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Cell Biology International (2006) 30, 977–982 (Printed in Great Britain)
Establishment of a mouse primary co-culture of endometrial epithelial cells and peripheral blood leukocytes: Effect on epithelial barrier function and leukocyte survival
Lok Sze Ho, Lai Ling Tsang, Yiu Wa Chung and Hsiao Chang Chan*
Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., HKSAR, Hong Kong


This study aimed to establish an in vitro co-culture model that would allow us to study the interaction between endometrial epithelial cells and immune cells. Flow cytometry analysis and cell surface marker staining were used to identify suitable immune leukocytes from a range of sources, such as intraepithelial lymphocytes (IEL), thymocytes, splenocytes and peripheral blood leukocytes. Optimizing culture conditions such as cell viabilities, cell seeding ratios and densities and co-culture methods were examined and determined. Results showed that co-culture of mouse endometrial epithelial cells (EEC) with peripheral blood leukocytes (PBL) at seeding densities of 3.0×106 and 1.0×106cells/ml, respectively, appeared to affect both the survival of leukocytes and epithelial barrier function. Cell viability counts of immune cells showed 95% and 72.5% cell survival after isolation and after 4days in co-culture with EEC, respectively, but only 11% cell survival when cultured alone for 4days without EEC. Short-circuit current (Isc) results also showed that EEC and PBL co-culture exhibited a four-fold increase in the transepithelial resistance (TER) as compared to EEC culture alone, indicating enhanced protective barrier function. Taken together, the currently established in vitro co-culture model of endometrial epithelial cells and immune cells may provide a means to investigate local cellular immune responses upon uterine infections.

Keywords: Co-culture, Endometrial epithelial cells (EEC), Immune cells, Peripheral blood leukocytes (PBL), Transepithelial resistance (TER).

*Corresponding author. Tel.: +852 2609 6839; fax: +852 2603 5022.

1 Introduction

In addition to forming a barrier, epithelial cells, together with immune cells, are actively involved in the host defense against microbe infections. However, the interaction between epithelial and immune cells upon infections remains largely unknown.

Bacterial infections in the uterus can cause changes in epithelial function and stimulate a large number of immune cells to infiltrate the epithelium as defense mechanisms. Early studies have observed an increase in electrolyte secretion as measured by the short-circuit current (Isc) in rat uterus after an oral infection with the parasite Trichinella spiralis, indicating increased fluid secretion upon infection (Castro and Harari, 1991). Other studies of the mucosa have shown the existence of immune cells in between epithelial cells of the endometrium of rats (Pace et al., 1991), including T-lymphocytes, B-lymphocytes, monocytes, macrophages, dendritic cells, neutrophils and intraepithelial lymphocytes (IELs). These cells exist in low numbers in normal endometrium during homeostasis, and their numbers can vary during different stages of the estrus cycles (Sawicki et al., 1988). Upon bacterial infections, cell recruitment to the site of the infection increases the number of immune cells in the epithelium. These findings suggest that immune cells at the site of the endometrium are important upon bacterial infections, and that cross-talk between epithelial cells and immune cells is likely to exist to regulate immune responses as well as electrolyte/fluid transport as defense mechanisms against bacterial infection.

The paucity of information on interaction between epithelial cells and immune cells is largely due to the complexity of this interaction in vivo. Therefore, there is a great need for an in vitro co-culture model whereby interaction between specific types of cells can be assessed. The present study aimed to establish a primary mouse co-culture model of endometrial epithelial cells with various sources of immune cells. This was based on a primary mouse endometrial epithelial culture previously established in our laboratory, which has been used extensively to investigate epithelial transport mechanisms and regulation (Chan et al., 1997a,b,1999,2000a–c,2001,2002; Fong et al., 1998a,b; Tsang et al., 2001; Wang et al., 2001, 2002). A co-culture model with peripheral blood leukocytes exhibiting strong T-cell characteristics was eventually established based on its significant effects on leukocyte survival and epithelial barrier function.

2 Materials and methods

2.1 Materials, chemicals and antibodies

Dulbecco's modified Eagle's medium/Ham's F12 (DMEM-F12), antibiotics penicillin-streptomycin and trypsin (porcine pancreas) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA); while phosphate buffered saline (PBS), fetal bovine serum (FBS), non-essential amino acid (NEAA) and pancreatin were from Gibco Laboratories (Grand Island, NY, USA). Millipore filters were purchased from Millipore Corp. (Billerica, MA, USA). Matrigel basement membrane matrix was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anesthetic ketamine (10%) and xylazine (2%) were from Alfasan International BV (Woerden, Holland). Anti-coagulating EDTA KE tubes were obtained from Sarstedt Ag. & Co. (Nürecht, Germany). Ficoll Paque-Plus and Percoll were purchased from Amersham Biosciences (GE Healthcare, NJ, USA). Mouse monoclonal anti-CD3+ FITC and anti-CD8+ PE antibodies were from BD Pharmingen (Franklin Lakes, NJ, USA), mouse monoclonal anti-cytokeratin 5 and 8 antibody was from Research & Diagnostic, Inc. (Flanders, NJ, USA) and mouse monoclonal anti-CD45+ PE antibody was purchased from Immunotech (Marselle Cedex 9, France).

2.2 Isolation of primary endometrial epithelial cells (EEC)

Endometrial epithelial cells were enzymatically isolated from mouse uteri according to the methods described (McCormack and Glasser, 1980), with slight modifications (Chan et al., 1997a). Briefly, uteri were obtained from 3-week-old immature ICR mice (body weight approximately 15g) to avoid the complication of the estrus cycle. Uteri obtained were washed in sterile 1× PBS (without Ca2+ and Mg2+). After removing the fatty and connective tissues, the uteri were sliced longitudinally before transferring to a 15ml centrifuge tube containing sterile 10ml of 1× PBS supplemented with 58.3mg/ml trypsin and 1ml of 4× USP pancreatin. Tissues were incubated on ice for 30min and then at room temperature for 45min. The enzyme-containing 1× PBS was discarded, and DMEM-F12 medium was added to stop enzymatic activities. The medium was replaced with 1× PBS and tissues were gently shaken for 30s. Uterine tissues were removed with forceps and epithelial cells were pelleted by centrifugation at 300×g for 5min. The supernatant was discarded and resuspended in DMEM-F12 medium. Cell counts were performed using a hemocytometer to determine cell concentration.

2.3 Isolation of peripheral blood leukocytes (PBL) and other immune cells

The concentration of anesthetic was prepared by mixing 0.75ml of ketamine with 0.5ml of xylazine in 0.75ml of distilled water. Adult mice at the age of 10weeks were weighed (approximately 30g) and anesthetized by injecting 0.075mg/g body weight (i.p.) for 5–10min. One milliliter syringes with 25×g 5/8″ gauge needle and anti-coagulant EDTA tubes were used for blood collection. Peripheral blood leukocytes (PBL) and other immune cells were collected by heart puncture on anesthetized mice using a 25×g 5/8″ gauge needle. Approximately 12ml of blood were collected and placed in the anti-coagulating EDTA KE tubes to avoid aggregation. Leukocytes and other immune cells were isolated from whole blood using density gradient centrifugation. The red blood cells (RBC) were first removed by Ficoll-Paque Plus. This is achieved by mixing 12ml of whole blood with 36ml 1× PBS and carefully layering 12ml of diluted blood on top of 3ml of Ficoll reagent in four 15ml centrifuge tubes. The blood was centrifuged without brakes at 400×g for 40min at RT. The top layers of lysed RBC and plasma were discarded and PBL were carefully collected at the interface. The pelleted RBC and granulocytes were discarded. PBL were washed once in 10ml of 1× PBS and cells were pelleted by centrifuging at 400×g for 5min at RT. Traces of RBC remaining were further removed by adding 2ml of sterile distilled water for 30s to lyse the RBC. An equal volume of 2× PBS was quickly added (1:1 ratio) to equilibrate the osmotic pressure. PBL were centrifuged at 400×g for another 5min and cell counts were performed using a hemocytometer to determine cell concentration.

Intraepithelial lymphocytes (IEL), residing in between epithelial cells, were isolated from mouse uteri by cutting the uteri longitudinally and enzymatically digesting the epithelium based on methods described for the isolation of IEL in rat gastrointestinal tracts (McKay et al., 1996; Kearsey and Stadnyk, 1996; Todd et al., 1999; Kerneis et al., 2000). IEL were further isolated using Percoll gradient centrifugation at 75% and 30% Percoll concentration diluted with 1× PBS and centrifuged at 400×g for 30min at RT. Thymocytes and splenocytes were collected from the thymus and spleen of the animals, washed in 1× PBS and the tissues were physically grounded using a glass rod. Isolated cells were filtered through a wire mesh (pore size 0.2mm) to remove traces of tissue fibers. Cells were centrifuged at 300×g for 5min at RT and washed again in 1× PBS.

2.4 Characterization of immune cells by flow cytometry

Immune cells of different sources including intraepithelial lymphocytes (IEL), thymocytes, splenocytes and peripheral blood leukocytes (PBL) were tested using flow cytometry (Beckman Coulter, USA). All isolated immune cells were stained with antibodies against CD3+ and CD8+ T-cell surface markers using anti-CD3+ FITC and anti-CD8+ PE antibodies (1:100) at RT for 1h for flow cytometric analysis.

2.5 Optimizing co-culture conditions

To establish the epithelial and immune cells co-culture, cell seeding ratios, cell viability and cell culture methods were examined. Cell seeding ratios were determined based on information suggested by references on rat vaginal histology and other gastrointestinal tract co-culture models (McKay et al., 1996; Kearsey and Stadnyk, 1996; Todd et al., 1999; Kerneis et al., 2000). Immune peripheral blood leukocyte viability was performed by trypan blue on cells right after isolation, after 4days of co-culture with epithelial cells or after 4days of culture in the absence of epithelial cells. Staining EEC with anti-cytokeratin 5 and 8 FITC fluorescent antibodies and PBL with anti-CD45+ PE fluorescent antibodies examined the distribution of co-cultured cells. Using a ‘mixed’ or ‘separate’ co-culture method, the effect of PBL on EECs’ transepithelial resistance (TER) was measured using short-circuit current (Isc) (Ussing and Zerahn, 1951). In the ‘mixed’ method, EEC were mixed with isolated PBL and seeded together in the apical compartments of permeable filters; whereas in the ‘separate’ method, EEC were seeded separately in the apical compartments of the permeable filters and the PBL were seeded in the basolateral compartments of the permeable filters. To examine the transepithelial resistance (TER) measurements, co-cultured cells were seeded on permeable filters with an area of 0.45cm2, pre-coated with 100μl of Matrigel basement membrane matrix, oven dried and UV sterilized for 1h. The filters were floated on V-shaped glass rods in DMEM/F12 medium in 90mm Petri dishes. Cultures were incubated at 37°C in 95% O2/5% CO2 for 4days until confluent.

2.6 Statistical analysis

Results were expressed as±standard error of the mean (SEM). Data with more than two groups were analyzed using one-way ANOVA using the GraphPad Prizm Software Inc. (San Diego, CA, USA), followed by Newman–Keuls post-hoc test to identify diverging means. A p value <0.05 was considered to be significant.

3 Results

3.1 Characterization of leukocytes by flow cytometry

Characterization of IEL by flow cytometry showed that only 5.9% of the IEL isolated exhibited both CD8+ and CD3+ T-cell characteristics in region E2 (Fig. 1a). Results of thymocytes showed high percentages of CD8+ and CD3+ T-cells (72.6%) in region E2, but most of these thymocytes were known to be immature (Fig. 1b). Flow cytometry results also showed that only very low numbers of splenocytes in region E2 (0.4%) exhibited CD8+ and CD3+ T-cell characteristics (Fig. 1c), while 67.5% of PBL in region E2 expressed both CD8+ and CD3+ characteristics, which were known to be mature T-cells (Fig. 1d). Based on the flow cytometry results, PBL were chosen for the subsequent co-culture experiments.

Fig. 1

Flow cytometric analysis of immune cells with T-cell surface markers labeled with anti-CD3+ FITC and anti-CD8+ PE monoclonal antibodies. Regions E1 and E4 represent cells that either express CD8+ or CD3+ cell surface markers, E2 represents cells that express both CD8+ and CD3+ surface markers and E3 represents cells that express neither of the surface markers. A total of 10,000 cells were analyzed. (a) Intraepithelial lymphocytes (IEL); (b) thymocytes of the thymus; (c) splenocytes of the spleen and (d) peripheral blood leukocytes (PBL).

3.2 Cells seeding ratio and cell viability

Ratios of 1 immune cell (1.0×106 cells/ml) to 3 epithelial cells (3.0×106 cells/ml) (ratio 1:3) were used in the co-culture, taking into account the fact that after 4days of incubation, the epithelial cells will proliferate, but the immune cells will not. Cell counts after 4days showed that this is approximately equivalent to a ratio of 1:10 (PBL:EEC). Immunofluorescent antibody staining against cytokeratin (a marker of epithelial cells) and cell surface marker CD45+ (a common receptor for leukocytes) showed co-localization indicating cell–cell contact of PBL (red) with EEC (green) in the ‘mixed’ co-culture (Fig. 2).

Fig. 2

Immunofluorescent staining showing cell–cell contact between endometrial epithelial cell (EEC) and peripheral blood leukocyte (PBL) co-culture. Anti-cytokeratin FITC (green) monoclonal antibodies were used to label EEC and anti-CD45+ PE (red) monoclonal antibodies was used to label PBL.

Cell viability of PBL cells after isolation showed 95% cell survival as compared to after 4days of EEC/PBL co-culture, which showed 72.5% (**p<0.01) cell survival. However, PBL cultured alone showed only 11% of cell survival (***p<0.001) (Fig. 3).

Fig. 3

Cell viability counts showing survival percentages of peripheral blood leukocytes after isolation (95%) compared to after co-culture with endometrial epithelial cells (EEC) for 4days (72.5%, **p<0.01) and leukocytes cultured alone without EEC for 4days (11%, ***p<0.001) (n=2).

3.3 Increased transepithelial resistance (TER) of EEC co-cultured with PBL

The effect of PBL on the EEC barrier function was examined by measuring the transepithelial resistance (TER) of the monolayers using short-circuit current (Isc). Results showed that when EEC and PBL were co-cultured in a ‘mixed’ configuration, where immune cells were allowed to have direct cell–cell contact with epithelial cells, there was a four-fold increase in TER in EEC/PBL co-culture (866.1±236.5Ω cm2, **p<0.01) as compared to EEC cultured alone (202.73±47.2Ω cm2) (Fig. 4a).

Fig. 4

Measurement of transepithelial resistance (TER) in ‘mixed’ and ‘separate’ co-cultures of endometrial epithelial cells (EEC) and peripheral blood leukocytes (PBL). (a) TER measurement of ‘mixed’ co-culture showing increased TER in EEC/PBL co-culture (**p<0.01) compared to EEC alone (n=8); (b) TER measurement of ‘separate’ co-culture showing no significant differences between EEC alone and EEC/PBL co-culture (n=8).

To test whether the effect of PBL on EEC could be mediated by leukocyte-released factors such as cytokines, PBL were co-cultured with EEC but separated by a permeable membrane to allow free flow of cytokines but avoid cell–cell contact between the two types of cells. The results showed that there was no significant TER change in the co-culture (222.99±31.97Ω cm2) compared to EEC cultured alone (202.73±47.2Ω cm2) (Fig. 4b).

4 Discussion

The present study explored the possibility of establishing an in vitro mouse co-culture model between endometrial epithelial cells (EEC) and immune cells of different sources. We made an effort to examine T-cell characteristics of the immune cells since they are thought to play a major role in local cellular immune response (Kelly et al., 2000; Robertson, 2000; Quayle, 2002; Johansson and Lycke, 2003; Kelly, 2003). The choice of IEL for the co-culture would have been ideal since they reside at the site of the endometrium, but it was experimentally impractical since only a very low number of IEL reside in the endometrial epithelium and to collect a significant amount of IEL for the co-culture would require an unreasonably large number of animals. Although the immune cells in the thymus and spleen were abundant, the thymus contains a large number of undeveloped T-cells and the spleen mainly possesses antibody-producing B-cells but not T-cells as shown by our flow cytometry results. The peripheral blood contains a large variety of circulating and maturing immune cells, with the majority of which exhibits T-cell characteristics shown by the flow cytometry results. During inflammation, recruitment of immune cells to the site of infection was also from the bloodstream. Therefore, the choice of PBL appeared to be suitable to mimic the endometrium environment for the future study of endometrial epithelial cell and immune cell interaction upon bacterial infections.

We based our current study on the ratios of immune cells to epithelial cells in the vagina of a rat, as reported by another study. The other study suggested that there was 1 immune cell per 100 epithelial cells (1:100) in the vagina of a rat under homeostasis, but this ratio quickly changes to 10 immune cells per 100 epithelial cells during bacterial infections (1:10) in the vagina (Sawicki et al., 1988). Based on this information, the EEC and PBL seeding ratio was determined to be 1:3 (PBL:EEC) during the initial cell culture, taking into account that EEC, but not PBL, will proliferate after 4days to give a final ratio of approximately 1:10. We observed that a very small number of immune cells were also isolated along with the epithelial cells during cell preparation, and this low number of immune cells did not affect any changes in the TER of the epithelial monolayers. This suggested that the ratio of immune cells used in the co-culture system was in fact very important as large numbers of localized immune cells can trigger a response similar to inflammation.

It is interesting to note that cell–cell contact plays a critical role in the interaction between endometrial epithelial cells and PBL since the effect of PBL on epithelial barrier function was diminished when they were co-cultured but separated by permeable support avoiding cell–cell contact. The effect of co-culture on leukocyte survival was also noted, which is likely to be mediated by cell–cell contact since we have previously observed that the co-culture effect on immune cell survival was greatly diminished when immune cells were co-cultured without cell–cell contact with intestinal epithelial cells (Xu et al., unpublished data). The present findings are consistent with the notion that cell–cell contact represents an important cross-talk mechanism between epithelial cells and immune cells, which is vital for immune cell survival and epithelial barrier function as demonstrated in the present study, as well as for cytokine release upon bacterial infection as demonstrated previously in the co-culture of intestinal epithelial cells and Peyer's patch lymphocytes challenged by Shigella lipopolysaccharide (LPS) (Chen et al., 2004).

In summary, the present study has established an in vitro co-culture model and demonstrated that endometrial epithelial-immune cell–cell contact is important in enhancing immune cell survival and epithelial protective barrier function. This co-culture model may enable future investigation of detailed cross-talk mechanisms between endometrial cells and immune cells upon a wide range of bacterial infections including Chlamydia trachomatis.


This work was supported by the Strategic Program of The Chinese University of Hong Kong.


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Received 25 November 2005/26 June 2006; accepted 19 July 2006


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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