Del Giudice, Francesco (2023) Long-lasting anastomosis on-a-chip: endogenous extracellular matrix as a key element for modeling stable and perfusable vascular network through a microfluidic device. [Tesi di dottorato]

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Abstract

Since tissue engineering has been developed, it represents one of the main strategies that bioengineering has to obtain in vitro substitutes for wounded tissues in human body, replicating the physiological environment with low risk of rejection after in vivo implantation. One of the main issues regarding tissue engineering is represented by nutrients diffusion during biological construct formation, which is insufficient in preventing necrosis of tissues in the scale of millimeters before in vivo application. The easiest way to increase the exchange of nutrients and metabolites in in vitro 3D cultures is providing a dynamic culture, which can be obtained in bioreactors or with a microfluidic approach. The custom design of a microfluidic device and the controlled use of cell media flows – with systems such as syringe pumps or hydrostatic pressure - can reduce the negative effects of scarce diffusion inside the extracellular matrix (ECM). Another solution to the problem may be vascularization, which consists in the process of formation of blood vessels. The presence of a perfusable network may switch the mechanism of molecule transport from diffusive to convective, leading to nutrients and oxygen convection through vessels lumen and exchange through the endothelial walls, reducing the impact of diffusion coefficient. The formation of a mature capillary network inside a biological construct may also better resemble the biological environment, and can provide a deeper understanding of physiological phenomena in a 3D model in vitro. Microfluidics can be used for inducing capillary formation inside a biological construct; a phenomenon which needs fine control over parameters such as flow rate, pressure and gradients for biochemical and mechanical stimulation of different cells, that microfluidic devices can guarantee. Once vascularization is obtained, the biological tissue may be implanted in vivo with a reduced time of adaptation to human body, or the vascular network may be kept perfusable by using the microfluidic device. Basically, an organ-on-chip can be obtained as a platform to investigate and analyze a circulatory system in vitro. Usually, in vitro vascular networks are generated by two distinct approaches: by endothelial-lined channels, or by self-assembled networks. The former strategy relies on the formation of a cavity network, which may be obtained in hydrogels or in synthetic materials, or in a polydimethylsiloxane (PDMS) device realized with soft-lithography techniques. Endothelial cells (ECs) are seeded inside the cavities and attach to the walls, with or without the presence of adhesion molecules coatings: these techniques guarantee high control over the geometry and the size of obtained vessels, but the vascular model clearly differs from the in vivo vasculature, since the formation of a real network is technically impossible. The latter strategy is based on the vasculogenesis phenomenon – the de novo vessel formation – which normally happens during embryogenesis in vivo. It has been demonstrated that seeding ECs inside a hydrogel in the presence of fibroblasts induces the self-assembly of ECs into vessel-like structure because of different growth factors secreted by fibroblasts: the architecture of such networks better resemble in vivo vasculature conformation, while the control over the network geometry is lost and the perfusability of the vessel network may be difficult to achieve or control. Geometrical cues also play a critical role in the endothelial behaviour in vitro. In this context, the aim of the presented project regards the formation of a perfusable, mature and long-lasting capillary network in a bio-engineered construct featured by endogenous extracellular matrix (ECM) inside a custom-designed microfluidic platform. By controlling parameters such as flow rate – thus velocity profiles and shear rate – the microfluidic platform aims to allow the perfusability of vascular vessels and, consequently, the transport of nutrients inside the stromal tissue, in a long-lasting manner. Briefly, the microfluidic device that will be designed and produced in this project will consist of two parts: two channels for the inlet and outlet and the control of flows, and one chamber for the introduction of a biohybrid that closely resembles physiological tissues. The biological construct will be a stromal tissue equivalent, whose most important features are the 3D structure and the endogenous ECM. At first, the integration of a macroscopic pre-vascularized bioengineered equivalent (disk of 1 mm in height and up to 1 cm in diameter) on-chip will be described. Afterwards, pre-vascularized micro-tissue units will be introduced into a different microfluidic platform to obtain tissue assembly and the long-lasting anastomosis on-chip. The approach in this project will rely on the combination of the two described strategies: the designed microfluidic device will feature channels, which will be lined with ECs, in direct communication to the tissue chamber through a series of interpillar pores. The channel-chamber interface will match with the lateral surface of the tissue itself, which will undergo endothelial lining as the channel walls of the chip. Controlling transendothelial flow at the communication pores, shear stress on the lining ECs, VEGF gradients – from the inlet or directly secreted by fibroblasts in the tissue – and flow rates, endothelial sprouts will depart from the channel-chamber interface and anastomose to the pre-vascular network inside the stromal tissue. The combination of tissue engineering and microfluidics may lead to prolonging the maturation of capillaries inside the tissue, thus determining a long-lasting vascularization on-chip model. This organ-on-chip configuration is increasingly relevant because of the different applications it is suitable for: building a small-scale version of functional human tissues and organs has shown to be a potential alternative to animal tests with a high throughput, a model for a deeper understanding of patho-physiological cell behaviours and a step forward towards personalized medicine, with the use of patient-derived cells to engineer the in vitro biological construct. Furthermore, the obtained platform may be used for the study of pathological angiogenesis phenomena correlated with tumors, as the biological construct inside the device can be engineered with tumor-related cells.

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