Treatment of vessel surfaces is common practice in many protein crystallization protocols. The research reported here shows that such treatments can have a significant effect on the resulting kinetics of protein crystallization. Untreated and silanized low-protein-binding polystyrene wells were used in batch isothermal crystallization experiments with the model protein lysozyme. An automated photomicroscopy apparatus was used to measure the induction time as a function of supersaturation, and the corresponding nucleation kinetics were determined and modeled using classical nucleation theory. The use of silanized wells led to a transition from homogeneous nucleation to heterogeneous nucleation that is not observed for the untreated polystyrene wells. The silanizing compounds are similar to those used in high-performance liquid chromatography stationary phases for protein separations. While the silanizing compounds beneficially modify the surface tension in the crystallization vessels, they also inadvertently create a surface conducive to heterogeneous nucleation, which affects the interpretation of crystallization experiments.

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion.

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A versatile and inexpensive microbatch crystallization apparatus with an integrated thermal gradient is introduced. The device is designed to fit onto automated microscope stages for timed image acquisition. The thermal gradient is established through an aluminum plate machined to house commercially available microplates with a standard 96-well footprint. The novelty of the design includes online monitoring of crystal growth without interfering with the established thermal gradient. The temperature stability and unidirectional temperature gradient were demonstrated. Hen egg white lysozyme (HEWL) was used as a model macromolecule to observe the effect of temperature variations on crystal habit and the number and size of crystals produced.

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