Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels
January 14th, 2021
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.
Our protocol allows researchers to study the vessel network formation process in a clear and easily trackable fashion, opening the doors to new studies and shedding light on blood vessel behavior. This technique presents the possibility of creating highly organized and repeatable vascular networks with respond to the surrounding environment. Endothelial cells from patients suffering from a vascular disease can be retrieved in using this system, making it possible to recreate the vascular disease and find an appropriate treatment.
The proposed method can be extended for studying vascular network behavior when paired with different cell types, allowing to observe the interaction of developing vessels with a specific tissue type. Begin by submerging the scaffolds in 70%ethanol for a minimum of 15 minutes, then wash them twice in PBS. Mix 1.5 microliters of fibronectin stock solution with 28.5 microliters of PBS per scaffold to be seeded.
Prepare one fibronectin dilution to be used for all scaffolds to avoid pipetting errors. Place the scaffolds sparsely on top of a hydrophobic surface and cover each scaffold with 30 microliters of the fibronectin dilution. Replace the plate's lid and put the fibronectin-covered scaffolds into an incubator set to 37 degrees Celsius and 100%humidity for a minimum of one hour.
After incubation, lightly rinse the scaffolds in PBS to remove fibronectin remnants. The scaffolds can be kept in PBS at four degrees Celsius for up to a week. Prepare endothelial cell or EC medium by mixing the basal medium with the corresponding medium kit components, including an antibiotic solution, FBS, and endothelial cell growth supplements as indicated by the manufacturer.
Make the human adipose microvascular endothelial cell suspension in EC medium with a concentration of four million cells per milliliter. Using forceps, place one fibronectin-coated scaffold per well in a non-TC 24-well plate. Cover each scaffold with 25 microliter droplets of the cell suspension, making sure not to let the suspension flow away from the scaffold.
Put the lid on the plate and place it in an incubator at 37 degrees Celsius, 5%carbon dioxide and 100%humidity for 60 to 90 minutes. After the incubation, fill each well with 700 microliters of EC medium. Incubate the endothelialized scaffolds until EC confluence can be observed using fluorescent microscopy or for three days.
Change the medium every other day. Prepare DPSC medium by mixing 500 milliliters of low glucose DMEM, 57.5 milliliters of FBS, 5.75 milliliters of non-essential amino acids, 5.75 milliliters of GlutaMAX, and 5.75 milliliters of penicillin-streptomycin-nystatin solution. Transfer the endothelialized scaffolds into a new non-TC 24-well plate.
Discard all media from the current plate using a pipette or vacuum suction, taking care not to apply vacuum directly to the scaffold. Place one scaffold into the center of each well, then dry the surrounding area with light vacuum. Dilute thrombin and fibrinogen stock solutions with PBS to a final concentration of five units per milliliter and 15 milligrams per milliliter respectively.
Prepare an eight million DPSC per milliliter suspension in the thrombin dilution and distribute 12.5 microliters of the suspension into individual microtubes per scaffold to be seeded. Set a five to 50 microliter pipette to 12.5 microliters and fill it with fibrinogen solution. Without removing the tip, set the pipette to 25 microliters.
The material in the tip should rise and leave an empty volume. Slowly press the plunger button until the liquid reaches the tip opening, but does not leak out. Hold the plunger in this position and put the tip into one of the microcentrifuge tubes containing the cells in thrombin suspension, making sure the tip is in contact with the liquid.
Gently release the plunger button and draw the cell suspension into the tip. Thoroughly mix both materials, avoiding bubble formation. Quickly dispense the mixed materials on top of an endothelialized scaffold.
Repeat the previous steps for each scaffold, making sure to change tips between uses to avoid unexpected fibrin gel formation within the tip. Replace the plate lid and incubate the scaffolds at 37 degrees Celsius, 5%carbon dioxide and 100%humidity for 30 minutes. After incubation, fill each well with one milliliter of one-to-one DPSC and EC medium.
Culture for one week, changing the medium every other day. At different time points during culture, remove the medium from the well and image the constructs using a confocal microscope to study the vascular development or any other parameter of interest. This protocol allows for the fabrication of tessellated scaffolds made of SU-8 photoresist.
Scaffolds with distinct compartment geometries and highly accurate and repeatable features were obtained. With traditional simultaneous seeding of both endothelial cells and support cells, the cells were homogeneously distributed over the scaffold resulting in unpredictable and disorganized vascular networks. Contrarily, stepwise cell seeding resulted in highly organized vascular networks.
When using fluorescent endothelial cells, the vessels can be imaged in real time. Red fluorescent protein expressing endothelial cells were cultured on hexagonal scaffolds and imaged. The support cells were added at day one and the vascular networks were imaged every other day to quantify vessel development.
For each time point, wide images of the whole scaffold were taken. Vessel growth was quantified as total vessel length and area. A confocal imaging time-lapse was performed to allow single vessel tracking using multi-colored endothelial cells.
The vessel path was generated from the time-lapse, making it possible to observe vessel migration. Vessel maturation was observed by the presence of smooth muscle actin and support cells. For the circular, hexagonal, and squared compartments, the cells multiply and organize into structures.
By day seven, all shapes showed a rich and complex vascular network. Higher magnification images revealed a denser smooth muscle actin and support cell presence co-localized with formed vessels, evidencing support cell recruitment and differentiation surrounding vascular structures. This technique can be used to study the behavior of three-dimensional vascular networks when exposed to different conditions, such as specific cell inhibitors or in the presence of additional cell types.
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Overview
This study presents a protocol for creating organized vascular networks using endothelial and support cells in a 3D environment. The method allows for the observation of vessel behavior and the potential recreation of vascular diseases for treatment exploration.
Key Study Components
Area of Science
- Vascular biology
- Tissue engineering
- Cell culture techniques
Background
- Proper vascular networks are essential for engineered tissues.
- Endothelial cells play a critical role in blood vessel formation.
- Understanding vessel behavior can lead to advancements in treating vascular diseases.
- A high-throughput platform enables controlled studies of vascular networks.
Purpose of Study
- To develop a stepwise seeding protocol for endothelial cells.
- To study the formation and behavior of vascular networks.
- To recreate vascular diseases using patient-derived cells.
Methods Used
- Submerging scaffolds in 70% ethanol for sterilization.
- Washing scaffolds in PBS.
- Mixing fibronectin with PBS for cell seeding.
- Utilizing a high-throughput platform for 3D culture.
Main Results
- Successful creation of organized vascular networks.
- Ability to track vessel formation in a controlled environment.
- Potential for studying interactions with different tissue types.
- Feasibility of modeling vascular diseases for research.
Conclusions
- The protocol provides a reliable method for vascular network studies.
- It opens avenues for research into vascular diseases.
- The technique can be adapted for various cell types and conditions.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables biopharma R&D teams to generate organized, repeatable vascular networks in 3D scaffolds, supporting mechanistic de-risking of angiogenic therapies. By isolating vascular behavior in a controlled, high-throughput system, it improves target validation and predictive confidence in preclinical angiogenesis models. The approach reduces biological variability in vessel formation studies, enhancing reproducibility across discovery and translational workflows.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by observing vessel sprouting in response to scaffold topography and compartment geometry.
- Operational Value: Provides a standardized system for functional target validation of angiogenic pathways using patient-derived endothelial cells.
- Predictive Value: Supports portfolio triage by quantifying vessel network formation as a functional readout for pathway modulation.
Screening & Assay Development
- Assay Readiness: Generates highly organized vascular networks that respond to microenvironmental cues, enabling reliable compound screening.
- Quantitative Outputs: Facilitates measurement of vessel length, area, and maturation markers for dose-response analysis.
- Platform Reuse: Supports adaptation to co-culture systems and mechanical stimulus studies for scalable screening campaigns.
Translational & Preclinical Research
- Disease Modeling: Allows recreation of vascular diseases using patient-derived cells to evaluate treatment effects in a disease-relevant system.
- Translational Continuity: Bridges discovery and preclinical work by providing quantitative vascular metrics for go/no-go decisions.
- Mechanistic De-risking: Clarifies the role of support cells in vessel maturation, reducing uncertainty in angiogenic mechanism interpretation.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target validation through lead identification to preclinical vascular model refinement, particularly for angiogenesis-focused programs.
- Discovery Biology: Supports hypothesis testing of angiogenic factors by enabling real-time tracking of vessel migration and network formation.
- Screening: Delivers reproducible, quantitative vascular outputs that allow comparison of compound effects across conditions.
- Analytics: Provides time-lapse imaging and morphometric data (vessel length, area) to inform statistical analysis of vascular responses.
- Translational Research: Connects to preclinical validation through observation of vessel maturation and support cell co-localization with formed networks.
- Enterprise Reuse: Establishes a reusable platform for studying vascular behavior under varied conditions, including inhibitor exposure and co-culture with tissue-specific cells.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in angiogenic target validation by reducing mechanistic ambiguity in vessel formation.
- Operational Value: Enhances standardization and reproducibility of vascular network generation across laboratories and projects.
- Strategic Value: Improves capital efficiency by enabling early de-risking of angiogenic candidates through quantitative vascular phenotyping.
- Portfolio Impact: Informs risk-adjusted advancement decisions by providing functional vascular network data for target prioritization.
Implementation Considerations
- Requires expertise in endothelial cell culture, confocal microscopy, and image analysis for vessel quantification.
- Depends on access to fabrication tools for tessellated SU-8 scaffolds and incubation systems with precise humidity and CO2 control.
- Necessitates cross-team standardization of seeding timing, medium formulations, and imaging protocols for consistent results.
- Involves adaptation considerations when extending the protocol to different cell types or extracellular matrix coatings.
- Limited by the need for specialized imaging equipment to perform time-lapse tracking and multi-color vessel tracing.
