Engineering complex in vitro microenvironments for a deeper understanding of the biophysical and biochemical cues during kidney development
Date
2025
Authors
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Publisher
University of Delaware
Abstract
Chronic kidney disease (CKD) affects over 850 million people globally, greater than 10\% of the total population, and is the third fastest growing cause of death making this a significant worldwide health burden. Central to kidney function are the 200,000 - 1.8 million nephrons, which are established during development and have limited capacity for repair in cases of injury or disease. Therefore, treatment options for end-stage kidney disease are limited to dialysis or transplantation, both of which are constrained by severe side effects, reduced quality of life, or critical donor organ shortages. An emerging strategy to overcome this shortcoming employs a bottom-up approach to engineer new functional tissues for more representative in vitro models and eventually as replacement organs by harnessing key processes which directs in vivo organ development. Many microenvironmental signals from biochemical soluble factors, cell-cell interactions between cellular populations, and cues from the extracellular cellular matrix composition or physical properties work together to coordinated the complex interconnected genetic programming which directs kidney organogenesis. However, the precise experimental controls over these microenvironmental factors required to understand the complete process using in vivo models is challenging. Microphysiological systems (MPSs), although reductionistic, offer an in vitro platform with greater control of key structural and functional components of a biological system. Yet many existing in vitro models lack the necessary microenvironmental complexity for mechanistic studies of these biochemical and biophysical cues while remaining accessible to non-engineering users. ☐ This thesis aims to address this lack of accessible, yet complex in vitro models through the development and validation of a series of MPSs designed to allow independent control over multiple biophysical and biochemical properties. These tools enable mechanistic studies of the interaction between multiple microenvironmental factors and their role in directing kidney development. In Aim 1 we developed and validated a cost-effective and easy-to-use parallel plate flow chamber with a polyacrylamide hydrogel substrate enabling independent control of substrate stiffness and fluid shear stress. Additionally, we measured a force-dependent increase in f-actin filament length and width, highlighting the model's capability for examining the effect of multiple concurrent forces on cellular behavior. Aim 2 describes the creation and validation of methodology to spatially pattern the ECM and interstitial cell population along a 3D microchannel. We confirmed the interface between ECM regions does not interfere with solute transport, microchannel structure, or epithelial lumen formation. Through the regional patterning of interstitial cells, we are able to induce a local response from the epithelial cells, illustrating spatial control of cell-cell interactions within this model. Finally, Aim 3 expands and advances our ability to model the complex microenvironment of the developing kidney through spatially patterning of the biophysical and biochemical factors. The stable patterning of interstitially perfused soluble factors through this model enables regional control and manipulation of cellular functions and the potential for directed differentiation of progenitor cells cultured within this model. ☐ Together these models provide a set of tools to better understand how individual and the combined microenvironmental cues can influence cellular functions and kidney development. The creation of models intentionally designed using simple and widely available materials, equipment, and techniques positions these models into the hands of biologists where they are being used to further our understanding of kidney development and provide the foundation for developing novel therapeutic strategies for kidney diseases.
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Keywords
In vitro models, Microfluidics, Microphysiological systems, Tissue engineering