪ࡑ ࡒ 㸪ࡇ ᮶ ࢫࢣ ࢩ พฝ ࡀ 㠃ほᐹࡉ ࡓ㸬㸫 ࡆἲ 㸪P(macPEG)-g-glass ᇶᯈ ᑐ ᛶࡀప 㒊ศࡀከࡃ㸪ᇶᯈ 㠃 ᒙࡀᆒ 㸪 ᪉ APMSi Ẽࡀᇶᯈ ᆒ ࡗࡓࡓ 㸪 ᅛᐃ ࡀ ࡌࡓ 㸬 ࡓ㸪㸫 ࡅἲ 㸪 P(macPEG-co-NAS)-g-glass㸪 P(macPEG)-g-glass macpeg 㙐ᅛᐃ ᇶᯈ 㸪ᾐₕἲ ᅛᐃ ᇶᯈ ᙎᛶኚ 㸪 พฝࡀᑡ ࡃ㸪ᾐₕἲ ㄆ㝽ᵝ พฝ ᑡ 㸬㠃 య ᆒ ᅛᐃࡀ ࡁࡓ 㸬ࡇ 㸪APMSi Ẽࡀᆒ ࢫᇶᯈ㠃 ᥋ゐࡋ㸪 APMSi SAM ࡀᙧᡂࡉ ࡓࡓ ᥎ᐃࡋ 㸬㓄 ᛶ SAM ᙧᡂ ࡗ 㔜 ሙ APMSi ᇶᮎ➃ࡀᇶᯈ 㠃 ᆒ ᅛᐃ ࡉ ࡓࡓ 㸪 ᅛᐃ㔞ཬ 㠃 พฝཬ ᙎᛶ ᆒ ᛶࡀ㧗 ࡗࡓ 㸬ࡉ 㸪㸫㟼 ἲ 㸪๓ ࡋࡓ ἲ 2 ἲ ẚ 㸪ᇶᯈ APMSi ࡀ 㞳㟼 ࡉ 㸪㠃 ᅛᐃ 㔞 ῶᑡ 㸪 ᅛᐃ㔞ࡀప ࡋ㸪 ᪉ 㠃ᛶ ኚ ࡋࡓ 㸬ᚑࡗ 㸪APMSi Ẽ ᇶᯈ ᆒ ᅛᐃ ᇶᯈ ᥋ゐࡉࡏ ࡓ 㸪ᇶᯈ APMSi 㓄 ཬ APMSi Ẽὶ ᛶ 㐺 ࡀᚲせ㸬 Fig.3. ᾐ ₕ ἲ ἲ స 〇 ࡋ ࡓ P(macPEG)g-glass SPM Fig.4. ᾐ ₕ ἲ ἲ స 〇 ࡋ ࡓ P(macPEG-coNAS)-g-glass SPM 62 Table 1. APMSi ᅛᐃ ἲ ㄪ〇ࡋࡓ PEG 㙐ᅛᐃ ࢫ 㠃 ᥋ゐゅ

5 3.1 APMSi - μay APMSi PEG μay μay 75 - g-μay μay SPM μay 3.1 SPM - μay PEG μay PEG HAC DNAsiRNA SPM PEG P(macPEG-co-NAS) P(macPEG) μay - APMSi μay μay PEG (1) S. Yamanaka et al, Cell, 126 (4), (2006), 663, 14(8), (2009), p8-14. (2) T. Kondo et al., Cell Stem Cell, 12, (2013), 487. (3) H. Chono et al., PLOS ONE, 6(8), (2011), e (4) T. Nakayama et al., RNA Biology, 10(9), (2013), (5) H. Mizuguchi et al., PNAS, 111(47), (2014), (6), ips,, (2009). (7) 82(9), (2010), 846. (8) M. Oshimura et al., PLoS ONE, 6(10), (2011), e (9) 52(11), (2012), (10) M.Oshimura et al., Molecular Therapy, 18(2), (2010), 386. (11) M. Oshimura et al., BMC Biotechnology, 10, (2010), 37. (12) 53, (2009), 11. (13) De St. Groth S.F et al., J. Immunol. Methods, 35, (1980), 1 (14) N. Naoki et al., Polymer Journal, 27(3), (1995), 211. (15) W. P. Thayer et al., Journal of Neuroscience Research, 93(4), (2015), 572. (16) Arnab Sen et al., Research in Plant Biology, 5(1), (2015), 20. (17) Cameron L. Ghergherehchi et al., Neural Regen Res., 11(2), (2016), 217. (18) T. Nakamura et al., Plant Cell Physiol, 20(7), (1979), (19) U. Zimmermann et al., Plant Physiol, 67(4), (1981), 849. (20) U. Zimmermann et al., J. Biol. Phys., 10, (1982), 43. (21) Alexander M. Christov et al., Plant Physiol, 100, (1992), (22) Eberhard Neumann et al., Journal of Cell Science, 126(9), (2013), (23),, 61(2), (2010),

< BDF91E58E9F90A291E38AEE94D595F18D908F E696E6464>

< BDF91E58E9F90A291E38AEE94D595F18D908F E696E6464> 近畿大学次世代基盤技術研究所報告 Vol. 9(2018)55-59 Additive Manufacturing *1 *2 *3 The Recent Trend on Additive Manufacturing Technology: A Review Hideki KYOGOKU *1, Toshi-Taka IKESHOJI, Makiko YONEHARA The recent trend