Cloning SU8 silicon masters using epoxy resins to increase feature replicability and production for cell culture devices

Abstract

In recent years, there has been a dramatic increase in the use of poly(dimethylsiloxane) (PDMS) devices for cell-based studies. Commonly, the negative tone photoresist, SU8, is used to pattern features onto silicon wafers to create masters (SU8-Si) for PDMS replica molding. However, the complexity in the fabrication process, low feature reproducibility (master-to-master variability), silane toxicity, and short life span of these masters have been deterrents for using SU8-Si masters for the production of cell culture based PDMS microfluidic devices. While other techniques have demonstrated the ability to generate multiple devices from a single master, they often do not match the high feature resolution (∼0.1 μm) and low surface roughness that soft lithography masters offer. In this work, we developed a method to fabricate epoxy-based masters that allows for the replication of features with high fidelity directly from SU8-Si masters via their PDMS replicas. By this method, we show that we could obtain many epoxy based masters with equivalent features to a single SU8-Si master with a low feature variance of 1.54%. Favorable feature transfer resolutions were also obtained by using an appropriate Tg epoxy based system to ensure minimal shrinkage of features ranging in size from ∼100 μm to <10 μm in height. We further show that surface coating epoxy masters with Cr/Au lead to effective demolding and yield PDMS chambers that are suitable for long-term culturing of sensitive primary hippocampal neurons. Finally, we incorporated pillars within the Au-epoxy masters to eliminate the process of punching media reservoirs and thereby reducing substantial artefacts and wastage.

FIG. 1.

Au-epoxy master production. (a) Schematic of the replica molding process starting from the SU8-Si master (step 1) to the Au-epoxy master (step 7) to the generation of a PDMS device (step 9). A PDMS replica of the SU8-Si master (steps 1–3) is used to mold the epoxy contained within a suitable container, such as a petri dish (dashed lines) (steps 4–6). The surface of the epoxy is then coated with Cr/Au to facilitate future PDMS demolding (steps 7–9). (b) Diagram of a PDMS microfluidic culture device for neurons which contains varying feature heights: ∼4 mm wells for loading solution into the channels; ∼100 μm high cell compartments which house the neurons; and ∼4 μm tall microgrooves which allow growth of axons and dendrites, but not cell bodies. We used this microfluidic device configuration as a model for the remainder of this study. (c) Photograph of a Au-epoxy master contained in a standard 100 mm × 50 mm petri dish for PDMS casting.

FIG. 2.

Feature replication precision from SU8-Si master to epoxy master and epoxy master durability. (a-i) SEM image of parallel SU8 microgrooves flanked by 2 SU8 compartments on a representative SU8-Si master. (a-ii) Close-up SEM image of the microgrooves and compartment side wall on the SU8-Si master. (b-i) SEM image of replicated parallel microgrooves on a representative epoxy master. (b-ii) Close-up SEM image of the microgrooves and compartment side wall features formed into the epoxy master. (c) Graph of the microgroove height measurements of the SU8-Si master 3.938 ± 0.11 μm (n = 8), low Tg Easy Cast Au-epoxy master 3.417 ± 0.11 μm (n = 12), and high Tg Epotek Au epoxy masters 3.785 ± 0.08 μm (n = 8). (d) Graph of microgroove width measurements of the SU8-Si masters 7.567 ± 0.21 μm (n = 24), low Tg Easy Cast Au-epoxy masters 6.883 ± 0.14 μm (n = 24), and high Tg Epotek Au-epoxy masters 7.533 ± 0.22 μm (n = 24). (e) Graph of compartment height measurements of the SU8-Si masters 129.9 ± 3.4 μm (n = 14), low Tg Easy Cast Au-epoxy masters 120.3 ± 7.39 μm (n = 14), and high Tg Epotek Au-epoxy masters 127.7 ± 4.85 μm (n = 10). (f) Graph of microgroove height measurements of an Epotek master before the first PDMS cast 3.785 ± 0.08 μm (n = 8) and after 50 PDMS castings 3.748 ± 0.02 μm (n = 12); p = 0.2274. (g) Graph of compartment height measurements of an Epotek master before the first PDMS cast 127.7 ± 4.85 μm (n = 10) and after 50 PDMS castings 130.5 ± 5.077 μm (n = 10); p = 0.2332. Both microgroove height measurements and compartment height measurements were obtained from a scanning profilometer, while microgroove width measurements were obtained using an optical microscope. For (c)–(e), two masters were measured for each condition. SEM images for the epoxy master were taken with uncoated samples, and thus appear to have bright regions due to the surface charging effects of SEM. Scale bars, 100 μm for low magnification SEM images and 20 μm for high magnification SEM images.

FIG. 3.

Batch-to-batch feature reproducibility of Au-epoxy masters compared with SU8-Si masters. (a) Graph of microgroove heights for a batch of 6 epoxy masters, mean height of 3.67 ± 0.071 μm (RSD 1.89%; n = 48 from the 6 masters). (b) Graph of compartment heights for the same batch of 6 masters; mean height of 127.5 ± 4.3 μm (RSD 3.3%; n = 24 from 6 masters). (c) Graph showing microgroove height variability from 3 different batches of SU8-Si masters and 2 different batches of Epotek masters produced from a single SU8-Si master. Mean heights of SU8-Si batch 1 (2 masters) was 3.786 ± 0.067 μm (RSD 1.74%; n = 24); SU8-Si batch 2 (1 master) was 4.358 ± 0.04 μm (RSD 0.87%; n = 12); SU8-Si batch 3 (1 master) was 2.932 ± 0.03 μm (RSD 0.89%; n = 12); Epotek batch 1 (2 masters) was 3.764 ± 0.06 μm (RSD 1.72%; n = 18); and Epotek batch 2 (12 masters) was 3.768 ± 0.06 μm (RSD 1.52%; n = 120). (d) Overall microgroove feature variation between SU8-Si batches (RSD 13.91%; n = 48) and Epotek batches (RSD 1.54%; n = 138).

FIG. 4.

Biocompatibility of PDMS devices cast from Au-epoxy masters. (a) Representative DIC micrographs of rat hippocampal neurons (6 DIV) within a cell compartment of PDMS devices cast from: (i) SU8-Si masters; (ii) epoxy masters coated with silane, and (iii) Au-epoxy masters. (b) and (c) Representative images of live neurons cultured within PDMS devices from SU8-Si masters (control) and the 26th cast of Epotek masters. Micrographs show (i) DIC, (ii) staining with the live cell marker CellTracker Green, and (iii) dead cell labelling using propidium iodide. Scale bars, 25 μm. (d) Quantification of the number of live cells relative to the total number of cells (live plus dead) for PDMS devices from the 1st, 20th, 26th, and 38th casts of Au-epoxy masters and normalized to controls from PDMS chambers molded from SU8-Si Masters for each cast tested. Two devices were used per condition and three frames per device. (e) and (f) Merged images of rat hippocampal neurons (6 DIV) within a PDMS device cast from Au-epoxy masters immunolabeled for β-tubulin III (green) and counterstained for DAPI (blue). Scale bar, 75 μm and 40 μm, respectively. (f) Neurons cultured with Au-epoxy-derived PDMS chambers extend axons into 4 μm tall microgrooves.

FIG. 5.

Tall pillar structures in Au-epoxy masters. (a) Photograph of a Au-epoxy master with pillars within a 100 mm petri dish. (b) SEM image of a section of the pillar and relief features on the Au-epoxy master. (c) PDMS device molded from a Au-epoxy master with pillars and assembled onto coverglass. Food coloring was used to highlight the cell compartments.