Cell Culture over Nanopatterned Surface Fabricated by Holographic Lithography and Nanoimprint Lithography

Eunhye Kim1, Jinwoo Lee1,2, Sungmo Ahn3, Heonsu Jeon3, Kyuback Lee1,*

1Nanomedicine and Nanobiointerface Lab.(Department of Biomedical Engineering, Korea University, South Korea) 2Display and Nanosystem Lab.(Department of Electrical Engineering, Korea University, South Korea)

3Micro and Nano Opto-Electronics Lab. (Department of Physics and Astronomy, Seoul National University, South Korea)

Abstract—Several nanoscale patterns were fabricated over a quartz substrate coated with an UV resin for cell culture. Holography lithography and nanoimprint lithography were applied for the development of the original patterns and replicas. Human osteoblasts were cultured over the patterned surface and showed interesting features including long extension of filopodia and lamellipodium formation.

Keywords-Holographic lithography; Nanoimprint lithography; Osteoblast; Nanopattern; cytoskeleton.

I. INTRODUCTION

Surface specific patterns have been known to influence the biological behaviors of various cells. Microscale patterns showed interesting guidance phenomena for neural cells or several blast cells. However, cells on nanoscale patterns showed something different from those on microscale patterns. Cells on micropatterns showed just the change of their motility over patterns[1], while cells on nanopatterns changed their cytoskeleton. For the investigation of the activities of cytoskeleton, various nanoscale patterns had been regarded to be very useful in providing new information on cellular activity with the resulting cell morphology. In fact, most of anchorage-dependent cells such as blast or neural cell types[2] show their characteristic biological behaviors in nanoscale including the

formation of focal adhesion, because cell-binding tripeptide of Arg-Gly-Asp (RGD) found from most extracellular matrices like fibronectin, vitronectin, laminin[3] and etc are distributed in nanoscale. For example, internal cytoskeletal structure was reorganized and more membrane protrusions were found over nanopatterns[4][5]. Chromatins inside the nucleus were rearranged by external nanopatterns[6]. Finally, gene regulations and apoptosis are also reported to be influenced by nanopatterns[7]. However, more clear understanding of the mechanism is not yet found in the literature. Several reports like above show that the barbed ends of filopodia are looked contact over nanospatterns either selectively or randomly, although specific mechanical activity of filopodia driven by the patterns are not clearly identified. In this study, we investigated the biological behavior of human osteoblast cells cultured over nanopatterns. Several nanotechnologies have introduced in the literature for those purposes including e-beam lithography, photo lithography, holography lithography, nanoimprint lithography, and etc. Among them, the latter two technologies are regarded to have strong potentials in the technological and economical point of views. Therefore, we introduced those two technologies for the fabrication of our characteristic surface nanopatterns.

This project was funded by Seoul R&BD Program and Korea Health 21R&d Project, Ministry of Health & Welfare, Republich of Korea (Projectcode: A0505750).

*Contact author:kblee@korea.ac.kr

Fig. 1 Schematic illustration of our nanopatterning system by laser interference lithography consisted of a laser source, a beamsplitter, two mirrors, two beam filters, two collimating lenses, and one target.

725978-1-4244-1908-1/08/$25.00 ©2008 IEEE.

Proceedings of the 3rd IEEE Int. Conf. on Nano/Micro Engineered and Molecular SystemsJanuary 6-9, 2008, Sanya, China

II. METHODS Our strategy is to fabricate several master stamps by holographic lithography and replicas of those stamps by nanoimprint lithography. Cells are cultured over the replicas, because once used nanopatterns cannot be recycled again for the same purposes. Fig. 1 shows our system of laser interference lithography consisted of one laser source (355 nm wavelength), one beam splitter, two reflecting mirrors, two beam filters, and two collimating lenses and one target. A quartz wafer (4 inch) was used for the stamp preparation. Chrome was at first deposited over the quartz substrate. Then a photoresist was deposited over the chrome layer. Two laser

beam separated by a beam splitter arrived over the photoresist and developed an interference phenomenon. The photoresist was patterned after the laser exposure and following lift-off process. Chrome layer and quartz surface were etched by chrome etchant and RIE (reactive ion etching) process, respectively. The fabricated nanopatterns (positive type) over the quartz substrate were transferred onto a resin (negative type) coated over a glass substrate by UV nanoimprint lithography technique. The transferred patterns were transferred again onto a different resin/quartz substrate to obtain a replica with original positive type pattern as shown in Fig. 2. For better detachment during pattern transfer, an anti-adhesion layer was coated over each resin by self-assembled

Fig. 2 Process flow for the replication of nanoscale patterns fabricated by Laser interference lithography and nanoimprint lithography. Two different resins are included and twice times of imprint processes are required

(a) (b) (c)

Fig. 3 SEM images of patterned UV curable polymer resists. (a) original master pattern, (b) 1st transferred pattern, and (c) final replica

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monolayer technique. Fig. 3 shows the fabricated original master, 1st transferred pattern, and final replica pattern. The width and interval of the pattern are 200 nm and 700 nm, respectively. Human osteoblast (hFOB1.19) was cultured over the replicas during three days. After the culture, cells were fixed and dehydrated for scanning electron microscope observation.

III. RESULTS AND DISCUSSIONThe results of cultured osteoblast over nanopatterns were

shown in Fig. 3. As shown in the figure, the cell shows several interesting features. The upper half of the cell extruded several filopodia, while no filopodia are shown from the lower half. Instead, a wide ruffle can be observed. In addition, the lengths

of filopodia are not uniform. The filopodia extruded toward right are much longer than those extruded toward left. Judging from the images, it can be estimated that the cell is moving from left to right by continuous probing the surface state with its filopodia[8].

Enormous amount of studies on the dynamics and activities of the two actin-related cytoplasmic protrusions have been reported. Signaling pathways accompanied with the cytoplasmic protrusions have been renewed every year in the literature, though the actual biochemical mechanism is not yet clearly understood. Two noticeable models for their genesis among them are dendritic-nucleation (or branching) model for lamellipodium genesis6 and convergent-elongation model for filopodium genesis.7 Based on the dendritic nucleation model, the genesis is initiated by forming a dendritic network of actin

(a) (b) (c)

Fig. 4 SEM images of cells over a nanopillar structure. (a) whole cell image (b) magnified leading edge with several filopodia, and (c) magnified leading edge with a lamellipodium.

Fig. 5 (a) ~ (e) a suggested mechanism of lamellipodium development. However, the mechanism of filopodia development in (f) is still not quite clear yet.

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filaments via series of actin polymerization, which is initiated by Arp2/3 complex bound to N-WASp or WAVE/Scar complexes following the binding with Cdc42, GTP, PIP2, and profilin.8-12 It is known that growth of each actin filament is inhibited by capping protein, however for the barbed ends closer to the leading edge of the plasma membrane, the capping protein is substituted with Ena/VASP, which promotes further actin polymerization through its anti-capping activity.13-15 Despite some disputes, this model is somewhat accepted. For filopodia genesis, no widely accepted model has been reported yet, even though well-controlled experimental results were accompanied. Recently, formin is also reported as an alternative promoter for actin filament assembly.20

IV. CONCLUSIONS We fabricated nanopatterns for osteoblast cell culture using

laser interference lithography and nanoimprint lithography. Over the nanopattern, cell showed a lot of filopodia protrusion into the direction of motility which was never found from the cells on flat surface without any nanopatterns.

ACKNOWLEDGMENT

This research was funded by Seoul R&BD Program and Korea Health 21 R&D Project (Project Code: A0505750), Ministry of Health and Welfare, Republic of Korea.

REFERENCES

[1] J. W. Lee, K. S. Lee, N. N. Cho, B. K. Ju, K. B. Lee and S. H. Lee, "Topographical guidance of mouse neural cell on SiO2 microtracks", Sens. Actuat. B. vol. 128, 2007, pp. 252-257.

[2] Z. Wen, and J. Q. Zheng, "Directional guidance of nerve growth cones", Curr. Opin. Neurobiol. vol. 16, 2006, pp. 52-58.

[3] P. Clark, S. Britland, and P. Connolly, "Growth cone guidance and neuron morphology on micropatterned laminin surfaces", J. Cell. Sci. vol. 105, 1993, pp.203-212.

[4] M. J. Dalby, D. McCloy, M. Robertson, C. D. W. Wilkinson, and R. O. C. Oreffo, "Osteoprogenitor response to define topographies with nanoscale depths", Biomaterials, vol. 27, 2006, pp.1306-1315.

[5] M. J. Dalby, M .O. Riehle, D. S. Sutherland, H. Agheli, A. S. G. Curtis, Biomaterials, vol. 25, 2004, pp.5415-5422.

[6] M. J. Dalby, "Topographically induced direct cell mechanotransduction", J. Med. Eng. Phys. vol. 27, 2005, pp.730-742.

[7] M. J. Dalby, M. O. Riehle, D. S. Sutherland, H. Agheli, and A. S. G. Curtis, "Use of nanotopography to study mechanotransduction in fibroblasts - methods and perspectives", Eur. J. Cell Biol, vol. 83, 2004, pp.159-169.

[8] T. J. Mitchison, and L. P. Cramer, "Actin-based cell motility and cell locomotion", Cell, vol. 84, 1996, pp.371-379.

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