Bioprinted Multi-Cell Type Lung Model for the Study of Viral Inhibitors

Abstract

Influenza A virus (IAV) continuously causes epidemics and claims numerous lives every year. The available treatment options are insufficient and the limited pertinence of animal models for human IAV infections is hampering the development of new therapeutics. Bioprinted tissue models support studying pathogenic mechanisms and pathogen-host interactions in a human micro tissue environment. Here, we describe a human lung model, which consisted of a bioprinted base of primary human lung fibroblasts together with monocytic THP-1 cells, on top of which alveolar epithelial A549 cells were printed. Cells were embedded in a hydrogel consisting of alginate, gelatin and collagen. These constructs were kept in long-term culture for 35 days and their viability, expression of specific cell markers and general rheological parameters were analyzed. When the models were challenged with a combination of the bacterial toxins LPS and ATP, a release of the proinflammatory cytokines IL-1β and IL-8 was observed, confirming that the model can generate an immune response. In virus inhibition assays with the bioprinted lung model, the replication of a seasonal IAV strain was restricted by treatment with an antiviral agent in a dose-dependent manner. The printed lung construct provides an alveolar model to investigate pulmonary pathogenic biology and to support development of new therapeutics not only for IAV, but also for other viruses.

Keywords: LPS stimulation; bioprinting; human lung model; influenza A virus.

Figure 1

Rheologic characterization of the printed tissue models cultured in the presence or absence of sodium citrate. (A) The elastic modulus was measured at a frequency of 1 Hz and 0.1% shear strain at 37 °C at the indicated time points. (B) Shear modulus of printed constructs at increasing frequencies (0.1–10 Hz). (C) Schematic representation of the multi-layer structure and photograph of the printed model. Results are shown as mean ± SEM of three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, ***, p ≤ 0.001, **** p ≤ 0.0001.

Figure 2

Metabolic activity and secretion of IL-8 from the bioprinted models. (A–C) Metabolic activity as determined by the tetrazolium hydroxide salt (XTT) assay at the indicated time points post-printing of models containing (A) A549 cells, THP-1 cells and human lung fibroblasts (NHLFb), as well as models containing either (B) only A549 cells or (C) only NHLFb. All models were printed in alginate/gelatin/collagen bioinks. (D) Quantitative enzyme-linked immunosorbent assay (ELISA) analysis of IL-8 from the three cell type model measured at indicated time points post-printing. Results are shown as mean ± SEM of five (A,D) or three (B,C) independent experiments. * p ≤ 0.05; ** p ≤ 0.01.

Figure 3

Cell viability staining of bioprinted lung models. Qualitative viability staining of living and dead cells in models containing (A) A549 cells, THP-1 cells and human lung fibroblasts (NHLFb), as well as in models containing either (B) only A549 cells or (C) only NHLFb printed in an alginate/gelatin/collagen bioink after indicated time points of cultivation post printing using calcein-AM (live in green) and ethidium homodimer-1 (dead in red). Scale bar: 200 µm.

Figure 4

Differential protein expression and morphology between printed cells. Bioprinted models containing either A549 cells, THP-1 cells and human lung fibroblasts or models containing only A549 cells were cultured for indicated time points, fixed, immunohistochemically labelled, cleared and analyzed by fluorescence microscopy. Type of model, culture time, side of view as well as stained proteins are mentioned in the respective panels (A–E). Nuclear counter staining was performed using DAPI (blue channel). Scale bar involves (A) (left column), (B,D,E): 200 µm; (A) (right column) and (C): 100 µm.

Figure 5

LPS-induced release of IL-1β from bioprinted multi-cell type lung models. (A,B) A549 cells were printed on top of a base containing THP-1 cells and NHLFb (fibroblasts). Additionally, constructs containing only NHLFb and THP-1 cells were printed (- A549). The printed constructs were cultured for 21 days. For maturation of THP-1 cells towards macrophage-like cells, the constructs were treated with 200 ng/mL PMA for 24 h. After a further 48 h, constructs were stimulated with 1 µg/mL lipopolysaccharide (LPS) for 16 h, followed by 30 min of treatment with 10 µM adenosine triphosphate (ATP). Centrifuged supernatants were assayed for the release of (A) IL-1β and (B) IL-8 by enzyme-linked immune assays. (C) THP-1 cells were seeded in 24-well plates and matured with PMA (100 ng/mL, 24 h) towards macrophage-like cells. After 48 h of culture without PMA, THP-1 cells were stimulated with LPS (1 µg/mL, 16 h), followed by 10 min of treatment with 5 µM ATP. Centrifuged supernatants were assayed for the release of IL-1β by enzyme-linked immune assays. Results are shown as mean ± SEM of three (A,B) and five (C) independent experiments.* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 6

Oseltamivir inhibition of IAV replication in bioprinted lung models. The A549 cells were printed on top of a base containing THP-1 cells and NHLFb (fibroblasts). Additionally, constructs containing only NHLFb and THP-1 cells were printed (- A549). The printed constructs were cultured for 21 days. For maturation of THP-1 cells towards macrophage-like cells, constructs were treated with 200 ng/mL PMA for 24 h. (A,B) After a further 48 h, constructs were infected with 4 × 10⁵ PFU/mL of human IAV [H3N2] and treated with different concentrations of oseltamivir. (A) Supernatants were taken 1, 16, 24 and 48 h post infection and titrated on MDCKII cells to determine IAV titers. (B) Centrifuged supernatants 48 h post IAV infection were assayed for release of IL-29 by enzyme-linked immune assays. Results are shown as mean ± SEM of three independent experiments. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. (C) Immunohistochemical analysis of IAV infection. Constructs were infected with 1 × 10⁶ PFU/mL of IAV for 24 h. Afterwards, samples were fixed and stained for viral nucleoprotein (red) and nuclei (DAPI, blue). Staining was analyzed by fluorescence microscopy. Scale bar: 200 µm.