Biol Cell (1992) 74, 255-265 © Elsevier, Paris 255 Original article Serum-free culture of stromal and functionally polarized epithelial cells of guinea-pig endometrium: a potential model for the study of epithelial-stromal paracrine interactions Abderrahim Mahfoudi, Sylvie Fauconnet, Jacqueline Bride, Laurent Beck, Jean-Paul Remy-Martin, Monique Nicollier, G6rard L Adessi* Unitd de Biochimie Hormonale et des Rdgulations, INSERM U 198, 240 route de Dole, 25000 Besan~on, France (Received 17 October 1991; accepted 15 January 1992) Summary - Stromal and glandular epithelial (GE) cells were isolated from guinea-pig endometrium and grown to near confluency (6-8 days) in primary culture on plastic surfaces in a serum-supplemented medium (SSM). The stromal cells were subcultured on plastic dishes and maintained for 72 h in SSM. Then SSM was replaced by a chemically defined medium (CDM) and the stromal cells grown to confluency (5- 7 days). The GE cells were subcultured in CDM, on a basement membrane matrix (Matrigel) applied to permeable Millicell-PC filters, and grown to confluency (5 days). Homogeneity of the subcultured endometrial cell populations was ascertained immunocytochemically. The filter-cultured GE monolayers were polarized morphologically, and displayed epithelial- specific specialized structures. These monolayers had functional tight junctions as verified by a measurable transepithelial resistance. The subcultured cell populations were distinguished by an analysis of their cellular and secretory proteins after labelling with [35S]- methionine and analysis by polyacrylamide gel electrophoresis. The filter-cultured GE monolayers allowed identification of the pro- teins released vectorially in the apical or the basal secretory compartment, thus demonstrating the functional polarization of GE cells in this bicameral culture system. Within the defined conditions of this culture system, the paracrine factors released by the two en- dometrial cell populations as well as the interplay of stromal-epithelial interactions and ovarian hormones could be investigated. endometrium / stromal cells / filter-cultured epithelial cells / functional polarization / serum-free medium Introduction It is well known that the uterine endometrium changes morphologically and biochemically in response to ovari- an steroid hormones [32, 52, 53]. Endometrium is deve- loped into a proliferative tissue by estrogens and, after ovulation, is changed by progesterone to a secretory tis- sue which easily accommodates ovum implantation [2, 48, 52]. These endometrial modifications involve two major cell types: the endometrial epithelium, either facing the uterine lumen or a glandular epithelium, supported by a network of mesenchymal cells commonly called endometri- al stroma. The establishment in vitro, and subsequent culture, of endometrial cells has proven an indispensable experimen- tal approach for the study of numerous aspects of en- dometrial cell biology. Unlike investigators who could only maintain endometrial cells as short-term primary cultures [I0, 14, 34, 51, 57], other authors have serially cultured the normal cells, particularly of epithelial origin [21, 54]. In general, the epithelial cell monolayers, obtained on clas- sic plastic surfaces, were found to have lost their in vivo hormone sensitivity. Recent reports in our laboratory demonstrated the suitability of guinea-pig uterine epithelial cell primary culture on plastic to investigate DNA synthe- sis and oncogene expression [3, 33]. In this model, while insulin and epidermal growth factor stimulated DNA syn- * Correspondence and reprints thesis, estradiol alone was unable to induce such a response [3]. A similar lack of response, and even an inhibitory ef- fect of cell proliferation by high levels of estradiol, was observed by Uchima et al [56] in primary culture of vagi- nal epithelial cells. These results are inconsistent with the mitogenic effect of estradiol established in vivo [48]. To our knowledge, no one has reported in vitro a convincing physiological response of the normal endometrial epithelial cell monolayers to sex steroid hormones. Interesting data are available concerning the stromal cell response to steroid hormones in vitro [7, 24, 55, 59], and that could be com- pared with the in vivo situation [39]. It would seem, then, that essential mediator(s) for this response is (are) miss- ing in the monolayer culture system of the uterine epithelial cells. At least part of the reason for this lack of response seems to be the use of monolayers on a plastic support per se, because studies with mammary [19, 47] thyroid [13] pancreatic [29] and tracheal [6] epithelial cell cultures have shown that the maintenance of the differentiated state, which includes hormone sensitivity, required the use of ex- tracellular matrix extracts as substrata for cell culture. Consequently, the expression of tissue specific functions in individual cells can be said to be governed at a fun- damental level by the interaction of the cells with their en- vironment [8, 9]. Such a dynamic nature of the cell-extracellular matrix interaction led Bissel and Aggel- er [9] to propose that: "the minimum unit of function in multicellular organisms is the cell plus its extracellular matrix". The natural basement membrane (Matrigel), ex- tracted from Engelbreth Holm Swarm mouse tumors [35], has been demonstrated to promote a differentiated mor-