Fabrication of Micro Patterned Fibronectin for Studying Adhesion and Alignment Behavior of Human Dermal Fibroblasts

Seung-Jae Lee; Youngsook Son; Chun-Ho Kim; Mansoo Choi

Macromolecular Research, Vol.15, No.4, 348-356, June, 2007

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Fabrication of Micro Patterned Fibronectin for Studying Adhesion and Alignment Behavior of Human Dermal Fibroblasts

Seung-Jae Lee, Youngsook Son, Chun-Ho Kim^†, and Mansoo Choi¹

Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Sciences, Seoul 139-240, Korea
¹School of Mechanical and Aerospace Engineering, Seoul National University, Seoul 151-742, Korea

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The aim of this study was to fabricate a submicro- and micro-patterned fibronectin coated wafer for a cell culture, which allows the positions and dimensions of the attached cells to be controlled. A replica molding was made into silicon via a photomask in quartz, using E-beam lithography, and then fabricated a polydimethylsiloxane stamp using the designed silicon mold. Hexadecanethiol [HS(CH2)15CH3], adsorbed on the raised plateau of the surface of polydimethylsiloxane stamp, was contact-printed to form self-assembled monolayers (SAMs) of hexadecanethiolate on the surface of an Au-coated glass wafer. In order to form another SAM for control of the surface wafer properties, a hydrophilic hexa (ethylene glycol) terminated alkanethiol [HS(CH2)11(OCH2CH2)6OH] was also synthesized. The structural changes were confirmed using UV and 1H-NMR spectroscopies. A SAM terminated in the hexa(ethylene glycol) groups was subsequently formed on the bare gold remaining on the surface of the Au-coated glass wafer. In order to aid the attachment of cells, fibronectin was adsorbed onto the resulting wafer, with the pattern formed on the gold-coated wafer confirmed using immunofluorescence staining against fibronectin. Fibronectin was adsorbed only onto the SAMs terminated in the methyl groups of the substrate. The hexa (ethylene glycol)-terminated regions resisted the adsorption of protein. Human dermal fibroblasts (P = 4), obtained from newborn foreskin, only attached to the fibronectin-coated, methyl-terminated hydrophobic regions of the patterned SAMs. N-HDFs were more actively adhered, and spread in a pattern spacing below 14 m, rather than above 17 m, could easily migrate on the substrate containing spacing of 10 m or less between the strip lines.

Keywords:submicro-pattern;fibronectin;human dermal fibroblast;self-assembled monolayer;lithography

[References]

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The American Heritage(r) Dictionary of the English Language, Third Edition (c) 1996 by Houghton Mifflin Company. Electronic version licensed from INSO Corporation
Kornberg L, Earp HS, Parsons JT, Schaller M, Juliano RL, J. Biol. Chem., 267, 23439 (1992)
Schaller MD, Parsons JT, Curr. Opin. Cell Biol., 6, 705 (1994)
Singhvi R, Kumar A, Lopez GP, Stephanopoulos GN, Wang DI, Whitesides GM, Ingber DE, Science, 264(5159), 696 (1994)
Tan J, Shen H, Saltzman WM, Biophys. J., 81, 2569 (2001)
Berry CC, Campbell G, Spadiccino A, Robertson M, Curtis ASG, Biomaterials, 25, 5781 (2004)
Dewez JL, Lhoest JB, Detrait E, Berger V, Dupont-Gillain CC, Vincent LM, Schneider YJ, Bertrand P, Rouxhet PG, Biomaterials, 19, 1441 (1998)
James K, Kohn J, MRS Bull., 21, 22 (1996)
Hubbell JA, TRIP, 2, 20 (1994)
Schakenraad JM, Busscher HJ, Colloids Surf., 42, 331 (1989)
Lan S, Veiseh M, Zhang M, Biosens. Bioelectron., 20, 1697 (2005)
Veiseh M, Wickes BT, Castner DG, Zhang M, Biomaterials, 25, 3315 (2004)
Wilkinson CDW, Riehle M, Wood M, Gallagher J, Curtis ASG, Mater. Sci. Eng., 19, 263 (2001)
Curtis ASG, Wilkinson CDW, Trends Biotechnol., 19, 97 (2001)
Xia Y, Whitesides GM, Angew. Chem.-Int. Edit. Engl., 37, 550 (1998)
Xia Y, Whitesides GM, Ann. Rev. Mater. Sci., 28, 153 (1998)
Park HS, Kang HJ, Kim CH, Kim CH, Kim TH, Gin YJ, Son Y, Tissue Eng., 10, 343 (2004)
Pale-Grosdemange C, Simon ES, Prime KL, Whitesides GM, J. Am. Chem. Sci., 113, 12 (1991)
Mrksich M, Dike LE, Tien J, Ingber DE, Whitesides GM, Exp. Cell Res., 235, 305 (1997)
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Dalby MJ, Riehle MO, Yarwood SJ, Wilkinson CD, Curtis AS, Exp. Cell Res., 284, 274 (2003)
Maniotis AJ, Chen CS, Ingber DE, Proc. Natl. Acad. Sci. USA, 94, 849 (1997)
Lewin B, Cassimeris L, Lingappa VR, Plopper G, Cells, Jones & Bartlett Publishers, Sudbury, 2006. p.4

[Referenced By]

[1] Williams D, ESB Satellite Consensus Conference, Sorrento, September 2005, p. 9, Outline Proposals for Definitions and Classifications

[2] The American Heritage(r) Dictionary of the English Language, Third Edition (c) 1996 by Houghton Mifflin Company. Electronic version licensed from INSO Corporation

[3] Kornberg L, Earp HS, Parsons JT, Schaller M, Juliano RL, J. Biol. Chem., 267, 23439 (1992)

[4] Schaller MD, Parsons JT, Curr. Opin. Cell Biol., 6, 705 (1994)

[5] Singhvi R, Kumar A, Lopez GP, Stephanopoulos GN, Wang DI, Whitesides GM, Ingber DE, Science, 264(5159), 696 (1994)

[6] Tan J, Shen H, Saltzman WM, Biophys. J., 81, 2569 (2001)

[7] Berry CC, Campbell G, Spadiccino A, Robertson M, Curtis ASG, Biomaterials, 25, 5781 (2004)

[8] Dewez JL, Lhoest JB, Detrait E, Berger V, Dupont-Gillain CC, Vincent LM, Schneider YJ, Bertrand P, Rouxhet PG, Biomaterials, 19, 1441 (1998)

[9] James K, Kohn J, MRS Bull., 21, 22 (1996)

[10] Hubbell JA, TRIP, 2, 20 (1994)

[11] Schakenraad JM, Busscher HJ, Colloids Surf., 42, 331 (1989)

[12] Lan S, Veiseh M, Zhang M, Biosens. Bioelectron., 20, 1697 (2005)

[13] Veiseh M, Wickes BT, Castner DG, Zhang M, Biomaterials, 25, 3315 (2004)

[14] Wilkinson CDW, Riehle M, Wood M, Gallagher J, Curtis ASG, Mater. Sci. Eng., 19, 263 (2001)

[15] Curtis ASG, Wilkinson CDW, Trends Biotechnol., 19, 97 (2001)

[16] Xia Y, Whitesides GM, Angew. Chem.-Int. Edit. Engl., 37, 550 (1998)

[17] Xia Y, Whitesides GM, Ann. Rev. Mater. Sci., 28, 153 (1998)

[18] Park HS, Kang HJ, Kim CH, Kim CH, Kim TH, Gin YJ, Son Y, Tissue Eng., 10, 343 (2004)

[19] Pale-Grosdemange C, Simon ES, Prime KL, Whitesides GM, J. Am. Chem. Sci., 113, 12 (1991)

[20] Mrksich M, Dike LE, Tien J, Ingber DE, Whitesides GM, Exp. Cell Res., 235, 305 (1997)

[21] Yim EKF, Reano RM, Pang SW, Yee AF, Chen CS, Leong KW, Biomaterials, 26, 5405 (2005)

[22] Dalby MJ, Riehle MO, Yarwood SJ, Wilkinson CD, Curtis AS, Exp. Cell Res., 284, 274 (2003)

[23] Maniotis AJ, Chen CS, Ingber DE, Proc. Natl. Acad. Sci. USA, 94, 849 (1997)

[24] Lewin B, Cassimeris L, Lingappa VR, Plopper G, Cells, Jones & Bartlett Publishers, Sudbury, 2006. p.4