Islet-on-a-chip for the study of pancreatic β-cell function

Diabetes mellitus is a significant public health problem worldwide. It encompasses a group of chronic disorders characterized by hyperglycemia, resulting from pancreatic islet dysfunction or as a consequence of insulin-producing β-cell death. Organ-on-a-chip platforms have emerged as technological systems combining cell biology, engineering, and biomaterial technological advances with microfluidics to recapitulate a specific organ’s physiological or pathophysiological environment. These devices offer a novel model for the screening of pharmaceutical agents and to study a particular disease. In the field of diabetes, a variety of microfluidic devices have been introduced to recreate native islet microenvironments and to understand pancreatic β-cell kinetics in vitro. This kind of platforms has been shown fundamental for the study of the islet function and to assess the quality of these islets for subsequent in vivo transplantation. However, islet physiological systems are still limited compared to other organs and tissues, evidencing the difficulty to study this “organ” and the need for further technological advances. In this review, we summarize the current state of islet-on-a-chip platforms that have been developed so far. We recapitulate the most relevant studies involving pancreatic islets and microfluidics, focusing on the molecular and cellular-scale activities that underlie pancreatic β-cell function.This review received financial support from the European Research Council program under grants ERC-StG-DAMOC (714317), the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa” Program for Centres of Excellence in R&D (SEV-2016-2019) and “Retos de investigación: Proyectos I+D+i” (TEC2017-83716-C2-2-R), the CERCA Programme/Generalitat de Catalunya (2017-SGR-1079), and Fundación Bancaria “la Caixa”- Obra Social “la Caixa” (project IBEC-La Caixa Healthy Ageing)

Role of keratin filaments on B-cell mitochondrial behavior and functionality

Keratin (K) intermediate filaments (IFs) are IFs of epithelial cell, and one of the most abundant IFs found in the cells, Keratins have various functions including stress protection, cell signaling and protein targeting. In pancreatic β-cells K8 and K18 are the main keratins expressed. Presence or absence of keratin IFs have shown to change the mitochondrial morphology in pancreatic β-cells which in turn likely influences the insulin production in the β-cells. Reduction in insulin production or insulin resistance causes diabetes mellitus (DM). Type I and type II DM was responsible for 1.6 million deaths according to study by World Health Organization (WHO) in 2016. The aim of this thesis work was to find the role of keratin IFs in mitochondrial morphology and dynamics to better understand the insulin production by using a cancerous pancreatic cell line, Murine Insulinoma 6 (MIN6) cells. Various microscopes were used to acquire images from the fluorescently labelled fixed and live cell samples followed by image analysis. In this study, it was found that the overexpression of wild type K8/K18 significantly decreased the mitochondrial motility and insulin vesicle count per unit area of the cell while mitochondrial count per unit cell area, mitochondrial fragmentation level and cell area were significantly increased. Likewise, the K18R90C mutation which disrupts keratins filaments, decreased the cell area, insulin vesicle count per unit cell area and mitochondrial count per unit cell area. The liver disease associated mutation K8G62 resulted in higher mitochondrial fragmentation and higher insulin vesicle count per unit area compared to the K8WT/K18WT overexpressing MIN6 cells. In conclusion, these results provide valuable insight to understand the role of keratin in the mitochondrial behavior and functionality which influence the insulin production in the pancreatic cells

Encapsulation of pancreatic beta cells

Immunoisolation of pancreatic beta cells is a promising approach for the treatment of type I diabetes. In this thesis, a vibrating nozzle technology was utilised to reproducibly generate 1% alginate microparticles with an average diameter of 200 μm±19 S.D. This technology further enabled the application of fluidised bed bioreactor owing to high uniformity of particles, an important parameter for achieving homogeneous fluidisation. Experimental data collected from the cultivation of cells in fluidised culture was shown to provide a promising solution for handling encapsulated cells from manufacturing phase to clinical sites, which is currently a challenging issue for cell-based therapies. A reduction in beta cells insulin-secreting ability was observed after two weeks of static culture. This problem was addressed by investigating a 3- dimentional culturing technique and a novel polyelectrolyte multilayer (PEM) coating approach. Concave agarose micro-wells were used to culture robust pancreatic beta cell spheroids that enhanced cell-cell contact. Additionally, the novel PEM coating using Ca2+ pre- conditioning improved cell function while providing immunoisolation from cytokines, and reducing the total volume of the graft. This work presented an effective immunoisolation and culturing system to improve cells survival rate, which hopes to bring a closer step towards therapeutic transplantation

Role of adipose tissue in the pathogenesis and treatment of metabolic syndrome

© Springer International Publishing Switzerland 2014. Adipocytes are highly specialized cells that play a major role in energy homeostasis in vertebrate organisms. Excess adipocyte size or number is a hallmark of obesity, which is currently a global epidemic. Obesity is not only the primary disease of fat cells, but also a major risk factor for the development of Type 2 diabetes, cardiovascular disease, hypertension, and metabolic syndrome (MetS). Today, adipocytes and adipose tissue are no longer considered passive participants in metabolic pathways. In addition to storing lipid, adipocytes are highly insulin sensitive cells that have important endocrine functions. Altering any one of these functions of fat cells can result in a metabolic disease state and dysregulation of adipose tissue can profoundly contribute to MetS. For example, adiponectin is a fat specific hormone that has cardio-protective and anti-diabetic properties. Inhibition of adiponectin expression and secretion are associated with several risk factors for MetS. For this purpose, and several other reasons documented in this chapter, we propose that adipose tissue should be considered as a viable target for a variety of treatment approaches to combat MetS

Investigating the differentiation and functional maturation of stem cell-derived β cells

Diabetes mellitus is a chronic and global disease rapidly growing in prevalence. Diabetes can be characterized by the dysfunction or death of the glucose sensing insulin secreting cell. cells are located within the islet of Langerhans (islet), a tissue within the pancreas. Human islets are critical for the study and treatment of diabetes. However, they can only be obtained from cadaveric organ donors. These cadaveric islets do not proliferate and can only be maintained in vitro for short periods of time, making their availability rare and fleeting. Stem cell-derived -like cells can be generated in indefinite amounts and are a potential alternative to cadaveric islet cells. Throughout this document stem cell-derived -like cells will be interchangeably referred as SC- cells or SC-islet. A major challenge towards applying SC- cells to disease modeling or cell replacement therapies is their lack of functional maturity and supporting technology. In this thesis, I am to investigate and improve the functional maturity of SC- cells and their supporting technologies. Chapter 1 serves as an introduction to the field of SC- cells. In Chapter 2, by temporally manipulating TGF signaling, I develop a novel differentiation protocol for generation of SC- cells with enhance function. These enhanced SC- cells are generated more efficiently and achieve dynamic insulin secretion with first and second phase insulin secretion kinetics, a critical hallmark of cadaveric islet function. When transplanted into immune compromised diabetic mice, their function is detected within two weeks and cure their diabetes. In Chapter 3, I elucidate the role of transcription factor SIX2 in SC- cells differentiations. Using gene knockdown and knockout techniques, I show SIX2’s necessity for the functional maturation of SC- cells. Importantly, I identify SIX2 positive and negative cell populations and postulate its use as a marker to guide future cell maturation efforts. In Chapter 4, I present a method for cryopreserving SC-islets and characterize them relative to their un-cryopreserved counterparts. Cryopreserved SC-islets functionally and transcriptionally resemble un-cryopreserved SC-islets. This advancement will facilitate biobanking of stem cells, a necessary step to increase their accessibility to the research and therapeutic research population. In Appendix A, I describe the application of a luciferase insulin secretion reporter in SC- cells. Luciferase co-secretes in correlation with insulin and a proof-of-concept compound screen is performed identifying several cell secretagogues. These secretagogues were then assessed with cadaveric islets verifying the potential of SC- cells as biologically relevant screening models. In Appendix B, I describe a method for co-culturing SC- cells with endothelial cells using a hydrogel system. The work in this thesis advances SC- cell technologies and facilitates their use as disease models and cell-therapeutics

Novel in vitro systems elucidating metabolic health and disease

The convoluted nature of biology warrants improved models that further insights into health and disease. Bioengineered features and platforms allow the modulation and study of a range of biological phenomena. However, there remains a lack of versatile and well-characterised organotypic models and microphysiological systems that recapitulate phenotypes of interest. We have developed and comprehensively characterised a chemically defined, high throughput and stable 3D human adipose model comprising adipocyte spheroids. Adipocyte spheroids exhibit physiologically relevant gene expression signatures and improved phenotypes compared to conventional monolayer counterparts, with 4704 genes being differentially expressed compared to 2D cultures. Moreover, the model closely resembles freshly isolated human in vivo mature adipocytes. Such organotypic models and cellular phenomena can be manipulated using nanotopographies and structured polymer devices. We have demonstrated that nanostructures fabricated via nanoimprint lithography enabled precise modulation of cellular attachment and behaviour. Specific and high resolution structuring of microfluidic platforms is achieved using a novel fabrication approach, NanoRIM, allowing high fidelity generation of systems for organotypic hepatic cultures. The physiological crosstalk between liver and human pancreatic islets was then efficiently captured in an original microfluidic system featuring reciprocal perfusion. The versatile nature of these ensuing models and platforms enables the provision of a toolbox with which biology can be manipulated and studied across the vast temporal and spatial scales on which it exists

Tissue Engineering Platforms for Cardiac Pathology in Diabetes: In Vitro and In Vivo Studies

425 million people have diabetes worldwide, and by 2045 this number is estimated to increase to 629 million. The risk for cardiovascular diseases such as cardiomyopathy, hypertension, and atherosclerosis increase 5 and 2-fold for diabetic women and men respectively. Diabetic cardiomyopathy (DCMP) is a ventricular dysfunction that occurs in patients with diabetes independent of coronary artery disease, hypertension or valvular abnormalities. Hyperglycemia and dyslipidemia cause metabolic disturbances that adversely affect myocardial cells and extracellular matrix. These modifications alter overall myocardial structure and cardiac function, which can lead to heart failure. As of now there is no specific marker for this disease and diagnosis is the same as other cardiomyopathies. Elucidating early stages of this disease is vital for early diagnosis, treatment, and possible therapy targets. Currently, rodent models and 2D cell cultures have been employed to analyze DCMP, however there are notable differences between rodents and humans that provide challenges when studying DCMP and cell cultures lack an extracellular matrix and dynamic environment crucial to the progression of this disease. Our overall goal was to use tissue engineering to bridge this gap by developing platforms to study pathological mechanisms at the cellular and extracellular level. We examined cardiac tissue engineered constructs in: (1) a perfusion 3D Kube minibioreactor and (2) an electromechanical bioreactor customized in our lab. Each platform contained decellularized myocardium seeded with human cardiomyocytes for two weeks; “diabetic” conditions were simulated by increased glucose concentration. We were able to better mimic physiological conditions with our electromechanical bioreactor, compared to static and non-diabetic conditions, as well as to 2D cell culture. Methods for detecting cellular and matrix changes associated with DCMP were validated in a type 1 diabetic rodent model. Our tissue engineering platform shows promise for unveiling early cellular and matrix modifications in DCMP. This system could also be useful for studying human cells in other cardiac diseases, test treatments, and precondition myocardial-like tissue prior to implantation