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. 2009 May 7;150(8):3921–3926. doi: 10.1210/en.2008-1674

Three-Dimensional Ovarian Organ Culture as a Tool to Study Normal Ovarian Surface Epithelial Wound Repair

Kevin S Jackson 1, Kari Inoue 1, David A Davis 1, Tyvette S Hilliard 1, Joanna E Burdette 1
PMCID: PMC2717856  PMID: 19423762

Abstract

Ovarian cancers are primarily derived from a single layer of epithelial cells surrounding the ovary, the ovarian surface epithelium (OSE). Ovarian surface proliferation is associated with ovulation and has been suggested to play a role in ovarian surface transformation and cancer progression. Aspects of ovarian surface repair after ovulation include proliferation, migration, and surface regeneration. To study ovarian surface repair, an organ culture system was developed that supports the proliferation, encapsulation, and repair of an artificially wounded surface. Wounded mouse ovaries embedded into an alginate hydrogel matrix have normal OSE cells as demonstrated by expression of cytokeratin 8, vimentin, N-cadherin, and a lack of E-cadherin. Normal OSE cells began proliferating and migrating around wounded surfaces after 1 d of culture. Organ cultures were propagated in medium supplemented with BSA and fetal bovine serum to determine optimal growth conditions. BSA cultured organs had OSE that proliferated significantly more than controls until d 4, whereas fetal bovine serum cultured organs had significantly more surface area encapsulated by OSE. Overall, a three-dimensional ovarian organ culture supports the growth of normal OSE in response to artificial wounding and provides a novel system for investigating wound repair as it relates to the possible role of ovulation and ovarian cancer.

Three-dimensional culture of artificially wounded ovaries in alginate allows for regrowth of normal ovarian surface epithelium.

Epithelial ovarian cancer is the fifth most common cancer among women and ranks first in terms of gynecological malignancy. A poor understanding of the early events responsible for ovarian cancer prolongs detection and increases the chance of poor prognosis (1). A paucity of suitable models for ovarian cancer contributes to the lack of information regarding early transformative events in the ovarian surface, the single layer of cells surrounding the ovary responsible for more than 90% of ovarian cancers (2,3). BRCA (breast cancer gene) mutation carriers suffer from a higher incidence of ovarian cancers (4). The prophylactic removal of fallopian tubes and ovaries from these women revealed a possible alternative source of cancer formation in the fimbria (5). In vitro organotypic models that mimic changes in specific types of epithelium will therefore be of critical importance to demonstrate the origins of ovarian cancers.

Ovulation is associated epidemiologically with the risk for ovarian cancers. In general, factors that reduce the lifetime ovulation rate are protective against ovarian cancers, such as parity, early menopause, breast-feeding, and the use of oral contraceptives (6,7). Studies suggest that ovulation increases the presence of dysplasia on the ovarian surface after fertility treatments, but overall evidence indicating an increased risk of cancer from superovulation fertility treatments is controversial (8,9). The process of ovulation involves an autoinflammatory reaction, oxidative stress, proliferation, and hormonal stimulation, all of which have been associated with aspects of malignant transformation (10,11). Ovulation increases proliferation within the ovarian surface epithelium generating potential DNA duplication errors (7,12,13). The gonadotropins have been suggested to increase ovarian surface proliferation and alter specific signaling pathways related to ovarian cancers (14,15). However, the individual contribution of each aspect of ovulation on initiating and progressing cancers in the surface epithelium is not fully understood.

Alginate is a natural product biomaterial that provides a three-dimensional context for growing tissues (16,17). We sought to develop a system of studying ovarian surface wound repair after artificial rupture using mouse ovaries embedded into alginate hydrogels. Most analyses of normal ovarian surface epithelium use human cell lines immortalized with a variety of genetic changes such as Simian virus 40 large T antigen, viral proteins, telomerase, and spontaneous immortalization (18,19). The purpose of this study was to investigate changes associated with ovarian surface after artificial wounding in a three-dimensional culture system (Fig. 1). The organ culture system can be used as a tool for studying changes in normal mouse ovarian surface cells as they relate to early events in ovarian cancers.

Figure 1.

Figure 1

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Schematic of mouse ovary organoid three-dimensional culture. A, Ovary with bursa intact (left) and ovary with bursa removed (right). B, Ovaries were bisected into eight tissues termed organoids. C, Organoids were placed into an alginate droplet. D, Alginate-encased organoid were dropped into a CaCl2 solution cross-linking the alginate into a gel. E, Alginate-organoids were incubated in culture medium. F, Organoids in alginate were processed, fixed, paraffin embedded, and hematoxylin and eosin stained. Black arrow, ovary; blue arrow, organoid; red arrow, alginate.

Materials and Methods

Animals

Ovaries were isolated from prepubertal, 16-d-old female F1 hybrids produced from the in-house animal colonies that cross C57B6 and CBA/J mice. CD1 d 16 pups were acquired from in-house breeding. Breeders were purchased from Harlan (Indianapolis, IN). Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the established institutional animal use and care protocol at the University of Illinois at Chicago. Animals were housed in a temperature and light controlled environment (12 h light, 12 h dark) and were provided food and water ad libitum.

Organ culture

Ovaries were dissected in dissection media composed of Liebowitz with l-glutamine, penicillin/streptavidin (10 μl/ ml medium; Invitrogen, Carlsbad, CA) and BSA (1 mg/ml medium; MP Biomedicals Inc., Solon, OH). The bursa was removed with forceps and ovaries were cut into eight pieces termed organoids using a scalpel. The process of bisecting the ovary into eight pieces is the artificial wound. No additional scraping or perturbation was performed. Each organoid was placed into a 0.5% alginate/PBS droplet formed on a mesh fiber. The alginate-organoid was then placed into 50 mm CaCl2 for 2 min to cross-link the alginate, causing the solution to solidify into a gel around the ovarian organoids. The gel-organoid was then placed in the appropriate growth media to be cultured for 1, 2, 4, and 8 d. The growth media consisted of α-MEM, insulin/transferrin/selenium (insulin 5 μg/ml; transferrin 5 μg/ml; selenite 5 ng/ml), and either BSA at 3 mg/ml or 10% fetal bovine serum (FBS) solution. Bromodeoxyuridine (BrdU; 10 μm) was added into the media to label proliferating cells during a 24 h period before fixing (Fig. 1).

Antibodies

The primary antibodies used in this study were raised against BrdU (rat; 1:200 dilution; Abcam, Cambridge, MA), cytokeratin-8 (CK8) rat; 1:200; CK8 TROMA-1 antibody; Developmental Studies Hybridoma Bank, Iowa City, IA), N-cadherin (rabbit; 20 μg/ml; Abcam), E-cadherin (rat; 100 μg/ml; Invitrogen), vimentin (goat; 1:100; Sigma, St. Louis, MO), activated caspase-3 (rabbit, 1:25; Cell Signaling, Beverly, MA) and were incubated overnight at 4 C. The proliferating cell nuclear antigen antibody (PCNA; rat; Zymed, San Francisco, CA) was used according to the manufacturer’s protocol (Zymed/Invitrogen PCNA staining kit). Terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining was performed according to the manufacturer’s protocol (Deadend; Promega, Madison, WI).

Immunohistochemistry

The organoids were removed from the culture media and placed into a 50 mm CaCl2 solution for 2 min to re-cross-link the alginate. They were fixed in 4% paraformaldehyde-cacodylate-Ca2+ overnight for 12–16 h.

All reagents were obtained from Vector Laboratories, Inc. (Burlingame, CA) unless otherwise indicated. Xylenes and ethanol were obtained from Surgipath Medical Industries, Inc. (Richmond, IL). Antigen retrieval was performed using 10 mm sodium citrate. When staining for BrdU, the tissue sections were treated with 4 m HCl for 10 min followed by sodium tetraborate for 10 min. Slides were washed in Tris-buffered saline with Tween 20 [20 mm Tris, 500 mm NaCl, 0.1% Tween 20 (pH 7.4)]. Standard immunohistochemistry was used and control slides received serum block instead of primary antibody as previously described (12).

Imaging and counts

Images were obtained on a Nikon E600 microscope using the DXM 1200 (Nikon Instruments, Evanston, IL) and Metavue software (version 5.0r7; Downington, PA). Using ImageJ (National Institutes of Health, Bethesda, MD), the percent ovarian surface epithelium (OSE) encapsulation was determined by measuring the perimeter of the organoid positive for CK8 divided by the circumference. In the time-point study, two separate investigators counted the total number of OSE (CK8 positive) and the total number of BrdU cells to determine the percentage. Similar counts were performed for PCNA, TUNEL, and caspase-3 staining.

Statistics

All values are expressed as the means ± sd. Tukey’s multiple comparison tests were used to assess differences between media groups and control samples. P < 0.05 was considered statistically significant. Student’s t test was only used in paired comparison and specifically noted.

Results

Three-dimensional ovarian organoids propagate OSE that retain normal characteristics

To investigate the impact of culturing ovarian organoids in a three-dimensional alginate suspension for 8 d (Fig. 1), normal OSE markers were examined by immunohistochemistry. CK8 is an epithelial cell marker and can be used to distinguish the OSE. The expression of CK8 indicates the presence of the OSE (Fig. 2A, red arrow). OSE coexpress epithelial and mesenchymal markers. Vimentin showed staining similar to that of CK8 in the OSE (Fig. 2B). N-cadherin is expressed in normal OSE and the acquisition of E-cadherin is considered an early marker of cancer (20). N-cadherin was present in the OSE (Fig. 2C). E-cadherin is not present in the OSE grown in the organ culture system (Fig. 2D). These data suggest that ovary organoids can be cultured in an alginate three-dimensional suspension and retain the expression profile of OSE in an intact ovary.

Figure 2.

Figure 2

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Ovarian organoids cultured for 8 d in BSA media maintain normal expressions of ovarian surface epithelial proteins. Antibody localization in the OSE stained with diaminobenzidine and counterstained with hematoxylin. Immunolocalization of CK8 (A), vimentin (B), N-cadherin (C), and absence of E-cadherin (D). Red arrows point toward OSE. Figure insets depict serial sections stained with CK8 used to confirm the presence of OSE.

Wounded surface is repopulated with OSE

Surface remodeling occurs after ovulation regenerating a layer of OSE. To investigate how wounding increases normal OSE proliferation, ovaries were artificially wounded and surface repair was measured. Organoids were cultured for 0 (isolated, placed in alginate, and fixed), 2, 4, and 8 d in both BSA and FBS supplemented media. The CK8 antibody was used to mark the OSE and determine the amount of encapsulation of the organoid. Overall there was an increase in encapsulation by the OSE when grown in both media conditions compared with d 0 (Fig. 3A). FBS-cultured organoids had significantly more surface area encapsulated by OSE after 2 and 4 d (P < 0.05). By d 8, surface encapsulation decreased and no significant difference between d 0 and d 8 in culture was reported in either condition (P > 0.05).

Figure 3.

Figure 3

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OSE repopulates the ovarian surface in response to wounding in culture. Organs cultured in both BSA and FBS media exhibited greater encapsulation of the ovarian surface over time. A, Encapsulation (percent) was obtained by measuring the perimeter of the ovary positive for CK8 and dividing by the circumference of the tissue. B, Organ cultures from C57B6/CBA mice labeled with CK8 depicting OSE (stained by diaminobenzidine) progressively encapsulating the ovarian surface over time. Magnification, ×200. Significant difference (*) from d 0 ovaries (P < 0.05) with symbol color related to strain.

Immunohistochemical analyses of the ovary organoids from each time point in both FBS and BSA showed the replacement of OSE by CK8 immunostaining (Fig. 3B). In d 8 BSA cultures, 81% of the OSE had multilayering, whereas 50% of FBS organoids had multilayered OSE. Thus, multilayering occurs more often in the BSA cultured samples, whereas FBS supported a single layer of surface cells.

Two strains of mice were examined to determine whether a different genetic background significantly altered OSE replacement of the wounded surface. The CD1 and C57B6/CBAJ strains did not differ in encapsulation rate at any time point measured, and both strains responded similarly to FBS and BSA conditions. These data suggest that OSE migrated and encapsulated wounded surfaces regardless of strain.

Wounding increased OSE proliferation

Ovulation increases OSE proliferation; therefore, the ability of the OSE to proliferate in response to artificial wounding was investigated using PCNA as a marker of mitosis. The percentage of dividing cells was defined as the number of PCNA-expressing cells in the OSE confirmed by CK8 expression. OSE proliferation was significantly higher (P < 0.05) in all samples at d 2 of culture in both BSA and FBS media (Fig. 4A). Samples grown for 4 d in BSA and FBS proliferated significantly more when compared with d 0 (P < 0.05). Cultures grown for 8 d in FBS were not significantly different when compared with d 0. BSA cultures at d 8 continued to proliferate at a higher rate (P < 0.05).

Figure 4.

Figure 4

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Wounding increases proliferation of the ovarian surface epithelium. A, Cells staining positive for PCNA were counted and compared with the total number of OSE cells stained by CK8 to determine percent OSE proliferation. Significant difference (*) from d 0 (P < 0.05). B, BrdU-labeled cells were counted and divided by the total number of CK8-labeled cells to generate the percent OSE proliferation. Ovaries that were wounded and cultured showed a significantly greater (*) rate of proliferation compared with that of d 0 ovaries (P < 0.05). C, Whole ovaries with and without bursa were cultured in BSA media with and without alginate for 24 h with continuous BrdU labeling. Significant difference (*) from whole ovary with bursa and without alginate (P < 0.05).

To further investigate proliferation after wounding, BrdU incorporation of the OSE was quantified in the ovary organoid cultures (Fig. 4). The organoids were grown in alginate, and BrdU was added to the culture medium 24 h before the day the tissue was fixed. The d 0 BrdU incorporation was determined by injecting d 15 mice ip and collecting the ovaries on d 16 of life (d 0 of culture). At d 0 only 3% of the OSE were dividing based on BrdU incorporation. Organoids cultured in both BSA and FBS demonstrated a significant increase in the number of proliferating cells by d 2 (P < 0.05) (Fig. 4B). After 4 d of culture, OSE grown in both conditions continued to divide significantly more compared with d 0 (P < 0.05). After 8 d in culture, organoids in BSA had OSE proliferating significantly, but FBS did not when compared with d 0.

The rodent ovary is surrounded by a protective membrane called the bursa. Whole ovaries with and without the bursa and with and without alginate were cultured to determine whether there was a difference in the proliferation from mechanical wounding or from gel encapsulation. Ovaries with the bursa intact and without alginate showed the lowest proliferation percentage when compared with the other groups (27 ± 10). Removal of the bursa regardless of the presence of the alginate scaffold significantly increased proliferation of the OSE (P < 0.05) (Fig. 4C). Whole ovaries cultured with the bursa also provide an unwounded control and indicate that proliferation significantly increases in wounded organoid cultures by d 2 compared with both in vivo unwounded and cultured in vitro unwounded controls (P < 0.05).

To investigate whether the differences in proliferation rates between FBS and BSA cultures were due to an increase in apoptosis, TUNEL and activated caspase-3 immunostaining was performed. No significant difference between groups was found between time points and media conditions (data not shown).

Alginate concentration and fetuin have limited effects on OSE proliferation

Culture systems using alginate indicate that gel rigidity influences organ culture growth (21). Two concentrations of alginate were used to determine whether OSE proliferation is enhanced in weaker gels. Fetuin is a glycoprotein that is frequently used as a tissue culture medium supplement contributing to the attachment and spreading of cells. No significant difference in proliferation was detected between the 0.5 and 0.25% concentration of alginate in either BSA or FBS medium (P > 0.05). Overall, the different percentages of alginate had no effect on the proliferation of the ovary organoids in culture for 8 d. The addition of fetuin to the FBS culture medium increased proliferation significantly when compared with the two other groups (P < 0.05).

Discussion

The lack of information regarding normal ovarian surface biology hinders progress toward understanding early events in ovarian cancer. The establishment of a three-dimensional ovarian organ culture provides a tool to study normal ovarian surface cells within their microenvironment. Ovarian surface cells retained normal morphology and protein characteristics and proliferated to repair wounded surfaces induced by incision. An alginate hydrogel scaffold retained epithelial-stromal interactions such that proliferation could be studied using an in vitro model. The culture medium containing FBS significantly increased surface encapsulation, suggesting enhanced migration, whereas BSA increased cellular proliferation independent of encapsulation. Induction of wounding either through scalpel incision or removal of the bursa resulted in significantly more OSE proliferation after 2 d in all conditions. OSE proliferation was induced after wounding, providing one mechanism for increased DNA damage and possible cellular transformation related to the process of ovulation.

The ovarian surface epithelium is thought to be the source of 90% of ovarian cancers. The study of normal OSE has been possible based on pioneering work to immortalize human cells obtained from surface scrapes on two-dimensional plastic (19). Normal OSE grown on plastic eventually undergoes an epithelial-to-mesenchymal transition, and this process has been speculated to relate to normal wound repair after ovulation (2,22). Ovarian organ cultures demonstrate that normal cells grown within the ovarian microenvironment maintain both epithelial and squamous appearance. Stromal cells are viable, and therefore, context-dependent cellular interactions are retained for a possible better understanding of the epithelial stromal interactions relating to cancer formation (23). The initiation of cancer transformation and tissue-specific remodeling responsible for cyst formation, stromal invasion, and tumor formation are areas that might be investigated using an ovarian alginate culture system.

OSE rapidly proliferates in response to wounding in a three-dimensional ovarian organ culture. Because previous studies in cycling primates have indicated that OSE can replace artificially wounded surfaces, these results confirm such a phenomenon using mouse tissue in an in vitro system (24). Although numerous studies pointed toward an increase in OSE proliferation after ovulation, the present study demonstrates that absent any hormonal stimulation, OSE proliferates rapidly both after scalpel generated wounding and removal of the bursa (12,25). The induced proliferation also takes place devoid of inflammation normally present at the time of ovulation (9). Cells tended to proliferate more significantly in the days immediately after wounding and then slow as the wounded surface was repaired with OSE.

The culture of mouse ovaries to study human ovarian cancer should provide information regarding early events in disease progression. Although rodents in general have a low incidence of ovarian cancer, several transgenic models have demonstrated that specific changes in the ovarian surface can produce ovarian cancers that resemble human disease (26,27,28,29). Only moderate changes in the strain-dependent growth characteristics of the OSE were detected using the alginate organ culture illustrating the usefulness of culturing transgenic and modified ovaries for understanding specific events after modulation with chemicals and other possible preventive strategies. Therefore, the use of an in vitro organ culture could help bridge a gap between developing transgenic mice to study specific changes critical for ovarian cancer and traditional two-dimensional cultures grown on plastic.

In summary, the ability to culture normal OSE cells within the ovarian microenvironment was supported using minimal medium and an alginate hydrogel scaffold. The organ culture system supports ovarian surface repair and epithelial-stromal interactions, independent of mouse strain, providing a tool for studying early events in ovarian surface epithelial cancers.

Acknowledgments

We thank the National Institutes of Health Building Interdisciplinary Research Careers in Women’s Health from the Office of Women’s Health Research and National Institute of Child Health and Human Development for their support of this research.

Footnotes

This work was supported by National Institutes of Health (NIH) Grant K12HD055892 Building Interdisciplinary Research Careers in Women’s Health from the Office of Women’s Health Research (to J.E.B.), NIH Grant C06RR15482 (to J.E.B.), and Human Development (to J.E.B.) and National Science Foundation Bridge to the Doctorate Fellowship provided by the Illinois Louis Stokes Alliance for Minority Participation (to T.S.H.).

Disclosure Summary: K.S.J., K.I., D.A.D., T.S.H., and J.E.B. have nothing to declare.

First Published Online May 7, 2009

Abbreviations: BrdU, Bromodeoxyuridine; CK8, cytokeratin-8; FBS, fetal bovine serum; OSE, ovarian surface epithelium; PCNA, proliferating cell nuclear antigen antibody; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling.

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