Poster J8 - Optimization of In-Vitro and In-Silico Modeling of Diffusion Assay in a Transwell System for Pro-Inflammatory Cytokine Permeability Profiles Through In Vitro Endothelial-Barrier Model

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Rudra Joshi

Student
Rutgers University — New Brunswick
Edison, New Jersey, United States

Co-Author(s)

VA

Vaidehi Apte

Graduate Student
Rutgers University — New Brunswick, United States
Rene Schloss, PhD

Associate Research Professor
Biomedical Engineering, Rutgers University
Piscataway, New Jersey, United States

Primary Investigator(s)

MY

Martin Yarmush

Paul & Mary Monroe Chair & Distinguished Professor
Biomedical Engineering, Rutgers University, United States

Introduction: The blood-brain barrier (BBB) is a critical regulatory interface that tightly controls the passage of substances between the bloodstream and the brain's neuro-microenvironment. Disruption of the BBB, often due to inflammatory agents, infections, or therapeutic interventions like CAR-T cell therapy, leads to increased permeability and subsequent neuroinflammation. This study aims to understand the progression of damage in endothelial cells, one of the primary components of the blood brain barrier, by using developing a permeability assay in an in-vitro transwell model. Prior to adding cells into the transwell for our permeability assay, we have developed the assay in a transwell without cells to determine a baseline diffusion profile using a specific tracer molecule (40 kDA Dextran).

Materials and

Methods: An in-vitro transwell diffusion assay was established using 12-well plates. Tracer molecules, specifically Dextran-FITC (40 kDa), were used to simulate permeability changes. High concentrations of Dextran-FITC (50 µg/mL) were placed in the donor region, while cell media with 2% fetal bovine serum (FBS) filled the acceptor region. Samples were incubated at 37°C, and fluorescence measurements using a fluorescence plate reader were taken at 1, 24, 48, 72, and 96 hours to determine concentration profiles. An in-silico model using COMSOL Multiphysics was developed to simulate the diffusion process, utilizing the Stokes-Einstein equation to calculate diffusion coefficients and match the in-vitro data.

Results, Conclusions, and Discussions:

Results:

Our study aims to determine the permeability and concentration profiles of pro-inflammatory cytokines through an endothelial barrier using in-vitro and in-silico models. As a proof-of-concept, we used Dextran-FITC (40 kDA) due to its fluorescent properties. An in-vitro diffusion assay with 12-well transwell systems showed that 40 kDA Dextran-FITC did not reach equilibrium within 96 hours (Figure 1). An in-silico model using COMSOL Multiphysics provided concentration profiles that matched our in-vitro data (Figures 2 & 3). The experimental setup involved Dextran-FITC in the donor region and cell media in the acceptor region, with samples collected up to 96 hours. Smaller Dextrans were hypothesized to reach equilibrium faster, confirmed by COMSOL simulations and diffusion coefficients (Figure 4). This integrated approach enhances our understanding of molecular transport mechanisms in blood-brain barrier dysfunction.

Conclusions:

Our study developed an in vitro diffusion assay to determine molecule permeability through a transwell membrane using 40 kDA Dextran-FITC. The system was unsteady-state within 96 hours, allowing for accurate in silico models using COMSOL Multiphysics that matched in vitro data for the first 48 hours. While the in vitro model represented tracer molecule permeability through a polycarbonate membrane, it did not capture the dynamic nature of endothelial barrier breakdown under injury. Future studies should include endothelial cells and examine permeability changes under non-injury and injury conditions. Refining models and performing further experiments will improve our understanding of permeability changes, potentially identifying therapeutic targets to prevent blood-brain barrier breakdown in neurological diseases and contributing to new treatments for these disorders.

Discussions:

While we successfully established proof-of-concept in vitro and in silico diffusion models, future work will focus on adapting this model to include endothelial cell monolayers derived from human umbilical vein endothelial cells (HUVECs) or rat brain endothelial cells (RBECs). This adaptation will allow for a more accurate representation of the BBB and its response to various conditions. Understanding the effects of various injuries on BBB permeability will be crucial in designing interventions to protect against neuroinflammatory damage, particularly for individuals with traumatic brain injuries.

Acknowledgements (Optional): Thank you Dr. Schloss, Dr. Yarmush, and Vaidehi Apte for your mentorship in developing this project.