Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

Summary

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

Abstract

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Introduction

The field of tissue engineering is rapidly progressing towards the fabrication of engineered constructs to replace missing or damaged organs and tissues¹. However, fully functional constructs have yet to be achieved, in part, since generating operational vascular networks for tissue nourishment remains an outstanding challenge. Without proper vascularization, engineered tissues are limited to a passive diffusion transport of oxygen and nutrients, constraining the maximum viable tissue thickness to the diffusion limit, approximately 200 µm². Such thicknesses are not suitable to repair large tissue defects or for full organ fabric....

Protocol

1. Tessellated scaffold fabrication

NOTE: Photolithography is a widespread technique that requires specialized equipment typically housed within a nanofabrication facility/laboratory. The method laid out in this protocol was generalized as much as possible for the audience; however, slight changes to procedures may be necessary depending on equipment available to the reader. We recommend performing these procedures in a clean room at a nanofabrication facility to ensure the highest process quality. Before beginning, obtain access to a mask-aligner (or some UV-exposure set-up), a spin coater, hot plates, a solvent washing station, a ....

Results

The presented protocol, using stereolithography techniques, allows for the fabrication of tessellated scaffolds made of SU-8 photoresist. Scaffolds with distinct compartment geometries (squares, hexagons, and circles), and highly accurate and repeatable features were obtained (Figure 1).

Fi.......

Discussion

The need for a rich vasculature within embedded in engineered tissues is critical for construct survival and proper function¹. Although engineering the vascular system has been the focus of a vast amount of research, much is left to investigate and understand²⁴. In particular, when recreating a specific tissue, the microvasculature should behave and organize accordingly¹². The most common approach for microvessels generation is co-seeding endothelial.......

Materials

Name	Company	Catalog Number	Comments
Angiotool freeware	NIH-CCR		Free download at https://ccrod.cancer.gov/confluence/display/ROB2/Home
Bovine albumin serum Probumin	Millipore	82-045-1
Dental pulp stem cells	Lonza	PT-5025
ECM media + bullet kit	Sciencell	#1001
Ethanol 96%	Gadot-Group	64-17-5
Evicel fibrin sealant	Johnson&Johnson	EVB05IL	Provides both thrombin and fibrinogen (BAC2) solutions
GlutaMAX	Gibco	35050061
Goat anti-mouse Cy3 antibody	Jackson	115-166-072
Goat anti-rabbit Alexa-Fluor 488	Thermo- Fisher Scientific	A11034
Human adipose microvascular cells	Sciencell	#7200
Human fibronectin	Sigma	F0895-5MG	Stock concentration: 1 mg/mL
ImageJ	NIH		Free download at https://imagej.nih.gov/ij/download.html
Isopropyl alcohol	Gadot-Group	67-63-0
Lift-off reagent	Kayaku Advanced Materials, Inc	G112850	Commercial name Omnicoat
Low-glucose DMEM	Biological Industries	01-050-1A
Mouse anti-SMA antibody	Dako	M0851
NEAA	Gibco	11140068
Paraformaldehyde solution 4% in PBS	ChemCruz	SC-281692
Penicillin-Streptomycin-Nystatin Solution	Biological Industries	03-032-1B
Phospate buffered saline (PBS)	Sigma	P5368-10PAK
Rabbit anti-vWF antibody	Abcam	ab9378
Silicon wafer	Silicon Valley Microelectronics (SVM)		Wafers 4", Type N-1-10, 500-550 microns thick
SU-8 2050 photoresist	Kayaku Advanced Materials, Inc	Y11058
SU-8 developer	Kayaku Advanced Materials, Inc	Y020100
Tryton-X 100	BioLab LTD	57836