Technical note Human monocyte-derived dendritic cells from leukoreduction system chambers after plateletpheresis are functional in an in vitro co-culture assay with intestinal epithelial cells Inés Tiscornia a , Viviana Sánchez-Martins a, 1 , Ana Hernández b , Mariela Bollati-Fogolín a, ⁎ a Cell Biology Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay b Cátedra de Inmunología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay article info abstract Article history: Received 17 January 2012 Received in revised form 14 July 2012 Accepted 16 July 2012 Available online 24 July 2012 The dendritic cells (DC) found in the intestine are involved both in the maintenance of tolerance towards commensal microbiota, and in the generation of protective immune responses against pathogens, thus contributing to gut immune homeostasis. There is an increasing interest in the use of lactic acid bacteria (LAB) as probiotics; among their beneficial effects we highlight the modulation of the immune system which is one of their fundamental properties. As these effects are strain-dependent, it is important to have in vitro systems that include DC and intestinal epithelial cells (IEC), which are crucial for intestinal homeostasis, to identify candidates by means of bacterial screening. Obtaining enough human cells, necessary to simultaneously test several bacteria, is a major challenge for researchers. In this study we analyzed the usefulness of the cellular fraction retained in leukoreduction system chambers following plateletpheresis (PP) as a source of DC. We compared the capacity of peripheral blood mononuclear cells (PBMC) from buffy coats (BC) or PP to generate DC using a short differentiation protocol. The functionality of the DC obtained was analyzed in co-cultures together with intestinal epithelial HT-29 cells, stimulating with LPS alone or with two LAB commonly used in the food industry, Streptococcus thermophilus and Lactobacillus delbrueckii. DC surface markers CD86, HLA-DR and cytokine production were measured. The behavior of DC derived from PP was similar to the behavior observed for DC derived from BC. When we tested the response of DC to bacteria, we found significant differences in cytokine secretion, especially for IL-10, suggesting that the system has the ability to discriminate LAB with different immunomodulatory properties. We also found that DC derived from both sources displayed a similar ability to phagocyte bacteria. In conclusion, we hereby propose a modification of the two-day protocol for obtaining human DC previously described, using PP as an alternative source of PBMC, to be used in co-culture systems with IEC. The novelty of this protocol is the combination of the blood monocyte source with a simple and fast differentiation method to obtain DC, and their use in a combined culture with IEC and LAB to model microbial-host interaction. Since the initial PP volume is ten times lower than that of BC, the use of PP minimizes biological residue generation and reagent consumption. In addition, monocyte-derived DC from PP were suitable for use in co-culture assays as a first screening step to study the immunomodulatory properties of LAB. © 2012 Elsevier B.V. All rights reserved. Keywords: Peripheral blood mononuclear cells Human dendritic cells Plateletpheresis Buffy coats Probiotic Journal of Immunological Methods 384 (2012) 164–170 Abbreviations: BC, buffy coats;CFSE, carboxyfluoresceinsuccinimidyl ester;CFU, colony-forming unit;DC, dendritic cells;IEC, intestinal epithelial cells;LAB, lactic acid bacteria;LPS, lipopolysaccharide;MFI, median fluorescence intensity;PP, cellular fraction retained in leukoreduction system chambers after plateletpheresis;WBC, white blood cells. ⁎ Corresponding author at: Cell Biology Unit, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay. Tel.: +598 2 5220910x126; fax: +598 2 5224185. E-mail address: mbollati@pasteur.edu.uy (M. Bollati-Fogolín). 1 Present address: Laboratorio de Control de Biofármacos, Institut Pasteur de Montevideo, Montevideo, Uruguay. 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2012.07.005 Contents lists available at SciVerse ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim