Building Biomimetic Potency Tests for Islet Transplantation.

Diabetes is a disease of insulin insufficiency, requiring many to rely on exogenous insulin with constant monitoring to avoid a fatal outcome. Islet transplantation is a recent therapy that can provide insulin independence, but the procedure is still limited by both the availability of human islets and reliable tests to assess their function. While stem cell technologies are poised to fill the shortage of transplantable cells, better methods are still needed for predicting transplantation outcome. To ensure islet quality, we propose that the next generation of islet potency tests should be biomimetic systems that match glucose stimulation dynamics and cell microenvironmental preferences and rapidly assess conditional and continuous insulin secretion with minimal manual handing. Here, we review the current approaches for islet potency testing and outline technologies and methods that can be used to arrive at a more predictive potency test that tracks islet secretory capacity in a relevant context. With the development of potency tests that can report on islet secretion dynamics in a context relevant to their intended function, islet transplantation can expand into a more widely accessible and reliable treatment option for individuals with diabetes.

Fattening chips: hypertrophy, feeding, and fasting of human white adipocytes in vitro.

Adipose is a distributed organ that performs vital endocrine and energy homeostatic functions. Hypertrophy of white adipocytes is a primary mode of both adaptive and maladaptive weight gain in animals and predicts metabolic syndrome independent of obesity. Due to the failure of conventional culture to recapitulate adipocyte hypertrophy, technology for production of adult-size adipocytes would enable applications such as in vitro testing of weight loss therapeutics. To model adaptive adipocyte hypertrophy in vitro, we designed and built fat-on-a-chip using fiber networks inspired by extracellular matrix in adipose tissue. Fiber networks extended the lifespan of differentiated adipocytes, enabling growth to adult sizes. By micropatterning preadipocytes in a native cytoarchitecture and by adjusting cell-to-cell spacing, rates of hypertrophy were controlled independent of culture time or differentiation efficiency. In vitro hypertrophy followed a nonlinear, nonexponential growth model similar to human development and elicited transcriptomic changes that increased overall similarity with primary tissue. Cells on the chip responded to simulated meals and starvation, which potentiated some adipocyte endocrine and metabolic functions. To test the utility of the platform for therapeutic development, transcriptional network analysis was performed, and retinoic acid receptors were identified as candidate drug targets. Regulation by retinoid signaling was suggested further by pharmacological modulation, where activation accelerated and inhibition slowed hypertrophy. Altogether, this work presents technology for mature adipocyte engineering, addresses the regulation of cell growth, and informs broader applications for synthetic adipose in pharmaceutical development, regenerative medicine, and cellular agriculture.

Synchronized stimulation and continuous insulin sensing in a microfluidic human Islet on a Chip designed for scalable manufacturing.

Pancreatic ß cell function is compromised in diabetes and is typically assessed by measuring insulin secretion during glucose stimulation. Traditionally, measurement of glucose-stimulated insulin secretion involves manual liquid handling, heterogeneous stimulus delivery, and enzyme-linked immunosorbent assays that require large numbers of islets and processing time. Though microfluidic devices have been developed to address some of these limitations, traditional methods for islet testing remain the most common due to the learning curve for adopting microfluidic devices and the incompatibility of most device materials with large-scale manufacturing. We designed and built a thermoplastic, microfluidic-based Islet on a Chip compatible with commercial fabrication methods, that automates islet loading, stimulation, and insulin sensing. Inspired by the perfusion of native islets by designated arterioles and capillaries, the chip delivers synchronized glucose pulses to islets positioned in parallel channels. By flowing suspensions of human cadaveric islets onto the chip, we confirmed automatic capture of islets. Fluorescent glucose tracking demonstrated that stimulus delivery was synchronized within a two-minute window independent of the presence or size of captured islets. Insulin secretion was continuously sensed by an automated, on-chip immunoassay and quantified by fluorescence anisotropy. By integrating scalable manufacturing materials, on-line, continuous insulin measurement, and precise spatiotemporal stimulation into an easy-to-use design, the Islet on a Chip should accelerate efforts to study and develop effective treatments for diabetes.

Measuring the activity of BioBrick promoters using an in vivo reference standard.

BACKGROUND: The engineering of many-component, synthetic biological systems is being made easier by the development of collections of reusable, standard biological parts. However, the complexity of biology makes it difficult to predict the extent to which such efforts will succeed. As a first practical example, the Registry of Standard Biological Parts started at MIT now maintains and distributes thousands of BioBrick standard biological parts. However, BioBrick parts are only standardized in terms of how individual parts are physically assembled into multi-component systems, and most parts remain uncharacterized. Standardized tools, techniques, and units of measurement are needed to facilitate the characterization and reuse of parts by independent researchers across many laboratories. RESULTS: We found that the absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments. We choose one promoter (BBa_J23101) to serve as an in vivo reference standard for promoter activity. We demonstrated that, by measuring the activity of promoters relative to BBa_J23101, we could reduce variation in reported promoter activity due to differences in test conditions and measurement instruments by approximately 50%. We defined a Relative Promoter Unit (RPU) in order to report promoter characterization data in compatible units and developed a measurement kit so that researchers might more easily adopt RPU as a standard unit for reporting promoter activity. We distributed a set of test promoters to multiple labs and found good agreement in the reported relative activities of promoters so measured. We also characterized the relative activities of a reference collection of BioBrick promoters in order to further support adoption of RPU-based measurement standards. CONCLUSION: Relative activity measurements based on an in vivoreference standard enables improved measurement of promoter activity given variation in measurement conditions and instruments. These improvements are sufficient to begin to support the measurement of promoter activities across many laboratories. Additional in vivo reference standards for other types of biological functions would seem likely to have similar utility, and could thus improve research on the design, production, and reuse of standard biological parts.