Indra Putra Wendi - Institutionen för experimentell medicinsk vetenskap

Title: Optimizing electrospun poly(ε-caprolactone) for scaffold-based in vitro lung models

Main supervisor: Darcy Wagner

Reviewers: Mattias Magnusson, Sara Rolandsson Enes

Abstract

Background

Historical records of pandemics within the last century have shown a disproportionate trend towards infectious airway diseases, during which air- liquid-interface (ALI) culture method has seen increased use. However, there has been limited innovation on these inserts after their initial development, and they do not morphologically or mechanically mimic airway basement membranes of the proximal or distal lung. In addition, global shortages of specialized ALI cell culture inserts were observed during the recent COVID- 19 pandemic. Therefore, there is a need to develop alternatives to commercial culture membranes. Here we explored electrospun poly(ε-caprolactone) (e- PCL) as a potential novel material for use as a lung epithelium culture substrate. Despite its potential, the natural hydrophobicity, opacity, and low melting point (~60º C) of PCL poses challenges for it to be utilized as culture scaffold material.

Research questions

1. Is the e-PCL membrane feasible to be used as a scaffold for proximal and distal lung epithelial ALI system culture?
2. Using the same scaffold material, is it possible to modify the culture system from static into a more dynamic one (i.e. lung-on-chip fluidic system) to model the in vivo situation?

Methodology and preliminary results

We determined that the membrane opacity can be overcome by utilizing fluorescence live-cell monitoring at far red wavelengths. Calu-3, an immortalized human airway model cell line, was used to evaluate different combination of surface treatments owing to its potential for forming monolayers with good tight junctions. Combination of polydopamine/collagen I or polydopamine treatment alone helped to functionalize the insert, allowing formation of sufficient barrier function to be airlifted (assessed using trans-epithelial electric resistance and diffusion experiments). Polydopamine coating was confirmed with attenuated total reflectance – fourier transform infrared (ATR-FTIR) spectroscopy. Histochemistry and immunostaining of the ALI culture cross section revealed phenotypical changes to the Calu-3 and mucus production increase after airlifting. Scanning electron microscopy (SEM) of the ALI culture from polydopamine/collagen I coated e-PCL showed rare patches of ciliated cells. After airlifting, the Calu-3 formed grape-like clusters reminiscent of serous tumors, similar to tumors from which these cells are derived. We also found that e-PCL could be used in a lung-on-a-chip device which supported growth of immortalized murine epithelial cells, MLE-12.

Significance

We have early proof of phenotypic characterization of Calu-3 when grown on e-PCL scaffold. Furthermore, we also established protocols to facilitate the use of e-PCL as an attractive alternative to commercially available membranes.

Manuscripts

Wendi, IP., Kispa, T., Gvazava, N., Alsafadi, H., Wagner, D.E. Leveraging large-pore electrospun PCL membrane to model upper airway epithelium at air-liquid interface. Manuscript

Tas S, Rehnberg E, Wendi, IP, Bölükbas DA, Beech JP, Kazado LN, Svenningsson I, et al. 3D printed lung on a chip device with a stretchable nanofibrous membrane for modeling ventilator induced lung injury. Manuscript