Advanced Morphology Analysis on the Biochemical and Mechanical Effect on Microfluidic Vessel Network
Year: 2024
Type: conference-paper
Abstract: Microfluidic vessel network is a promising tool in various area of study including modeling of vascularized organs and vascular diseases.[1] The common supporting cell for vessel network formation, fibroblasts, grow excessively in 3D environment and cause significant vessel morphology change, render the vessel network unstable for long term culture.[2] We propose to profile the vessel morphology of various culture set-ups, with the aim of replacing fibroblast with its conditioned media or a set of angiogenic factors and testing the effect of flow on vessel morphology.Current analyses on microfluidic vessel networks were mostly conducted at 1 to 3 far apart time points.[3] Since the day-to-day morphology change is significant and can affect result interpretation, we propose and report here in-depth morphology analysis comprising continuous temporal profiles to highlight dynamic remodeling and aid time point selection.Because the traditional branch-based image analysis tools [4] and metrics [5] failed to capture the wide range of morphology features presented in in vitro vessel network culture, we proposed new void-based metrics to capture and compare those morphological features.Microfluidic device was fabricated with polydimethylsiloxane and consists of 3 rectangular channels [Fig 1a,b].Vessel network was cultured in the middle channel from endothelial cells (EC) self-assembly in 3D hydrogel for 7 days.The side channels were coated with EC for perfusion.[Fig 1c] Tested conditions include two fibroblast-EC co-cultures at different ratio (FB1:EC5 and FB5:EC5), EC single culture with fibroblast conditioned media (FB-CM) or angiogenic factors (VEGF, FGF and sphingosine-1-phosphate, labeled as Ang-V50) and a control group of EC single culture in culture media (EGM-2).Four concentrations of VEGF in the angiogenic factors were tested.To examine effect of perfusion on vessel morphology, flow was established by setting 5mmH2O hydrostatic pressure from media in reservoirs.[Fig 1d] Images were taken daily and converted to binary to isolate voids using ImageJ particle analyzer.Seven morphology metrics were measured including %vessel area, vessel diameter, void number, and area, roundness and alignment of voids.Vessel networks were successfully cultured for 7 days and tested for perfusability using fluorescent beads.[Fig 2] Each condition showed distinct morphology and significant temporal dynamics.[Fig 3a] Temporal curves were constructed for morphology metrics for Day 3 to Day 7. [Fig 3b-f] The slopes of temporal curves indicate difference in average growth rates across culture days for different conditions.Directions of slopes collectively indicate different dominate growth event in each condition, including new branch formation, vessel fusion and vessel pruning.Fibroblasts cannot be fully replaced by conditioned media or angiogenic factors to produce the same vessel morphology.Flow affected morphology change of conditions differently.And VEGF concentration is not the morphology determining factor in angiogenic factor combination.We provided morphology temporal profile of various vessel network culture set-ups to aid future experiment design.We demonstrated the use of 1) temporal profile to gain insight into vessel network growth process, including growth rate and detailed biological event and 2) a set of newly proposed void-based metrics to summarize vessel network morphology, which will be useful for reducing data dimensions for future image analysis by machine learning.
DOI: 10.70477/ultl3417
Authors: Han Shao, Edmond Young
Institution: University of Toronto
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: gold
OpenAlex ID: W4408828187