Monolayer Culture Systems with Respiratory Epithelial Cells for Evaluation of Bacterial Invasiveness

Monolayer Culture Systems with Respiratory Epithelial Cells for Evaluation of Bacterial Invasiveness

YOICHI HIRAKATA,^1,2 HISAKAZU YANO,^1,2 KAZUAKI ARAI,^1,2 SHIRO ENDO,¹ HAJIME KANAMORI,¹ TETSUJI AOYAGI,¹ AYAKO HIROTANI,¹ MIHO KITAGAWA,¹ MASUMITSU HATTA,¹ NATSUO YAMAMOTO,¹ HIROYUKI KUNISHIMA,¹ KAZUYOSHI KAWAKAMI³ and MITSUO KAKU^1,2

¹Department of Infection Control and Laboratory Diagnostics, Tohoku University Graduate School of Medicine, Sendai, Japan
²Department of Clinical Microbiology with Epidemiological Research & Management and Analysis of Infectious Diseases (C-MERMAID) , Tohoku University Graduate School of Medicine, Sendai, Japan
³Department of Medical Microbiology, Mycology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan

Pseudomonas (P.) aeruginosa is a major opportunistic pathogen especially in immunocompromised patients. To evaluate the invasiveness of respiratory pathogens, we developed monolayer culture systems and examined the degree of invasion by P. aeruginosa and invasive Salmonella (S.) typhimurium strains using human respiratory cell lines: A549 (derived from lung cancer), BEAS-2B (normal bronchial epithelium), and Calu-3 (pleural effusion of a patient with adenocarcinoma of the lung). Cells were seeded into filter units containing 0.33 cm² filter membranes with 3.0 μm pores, and were incubated at 37°C under 5% CO₂ for 4-10 days. By monitoring the trans-monolayer electrical resistance (TER), we judged that BEAS-2B cells (TER values: 436.2 ± 16.8 to 628.8 ± 66.3 Ω cm²) and Calu-3 cells (TER values: 490.5 ± 25.2 to 547.8 ± 21.6 Ω cm²) formed monolayers with tight junctions, but not A549 cells. On day 8 of culture, monolayer cultures were infected with bacteria, and the number of microorganisms penetrating into the basolateral medium was counted. Wild-type P. aeruginosa PAO1 (PAO1 WT) and S. typhimurium SL1344 were detected in the basolateral medium of BEAS-2B monolayer system by 3 h after inoculation, while only P. aeruginosa PAO1 WT was detected in the basolateral medium of Calu-3 monolayer, indicating poor invasiveness of S. typhimurium SL1344 in the Calu-3 system. These findings suggest that BEAS-2B or Calu-3 monolayer system could be useful for evaluating the invasiveness of respiratory pathogens. Because of the difference in bacterial invasiveness, we may need to choose a suitable cell system for each target pathogen.

keywords —— Pseudomonas aeruginosa; invasiveness; MDCK; BEAS-2B; Calu-3

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Tohoku J. Exp. Med., 2010, 220, 15-19

Correspondence: Yoichi Hirakata, M.D., FJSIM, Ph.D., Department of Clinical Microbiology with Epidemiological Research & Management and Analysis of Infectious Diseases (C-MERMAID), Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

e-mail: hiraichi@mail.tains.tohoku.ac.jp