Article pubs.acs.org/journal/abseba
Multi-Component Microscaffold With 3D Spatially Defined Proteinaceous Environment Daniela Serien†,‡ and Shoji Takeuchi*,†,‡ †
Center for International Research on Integrative Biomedical Systems, Institute of Industrial Science (IIS), The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan ‡ ERATO Takeuchi Biohybrid Innovation Project, Japan Science and Technology Agency, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan S Supporting Information *
ABSTRACT: In this paper, we present a multicomponent microenvironment consisting of proteinaceous networks with submicron-sized features optionally embedded into a photoresist microscaffold. By two-photon direct laser writing, free-standing 3D proteinaceous microstructures were fabricated for cell culture application, demonstrated with NIH/3T3 fibroblast cells. A Young’s modulus of megapascal-order contributes to the challenge of structural sustainability of the proteinaceous microstructures for experiments as well as sequential fabrication steps. We propose to embed proteinaceous networks into a mechanically robust photoresist microscaffold. We investigate the limits of this 3D microfabrication of embedded proteinaceous networks and demonstrate the embedment of two different proteinaceous networks within one microscaffold. Performing cell culture of PC12 cells, we observe cell adhesion and cell motility on embedded proteinaceous networks of collagen type-IV mixed with bovine serum albumin into a photoresist microscaffold. The ability to structure proteinaceous elements for 3D spatial control of microenvironment might be a key feature in cell culture to decouple environmental cues to control cellular behavior. KEYWORDS: cell culture, direct laser writing, cross-linking
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INTRODUCTION Several 3D printing technologies have been developed to address a range of scales from millimeter sizes1,2 down to micrometer sizes3,4 utilizing different deposition technologies and appropriate materials. Utilization of biocompatible materials5−7 or biomaterials,6−8 3D printing of scaffolds pursues to mimic tissue architectures5,6,8 or cellular microenvironments.7,9,10 Mimicking cellular microenvironment has been used to study cellular communication and mechanobiological processes depending on spatially provided stimuli.10 Recently, direct laser writing (DLW) is gaining relevance for 3D microscaffold fabrication because of its high precision11 and variable fabrication modes that approach super-resolution11 on the one side and mesoscale-sized fabrication dimensions with modified material properties on the other side.12 In particular, proteinaceous microstructures with submicron feature sizes13−15 fabricated by direct laser writing are promising for cell culture applications because photoinitiated cross-linking of protein16,17 preserves the protein binding affinity.18,19 Proteins play an important role in providing spatiotemporal and biophysical stimuli for cellular responses within the cellular microenvironment.20,21 Multicomponent scaffolds fabrication might be a relevant technology to provide spatially separate stimuli for appropriately mimicking cellular microenviron© XXXX American Chemical Society
ments; a previously reported two-component scaffold that consists of two different proteins has illustrated the potential for cell migration studies.22 However, low mechanical strength of proteinaceous microstructures of 0.2−12 MPa23 currently restricts complexity and 3D spatial precision of multicomponent proteinaceous microscaffolds. Here, we present a method for 3D patterning of proteinaceous elements or proteinaceous networks with submicronsized features by DLW, which are embedded within a photoresist backbone scaffold of high mechanical strength,24 in order to provide 3D spatially defined protein stimuli (Figure 1). DLW is applied to a drop-cast of protein prepolymer solution on a cover glass and by two-photon cross-linking of protein free-standing or embedded proteinaceous microstructures are fabricated along the PC-guided laser movement (Figure 1a). On the basis of an icosahedral unit, a free-standing entirely proteinaceous microstructure is easily fabricated (Figure 1b). The pattern freedom of free-standing microstructures is restricted to ensure structure sustainability for all subsequent steps. In comparison, embedding proteinaceous Received: November 10, 2016 Accepted: January 13, 2017
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DOI: 10.1021/acsbiomaterials.6b00695 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Microscaffold Fabrication by Direct Laser Writing. A protein prepolymer solution contains protein BSA and photosensitizer FAD dissolved in an organic-aqueous solvent, optionally further photosensitizer or another protein of interest were added. Details are described in the Supporting Information, similar to previous reports.13−15,17,18 Protein prepolymer solution was drop-cast on cleaned cover glasses (CO13001, Matsunami Glass Ind., Ltd., Japan) and is exposed to a fs-pulsed 780 nm laser light via a 100× N.A. = 1.4objective lens by Photonic Professional (Nanoscribe, Germany), as previously described.14,15 For multicomponent microscaffolds, cleaned cover glasses were coated with protein repellent 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer.25 Photoresist microscaffolds were fabricated with IP-L 780 photoresist (Nanoscribe, Germany) using the Photonic Professional (Nanoscribe, Germany). Immediately after mild O2plasma treatment, proteinaceous prepolymer solution is drop-cast and proteinaceous elements were fabricated as embedment into the photoresist microscaffold. Cell Culture. NIH/3T3 cells were seeded at a concentration of approximately 5 × 104 cells/mL in DMEM medium (D5796, SigmaAldrich) and cultured at 37 °C with 5% CO2 for up to 1 week. Cell culture scaffolds were incubated in 50 μg/mL fibronectin (FN, BT226S, Biomedical Technologies, Inc.) for 20 min and thoroughly rinsed prior to experiments. When required, cells were stained during experiments with cell tracker (cell tracker Green, life technologies). Rat adrenal pheochromocytoma PC12 cells (RCB0009, RIKEN Bio Resource Center) were incubated in growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM, D5796, Sigma-Aldrich) containing 5% (v/v) fetal bovine serum (FBS, FB-1061/500, Biosera), 10% (v/v) Horse Serum (HS, S900−500, Japan Bio Serum), and 1% (v/v) penicillin-streptomycin (P4333, Sigma-Aldrich) at 37 °C and 5% CO2. Differentiation was initiated by applying serum-free DMEM with 1% (v/v) penicillin-streptomycin and 50 ng/mL nerve growth factor (NGF, N6009 NGF-2.5S, Sigma-Aldrich) as reported previously.26,27 We identify potential neurites of differentiation-induced neuritogenesis by their thin and elongated morphology of lengths over 30 μm, i.e., at least 1.5 times a cell body, as suggested previously.28 Microscopy. Cell culture was observed by Biorevo BZ-9000 (Keyence, Japan) in an hourly time lapse. Cell culture was also observed by confocal laser scanning microscopy (LSM 780, Zeiss, Germany) and bright-field microscopy (Olympus IX, Olympus, Japan). After experiments, samples were fixated in preparation for scanning electron microscopy (SEM) with SU-8000 (Hitachi, Japan). Images were subsequently analyzed with ImageJ and Fiji (U.S. National Institutes of Health, USA). Fluorescence imaging, green channel emission fluorescence profiles and 3D reconstruction were obtained using an oxygen scavenging system as reported elsewhere29,30 with confocal laser scanning microscope LSM 710 (Zeiss, Germany) and processing software ZEN (Zeiss, Germany). AFM microscopy was performed and evaluated with JPK NanoWizard SPM and DP Software (JPK instruments AG, Germany). Contact-G cantilever with a rotated monolithic silicon probe with tip radius
