Helicobacter ISSN 1523-5378 © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Helicobacter 12: 341–353 341 Blackwell Publishing Ltd Oxford, UK HEL Helicobacter 1083-4389 Blackwell Publishing Ltd, 2007 XXX Original Articles Improved In vitro Infection Models Lindén et al. Improved In vitro Model Systems for Gastrointestinal Infection by Choice of Cell Line, pH, Microaerobic Conditions, and Optimization of Culture Conditions Sara K. Lindén, Kim M. Driessen and Michael A. McGuckin Mucosal Diseases Program, Mater Medical Research Institute, Mater Health Services, Level 3, Aubigny Place, South Brisbane, Queensland 4101, Australia Abstract Background: Commonly used in vitro infection cultures do not mimic the human gastrointestinal tract with regard to pH and microaerobic conditions. Furthermore, despite the importance of mucin– Helicobacter interactions, the cell lines used have not been selected for appropriate mucin expression. To make in vitro studies more applicable to human disease, we have developed coculture methods taking these factors into account. Materials and methods: Nine human gastrointestinal epithelial cell lines (MKN1, MKN7, MKN28, MKN45, KATO3, HFE145, PCAA/C11 Caco-2, and LS513) were investigated. Expression and glycosylation of mucins (MUC1, 2, 3, 4, 5AC, 5B, 6, 12, 13, and 16) were determined by immunohistochemistry. We analyzed the effect of microaerobic conditions and acidic pH on cell proliferation, viability, and apoptosis. Results: Microaerobic culture, which is more physiological for the bacteria, did not adversely affect mammalian cell viability, proliferation, or induce apoptosis The cell lines varied in mucin expression, with MKN7 and MKN45 being most similar to gastric mucosa and Caco-2 and LS513 to intestinal mucosa, although none exactly matched normal mucosa. However, changes in culture conditions did not cause major changes in the mucin expression within cell lines. Conclusions: Culture conditions mimicking the natural environment and allowing the bacterial cells to thrive had no effect on cell viability or apoptosis, and very little influence on mucin expression of human gastrointestinal cells. Thus, it is feasible, using the simple methods we present here, to substantially improve bacterial–mammalian cell in vitro coculture studies to make them more reflective of human infection. Keywords Mucin, microaerobic culture, bacterial–mammalian co-culture. Reprint request to: Dr Sara Linden, Mucosal diseases program, Mater Medical Research Institute, Mater Health Services, Level 3, Aubigny Place, South Brisbane, Queensland 4101, Australia. Tel.: +61 7 3840 2582; Fax: +61 7 3840 2550; E-mail: slinden@mmri.mater.org.au Introduction In vitro bacterial–mammalian cocultures are used exten- sively to elucidate the mechanisms by which Helicobacter species and other microaerophilic microorganisms adhere, invade, and signal to the host, and to examine ensuing mammalian cell responses. These complex interactions are reliant on appropriate gene expression and cellular functioning of both the bacteria and the mammalian cells. It is therefore critical that appropriate bacterial and mam- malian cells are used, and that the environment created experimentally is as similar to the human gastrointestinal environment as possible. Despite it being well known that Helicobacter pylori and other microaerophilic organisms need microaerobic condi- tions for sustained survival as well as growth [1], 20% O 2 and 5% CO 2 are currently the standard condition in which most in vitro coculture experiments are conducted. Induc- tion of MCP-1, GRO-α, and RANTES, which are present in H. pylori-infected biopsies, can only be reproduced under microbial microaerobic conditions, as previously shown using an asymmetrical culture chamber that supplies the mammalian cells 20% O 2 basolaterally [1]. In the gastric and intestinal mucosa, the pO 2 is 20 – 45 mmHg and the pCO 2 is 60 mmHg, which correspond to 3 –5% O 2 and 9% CO 2 [2,3], indicating that the microaerobic conditions in