Searching for Optimal Models for Endothelial Cell 3D Culture

Endothelial Cells and the Extracellular Matrix (ECM)

Endothelial cells line the vessels of the circulatory system and provide important support for vessel homeostasis, wound repair, and the inflammatory response. The ECM is a critical part of all tissues of the body, providing structural support and mediating cell adhesion and cell-cell interactions. Underlying the layer of endothelium is the basement membrane, a thin sheet of ECM that anchors endothelial cells to the underlying tissue structure. Endothelial cell interaction with the basement membrane and surrounding ECM drives tissue homeostasis and contributes to the overall functioning of the endothelium, supporting endothelial barrier function and regulating endothelial cell proliferation and survival.

Lifeline® HUVEC in Hydrogel Bioengineering

Studying cell behavior in culture has always been a challenge. When cells are removed from the 3D context of a tissue, they lose contact with the ECM and basement membrane; this can alter cell behavior in a way that does not reflect the same cell’s behavior in a tissue. Because of this, researchers have long been concerned that studying cells on plastic culture dishes is not the most optimal way to replicate the in vivo context. Therefore, optimization of 3D culture systems that more closely mimic what cells might experience in vivo has long been of considerable interest.

In a study this year, Su and colleagues set out to develop a better 3D culture substrate for modeling the basement membrane in vitro, particularly for the study of endothelial cells. They used the PEGX method (polyethylene glycol [PEG] crosslinking) to make a hydrogel substrate, to which they conjugated two peptides: YIGSR (derived from laminin, a basement membrane protein) and QK (a vascular endothelial growth factor [VEGF] mimetic). They hypothesized that addition of these two bioactive peptides to the hydrogel would support endothelial cell attachment and signaling.

The authors first determined the optimal concentration of YIGSR and QK peptides using conditionally immortalized glomerular endothelial cells (GEnCs), which formed a maximal number of cord-like structures on hydrogels with 100 mM QK and 12 mM YIGSR. Quantification of the free amine content of the hydrogels, as well as rheological characterization (characterization of how a material deforms under force), demonstrated that crosslinking of the hydrogel and conjugation of the bioactive peptides were successful. Additionally, scanning electron microscopy illustrated that there was little structural variation across different hydrogels.

Next, the authors tested the bioactivity of the QK peptide within hydrogels. They cultured Lifeline® human umbilical vein endothelial cells (HUVECs) on QK hydrogels and measured phosphorylation of the VEGF receptor, VEGFR2, which should increase upon binding of QK. As expected, the authors observed increased VEGFR2 phosphorylation in HUVECs and GEnCs grown on QK hydrogels.

Further analysis demonstrated that HUVECs grown on QK hydrogels formed cord-like structures that were maintained over time. This was in contrast to culture on Matrigel (a commonly used 3D culture substrate), whereby these cord-like structures formed, but eventually regressed. The authors also found that gene expression of multiple genes involved in cell-cell interactions, cell surface receptor signaling, and endothelial cell function were upregulated after 5 days in culture on QK/YIGSR hydrogels. In contrast to the behavior of HUVECs, GEnCs formed less robust cord-like structures on QK, YIGSR, and QK/YIGSR hydrogels, and in addition, the greatest increase in GEnC gene expression occurred on Matrigel, rather than the investigational hydrogels.

Together, the results of this study demonstrate that hydrogels developed using the PEGX method and conjugated with bioactive peptides are c, although responses of different cell types to growth on the hydrogels may differ.

Interested in learning more about Lifeline® endothelial cell systems? Check out our catalog, which includes the following endothelial cell types:

• Aortic endothelial cells
• Coronary endothelial cells
• Lung microvascular endothelial cells
• Dermal microvascular endothelial cells (adult and neonatal)
• Cardiac microvascular endothelial cells
• Iliac artery endothelial cells
• Pulmonary artery endothelial cells
• HUVECs

We love hearing from you! Let us know how you are using Lifeline® products in your research and your published study could be featured on our next blog. In the meantime, check in every other week to see which cell systems will be featured next!