Primary culture of ovarian surface epithelial cells and

PROTOCOL

Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients Trevor G Shepherd, Brigitte L The´riault, Elizabeth J Campbell and Mark W Nachtigal Department of Pharmacology, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5. Correspondence should be addressed to M.W.N. ([email protected]).

© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 11 January 2007; doi:10.1038/nprot.2006.328

Our laboratory has refined the technique for isolating primary cultures of normal human ovarian surface epithelial (OSE) cells by combining two different protocols involving the enzymatic and mechanical removal of OSE cells from ovarian biopsies. A simple protocol of obtaining primary epithelial ovarian cancer (EOC) cells from the ascites fluid removed from patients with high-grade ovarian cancer is also described. These methods allow for the direct application of many molecular and cellular analyses of normal versus cancer cells isolated freshly from patients, with the added potential for retrospective analyses of archived cells and tissues. Thus, we have included optional steps for the immediate preparation of ascites-derived EOC cells to be used for subsequent cytological analyses. Initial isolation of OSE or EOC cells can be completed in 1 h, and primary cells are further expanded in culture for several weeks.

INTRODUCTION Ovarian cancer is the most lethal of the gynaecological malignancies. The majority of epithelial ovarian cancers are thought to arise from the ovarian surface epithelium, although the etiological mechanisms remain to be clearly elucidated1–3. To this end, we employ a set of techniques to isolate normal human ovarian surface epithelial (OSE) cells and ascites-derived epithelial ovarian cancer (EOC) cells directly from patients, and propagate these primary cells in culture4 (Figs. 1 and 2). This approach of culturing normal OSE and pathologic EOC cells under identical conditions is crucial for performing accurate, parallel analyses at the molecular and cellular levels to better understand the development of ovarian cancer5,6. In addition, important functional assays can be performed to address the involvement of specific signalling pathways present in both cell types7, and quite possibly leading to the early development of targeted therapeutic approaches for the disease. To date, much of our current knowledge of human ovarian cancer has been uncovered through the use of established, immortalized cell lines. Many well-characterized ovarian cancer cell lines exist, and these cells possess the obvious advantages of high proliferative capacity, extended lifespan in culture and clonogenicity. In addition, several groups have successfully established

MATERIALS REAGENTS . Ovarian tissue samples or ascites fluid ! CAUTION Institutional informed patient consent and appropriate biohazard authorization is required for experimentation with human tissue. Depending upon institutional screening procedures, patient samples should be treated as potentially positive for contagious pathogenic viruses (e.g., HIV or hepatitis viruses), as well as methicillin resistant Staphylococcus aureus (MRSA)-positive bacteria in rare circumstances. . MCDB 105 medium (Sigma) . Medium 199 (Invitrogen) . Fetal bovine serum (FBS; stocks from different suppliers have been tested and used with equivalent efficacy) . 100 penicillin-streptomycin, liquid (Invitrogen) . Dispase II (Roche) . Phosphate-buffered saline (PBS), pH 7.4, sterile . 0.25% w/v trypsin-EDTA (Invitrogen)

immortalized human OSE cell lines, adding to the repertoire of available tools to further our understanding of ovarian cancer pathogenesis8. However, both OSE and EOC cell lines possess a plethora of genetic and biochemical abnormalities that support immortality, which remind us of how far-removed these cell lines are from the patient. The ability to culture and characterize freshlyisolated OSE and EOC cells from patients provides a very important experimental system that has the potential to resemble the patient situation more closely. Several methods have been described for the primary culture of either human OSE cells from benign ovaries or EOC cells isolated from the ascites fluid of ovarian cancer patients9–12. Some of these techniques employ additional procedures to purify EOC cells from other cell types present in ascites fluid, namely erythrocytes. In addition, the culture medium can contain added factors that, although they aid in growth promotion, may alter primary human epithelial cell morphology significantly9,13. Taken together, the primary OSE and EOC cell isolation and culture techniques, as described herein, are relatively simple and rapid, yet allow for the direct comparison of normal human OSE cells and their pathologic EOC counterparts under identical culture conditions.

. Percoll (GE Healthcare) . (Ethylenedinitrilo)-tetraacetic acid (EDTA) . 95% v/v ethanol/5% v/v acetic acid, prepared fresh . Dimethyl sulphoxide (DMSO) . Isopropanol . Liquid nitrogen EQUIPMENT

. Small fine-tip forceps and scalpel blade, sterile . Rubber policeman, optional . Cytobrush Plus (Medscand, Inc.), optional . T-75 tissue culture-treated flasks with 0.2 mm vented cap . 6-well tissue culture-treated dish . Refrigerated tabletop centrifuge with swinging bucket rotor and adaptors for 15 ml conical tubes

. 15 ml conical capped tubes, sterile . Certified laminar flow hood NATURE PROTOCOLS | VOL.1 NO.6 | 2006 | 2643

PROTOCOL . Cell culture incubators (37 1C, humidified, 95% air/5% CO2)

. Nalgene cryo 1 1C freezing container . Coated Shandon cytoslides (Thermo Scientific) . Shandon Cytospin 3 cytocentrifuge (Thermo

a

b

c

Scientific)

. Shandon disposable sample chambers (Thermo Scientific)

80 1C freezer, liquid nitrogen, or -150 1C freezer) . Light microscope REAGENT SETUP Complete MCDB/M199 medium Make equal d e f volumes of each medium separately as per manufacturer’s instructions (MCDB 105 medium; Medium 199), then combine and pH accordingly (usually pH 7.2 to give pH 7.4 after filter sterilization). Store at 4 1C and protect from light. Add 50 ml of FBS to 450 ml of MCDB/M199 medium and 5 ml of 100 units penicillin-streptomycin. Store medium at 4 1C and warm to 37 1C prior to use. 0.06% v/v trypsin/EDTA Dilute 0.25% w/v trypsin-EDTA in sterile 1 PBS/0.5 mM EDTA. Store at 4 1C, and pre-warm to room temperature (20–24 1C) prior to use. Figure 1 | Primary cultures of normal human OSE cells. (a) Small clusters of OSE cells freshly isolated from Dispase II Aliquot Dispase II into separate an ovarian tissue sample by Dispase II treatment and gentle scraping. Several erythrocytes are visible volumes of 5–10 ml to avoid multiple freeze thaws, (arrowhead). (b) A colony of OSE cells have attached and started to spread on the tissue culture plastic and store at -20 1C in a non-frost-free freezer after four days. (c) Outgrowth of OSE cells from a cluster. (d) Confluent monolayer of OSE cells depicting until enzymatic activity is noticeably reduced. typical epithelial cobblestone morphology. (e) Senescent OSE cells at passage-4 illustrating irregularlym CRITICAL If a pale yellow precipitate forms in the shaped cells that have ceased dividing. (f) Confluent monolayer of contaminating fibroblasts that have Dispase II solution, especially if greater than 6 months overtaken the OSE cell culture. from receipt of product, then the yield and integrity of the primary OSE cultures will be compromised. Freezing medium 70% v/v MCDB/M199 50% v/v Percoll Make a fresh solution each time by diluting Percoll in 10 medium, 20% v/v FBS, 10% v/v DMSO, store indefinitely in a non-frost-free PBS at a 1:1 v/v ratio and mix well. freezer at -20 1C, when thawed, mix thoroughly by inversion.

PROCEDURE 1| Cultures can be derived from patients’ ovarian tissue samples (A), or ascites-derived ovarian cancer cells (B). Follow Steps A and/or B according to your experimental needs. (A) Primary human OSE cell culture TIMING 2–3 weeks Isolation of OSE cells TIMING 1 h (i) Receive ovarian tissue samples from surgical staff in sterile vessel containing saline. (ii) Working in a tissue culture laminar flow hood, remove the ovarian tissue using sterile forceps and place OSE-side (i.e., smooth side) down into 2.0–2.5 ml of pre-warmed (37 1C) Dispase II in 35 mm wells of a 6-well tissue culture dish. If only a small portion of the ovary is received, it is important to correctly distinguish the ovarian surface from the cut interior of the ovary to minimize contamination with unwanted cell types. ? TROUBLESHOOTING (iii) Place the dish in a 37 1C incubator and swirl every 10 min for a 30 a b c EOC total incubation time of 30 min. 25 SkOV3 (iv) Pre-warm complete MCDB/M199 20 media at 37 1C and place in a 15 tissue culture laminar flow hood, 10 along with a sterile scalpel blade 5 ready for use. Dispense 2.0–2.5 ml 0 of medium into the 35 mm wells 0 1 2 3 4 5 6 adjacent to the wells containing Time (days) the ovarian tissue/Dispase II. Figure 2 | Primary cultures of human EOC cells from patient ascites. (a) Cytospin preparation of freshlyUsing sterile forceps, transfer isolated EOC cells following Percoll centrifugation to remove erythrocytes. (b) Confluent monolayer of the ovarian tissue to the wells primary human EOC cells illustrating epithelial cobblestone morphology. (c) Representative growth curve containing the medium. Gently assays of primary EOC cell sample as compared with the established EOC cell line, SkOV3. Primary EOC cells swirl the tissue OSE-side down are seeded at twice the number of cells versus cell lines as a result of their poor seeding efficiency and in the medium. slow growth rates.

Cell number (×10–4)


. Ultralow temperature storage space (

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PROTOCOL m CRITICAL STEP Lift the tissue so it remains partially submerged in medium, and gently scrape the surface of the ovary towards and into the media with the scalpel blade. Swirl the scalpel blade in the media as well. Place tissue back into the original sterile container of saline. ? TROUBLESHOOTING (v) Transfer the used Dispase II into a sterile 15 ml conical centrifuge tube and dilute with complete MCDB/M199 medium to its full capacity. m CRITICAL STEP Unlike trypsin, Dispase II will not be inactivated by the serum present in the medium, so adequate dilution is critical to grow healthy OSE cells after Dispase II treatment. ? TROUBLESHOOTING (vi) Centrifuge the diluted Dispase II at 100g for 5–10 min at room temperature. Carefully aspirate the media until less than 1 ml remains. Resuspend the very small pellet of cells (almost not visible) in 2.0–2.5 ml of complete MCDB/M199 medium and transfer to another 35 mm well of the 6-well tissue culture dish. (vii) Clusters of OSE cells can be visualized floating in the medium in each of the wells (Fig. 1a). In our experience, the well in which OSE cells are scraped directly into the medium yields the best preparations. Occasionally, additional OSE cells will be recovered in the pelleted and resuspended sample well (Step Avi). Erythrocytes will likely be present, but usually in very small amounts and they will be removed with the first medium change. ? TROUBLESHOOTING Propagation of OSE cells TIMING 2–3 weeks (viii) Leave the plate of cells in the incubator for at least 3–4 d prior to the first medium replacement. m CRITICAL STEP The clusters of OSE cells are very slow to attach and spread on the tissue culture plastic (Fig. 1b). ? TROUBLESHOOTING (ix) Once 40–50% of the well is covered in expanding OSE clusters (Fig. 1c), trypsinize cells using 0.06% w/v trypsin/EDTA for 2–3 min at 37 1C, disaggregate within the well, then resuspend within the same well with complete MCDB/M199 medium. m CRITICAL STEP Replating in the same well increases the yield of healthy, growing OSE cells, because some cells and perhaps newly-deposited extracellular matrix components may be left behind by transferring OSE cells to a new well too soon. ? TROUBLESHOOTING (x) Replace medium every 2–3 d until OSE cells have reached close to 100% confluence (Fig. 1d). At this time, trypsinize the cells using 0.06% w/v trypsin/EDTA for 2–3 min at 37 1C, resuspend in complete MCDB/M199 medium, and plate into two or three 35 mm wells, or one 60 mm dish, depending on the downstream experiments to be performed. m CRITICAL STEP OSE cells should not be split at a lower density than a 1:3 dilution, since their growth can be seriously compromised. In our experience, this can induce premature growth arrest and cell senescence. ? TROUBLESHOOTING (xi) Primary cultures of normal human OSE cells can be propagated for up to 3–4 passages, as performed according to Step Ax, before the cells senesce (Fig. 1e). m CRITICAL STEP It is crucial that specific experiments be well defined and prepared-for in advance of obtaining samples and culturing cells. It is also important to monitor for possible fibroblast contamination during the subculture of primary OSE cells (Fig. 1f) and to modify the protocol for subsequent isolations, if necessary. ? TROUBLESHOOTING (B) Primary human ascites-derived EOC cell culture TIMING 4–6 weeks Isolation of ascites-derived EOC cells TIMING 0.5–1 h (i) Receive freshly-isolated ascites fluid from surgical staff in a sterile vacuum container or evacuated bottle(s). (ii) Working in a tissue culture laminar flow hood, aseptically transfer 25 ml of ascites fluid to tissue culture flasks (we typically use 6–10 T-75 flasks with 0.2 mm vented caps). Add an equal volume of complete MCDB/M199 medium to each flask. (iii) Transfer additional ascites fluid to sterile tubes and centrifuge at 3,200g for 10 min at 4 1C. Transfer the supernatant (clarified ascites fluid) to multiple tubes/vials and freeze at -80 1C for archival purposes (it is unknown what the upper storage limit might be). (iv) An optional step is to prepare ascites-derived ovarian cancer cells for subsequent cytological analysis (Step Bv–Bxvi) according to your experimental needs. If not needed, go to Step Bxvii. Cytospin preparation TIMING 2 h (v) Transfer 50–100 ml of ascites fluid from the vacuum container (Step Bi) to 50 ml tubes. m CRITICAL STEP Purify ascites samples that appear bloody by centrifugation over a Percoll cushion to remove erythrocytes (follow Steps Bv–Bxii). If this is not the case, proceed to Step Bxiii. (vi) Aliquot 5 ml of 50% v/v Percoll into each of 16–20 15 ml conical centrifuge tubes.

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PROTOCOL (vii) Mix aliquot(s) of ascites sample by inversion. Carefully layer 5 ml of ascites on to the 50% v/v Percoll cushion in each tube. m CRITICAL STEP To avoid mixing ascites and Percoll, rest the pipet on the inside of the tube while the ascites is slowly expelled down the side. It is important not to rush this step so that the integrity of the two layers is maintained. (viii) Centrifuge at 1,200g for 20 min at 4 1C using a swinging bucket rotor. (ix) Erythrocytes will be at the bottom of the tubes, while the desired EOC cells will be at the interphase between the ascites fluid and Percoll. Using a 5 ml pipet, carefully enter the interphase to withdraw the cells, and pool the cells into new 15 ml conical centrifuge tubes so that each tube contains up to 6 ml of recovered cells. m CRITICAL STEP Do not try to remove the top layer of ascites fluid directly. Remove as small a volume of liquid containing the EOC cells as possible to limit the transfer of contaminating Percoll and to minimize subsequent tube numbers. (x) Dilute cells to 12 ml using ice-cold 1 PBS and mix by inversion. (xi) Centrifuge at 1,800g for 30 min at 4 1C using a swinging bucket rotor. Cells should be pelleted and the supernatant should appear clear. (xii) Carefully discard supernatant with a pipet. Wash cells by resuspending in 3 ml of ice-cold 1 PBS, and transfer to several 1.5 ml Eppendorf tubes. Centrifuge at 1,500g for 5 min at 4 1C. Finally, resuspend and pool cells to a final volume using 1.2 ml of ice-cold 1 PBS in a single Eppendorf tube. (xiii) Perform cytospins using a Shandon Cytospin 3 cytocentrifuge (or similar equipment) at 500 r.p.m. with medium acceleration for 5 min, and transfer cells to coated Shandon cytoslides. Load 100 ml of cell suspension into the assembled slide and disposable sample chamber. m CRITICAL STEP Prepare a test slide and use a microscope to verify if EOC cells are at an appropriate density (Fig. 2a), and dilute cell suspension with 1 PBS as appropriate. Prepare the desired number of slide assemblies and complete cytospins. (xiv) Allow slides to air-dry at room temperature. (xv) Fix cells on slides in fresh 95% v/v ethanol/5% v/v acetic acid for 1 min, and allow slides to air-dry at room temperature. (xvi) Store slides at 4 1C until subsequent analysis. It is unknown how long these slides can be stored for. Propagation of EOC cells TIMING 4–6 weeks (xvii) Place in an incubator undisturbed for 3–4 d prior to the first change of complete medium. m CRITICAL STEP The amount of erythrocytes and the viscosity of the fluid will vary tremendously from patient to patient, but additional processing steps, such as the removal of the erythrocytes by centrifugation over a Percoll cushion (see optional cytospin preparation, Step Biv) or hypotonic lysis, are not necessary. The EOC cells will eventually bind to the tissue culture plastic, though it may not be clearly visible, and the erythrocytes will be removed after the first set of medium changes. Furthermore, the complete MCDB/M199 medium used to propagate EOC cells does not support growth of hematopoietic cell contaminants. ? TROUBLESHOOTING (xviii) Replace media after approximately 3–4 d, and continue to change media every 2–3 d until the flasks are confluent (Fig. 2b). To passage EOC cells, wash cells once with sterile 1 PBS then trypsinize using a minimal volume of 0.06% w/v trypsin/EDTA for 2–3 min at 37 1C. Add complete MCDB/M199 medium and transfer cells to fresh T-75 flasks. m CRITICAL STEP Split cells at a 1:2 dilution, and no more than 1:3 for maintaining adequate growth of the cells over time. It should be noted that primary EOC cells have a reduced growth rate as compared with established EOC cell lines (Fig. 2c). We typically split one flask of passage-0 cells at a 1:2 dilution and monitor over the next 24–48 h to observe seeding efficiency, growth and general morphology of the cells. Based on these observations, we then choose whether to proceed with freezing numerous vials of passage-0 cells for archival purposes. (xix) To freeze stocks of primary EOC cells, wash cells once with sterile 1 PBS then trypsinize using a minimal volume of 0.06% w/v trypsin/EDTA for 2–3 min at 37 1C. Add complete MCDB/M199 medium and transfer cells to sterile 15 ml conical centrifuge tubes. Centrifuge at 500g for 5 min at room temperature. Carefully discard the supernatant and gently resuspend cells with appropriate volume of freezing medium (i.e., 1.0–1.5 ml per T-75 flask of cultured cells prior to trypsinization). Transfer cells to labelled, sterile cryovials, cap tightly, and place in a Nalgene cryo 1 1C freezing container containing isopropanol (according to manufacturer’s instructions). Place into a -80 1C freezer for at least one day prior to transferring indefinitely to liquid nitrogen or a 150 1C freezer. m CRITICAL STEP A slow cooling rate of B1 1C min 1 increases the viability of cells. Freeze as many passage-0 cells as possible and store indefinitely in liquid nitrogen or a 150 1C freezer. Higher passage number cells can be frozen as well, but be aware that recovery and lifespan of experimentally serviceable EOC cells will be compromised (especially for EOC cells Z passage-4). Experiments are typically performed using cells at passage-2 through -6, because many cell samples will stop growing, or senesce, shortly thereafter. ? TROUBLESHOOTING


PROTOCOL (xx) To thaw a vial of frozen primary EOC cells, place at 37 1C until just thawed, and while working in a laminar flow hood aseptically transfer to a sterile 15 ml conical centrifuge tube. Dilute cells and freezing medium with a maximum volume of complete MCDB/M199 medium (13–14 ml) and centrifuge at 500g for 5 min at room temperature. Carefully discard supernatant, gently resuspend cells with complete MCDB/M199 medium, and transfer to a sterile T-75 tissue culture flask. Culture cells according to Step Bxviii.


TIMING Isolation of primary OSE cells: 1 h Propagation of primary OSE cells: 2–3 weeks Isolation of EOC cells from ascites: 0.5–1 h Propagation of EOC cells: 4–6 weeks Cytospin preparation of EOC cells: 2 h (optional) ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. TABLE 1 | Troubleshooting table. Problem Primary OSE cells Little or no yield of cells

Contamination with fibroblasts

Premature senescence Primary EOC cells Little or no yield of cells

Premature senescence

Possible reason

Solution

Old Dispase II or stock freeze-thawed too often

Aliquot Dispase II into separate volumes of 5–10 ml, and store in a non-frost-free 20 1C freezer

Surface of ovary not exposed to Dispase II

Ensure smooth surface of ovary remains in Dispase II for full 30 min incubation

Too much manipulation of tissue

Transfer tissue carefully and do not try to further process larger samples

Medium changed too soon after initial plating, cells not attached well enough

Leave cells undisturbed for first 3–4 d

Residual Dispase II

Increase volume of medium to dilute Dispase II

Too forceful when scraping ovarian surface

Gently rub surface of ovary with sterile scalpel; if still a problem, use a rubber policeman or sterile cytology brush instead of scalpel blade, and pre-plate disaggregated cells on non-tissue culture polystyrene (Petri plastic) for 24 h before transferring to tissue culture plates

Too much manipulation of tissue

Transfer tissue carefully and do not try to further process larger samples

Wrong surface of sample exposed to Dispase II

Ensure smooth surface of ovary is placed directly in Dispase II

Cells split at too low a density

Expand cells at 1:2 or 1:3 dilution only

Medium changed too soon after initial plating, cells not attached well enough

Leave cells undisturbed for first 3–4 d

Patient has undergone chemotherapy

Try to obtain ascites samples from chemotherapy-naı¨ve ovarian cancer patients

Cells split at too low a density

Expand cells at 1:2 or 1:3 dilution only

Cells have been frozen down at a higher passage number

Freeze down as many passage-0 cells as possible



PROTOCOL ANTICIPATED RESULTS a b In our hands, initial isolates of primary human OSE yield approximately 500–2,000 cells. OSE cells will usually reach first confluence in 7–10 d (B3 105 cells), and can be further expanded in culture to yield upwards of several million cells by passage-3 or -4. This will, however, vary greatly from patient sample to patient sample and cannot be predicted very well when based on the amount of tissue originally received. A much larger starting yield is obtained with ascites-derived primary EOC cells, where we are able to obtain several million Figure 3 | Downstream applications using primary human EOC cells. (a) EOC EOC cells from a starting volume of B200 ml patient ascites cells growing as a spheroid in suspension. (b) Efficient transduction of EOC fluid. This yield can be expanded greatly depending on the cells with GFP-expressing adenovirus. initial volume of ascites received and processed (range: 50 ml to 4 l). Primary EOC cells will typically reach first confluence by 3–7 d, at which time the majority of cells should be frozen as passage-0 cell stocks, and the remaining cells can be rapidly expanded for immediate experimental use. Other groups have described alternative culture conditions for both OSE and ascites-derived EOC cells10,12. It should be noted that the addition of supplemental factors to the culture medium to extend the growth potential of the primary OSE and EOC cells may induce altered cellular characteristics9, such as epithelial to mesenchymal transition13. Several immunocytochemical markers are available to distinguish the characteristics of OSE and EOC cells from those of other contaminating ovarian and extra-ovarian cell types1,14. As specified in this protocol, fibroblast cell contamination is sometimes observed in primary OSE cultures. This problem probably occurs if ovarian tissue samples are scraped too firmly, resulting in the removal of underlying ovarian stromal cells, or when there has been excessive manipulation of the tissue. Although we have not directly compared our protocol with mechanical scraping alone, in general we find that the extra step of enzymatic disaggregation of OSE cells produces monolayer cultures of OSE that rarely contain stromal contaminants. Although fibroblasts can outgrow the OSE cells, we have occasionally observed the loss of fibroblast-like cells when maintained in the MCDB/M199 medium used to culture OSE cells over 1–2 weeks of continuous culture. It should also be noted that fibroblast contamination of primary EOC cultures isolated directly from patient ascites fluid has rarely been observed. Because fibroblasts efficiently adhere to non-tissue culture polystyrene (i.e., sterile Petri plastic), a pre-clearing step may be incorporated into the protocol prior to final plating on treated tissue culture plastic to reduce potential contamination by fibroblasts (Table 1). Human EOC consists of several different histological subtypes, and we commonly receive samples that reflect the expected prevalence observed in patients. We have cultured serous, mucinous, clear cell and poorly-differentiated EOC cells successfully, but have received very few of the endometrioid subtype, because this histotype rarely occurs and development of ascites fluid is infrequent. The majority of primary EOC cells display a similar epithelial cobblestone morphology and growth rate regardless of histotype when using the culture conditions described in this report. The effect of chemotherapy is probably the most significant factor that impacts the ability to isolate and grow EOC cells from patient ascites fluid. We have rarely succeeded in growing EOC cells from patients who are concurrently undergoing chemotherapy for their disease. Utilization of these protocols has allowed for the establishment of primary normal OSE and EOC cell cultures that facilitate comparative analyses using modern molecular and cellular investigative techniques5–7. For example, assays involving threedimensional culture systems can be established for primary EOC cells (Fig. 3a). In addition, both primary OSE and EOC cells can be transduced efficiently with recombinant adenoviral vectors, thus greatly expanding the capacity for experimental manipulation using these cells (Fig. 3b).

ACKNOWLEDGMENTS This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society Grant No. 15303. T.G.S. is supported by a fellowship from the NCIC with funds from the Terry Fox Foundation. B.T. is supported by a fellowship from the Nova Scotia Health Research Foundation. M.W.N. is a Canadian Cancer Society Scientist of the NCIC. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.natureprotocols.com Rights and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Auersperg, N., Wong, A.S., Choi, K.C., Kang, S.K. & Leung, P.C. Ovarian surface epithelium: biology, endocrinology, and pathology. Endocr. Rev. 22, 255–288 (2001).


2. Murdoch, W.J. & McDonnel, A.C. Roles of the ovarian surface epithelium in ovulation and carcinogenesis. Reproduction 123, 743–750 (2002). 3. Wong, A.S. & Auersperg, N. Ovarian surface epithelium: family history and early events in ovarian cancer. Reprod. Biol. Endocrinol. 1, 70 (2003). 4. Dunfield, L.D., Shepherd, T.G. & Nachtigal, M.W. Primary culture and mRNA analysis of human ovarian cells. Biol. Proced. Online 4, 55–61 (2002). 5. Fu, Y., Campbell, E.J., Shepherd, T.G. & Nachtigal, M.W. Epigenetic regulation of proprotein convertase PACE4 gene expression in human ovarian cancer cells. Mol. Cancer Res. 1, 569–576 (2003). 6. Matei, D. et al. Gene expression in epithelial ovarian carcinoma. Oncogene 21, 6289–6298 (2002). 7. Shepherd, T.G. & Nachtigal, M.W. Identification of a putative autocrine bone morphogenetic protein-signaling pathway in human ovarian surface epithelium and ovarian cancer cells. Endocrinology 144, 3306–3314 (2003).

PROTOCOL 12. Evangelou, A., Jindal, S.K., Brown, T.J. & Letarte, M. Down-regulation of transforming growth factor beta receptors by androgen in ovarian cancer cells. Cancer Res. 60, 929–935 (2000). 13. Salamanca, C.M., Maines-Bandiera, S.L., Leung, P.C., Hu, Y.L. & Auersperg, N. Effects of epidermal growth factor/hydrocortisone on the growth and differentiation of human ovarian surface epithelium. J. Soc. Gynecol. Investig. 11, 241–251 (2004). 14. Ueda, J., Iwata, T., Ono, M. & Takahashi, M. Comparison of three cytologic preparation methods and immunocytochemistries to distinguish adenocarcinoma cells from reactive mesothelial cells in serous effusion. Diagn. Cytopathol. 34, 6–10 (2005).


8. Auersperg, N. et al. E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium. Proc. Natl Acad. Sci. USA 96, 6249–6254 (1999). 9. Auersperg, N., Maines-Bandiera, S.L., Dyck, H.G. & Kruk, P.A. Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes. Lab. Invest. 71, 510–518 (1994). 10. Kruk, P.A., Maines-Bandiera, S.L. & Auersperg, N. A simplified method to culture human ovarian surface epithelium. Lab. Invest. 63, 132–136 (1990). 11. Hirte, H.W., Clark, D.A., Mazurka, J., O’Connell, G. & Rusthoven, J. A rapid and simple method for the purification of tumor cells from ascitic fluid of ovarian carcinoma. Gynecol. Oncol. 44, 223–226 (1992).
