Evaluation of different media for ex vivo porcine lung culture model

Abstract

Developing drugs targeting respiratory pathogen is essential to control respiratory diseases. Many experiments have been performed under in vivo situation. However, in vivo experiments have economical and ethical issues. The objective of this study was to determine the possibility of developing an ex vivo lung culture system with possible application for respiratory infection studies. After isolating lungs from naïve pigs, agarose-inflated lung tissues were prepared and sliced manually. These sliced lung tissues were then subsequently placed on 24-well plates. Eight different combinations of media were used to determine the optimum ex vivo lung culture condition. In addition, lung tissues were infected with porcine reproductive and respiratory syndrome (PRRS) virus at a titer of $1{\times}10^4\;TCID_{50}/mL$. Virus growth was confirmed by titration in MARC-145 cells at 2, 4, 6 days post infection (dpi). We found that ex vivo lung culture in physiological environment was not media specific based on histopathology and cytotoxicity. However, under virus-infected condition, thickened alveolar walls in the lung tissues and stable virus titers at 2, 4, 6 dpi were shown in F12K medium suggesting that it was useful for tissue maintenance and virus infection using PRRS virus infected lung tissues. The present study shows the possibility of using porcine ex vivo lung model for respiratory infection studies.

Fig. 3. Virus titer measured in the medium of PRRS virus infected tissue. The supernatants from infected tissues were collected at 2, 4, 6 dpi. The plaque titers were determined at appropriate time points on MARC-145 cells.

Fig. 1. (A) Lung tissues from ex vivo culture in physiological conditions were stained with H&E. Histopathological examinations were performed and compared between 1 hour and 6 days after cultivation. Scale bar is 50 μm. (B) Lung tissues from ex vivo culture in physiological conditions were performed with TUNEL assay and compared between the groups on 6 days after cultivation. 3,3'-Diaminobenzidine (DAB) was used for visualizing the apoptosis as brown color. Counterstaining was performed with hematoxylin. Scale bar corresponds to 50 μm. (C) Percentage of positive area with TUNEL assay on day 6 lung tissue in physiological condition. X axis represented the each group and Y axis represented the percentage of positive area. Different letters indicate significant differences according to Duncan’s multiple range test. Value of P <0.05 was considered statistically significant based on results of one-way ANOVA. (D) Results of LDH released from lung tissues with physiological conditions on each time point. The supernatants from each group were collected at 1 hour and 2, 4, 6 days post cultivation.

Fig. 2. (A) Lung tissues which were infected with PRRS virus from ex vivo culture were stained with H&E. Histopathological examinations were performed and compared between the groups on 6 dpi. Scale bar is 100 μm. (B) Lung tissues infected with PRRS virus from ex vivo culture were performed with TUNEL assay and compared between the groups on 6 dpi. 3,3'-Diaminobenzidine (DAB) was used for visualizing the apoptosis as brown color. Counterstaining was performed with hematoxylin. Scale bar corresponds to 50 μm. (C) Percentage of positive area with TUNEL assay on day 6 lung tissue infected with PRRS virus. X axis represented the each group and Y axis represented the percentage of positive area. Different letters indicate significant differences according to Duncan’s multiple range test. Value of P <0.05 was considered statistically significant based on results of one-way ANOVA. (D) Results of LDH released from lung tissues with virus-infected conditions on each time point. The supernatants from each group were collected at 1 hour and 2, 4, 6 dpi.