4D Control of Tissue Development and Blood Vasculature using Photo-mediated Chemistries
Arakawa, Christopher Kenji
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Organ function relies on the synergistic interactions and conserved organization between all its system components. Blood, blood vessels, extracellular matrix (ECM), and parenchyma interact dynamically, existing in a state of constant cross-talk and regulation. Yet for all we have discovered about physiologic and pathophysiologic organ function, our capacity to recapitulate these events has been limited. Currently, there does not exist the necessary set of tools required to create de novo tissues or organs. This thesis aims to address these pertinent hurdles in tissue engineering by exploring new means to generate complex tissues using a combination of tunable synthetic hydrogels and photo-mediated chemistries. A preliminary data section followed by two main aims outline these goals. In the preliminary data section, the interactions between matrix and parenchyma are investigated by creating 3D tissues from human pluripotent stem cells (hPSC) encapsulated within tunable cytocompatible poly(ethylene glycol) (PEG) hydrogels formed via strain-promoted azide-alkyne cycloaddition (SPAAC). In this section, material and cellular variables are optimized to both create functional cardiac tissues from hPSC-derived cardiomyocytes (CM) using PEG hydrogels as well as first maintain and then differentiate hPSCs into mesodermal-derived tissues within fully defined synthetic matrices. As these tissues require vascularization to be supplied with adequate oxygen and nutrients, we were then motivated to explore of the interactions between vasculature and parenchyma. In aim 1, a novel photodegradation-based vascular fabrication methodology is developed, wherein PEG hydrogels are modified with a photodegradable ortho-nitrobenzyl moiety. Combining this novel material with a programmable multiphoton laser, blood vessels are generated with complete 4D control to create multicellular vascularized tissues with some of the most complex vessel networks to date. These devices feature single-micron fabrication resolution, intraluminal structures, and interconnected hierarchical branching networks. To expand our methodology to naturally occurring polymers and to investigate the interactions between blood and vasculature, in aim 2, we develop a multiphoton photoablation fabrication technique and in the process create the first in vitro engineered human capillaries. Utilizing these devices, we then study the physiological and pathophysiological hemodynamics associated with severe malaria, probing the mechanisms that give rise to microvascular occlusion and sequestration during infection. Findings here demonstrate the first human model of microvascular sequestration with insights into the biophysical and biomolecular contributions of infected red blood cells to malarial disease. Together this work systematically identifies the gaps in our knowledge to create complex tissues and addresses them by inventing and rigorously testing new photo-mediated fabrication techniques. Work here to construct 3D tissues with complete synthetic control and to generate complex vascular networks permits the study of intricate interactions between all organ system components. These findings represent the first of many mechanisms once observed only in in vivo systems which can now be recapitulated in a dish with greater control of all cellular and structural variables.
- Bioengineering