Researcher(s)
- Rylie Bird, Biomedical Engineering, University of Delaware
Faculty Mentor(s)
- Jason Gleghorn, Biomedical Engineering, University of Delaware
Abstract
Congenital Diaphragmatic Hernia (CDH) is a fetal developmental disorder affecting one in 2,500 – 4,000 infants each year [1]. Dysregulation of diaphragm formation produces a hole through which the abdominal organs herniate into the thoracic cavity, impairing lung growth and resulting in both pulmonary hypoplasia and pulmonary hypertension (CDH-PH) [2]. CDH-PH is associated with changes in the pulmonary vasculature architecture, including increased vascular extracellular matrix deposition [3], vessel wall thickening [4], and vascular rarefication [2]. There is a higher incidence rate of CDH in males in some clinical cohorts, but the impact of sex is not well understood [5]. Previously, we found that sex differences in healthy and CDH endothelial cells could be studied via a three-dimensional (3D) in vitro angiogenesis assay. We sought to optimize this assay by varying the seeding density to ensure maximum coating of our collagen beads. Human Umbilical Vein Endothelial Cells (HUVECs) from both CDH and healthy patients were coated on collagen beads at 40,000 and 250,000 cells/mL and embedded in a fibrin clot, where they proliferated and sprouted for 3 days in EGM2 medium. The wells were fixed, stained, and imaged then processed through a custom MATLAB image analysis software to determine the number of sprouts per bead, length of the longest sprout, and percentage of beads with sprouts. Female cells from healthy and CDH donors had longer sprouts with higher seeding density, but only female control cells had increased sprout numbers. Male cells from control and CDH donors produced the same number of sprouts regardless of seeding density, but with increased seeding densities, both groups had longer sprouts. At a higher density, female cells from control donors had significantly more sprouts than female cells from CDH donors and longer sprouts than all other groups. These studies show successful optimization of this in vitro assay by using a higher cell seeding density. The optimized assay can now be used to investigate possible mechanisms that underpin differences observed in vascular development in CDH patients from clinical studies.
References:
[1] W.-Y. Teo, et al. PEDN. 2020 [2] P. K. Chandrasekharan, et. al. Matern. Health Neonatol. 2017 [3] M. N. Monroe, et. al. Pediatr. Pulmonol. 2020 [4] R. Tenbrinck, et. al. PEDN. 1992 [5] S. R. Sferra, et. al. J Pediatr. 2023