Precisely controlling spatio-temporal environmental parameters has become increasingly important in the field of biotechnology. For this reason, biomedical research has increasingly moved toward the design and implementation of microfabricated systems to efficiently improve technologies such as drug delivery,[1] diagnostics,[2] and tissue engineering.[3] Technologies featuring micron length-scales tailored specifically for biomedical applications, termed Bio-microelectrical mechanical systems (BioMEMS), are able to interact with biological systems such as cells[4] or even single biomolecules.[5] Strategies for developing BioMEMS typically involve adapting traditional microfabrication materials and processes resulting in systems fabricated using non-degradable materials including silicon[6] and polydimethylsiloxane (PDMS).[7] Using biodegradable polymers allows for implantable Bio-MEMS to satisfy the growing demand for in vivo applications such systems for drug delivery systems[8] or tissue engineering. To address these potential applications, BioMEMS devices have been fabricated using biopolymers, both natural and synthetic, including gelatin,[9] alginate,[10] poly(l-lactic acid) (PLA),[8] poly(l-lactic-co-glycolic) acid (PLGA),[11] and poly(glycerol-co-sebacate) (PGS).[12] An ideal biomaterial for BioMEMS fabrication from a material properties standpoint is one that; 1) can be processed using mild conditions to facilitate protein or growth factor incorporation; 2) naturally promotes adhesion and normal function of seeded cells; 3) contains moieties for potential chemical modification of the surface; 4) exhibits slow and predictable degradation rates to maximize duration of functional implanted devices; 5) has robust, yet flexible mechanical properties; 6) is relatively inexpensive. One class of natural biomaterials that could potentially meet these material requirements is silk fibroin.[13–20] Silk fibroin protein from the Bombyx mori silkworm is FDA approved and has been used in medicine for a wide variety of applications including surgical, drug delivery,[14] and tissue engineering.[13] Silk fibroin exhibits in vitro and in vivo biocompatibility,[13,15] robust mechanical properties including high mechanical modulus and toughness,[16] and relatively slow proteolytic biodegradation.[17] In this report, we describe techniques and materials processing strategies utilized in the fabrication of cell-seeded silk fibroin microfluidic devices. We have developed material-specific processes for silk fibroin micromolding and device assembly that is analogous to soft-lithographic techniques. By implementing aqueous casting of regenerated aqueous silk fibroin solutions to produce microfabricated silk films, we avoid the use of toxic solvents and harsh processing conditions. Micro-fluidic devices were produced by laminating water-stable micromolded silk fibroin membranes, which were modified with macroscopic fluidic connections. Biocompatibility and functionality of patent devices with cells was studied by seeding and perfusion of a model human hepatocarcinoma cell line for up to five days. Hepatocytes cultured in silk fibroin-based microfluidic devices exhibited similar morphology and cell functions to those grown on other widely used biomaterials. Silk films derived from regenerated aqueous silk solutions exhibited FT-IR absorbance peaks that are characteristic of amorphous silk I structure, such as the amide I peak at 1656.6 cm−1 and the amide II peak at 1541.5 cm−1. Treatment with aqueous-methanol solution shifted the peaks from silk I configuration to the crystalline silk II configuration, as amide I and amide II peaks shifted to 1616.3 cm−1 and 1515.6 cm−1 for post-methanol treated films, respectively (Supporting Information, Fig. 1). These peak shifts suggest an increase in the percentage of crystalline structure within the bulk, which has been demonstrated by others.[21] Fully hydrated water-stable films processed in this manner have been shown to increase β-sheet formation,[22] which result in increased stiffness, as determined by tensile Young’s modulus, increased toughness modulus, and an increased ultimate tensile strength over thermally crosslinked PGS films (Supporting Information, Fig. 1; Table 1). Table 1 Comparison of Mechanical Properties of Regenerated Silk Fibroin and PGS Films. The value n represents the number of samples in each dataset. Data reported as mean ± s.d. Devices were fabricated from regenerated silk fibroin films that measured approximately 200 microns in thickness, which was controlled by the volume to surface area ratio during casting. The lamination strategy utilized aqueous silk fibroin solution to bond replica-molded water-stable silk films (Fig. 1). The average and root-mean-squared surface roughness of silk films after the lamination process was 216.6 nm and 267.4 nm, respectively (Supporting Information, Fig. 2). Replica-molded silk fibroin films cast on PDMS negative molds could be produced in rapid succession while maintaining a high degree of feature fidelity. Features as small as 400 nm could be produced using this method (Fig. 2a). Micro-molded films (Fig. 2b) were bonded to flat films to produce microfludic devices (Fig. 2c and d) that could support flow (Fig. 2e and f). Occlusion of microchannels from excess aqueous silk fibroin solution during the bonding process at the inlet/outlet both contributed to reduced device yield. The microfluidic layout used in this study has been designed to produce a constant maximum wall shear stress within all microchannels in the device, given a steady volumetric flow rate.[23] This device geometry assisted in initial cell seeding by allowing cells to be evenly distributed throughout the device during attachment. Furthermore, the constant maximum wall shear stress design facilitates rapid perfusion, while minimizing the potential detachment of seeded cells from shear forces. The high cell seeding density resulted in the formation of HepG2 aggregates, which increased the opportunity for adhesion of cells to the microchannels. Suitable perfusion rates were characterized for the perfusion culture of HepG2 cells cultured in PGS microfluidic devices with similar length scales.[12] The morphology of HepG2 cells seeded and perfused in silk fibroin microdevices (Fig. 3) was similar to that of HepG2 cells cultured on other biomaterials including PGS[12] (Supporting Information, Fig. 3). Viability and liver-specific function of HepG2 cells cultured on silk fibroin were also determined to be similar across static and dynamic cultures. HepG2 cells cultured on silk fibroin films exhibited similar albumin secretion rates as those cultured on other typical biomaterials. Additionally, HepG2 cells cultured statically on films had similar albumin secretion levels to those cultured in dynamically perfused silk fibroin microfluidic devices (Supporting Information, Fig. 2). An increase in albumin secretion levels was observed from day 3 through day 5, which was likely due to increasing cell densities within the microchannels. Figure 1 Fabrication Strategy for Silk Fibroin Microfluidic Devices Figure 2 Silk Fibroin-based Microfluidic Devices Figure 3 Cell-Seeded Silk Microfluidic Devices The device fabrication strategy in this study allows for the rapid and scaleable production of silk-fibroin-based microfluidic devices without the need for harsh processing conditions or cytotoxic compounds. The techniques employed in this strategy are scalable by designing systems with increased surface area and lamination of multiple layers. Although the device yield for patent devices in this study was relatively low, the success rate could be increased by designing flow layouts with redundant microchannel connectivity and by employing additional covalent bonding agents such as (1-ethyl-3-[3-dimehtylaminopropyl] carbodiimide hydrochloride) (EDC) and N-hydroxysuccinimide (NHS), an established chemical route for bioconjugation of amines to carboxylates.[13,24] Similar techniques could be used to covalently link peptides or other bioactive molecules both on the surface and throughout the bulk of the material of the device as previously shown for cell binding peptides and morphogens.[19,28] The aspects of device scalability and incorporation of biomolecules may be important in the design and fabrication of tissue engineering scaffolds for highly vascularized tissue. These biodegradable microfluidic systems can also be integrated with existing biomaterial systems and technologies for tissue-specific applications and increased functionality. For example, drug delivery systems,[25] cell patterning techniques,[26,27] contact guidance cues,[28] and co-culture systems for hepatocytes[29] can be integrated within the microchannels to promote the organization of seeded cells into complex tissues. The robust properties including a high toughness modulus and ultimate tensile strength could permit the use of silk fibroin-based devices in dynamic mechanical environments associated with in vivo applications. In addition to the fabrication of microfluidic systems, strategies for micromolding silk fibroin could be potentially useful in other Bio- MEMS devices including biodegradable drug delivery devices, scaffolds, or biosensors.[8,11–12] The techniques in this report are general and could be used for advancement of the field of implantable and resorbable microfabricated systems, further expanding the impact of related technologies in biomedical applications.