Commentary Lessons learned from conventional animals: Encouragement to use specific-pathogen-free animals Tsunetaro Morino a, b , Shun Kikuchi a, b , Taro Inagaki a , Manabu Komori b , Kazuhisa Yamamoto b , Hiromi Kojima b , Masayuki Yamato a, * a Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan b Department of Otorhinolaryngology, Jikei University School of Medicine, Tokyo, Japan article info Article history: Received 13 March 2020 Received in revised form 19 March 2020 Accepted 25 March 2020 Keywords: Non-specific-pathogen-free rabbits Mucosal regeneration study Pasteurella multocida contamination Otitis media Sinusitis Vigilant microbiological monitoring abstract Experimental rabbits provide evidence for translational research regarding the pathogenies or treatment of human diseases. We developed a novel method for regenerating the middle ear mucosa using autologous cultured nasal mucosal epithelial cell sheets, and evaluated the wound healing process in the middle ear mucosa of experimental rabbits. Nonetheless, vigilant microbiological monitoring of exper- imental animals is essential to effectively prevent a decline in their health conditions, which may affect the research results. We experimented with contamination of Pasteurella multocida in non-specific- pathogen-free (SPF) rabbits (without microbiological monitoring). Most non-SPF rabbits had otitis media, whereas SPF rabbits did not, which affected their results during the mucosal regeneration study. The contamination was resolved by changing the experimental design from using non-SPF rabbits to that using SPF rabbits. It is crucial to use the SPF animals for any surgical intervention studies. © 2020, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/). In pre-clinical regenerative medicine and tissue engineering, mice may not be used for transplant surgery due to their small size. Rats are often used in preclinical medicine, and rabbits are used when dealing with smaller organs such as eyes [1], knee joints [2], or the trachea [3]. We have studied regenerative strategies for the middle ear mucosa as a subset of the respiratory epithelia, with an aim to improve the post-operative middle ear mucosa regeneration on the middle ear bone surfaces using autologous cultured nasal mucosal epithelial cell sheets applied after surgery. A successful rabbit preclinical study regarding wound healing of the middle ear [4] led to the publication of a human clinical study [5]. However, when conventional rabbits were used as experimental models, the preclinical study did not work at all. We pre-screened rabbits for infections, such as sinusitis and otitis media, through nasal examination and endoscopic examina- tion of the eardrum, and those with an evidence of infection were excluded (Fig. 1). However, an acute suppurative otitis media almost always developed in the middle ear mucosa wound-healing rabbit model (Fig. 2a and b). Some rabbits exhibited subcutaneous ab- scesses near the skin incision (Fig. 2c). Bacterial tests of nasal mucin, ear discharge, and subcutaneous abscesses detected P. multocida. By contrast, all SPF rabbits undergoing a routine microbiological monitoring did not exhibit any ear discharge (Fig. 2d and e). As is frequently the case, conventional animals screened before operation were not the same as the SPF animals. P. multocida is a pathogen commonly infecting diverse animal hosts worldwide. In rabbits, infections due to P. multocida are commonly known as “snuffles”, that is, rabbit rhinitis. P. multocida can also infect humans bitten or licked by various carrier animals [6e8], and can cause sepsis in immuno-compromised individuals [9, 10]. The prevalence of P. multocida infection in rabbits housed under laboratory conditions or on breeding farms has been re- ported to be between 15.8% and 94% [11]. P. multocida can infect rabbits asymptomatically and develop into an opportunistic infec- tion when the host's immune system is compromised [12]. The infection route can be through droplet infection, contact infection, Abbreviations: SPF, specific-pathogen-free; P. multocida, Pasteurella multocida. * Corresponding author. Institute of Advanced Biomedical Engineering and Sci- ence, Tokyo Women's Medical University, 8-1 Kawada-cho Shinjuku-ku, Tokyo,162- 8666, Japan. E-mail addresses: kikuchi.shun@twmu.ac.jp (S. Kikuchi), inagaki.taro@twmu.ac. jp (T. Inagaki), m_komori@jikei.ac.jp (M. Komori), kazu1109@jikei.ac.jp (K. Yamamoto), kojimah@jikei.ac.jp (H. Kojima), yamato.masayuki@twmu.ac.jp (M. Yamato). Peer review under responsibility of the Japanese Society for Regenerative Medicine. Contents lists available at ScienceDirect Regenerative Therapy journal homepage: http://www.elsevier.com/locate/reth https://doi.org/10.1016/j.reth.2020.03.003 2352-3204/© 2020, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Regenerative Therapy 14 (2020) 296e298