A high-throughput robotic system has been developed for crystallizing membrane proteins using lipidic mesophases. It incorporates commercially available components and is relatively inexpensive. The crystallization robot uses standard automated liquid-handlers and a specially built device for accurately and reproducibly delivering nanolitre volumes of highly viscous protein/lipid mesophases. Under standard conditions, the robot uses just 20 nl protein solution, 30 nl lipid and 1 microl precipitant solution. 96 wells can be set up using the robot in 13 min. Trials are performed in specially designed 96-well glass plates. The slim (<2 mm high) plates have exquisite optical properties and are well suited for the detection of microcrystals and for birefringence-free imaging between crossed polarizers. Quantitative evaluation of the crystallization progress is performed using an automated imaging system. The optics, in combination with the slim crystallization plates, enables in-focus imaging of the entire well volume in a single shot such that a 96-well plate can be imaged in just 4.5 min. The performance characteristics of the robotic system and the versatility of the crystallization robot in performing vapor-diffusion, microbatch and bicelle crystallizations of membrane and soluble proteins are described.

Silicon membranes with highly uniform nanopore sizes fabricated using microelectromechanical systems (MEMS) technology allow for the development of miniaturized implants such as those needed for renal replacement therapies. However, the blood compatibility of silicon has thus far been an unresolved issue in the use of these substrates in implantable biomedical devices. We report the results of hemocompatibility studies using bare silicon, polysilicon, and modified silicon substrates. The surface modifications tested have been shown to reduce protein and/or platelet adhesion, thus potentially improving biocompatibility of silicon. Hemocompatibility was evaluated under four categories—coagulation (thrombin–antithrombin complex, TAT generation), complement activation (complement protein, C3a production), platelet activation (P-selectin, CD62P expression), and platelet adhesion. Our tests revealed that all silicon substrates display low coagulation and complement activation, comparable to that of Teflon and stainless steel, two materials commonly used in medical implants, and significantly lower than that of diethylaminoethyl (DEAE) cellulose, a polymer used in dialysis membranes. Unmodified silicon and polysilicon showed significant platelet attachment; however, the surface modifications on silicon reduced platelet adhesion and activation to levels comparable to that on Teflon. These results suggest that surface-modified silicon substrates are viable for the development of miniaturized renal replacement systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s10439-011-0256-y) contains supplementary material, which is available to authorized users.

In this study, we investigated how matrix nanotopography affects corneal fibroblast phenotype and matrix synthesis. To this end, corneal fibroblasts isolated from bovine corneas were grown on collagen nanofiber scaffolds of different diameters and alignment – 30 nm aligned fibrils (30A), 300 nm or larger aligned fibrils (300A), and 30 nm nonaligned fibrils (30NA) in comparison to collagen coated flat glass substrates (FC). Cell morphology was visualized using confocal microscopy. Quantitative PCR was used to measure expression levels of six target genes: the corneal crystallin - transketolase (TKT), the myofibroblast marker - α-smooth muscle actin (SMA), and four matrix proteins - collagen 1 (COL1), collagen 3 (COL3), fibronectin (FN) and biglycan. It was found that SMA expression was down-regulated and TKT expression was increased on all three collagen nanofiber substrates, compared to the FC control substrates. However, COL3 and biglycan expression was also significantly increased on 300A, compared to the FC substrates. Thus matrix nanotopography down-regulates the fibrotic phenotype, promotes formation of the quiescent keratocyte phenotype and influences matrix synthesis. These results have significant implications for the engineering of corneal replacements and for promoting regenerative healing of the cornea after disease and/or injury.