(J-379) A microphysiological model of vascularized human lung tissue to investigate vascular contributions to idiopathic pulmonary fibrosis

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row J - Poster # 379

Presenting Author(s)

AS

Austin Summers (he/him/his)

Undergraduate Student
Department of Biological Sciences, Duquesne University, Pittsburgh, PA 15282, USA., NSF Science and Technology Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA 19104, USA
Pittsburgh, Pennsylvania, United States

Co-Author(s)

NM

Nikolaos Matisioudis

PhD Student
Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA, United States

Primary Investigator(s)

DH

Dan Dongeun Huh

Associate Professor
NSF Science and Technology Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA 19104, USA., Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA., Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA, United States

Introduction::

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic interstitial lung disease (ILD) of unknown origin that can lead to respiratory failure. The current pathogenesis model suggests that recurrent, unresolved epithelial injury leads to aberrant wound healing in which injured lung epithelial cells communicate with subepithelial fibroblasts to activate them and induce their differentiation into myofibroblasts that can deposit excessive amounts of matrix proteins, resulting in stiffening and architectural distortion of lung tissue with the formation of fibrotic foci. Over the last decade, considerable progress has been made in our understanding of how the epithelial-mesenchymal crosstalk contributes to the development and progression of the disease. Despite well-documented anatomical evidence of abnormal changes in the blood vessels of IPF lungs, however, little is known about whether and how the pulmonary vasculature plays a role in the pathophysiology of IPF. Motivated by this knowledge gap, our study aims to develop a microengineered in vitro platform to model the vasculature of human lung tissue with IPF and use this microphysiological system to study the interaction of the pulmonary endothelium with effector cell populations that drive the process of dysregulated fibrogenesis.

Materials and Methods::

For this study, we created a compartmentalized three-dimensional (3D) microfluidic device consisting of three parallel channels partially separated by two microfabricated rails (Fig. 1). This device was fabricated in poly(dimethylsiloxane) (PDMS) using standard soft lithography methods. Briefly, PDMS base was mixed with curing agent at a weight ratio of 10:1 (base:curing agent) and poured over a 3D printed master containing microchannel features. After curing, the micropatterned PDMS slab was peeled off of the master and bonded to another thin blank PDMS layer using plasma treatment to create a fully enclosed system. For cell culture, we used primary normal human lung fibroblasts (NHLFs), human lung fibroblasts isolated from diseased lungs with IPF, and human lung microvascular endothelial cells. As a first step to engineer vascularized stroma of the lung mucosa in our system, a mixture of fibrinogen, thrombin, aprotinin, fibroblasts, and endothelial cells was gently injected into the middle cell culture channels on Day 0 (Fig. 2a). After gelation, Microvascular Endothelial Cell Growth Medium-2 (EGM-2 MV) was introduced to the side channels to provide nutrient supply to the cells embedded in the fibrin scaffold. On the following day (Day 1), lung microvascular endothelial cells were seeded into the side channels and grown up to Day 7 to form an endothelial lining on the channel surfaces (Fig. 2b). The process of blood vessel development in this system was visualized by immunofluorescence staining of CD31 expressed by endothelial cells.

Results, Conclusions, and Discussions::

Our data showed that the 3D culture configuration in our device enabled vascular development. To investigate vascular features of IPF lung tissues, we formed blood vessels using patient-derived IPF fibroblasts whereas NHLFs were used as a control model. In this preliminary experiment, the IPF fibroblasts were found to be supportive of vasculogenesis in the 3D culture environment of our device, permitting the development of a 3D blood vessel network. Specifically, the endothelial cells grown in the fibrin gel began to form hollow tubular structures within the first 3-4 days of culture, which then gradually assembled into a 3D network of interconnected microvessels throughout the scaffold as shown in Fig. 3 and Fig. 4. Importantly, the 3D vasculature in the fibrin gel was observed to anastomose with the endothelium in the side channels and as a result, the vascular network became directly accessible and perfusable from the side channels. Vascular perfusability of the engineered tissue construct was illustrated by the flow of 1-µm fluorescent microbeads throughout the network (Fig. 5). In ongoing work, efforts are being made to quantify the architectural and morphological characteristics of the vascular network in the IPF fibroblast-containing tissues and compare the results to those obtained from another model containing NHLFs. Studies are also underway to systematically and quantitatively compare the barrier function of the endothelium between the normal and IPF models, as well as the phenotype of fibroblasts, especially their responsiveness to fibrogenic insults.

Although preliminary, this work demonstrates the feasibility of developing a microengineered system that represents a promising strategy to model the pulmonary microvasculature in normal and IPF lungs. Much work remains to be done but we believe that this model may provide an enabling platform for in-depth investigation of how vascular remodeling occurs in IPF and whether and how the altered vasculature affects disease progression. Establishing a mechanistic understanding of these important yet understudied intercellular interactions will advance our fundamental knowledge of IPF and may also provide opportunities to identify new therapeutic targets and effective treatment strategies that are urgently needed.

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