Exploring Endothelial Cell Sources for Brain Vasculature Modeling in 2D and 3D Cultures Restricted; Files Only
Taylor, Kyle (Spring 2025)
Abstract
Brain organoids present a novel platform for studying aspects of the human brain, including neurodevelopment, evolutionary changes, and neurological disorders. However, brain organoids lack vasculature inherent to the human brain, which serves many important functions including providing nutrients and oxygen and removing waste. Therefore, this project seeks to address this limitation by exploring a model that incorporates endothelial cells into 3-dimensional bioprinted (3DBP) scaffolds as guided support structures. We hypothesize that human brain endothelial cells (HBECs) will display greater branching and coverage of 3DBP scaffolds than other cell types. In this study, we use qualitative and qualitative image analysis to characterize three endothelial cell types in 2D and 3D, human umbilical vein endothelial cells (HUVECs), induced pluripotent stem cell endothelial cells (iECs), and human brain endothelial cells (HBECs). Additionally, we show that HBECs can culture in 3DBP scaffolds with similar coverage to our previously validated endothelial cell line, HUVECs. Lastly, we started incorporating perfusion into our model system to further its physiological relevance. By exploring different endothelial cell sources to incorporate into our scaffolds, we strive to advance the brain organoid model and provide a platform for studying the interaction between neural and endothelial cells. Incorporation of dynamic flow in particular aims to advance research on the blood brain barrier and has applications in drug discovery and treatment.
Table of Contents
Table of Contents
1. Introduction 1
2. Background 4
2.1 Brain Organoids as Models for Neurodevelopment 4
2.2 The Importance of Organoids in Studying Brain Evolution 4
2.3 The Role of Brain Organoids in Studying Diseases 6
2.4 Vasculature and the Blood Brain Barrier 8
2.5 Evolution of Increased Human Brain Size 11
2.6 3D Bioprinting 12
2.7 Past Research 14
3. Methods 16
3.1. Bioprinting Materials 16
3.1.1 GelMA 16
3.1.2 Cytoink6000 16
3.1.3 Carbopol 18
3.1.4 Gelatin Microparticles 18
3.2 Bioprinting 19
3.2.1 Extrusion Bioprinting with BioX 20
3.2.2 DLP Bioprinting with LumenX 21
3.3 Cell Culture 21
3.3.1 Human Brain Microvascular Endothelial Cells (HBECs) 21
3.3.2 Human Umbilical Vein Endothelial Cells (HUVECs) 23
3.3.3 induced Pluripotent Stem Cell-Endothelial Cells (iECs) 24
3.4 Tube Formation Assay 28
3.5 2D Experiment 28
3.6 3D Culture Experiment 29
3.7 Perfusion Experiment 29
3.7 Fixing and Sectioning 30
3.8 Immunohistochemistry 30
3.9 Quantification 31
3.9.1 2D Culture EdU Quantification 31
3.9.2 3D Culture Surface Area Coverage Quantification 32
3.9.3 3D Culture Channel Coverage 32
3.10 Statistics 32
4. Results 33
4. 1 Differentiation of iPSCs into iPSC derived Endothelial Cells Yielded Cells with EC Morphology 33
4.2 Tube Formation Assay Demonstrates Intrinsic Tube Formation Properties of ECs 34
4.3 2D Culture of ECs Reveals Distinctive Morphologies and Presence of Endothelial Cell Marker CD31 36
4.4. HUVECs and HBECs demonstrate similar area coverages of 3DBP channels and significantly more coverage than iECs 38
5. Discussion 46
6. Conclusion 49
References 50
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File download under embargo until 22 May 2026 | 2025-04-09 20:08:41 -0400 | File download under embargo until 22 May 2026 |
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