Please cite this article in press as: Razzuoli, E., et al., Isolation and culture of pig tonsil lymphocytes. Vet. Immunol. Immunopathol. (2012), http://dx.doi.org/10.1016/j.vetimm.2012.04.022 ARTICLE IN PRESS G Model VETIMM-8795; No. of Pages 6 Veterinary Immunology and Immunopathology xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Veterinary Immunology and Immunopathology j ourna l ho me pag e: www.elsevier.com/locate/vetimm Short communication Isolation and culture of pig tonsil lymphocytes E. Razzuoli a , E. Faggionato b , S. Dotti a , R. Villa a , T. Lombardo a , L. Boizza b , M. Ferrari a , M. Amadori a,∗ a Laboratory of Cellular Immunology, IZSLER, Brescia, Italy b Laboratory of Chemical Residues in Food of Plant Origin, IZSLER, Brescia, Italy a r t i c l e i n f o Article history: Received 24 February 2012 Received in revised form 18 April 2012 Accepted 23 April 2012 Keywords: Tonsil Pig Primary culture B cells Immunoglobulin a b s t r a c t Tonsils are secondary lymphoid organs that play an important role in host defense. The aim of our study was to develop reliable procedures for isolation and culture of pig ton- sil cells, and to validate their possible use in functional immunoassays. Using our isolation procedure, we recovered on average 238.7 ± 107.1 × 10 6 cells per tonsil couple with a mean vitality of 89.8 ± 2.7%. These values significantly decreased 8 months after freezing at -80 ◦ C along with the subsequent spontaneous release of both IgA and IgG in culture. These results suggest to use pig tonsil cells within 2 months from thawing to maintain suitable condi- tions in terms of recovery, vitality and release of antibody in vitro. Tonsil mononuclear cells also showed the ability to secrete antimicrobial peptides and to respond in vitro to immunological stimuli. On the whole, our study has defined operating conditions for tonsil processing, control of bacterial contaminations, time limits of storage at -80 ◦ C, as well as for evaluating polyclonal Ig production in vitro. Such procedures are likely to be of some importance in studies on regional immunity and in the development of large animal models for biomedical sciences. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Tonsils are secondary lymphoid organs which repre- sent a major component of mucosa-associated lymphoid tissue (MALT). This consists of lymphoid cell aggregates widely distributed in the mucosal surfaces (e.g. nasophar- ynx, oropharynx and laryngopharynx). On the basis of the anatomical localization, MALT can be subdivided into regional structures, including the lymphoid tissues of the Waldeyer’s ring (Horter et al., 2003; Liebler-Tenorio and Pabst, 2006). This is located at the opening of both digestive and respiratory tracts and consists of lymphoid structures named tonsils; in pigs are present five different tonsils, i.e. pharyngeal, paraepiglottic, lingual, tubal and soft palate ∗ Corresponding author at: Laboratory of Cellular Immunology, Isti- tuto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna (IZSLER), Via A. Bianchi, 9, 25124 Brescia, Italy. Tel.: +39 030 2290632; fax: +39 030 2290392. E-mail address: massimo.amadori@izsler.it (M. Amadori). tonsils. The latter are the most developed ones, consisting of bilateral oval plaques of lymphoid tissue with a size of approximately 5 cm length and 3 cm width in adult pigs. Many tonsilar fossules are visible on the surface, leading to crypts located in the center of tonsillar follicles, sepa- rated in turn by diffuse lymphoid tissue (Casteleyn et al., 2011). Tonsils can be infected by viral agents which sustain e.g. classical swine fever, pseudorabies, foot-and-mouth dis- ease and porcine respiratory and reproductive syndrome, to cite a few (Alexandersen et al., 2001; Romero et al., 2003; Rowland et al., 2003). In addition to these, various non- pathogenic and pathogenic bacteria like Salmonella spp., Yersinia enterocolitica, Pasteurella multocida, Haemophilus parasuis, Mycoplasma hyopneumoniae can be isolated from porcine tonsils (Lowe et al., 2011; O’Sullivan et al., 2011). Moreover, there is evidence of an important role in the host’s defense played by tonsils, which often represent the first site of interaction between microbial agents and lymphoid tissues. This is the reason why the isolation 0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2012.04.022