Polymer Micro- and Nanofabrication for On-Chip Immune Cell Handling

Abstract

There is an increasing interest in combining micro- and nanotechnology with mass-fabrication techniques for clinical applications such as diagnostics and therapeutics. One of the most promising strategies for developing a cancer vaccine is immunotherapy based on dendritic cells (DCs). However, major challenges such as high cost and low efficiency still limit its widespread use. The cost per treatment could be reduced by using closed and automated cell culture systems to eliminate infections and reduce manual labor. Furthermore, the efficiency could be increased by using lowcost isolation methods for DC precursor cells, such as surface-immobilized capture antibodies, as well as less invasive methods for harvesting cell cultures, such as cell cultureware with nanoscale surface topography to reduce cell adhesion. In this thesis we demonstrate the replication of sub-250 nm pillars in cyclic olefin copolymer (COC) over large surface areas by injection molding, using nanostructured mold inlays patterned by high-throughput deep-UV stepper photolithography. Injection molding at constant mold temperature below the glass transition point was significantly improved using nanostructured ceramic hydrogen silsesquioxane (HSQ) coatings on stainless steel mold inserts, compared to more traditional Ni mold inlays formed by electroplating. Numerical simulations suggested that the thermal isolation of HSQ films retards the cooling of the polymer melt, thus allowing more time to fill nanoscale cavities on the mold. In addition, the homogeneity of low surface-energy mold coatings could be improved by coating molds with silicon dioxide prior to deposition of fluorinated silanes. We successfully demonstrated the transfer of functional proteins from a mold surface to thermoplastic replicas using injection molding in a process compatible with mass production of single-use devices for molecular analysis and cell culture. The transfer process was highly efficient, as verified by atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS) of the mold and replica surfaces. Both ink-jet printed sub-100 μm homogeneous spots of avidin and patches of capture antibodies were transferred using this method. Transferred avidin retained its biotinaffinity as shown by fluorescence microscopy and monocyte-capture from biotinylated anti-CD14 antibodies in a microfluidic channel. Injection molded rabbit anti-mouse IgG showed similar affinity for mouse IgG in sandwich enzyme-linked immunosorbent assay (ELISA) as capture antibodies deposited by passive adsorption to bare thermoplastic replica. The transferred proteins were stable during incubation in serumcontaining cell medium for >1 week. Finally, disposable polymer chips were fabricated by injection molding and ultrasonic welding for the generation of a large number of mature DCs in a closed microfluidic perfusion culture. By using low gas permeable tubings and chip materials, a constant pH and bubble-free culture medium was maintained for 7 days outside a CO2 cell incubator. Numerical simulations of oxygen transport were performed to establish guidelines for medium exchange rates in an impermeable culture system. Maturation of CD83+ mature DCs generated from CD14+ monocytes was demonstrated inside the disposable culture chip, with a yield almost equal to standard culture procedures in an open Petri dish. This indicates that closed chip-culture systems, with further optimization of the perfusion parameters, are a promising strategy to increase automation and reduce cost of currently used procedures for cancer immunotherapy.