A protocol for differentiation of human intestinal Caco-2 cells in asymmetric serum-containing medium Simonetta Ferruzza, Carlotta Rossi, Maria Laura Scarino, Yula Sambuy ⇑ National Research Institute on Food and Nutrition (INRAN), Rome, Italy article info Article history: Available online xxxx Keywords: Foetal bovine serum Basolateral medium Serum reduction Alkaline phosphatase Permeability Differentiation protocol 3Rs Enterocytes abstract The human intestinal Caco-2 cell line still represents the best available in vitro model of absorptive enterocytes, despite its origin from a colon adenocarcinoma. Caco-2 cells seeded on filter inserts undergo in culture a process of spontaneous differentiation that leads to the formation, after two to three weeks, of a monolayer of polarized cell, coupled by tight junctions and expressing several morphological and functional features of small intestinal enterocytes. The medium normally used for differentiation of Caco-2 cells contains a supplement of foetal bovine serum (FBS) in both the apical (AP) and basolateral (BL) compartments. The use of FBS as cell culture media supplement has been frequently and increasingly questioned on scientific and also on ethical grounds. We have shown that addition of serum only to the BL medium (asymmetric protocol) appears to be sufficient to allow differentiation of Caco-2 cells, as monitored by morphology, monolayer permeability and alkaline phosphatase activity, compared to stan- dard conditions using 10% FBS supplement in both AP and BL media (asymmetric protocol). Although not eliminating the use of FBS, its addition only in the BL medium results in more physiological conditions for differentiation and in a significant reduction of its use. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The human intestinal Caco-2 cell line originally isolated by J. Fogh (Sloan Kettering Institute, New York, NY) from a human co- lon adenocarcinoma (Fogh et al., 1977), is still the best available cell culture model of absorptive small intestinal enterocytes (Delie and Rubas, 1997; Le Ferrec et al., 2001; Sambruy et al., 2001) and it is extensively utilized for toxicological and pharmacological stud- ies. However, in recent years improvements to Caco-2 cell culture methods have been suggested to overcome the variability and het- erogeneity emerging from the literature in the performance and differentiation of this cell line (Natoli et al., 2011; Sambuy et al., 2005). Standard methods used to differentiate Caco-2 cells involve seeding them on culture inserts fitted with polycarbonate filters, and allowing spontaneous differentiation to proceed for two to three weeks in culture medium containing 10% or 20% foetal bo- vine serum (FBS) in both the apical (AP) and basolateral (BL) com- partments (symmetric protocol). In vivo the serosal or BL side of the enterocytes is in contact with interstitial fluid, while the AP side comes in contact with the rapidly changing luminal fluid, which is strongly influenced by the state of food digestion. To bet- ter reproduce this asymmetry existing in vivo, we investigated the effects of removing the serum supplement form the AP medium of Caco-2 cells undergoing in vitro differentiation, while maintaining a 10% FBS supplement in the BL medium (asymmetric protocol). Cell differentiation was monitored by morphology with confocal laser scanning microscopy, cell monolayer permeability using the well established assays trans epithelial electrical resistance (TEER) and 14 C-mannitol passage, and activity of the brush border enzyme alkaline phosphatase (ALP), a marker of enterocyte differentiation (Le Ferrec et al., 2001; Zucco et al., 2005). A similar asymmetric protocol has previously been used in investigations of intestinal lipids absorption and metabolism in Caco-2 cells (Beaslas et al., 2009; Morel et al., 2004), and sucrase-isomaltase, a good and spe- cific marker of enterocyte differentiation, was shown to be strongly expressed in the AP brush-border of the cells when using asym- metric culture condition (Beaslas et al., 2009). However, the effects of the asymmetric protocol on the in vitro differentiation of Caco-2 cells were not compared with those obtained after a symmetric supply of serum-containing medium. 2. Materials and methods 2.1. Cell maintenance Caco-2 cells were sub-cultured at low density as described in (Natoli et al., 2011). Briefly, cells were routinely sub-cultured at 0887-2333/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2012.01.008 Abbreviations: ALP, alkaline phosphatase; AP, apical; BL, basolateral; FBS, foetal bovine serum; pNP, p-nitrophenol; pNPP, p-nitrophenyl phosphate; TEER, trans epithelial electrical resistance. ⇑ Corresponding author. Address: INRAN, via Ardeatina 546 – 00178 Roma, Italy. Tel.: +39 06 51 494 497; fax: +39 06 51 494 550. E-mail address: sambuy@inran.it (Y. Sambuy). Toxicology in Vitro xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit Please cite this article in press as: Ferruzza, S., et al. A protocol for differentiation of human intestinal Caco-2 cells in asymmetric serum-containing med- ium. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2012.01.008