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Diabetes Approach by Multi-Organ-on-a-Chip

Periodic Reporting for period 2 - DAMOC (Diabetes Approach by Multi-Organ-on-a-Chip)

Reporting period: 2018-07-01 to 2019-12-31

Diabetes is the most prevalent and epidemic metabolic disorder throughout the world. Type 2 diabetes (T2D) is the most common form , and is characterized by hyperinsulinemia and insulin resistance. Its prevalence has dramatically increased in last decades, establishing a good correlation with the incidence of obesity and sedentary. Pancreas is the organ which has a key role in T2D. In addition, insulin resistance in skeletal muscle is considered to be the initiating of T2D. The maintenance of normal glucose homeostasis depends on a finely balanced dynamic interaction between skeletal muscle sensitivity to insulin and insulin secretion. Thus, the evolution of T2D requires the presence of defects in both insulin secretion and insulin action and it requires the simultaneous study of both tissues which results difficult, expensive and time consuming with conventional methods.

The pharmaceutical industry relies heavily on in vivo animal models and in vitro two-dimensional (2D) cell cultures to develop therapeutic strategies. There are many ethical issues surrounding animal studies and serious concerns also exist regarding their biological relevance to humans. Current in vitro tissues are also useful for studying the molecular and cellular basis of physiological and pathological responses of biological processes. However, due to their 2D structure they do not take into account the complexity of physiological microenvironment in which cells grow. There is thus growing interest in developing fully functional three-dimensional (3D) tissues. Organoids are a miniaturized and simplified version of an organ produced in vitro. Here, for the first time, a multi-organ-on-chip device combining 3D engineered skeletal muscle tissue with engineered 3D pancreatic islets will be fabricated. Integration of biosensor technology in the device will allow the real-time monitorization of glucose uptake and biomarkers secreted by skeletal muscle and insulin secretion from pancreatic islets. We will add electrical systems to mimick an exercising condition. This engineered multi-organ-on-a-chip will be an important enabling step for disease modelling, study of the insulin resistance, and investigation of drug candidates for treatment of T2D, which have usually been performed by long time and expensive animal experiments.

The aim of this project is to overcome these limitations in a revolutionary technological approach, that allow us to engineer skeletal muscle tissues and pancreatic islets in a multi-organ-on-a-chip to open new areas of research on human T2D disease.
The specific objectives are to engineer a functional skeletal muscle tissues using 3D tissue technology fabrication combining precursor cells with hybrid biomaterials as scaffold. We will develop a fully functional pancreatic islets using 3D bioprinting as microscale technology and integrate them with biosensors systems on a microscale chip for in vitro real-time monitoring of their functionality. Furthermore, we will integrate the pancreatic islets with skeletal muscle in a subdivided bioreactor with biosensors systems. This multi-organ-on-a-chip will be used for the study of several targets to prevent and reverse amyloid deposition, to check toxicity of amyloid formation in the pancreatic islets and to study the effect of secretion of stress markers (myokines) on skeletal muscle and pancreatic islets.
This last year, we have studied three different composite biomaterials as bioinks to produce 3D engineered skeletal muscle myotubes. We formulated a library of composite hydrogel prepolymers solutions with different prepolymers concentrations, different degree of methacrylation and two different UV photoinitiators. We used those formulations for the preparation of cell-laden hydrogels in combination with C2C12 murine myoblasts and we evaluated the maturation of myotube structures by fluorescence microscopy. We have demonstrated that these composite materials allow the bioprinting of 3D constructs of skeletal muscle that can be used in in vitro applications promoting their functionality and preserving their structure. In this study it is vital not only to match the morphology of the functional skeletal muscle fibers, but also the cellular arrangement, controlling hydrogel properties,which are critical forproper cellular function and tissue morphogenesis. In parallel, we worked in the production of 3D beta-cells aggregates with controllable sizes in hydrogels using 3D bioprinter technology. The pancreatic islets encapsulated within a hydrogel prepolymer have been produced by bioprinting, upon applying UV light crosslinking the hydrogel, form 3D pancreatic islets aggregates. Scaffolds are the key part on the development of engineered tissues. They support growth and differentiation of progenitor cells and provide 3D environment. We have developed biocompatible, hybrid biomaterials with tuneable mechanical and porosity properties that give structure and support to the cells. In this project, the 3D architecture of pancreatic islets will be reproduced by UV photopolymerization of hydrogels through 3D bioprinting in 3D spherical structures. Insulin release has been measured to determine functionality and maturation of encapsulated cells aggregates. Insulin secretion will be also monitored.
The upcoming of engineered microtissues represent a new paradigm in the field of in vitro assays, providing in vitro systems with tissue-like in vivo functionality. Engineered microtissues are available because of the combination of microfabrication, microfluidics, tissue-engineering components (scaffolds and bioreactors), biosensors and specific functional cells. The main objective of DAMOC is to create a microengineered system that can act as an in vitro model of the pancreas tissue and skeletal muscle, providing the biological, biochemical and biomechanical properties found in the in vivo tissue.
We have screened and optimized the physical properties of different composite hydrogels for the growth and development of muscle fibers. They have shown good biocompatibility and bioactivity and, in contrast with other hydrogels, they are long-lasting materials. Thus, they are good candidates as biomaterials for in vitro applications and as bio-actuators. These hydrogels have the potential to meet an assortment of cellular and mechanical demands required for engineering tissues. The combination of hydrogel composites with bioprinting methods allowed us to efficiently obtain 3D structures of differentiated and aligned muscle fibers, which is of high interest in skeletal muscle tissue engineering. Using a similar composite biomaterial and 3D bioprinting technology we have obtained pancreatic cells of the controllable size. The proposed technique has the potential to be used in cell therapy and tissue regeneration applications.
We have identified a number of results of the project that can be subject to intellectual protection or that offer a potential for future technology transfer and exploitation. Among them, 3D engineered microtissues. More specifically, the protocol of fabrication of pancreatic islets and skeletal muscle fibers by 3D bioprinting technology (all biomaterials and bioinks used in the protocol included) with a controllable size and maintaining a long-lasting functionality could attract not only in vitro applications but also in vivo transplants for patients with type I diabetes.
Pancreatic islets incorporated in a microporous scaffold
Bioprinted scaffold with the precursor muscular cells encapsulated after 1 day of incubation
Schematic representation of pancreatic cells microencapsulation
Design and fabrication of microdevice