Micropatterned photodegradable hydrogels for the sorting of microbeads and cells.

Microarrays of biomolecules and cells have played important roles in genomics, diagnostics, and drug screening. Live-cell capture on micropatterned surfaces is particularly valuable for diagnostic or biosensing applications, as cell arrays are amenable to rapid characterization and sorting. For example, antibody-coated micropost arrays have been used to detect and isolate rare circulating tumor cells from peripheral blood with high fidelity. Microwell arrays have been used for the capture and analysis of single immune cells. While it is often important to release or sort subsets of cells from these arrays for more thorough downstream analysis (e.g. by PCR or Western blot), methods for the retrieval of specific cells from micropatterned surfaces remain limited. Electrochemical stimulation can be exploited to release cells from particular regions within micropatterns, but these approaches require that cell arrays be registered with electrodes, and they can become somewhat complicated for larger arrays. Arguably, light-activated cell-release strategies provide more flexibility, particularly for retrieval from highly dense arrays. Laser capture microdissection or laser catapulting can be used to remove specific cells from micropatterned surfaces, but the former approach works with fixed cells while the latter involves harsh conditions, often resulting in cell injury. Another example of light-activated cell sorting is the micropallet array developed by Albritton et al., in which cells are cultured on polymer microstructures (e.g. photoresist patterns) that can later be dislodged from the surface by laser pulses. The goal of this study was to employ photocleavable hydrogels for capture and release of cells on microbeads. Hydrogels in general may be bioactive or nonfouling depending on their composition and may be microfabricated using photolithography or soft lithography. Photodegradable hydrogels have garnered considerable attention recently for tissue-engineering applications because of new possibilities for modulating matrix properties or delivering signals in 3D scaffolds. However, we are only aware of one report describing the use of a photocleavable gel for cell capture and release. In this report, Yamaguchi et al. describe a hydrogel comprised of poly(ethylene glycol) connected to lipid moieties by photolabile linkers. Cells bound to lipid moieties on the gel were released upon cleavage of lipid groups by UV light. In contrast, we describe the incorporation of photocleavable groups into the bulk of the hydrogel and demonstrate the capture and release of cells and beads based on photoinitiated degradation of the gel. Microand nanobeads are commonly chosen as solid substrates for diagnostic immunoassays, target DNA detection, and cell-sorting applications because of their large surface area-to-volume ratio, broad range of both coding strategies and functional surface chemistries, and ease of manipulation in microfluidic devices (acoustic, magnetic, mechanical, etc.). In one example, Lam and colleagues developed the one-bead-one-compound (OBOC) combinatorial method to synthesize vast peptide libraries on polystyrene microbeads. High-throughput screening of cell binding on OBOC libraries has been used to identify high-affinity peptide ligands against several cancer cell surface receptors. However, sorting cell-containing beads from cell-free beads requires labor-intensive manual picking of single beads. The goal of the present study was to demonstrate the use of photodegradable hydrogels for cell sorting while addressing a specific technical challenge: the screening cell– microbead interactions. The above-mentioned hydrogel material, herein referred to as photogel, consists of poly(ethylene glycol) (PEG) and a photolabile linker (PLL) containing o-nitrobenzyl groups, which are sensitive to near-UV light (l= 365 nm). Recently, Anseth and co-workers reported a method for photolabile crosslinker (PCL) synthesis, in which o-nitrobenzyletherbased acrylate monomers were synthesized in an eight-step procedure before coupling to PEG-bis(amine). Here, we developed a novel PCL synthesis route that does not require chromatographic separation and may be accomplished in three steps : 1) Fmoc-PLL coupling to terminal amino groups of O,O’-bis(2-aminopropyl) poly(propylene glycol)-blockpoly(ethylene glycol)-block-poly(propylene glycol) 1900 (Jeffamine ED-2001), 2) removal of Fmoc groups, and 3) coupling of methacryl groups. These Jeffamine-containing products can be easily isolated in each step by ether precipitation using a liquid-phase polymer-supported synthesis method. The resulting structure of the PCL is a linear [*] C. Siltanen, Dr. D.-S. Shin, Prof. J. Sutcliffe, Prof. A. Revzin Department of Biomedical Engineering University of California Davis One Shields Ave, Davis, CA 95616 (USA) E-mail: [email protected] [email protected]