A Tissue-Engineered Tracheobronchial In Vitro Co-Culture Model for Determining Epithelial Toxicological and Inflammatory Responses

Abstract

Translation of novel inhalable therapies for respiratory diseases is hampered due to the lack of in vitro cell models that reflect the complexity of native tissue, resulting in many novel drugs and formulations failing to progress beyond preclinical assessments. The development of physiologically-representative tracheobronchial tissue analogues has the potential to improve the translation of new treatments by more accurately reflecting in vivo respiratory pharmacological and toxicological responses. Herein, advanced tissue-engineered collagen hyaluronic acid bilayered scaffolds (CHyA-B) previously developed within our group were used to evaluate bacterial and drug-induced toxicity and inflammation for the first time. Calu-3 bronchial epithelial cells and Wi38 lung fibroblasts were grown on either CHyA-B scaffolds (3D) or Transwell^® inserts (2D) under air liquid interface (ALI) conditions. Toxicological and inflammatory responses from epithelial monocultures and co-cultures grown in 2D or 3D were compared, using lipopolysaccharide (LPS) and bleomycin challenges to induce bacterial and drug responses in vitro. The 3D in vitro model exhibited significant epithelial barrier formation that was maintained upon introduction of co-culture conditions. Barrier integrity showed differential recovery in CHyA-B and Transwell^® epithelial cultures. Basolateral secretion of pro-inflammatory cytokines to bacterial challenge was found to be higher from cells grown in 3D compared to 2D. In addition, higher cytotoxicity and increased basolateral levels of cytokines were detected when epithelial cultures grown in 3D were challenged with bleomycin. CHyA-B scaffolds support the growth and differentiation of bronchial epithelial cells in a 3D co-culture model with different transepithelial resistance in comparison to the same co-cultures grown on Transwell^® inserts. Epithelial cultures in an extracellular matrix like environment show distinct responses in cytokine release and metabolic activity compared to 2D polarised models, which better mimic in vivo response to toxic and inflammatory stimuli offering an innovative in vitro platform for respiratory drug development.

Keywords: 3D in vitro models; air-liquid interface; bleomycin; co-culture; collagen; epithelium; lipopolysaccharide; respiratory tissue engineering; toxicology.

Figure 1

Evaluation of epithelial barrier integrity in air liquid interface (ALI) culture models used for exposure studies. (A) Calu-3 epithelial monocultures, Wi38 fibroblast monocultures, and Calu-3 Wi38 epithelial co-cultures on Transwell^® inserts and CHyA-B scaffolds grown under ALI conditions for 14 days. (B) Average TEER values of Calu-3 epithelial cell barriers following plateau of electrical resistance (day 10, 12 and 14). (* TEER measurements for fibroblast monocultures on Transwell^® inserts and CHyA-B scaffolds were performed as n = 2) Results displayed as mean ± SEM (n = 7).

Figure 2

Cell viability of epithelial monocultures and co-cultures grown on Transwell^® inserts and CHyA-B scaffolds following bacterial challenge with 10 μg/mL of P. aeruginosa derived LPS for 24 h. Apical (A) and basolateral (B) cell metabolic activity of Calu-3 epithelial monocultures, Wi38 fibroblast monocultures and Calu-3 Wi38 epithelial co-cultures on Transwell^® inserts and CHyA-B scaffolds. Apical and basolateral LDH release from Calu-3 epithelial monocultures (C) and Calu-3 Wi38 epithelial co-cultures (D) on Transwell^® inserts and CHyA-B scaffolds following LPS exposure. Changes in metabolic activity and LDH release are relative to untreated samples for each culture condition. Results displayed as mean ± SEM (n ≥ 3).

Figure 3

Pro-inflammatory cytokine release profile from epithelial cultures following LPS exposure. Apical and basolateral IL-8 release from Calu-3 monocultures (A) and Calu-3 Wi38 co-cultures (B) on Transwell^® inserts and CHyA-B scaffolds following LPS exposure with 10 μg/mL of P. aeruginosa derived LPS for 24 h. Apical and basolateral IL-6 release from Calu-3 epithelial monocultures (C) and Calu-3 Wi38 co-cultures (D) on Transwell^® inserts and CHyA-B scaffolds following LPS exposure. Results displayed as mean ± SEM (n = 3; * p < 0.05, ** p < 0.01).

Figure 4

Cell viability on Transwell^® inserts and CHyA-B scaffolds following drug-mediated challenge. Apical (A) and basolateral (B) cell metabolic activity of Calu-3 epithelial monocultures, Wi38 fibroblast monocultures, and Calu-3 Wi38 epithelial co-cultures on Transwell^® inserts and CHyA-B scaffolds following exposure to 100 μg/mL of bleomycin for 24 h and measured at day 23 in culture. Apical and basolateral LDH release from Calu-3 epithelial monocultures (C) and Calu-3 Wi38 epithelial co-cultures (D) on Transwell^® Inserts and CHyA-B scaffolds following bleomycin exposure. Changes in metabolic activity and LDH release are relative to untreated samples for each culture condition. Results displayed as mean ± SEM (n ≥ 3; * p < 0.05).

Figure 5

TEER measurements following drug-mediated challenge. TEER recovery of Calu-3 epithelial monocultures (A) and Calu3 Wi38 epithelial co-cultures (B) on Transwell^® inserts and CHyA-B scaffolds following challenge with 100 μg/mL of bleomycin for 24 h measured at day 15, 17, 19, 21 and 23 in culture. TEER recovery values normalised to the recovery of untreated samples for each culture condition. Results displayed as mean ± SEM (n = 3; * p < 0.05, ** p < 0.01).

Figure 6

Pro-inflammatory cytokine release profile from epithelial cultures following drug-mediated challenge. Apical and basolateral IL-8 release from Calu-3 epithelial monocultures (A) and Calu-3 Wi38 co-cultures (B) on Transwell^® inserts and CHyA-B scaffolds following challenge with 100 μg/mL of bleomycin for 24 h measured at day 23 in culture. Apical and basolateral IL-6 release from Calu-3 epithelial monocultures (C) and Calu-3 Wi38 co-cultures (D) on Transwell^® inserts and CHyA-B scaffolds following bleomycin exposure. Results displayed as mean ± SEM (n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).