Chemical Bionics - a novel design approach using ion sensitive field effect transistors

In the late 1980s Carver Mead introduced Neuromorphic engineering in which various aspects of the neural systems of the body were modelled using VLSI1 circuits. As a result most bio-inspired systems to date concentrate on modelling the electrical behaviour of neural systems such as the eyes, ears and brain. The reality is however that biological systems rely on chemical as well as electrical principles in order to function. This thesis introduces chemical bionics in which the chemically-dependent physiology of specific cells in the body is implemented for the development of novel bio-inspired therapeutic devices. The glucose dependent pancreatic beta cell is shown to be one such cell, that is designed and fabricated to form the first silicon metabolic cell. By replicating the bursting behaviour of biological beta cells, which respond to changes in blood glucose, a bio-inspired prosthetic for glucose homeostasis of Type I diabetes is demonstrated. To compliment this, research to further develop the Ion Sensitive Field Effect Transistor (ISFET) on unmodified CMOS is also presented for use as a monolithic sensor for chemical bionic systems. Problems arising by using the native passivation of CMOS as a sensing surface are described and methods of compensation are presented. A model for the operation of the device in weak inversion is also proposed for exploitation of its physical primitives to make novel monolithic solutions. Functional implementations in various technologies is also detailed to allow future implementations chemical bionic circuits. Finally the ISFET integrate and fire neuron, which is the first of its kind, is presented to be used as a chemical based building block for many existing neuromorphic circuits. As an example of this a chemical imager is described for spatio-temporal monitoring of chemical species and an acid base discriminator for monitoring changes in concentration around a fixed threshold is also proposed

Three-dimensional printed polymeric system to encapsulate human mesenchymal stem cells differentiated into islet-like insulin-producing aggregates for diabetes treatment

Diabetes is one of the most prevalent, costly, and debilitating diseases in the world. Pancreas and islet transplants have shown success in re-establishing glucose control and reversing diabetic complications. However, both are limited by donor availability, need for continuous immunosuppression, loss of transplanted tissue due to dispersion, and lack of vascularization. To overcome the limitations of poor islet availability, here, we investigate the potential of bone marrow–derived mesenchymal stem cells differentiated into islet-like insulin-producing aggregates. Islet-like insulin-producing aggregates, characterized by gene expression, are shown to be similar to pancreatic islets and display positive immunostaining for insulin and glucagon. To address the limits of current encapsulation systems, we developed a novel three-dimensional printed, scalable, and potentially refillable polymeric construct (nanogland) to support islet-like insulin-producing aggregates’ survival and function in the host body. In vitro studies showed that encapsulated islet-like insulin-producing aggregates maintained viability and function, producing steady levels of insulin for at least 4 weeks. Nanogland—islet-like insulin-producing aggregate technology here investigated as a proof of concept holds potential as an effective and innovative approach for diabetes cell therapy

The Effects of Cell Clustering on Cell Differentiation in Human Pancreatic Ductal Epithelial Cancer Cells

The thesis was funded by the College of Engineering by the Undergraduate Research Scholarship, when the project proposal was approved in April 2011.For diabetes mellitus type I, regular insulin therapy is not enough to completely control the disease. Ineffective control of blood glucose levels can lead to complications in other parts of the body, including kidney failure, neuropathy, cardiovascular disease, blindness, and acute hypoglycemic attacks. Although pancreatic transplants are one possible solution to reduce the effects of diabetes, it is not the best option. The proposed work of this research project is new and innovative with respect to hypothesis and methodology. It follows the goal to identify the role of cell clustering for differentiation of β cells so that the option of pancreatic islet transplantation can be substituted completely. Work by Boretti and Gooch in their paper “Induced Cell Clustering Enhances Islet β Cell Formation from Human Cultures Enriched for Pancreatic Ductal Epithelial Cells” was relevant to this project as it established the importance of clustering for differentiation of precursors cells into β cells. I used this work as a motivation to further research the amount of clustering required for the expression of insulin. My project speculates that the number of cells per cluster is essential for differentiated β cells to form. Further, there also must be a certain number of cells per cluster in order for proper insulin expression to take place. Upon experimentation, the results showed that cluster sizes of at least 4 cells per cluster were successful in expressing insulin as differentiated β cells. Whereas, small cluster sizes with only 1-2 cells per cluster were not able to express insulin. Further an area-to-perimeter analysis showed that ratios greater than 4.5 were common among insulin-expressing large cluster sizes. Although the results are quite preliminary, the investigation of the role that cluster size has on differentiation of β cells has a lot of potential for further research.The Ohio State University, College of Engineering, Undergraduate Research Scholarship (URS)No embarg

Recombinant Incretin-Secreting Microbe Improves Metabolic Dysfunction in High-Fat Diet Fed Rodents

peer-reviewedThe gut hormone glucagon-like peptide (GLP)-1 and its analogues represent a new generation of anti-diabetic drugs, which have also demonstrated propensity to modulate host lipid metabolism. Despite this, drugs of this nature are currently limited to intramuscular administration routes due to intestinal degradation. The aim of this study was to design a recombinant microbial delivery vector for a GLP-1 analogue and assess the efficacy of the therapeutic in improving host glucose, lipid and cholesterol metabolism in diet induced obese rodents. Diet-induced obese animals received either Lactobacillus paracasei NFBC 338 transformed to express a long-acting analogue of GLP-1 or the isogenic control microbe which solely harbored the pNZ44 plasmid. Short-term GLP-1 microbe intervention in rats reduced serum low-density lipoprotein cholesterol, triglycerides and triglyceride-rich lipoprotein cholesterol substantially. Conversely, extended GLP-1 microbe intervention improved glucose-dependent insulin secretion, glucose metabolism and cholesterol metabolism, compared to the high-fat control group. Interestingly, the microbe significantly attenuated the adiposity associated with the model and altered the serum lipidome, independently of GLP-1 secretion. These data indicate that recombinant incretin-secreting microbes may offer a novel and safe means of managing cholesterol metabolism and diet induced dyslipidaemia, as well as insulin sensitivity in metabolic dysfunction

Mouse and human islets survive and function after coating by biosilicification

Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to medium containing saturating silicic acid levels for 9-15 min. The resulting tissue constructs were then cultured for up to 4 wk under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. A cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function were assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice

Surface Optochemical Sensors

The objective of my research is to develop new surface optochemical sensors for studying cellular processes by investigating techniques to modify surface properties. The spectral characteristics of the modified surfaces and coatings are designed to show remarkable changes after interaction with analytes from biological fluids and cells. My studies focused on pancreatic cells and addressed the need for improved techniques to measure zinc release from pancreatic cells (chapter 3, 4) and to measure the metastasis potential of cancerous pancreatic cells (chapter 5). Chapter 3 describes the development of zinc sensing glass slides by conjugating a carboxylmodified ZnAF-2 to an amino functionalized glass surface. The sensor was used for the measurement of glucose-stimulated zinc ion release from cultured beta pancreatic cells with impact in diabetes research. In chapter 4 is described conjugation of the carboxyl-modified ZnAF-2 to antibody molecules (A2B5) that specifically recognize pancreatic cells. This enabled for the first time the use of targeted zinc sensors to monitor zinc release events from pancreatic cells. Chapter 5 describes development for the first time of a fluorescence sensor to measure the proteolysis activity of pancreatic cancer cells in microfluidic systems. The sensor was fabricated using a Layer by layer (LbL) deposition of polyelectrolyte. The sensor was based on Fluorescence Resonance Energy Transfer (FRET) between luminescent quantum dots (serve as donors) and rhodamine molecules (serve as acceptors) that are separated by multi-layers of polyelectrolytes. The microfluidic platform enables precise delivery of reactants to assemble the sensor and facilitate unique cellular assays of enzymatic activity and enzymatic expression on pancreatic cancer cells

Fibronectin and Collagen IV Microcontact Printing Improves Insulin Secretion by INS1E Cells

Extracellular matrix (ECM) molecules play significant roles in regulating β-cell function and viability within pancreatic islets by providing mechanical and biological support, stimulating cell survival, proliferation, and their endocrine function. During clinical islet transplantation, the β-cell's ECM environment is degraded by enzymatic digestion. Literature suggests that interactions between islet cells and ECM molecules, such as fibronectin (FN), collagen type IV (Col4), and laminin (LN), are essential for maintaining, or stimulation of islet function and survival, and can effect differentiation and proliferation of the endocrine cells. It is also thought that three-dimensional (3D) culture of β-cells can improve glucose responsiveness by providing a specific niche, in which cells can interact with each other in a more natural manner. Conventional suspension cultures with β-cells results generally in a heterogeneous population with small and large aggregates, in which cells experience different nutrient diffusion limitations, negatively affecting their physiology and function. In this study, we have explored the effect of FN, Col4, and LN111 on INS1E insulinoma cells by using microcontact printing (μCP) to investigate whether a controlled environment and aggregate dimensions would improve their endocrine function. Using this method, we produced a pattern of well-defined circular spots of FN, Col4, and LN111 on polydimethylsiloxane with high spatial resolution. Cell seeding of the INS1E cells on these ECM protein spots resulted in the formation of 3D β-cell aggregates. We show that these INS1E aggregates have very reproducible dimensions, and that the cell culture method can be easily adjusted, leading to a highly accurate way of forming 3D β-cell aggregates on an ECM-functionalized substrate. In addition, we show that ECM molecules can act as anchoring points for β-cells on an otherwise non-cell-adherent material, and this can improve both the endocrine function and viability. We found a significant increase in the secretion of insulin by INS1E cells cultured on μCP FN and Col4 substrates, in comparison to cells that were growing in monolayers on substrates without ECM molecules. Moreover, INS1E cells growing on circular ECM spots in a 3D manner showed improved endocrine function in comparison to their two-dimensional counterparts. This research deals with finding a proper bioengineering strategy for the creation of improved β-cell replacement therapy in type 1 diabetes. It specifically deals with the microenvironment of β-cells and its relationship to their endocrine function

Implantable medical devices for drug and cell release

This work is focused on the research on how to leverage 3D printing technology in the field of cell transplantation. More specifically, the study of an artificial organ for hormone replacement therapies thanks to the close collaboration between the Methodist Hospital Research Institute, Houston, Texas and Politecnico di Torino, Turin, Italy. Cell transplantation offers an attractive therapeutic approach for many endocrine deficiencies. Transplanted endocrine cells or engineered cells encapsulated in the here presented 3D printed device, can act as biological sensors detecting changes in hormonal levels and secrete molecules in response to maintain homeostasis. The major advantage of this technology is that patients affected by endocrine disorder could potentially avoid the need of frequent hormone injections, such as insulin or testosterone, resulting in an improved quality of life and lower chronic side effects associated to external hormone supplementations. This implant was extensively tested both in vitro and in vivo condition, providing remarkable results that lead to several publications. The cell encapsulation system was fabricated via 3D printing technology adopting an FDA approved polymeric material. The structure, composed by an array of micro and macro channels, was specifically designed in order to allow vasculature formation within the device and for housing cells while avoiding cell clustering. Over the course of the Ph.D., the technology was designed, fabricated and tested for the encapsulation of several cell lines and for small and large animal models. According to the in vivo results, we demonstrated that our 3D printed device exemplifies a clinically translatable strategy for preserving viability and function of transplanted cells. Currently, is ongoing an experiment in Non-Human Primates (data not shown), last pre- clinical study before the possibility to move to the clinical development in humans. The pre-vascularization approach to achieve an ideal intra-device milieu prior to transplantation, transcutaneous cell loading and refilling capabilities, as well as the potential for rapid device retrievability, addresses current challenges in transplantation. This technology may offer exciting potential for clinical adoption in relevant medical areas of diabetes, hypogonadism, hypothyroidism, cancer, and neurological diseases among others