Development of poly(lactic-co-glycolic acid) electrospun membranes for incorporation into 3-D co-culture models of the airway-blood barrier

Abstract

The lung epithelium forms an essential protective barrier, maintaining lung homeostasis and limiting the effects of inhaled environmental agents. The vascular endothelium is in close contact with the epithelial cells of the lung, separated by a basement membrane (BM) allowing cellular crosstalk, providing structural support and promoting epithelial polarity. In response to environmental challenges, the epithelium co-ordinates localized crosstalk with endothelial cells of the microvascular network to mount an inflammatory response, recruiting neutrophils to the affected area. This project sought to form a 3-D co-culture model that more fully recapitulates the airway-blood barrier. Current models underperform due to sub-optimal semi-porous film BMs that fail to accurately recreate the in vivo BM structure, potentially affecting the accurate modelling of epithelial barrier development, co-culture crosstalk and neutrophil migration. It was hypothesised that electrospinning PLGA membranes could recreate the nanofibrous network structure of the in vivo BM: improving cell attachment, encouraging epithelial polarisation (creation of apical and basolateral regions with specific protein compositions supporting functions) and differentiation (transition to highly specialised cell type determined by cellular microenvironment), stimulating crosstalk between airway epithelium and endothelium, and permitting neutrophil migration. To address this, bespoke, biodegradable PLGA membranes were produced which balance a membrane which is (77 ± 3%) porous (total volume of pores (empty space) in membrane) with a relatively low thickness (81±16µm) that provides support of an epithelial layer capable of supporting epithelial and endothelial barrier formation. These membranes possessed 1.0 ± 0.3µm diameter fibres and a pore size distribution of 7.7 to 1.6µm that facilitated epithelial and endothelial surface retention whilst permitting neutrophil migration into the membranes. vIn-house and Transwell®-based inserts were designed and optimised for media infiltration and to support electrospun membranes during cell culture, allowing accurate ionic barrier integrity readings through Transepithelial electrical resistance (TEER) to be taken. The 16HBE 14o- and BCi-NS1.1 cell line successfully polarised on PLGA membrane showing similar TEER readings (16HBE 14o- on Transwells®: 875±245W.cm2 and ES membrane: 746±175W.cm2, BCi NS1.1 on Transwells®: 688±3W.cm2 and ES membrane: 732 ±56W.cm2) and strong tight junction (occluding) staining. BCi-NS1.1 also showed cilia and secretory cell differentiation over 28days in culture. 16HBE 14ocells showed a 70% reduction in TEER after dysregulation by poly(I:C) mimic on both electrospun membranes and Transwells®. Endothelial (HUVEC and microvasculature (MV)) cells showed adherence to the basolateral surface of electrospun membranes through CD31 immunocytochemistry. Neutrophils and differentiated HL60 (dHL60) neutrophil-like cells showed migration across electrospun membranes towards 10ng/ml IL-8 and 10nmol/l fMLP stimulation respectively. Co-cultures were formed showing crosstalk between 16HBE 14o- and MV cells which were seeded on opposite sides of electrospun membrane. Preliminary crosstalk experiments suggested that there was a different cytokine release profile for IL-8, IP-10, TNF-α and Fractalkine in response to poly(I:C) stimulation, and these profiles did not match that seen in Transwells® , indicating that the structure of nanofibrous membranes may affect the co-culture cytokine release profile. The pore size and porosity of these membranes supported the migration of dHL60 cells through a layer of MV cells into and through electrospun membrane towards fMLP. Finally, transmission electron microscopy of BCi-NS1.1/MV electrospun membrane co-cultures showed the formation of tight junctions, secretory and ciliated cell differentiation, and MV cell integration into the model which has not been previously shown on PLGA membranes or any electrospun membrane that was also capable of supporting neutrophil migration. This project has shown that electrospun membranes provide a valuable alternative to commercial film-based inserts, successfully forming polarised and differentiating epithelial-endothelial co-cultures, showing potential for crosstalk analysis, and permitting immune cell migration through these highly porous membranes. Developing in vivo-like nanofibrous mesh membranes that more accurately recapitulate the physiological basement membrane allows novel testing of neutrophil infiltration in response to pathogenic stimuli and improved accuracy of chemokine and cytokine release provi files across multiple human and non-human physiological barrier models. These PLGA membranes could be integrated into the next generation of Lung-on-Chip models, improving drug discovery, disease modelling and replacing animal models.

More information

Identifiers

ORCID for Cornelia Blume: orcid.org/0000-0001-6133-7318

ORCID for Emily Swindle: orcid.org/0000-0003-3644-7747

ORCID for Donna Davies: orcid.org/0000-0002-5117-2991

ORCID for Timothy M Millar: orcid.org/0000-0002-4539-2445

Catalogue record

Export record