From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid

Diseases impacting the female reproductive tract pose a critical health concern. The establishment of in vitro models to study primary endometrial cells is crucial to understanding the mechanisms that contribute to normal endometrial function and the origins of diseases. Established protocols for endometrial stromal cell culture have been in use for decades but recent advances in endometrial organoid culture have paved the way to allowing study of the roles of both epithelial and stromal endometrial cells in vitro. Due to inter-individual variability, primary cell cultures must be established from numerous persons. Generally, endometrial epithelial and stromal cells can be isolated from an endometrial biopsy, however, this is collected in a clinical setting by an invasive transcervical procedure. Our goal was to develop a non-invasive method for the isolation of paired endometrial epithelial organoids and stromal cells from menstrual fluid collected from individual women, based on recent reports describing the isolation of endometrial epithelial organoids or endometrial stromal cells from menstrual fluid. Participants recruited by the NIEHS Clinical Research Unit were provided with a menstrual cup and instructed to collect on the heaviest day of their menstrual period. Endometrial tissue fragments in the menstrual fluid samples were washed to remove blood, minced, and digested with proteinases. Following digestion, the solution was strained to separate epithelial fragments from stromal cells. Epithelial fragments were washed, resuspended in Matrigel, and plated for organoid formation. Stromal cells were separated from residual red blood cells using a Ficoll gradient and then plated in a flask. Once established, estrogen responsiveness of endometrial epithelial organoids was assessed and the decidual response of stromal cells was evaluated. Following treatments, qPCR was performed on organoids for genes induced by estradiol and on stromal cells for genes induced by decidualization. In this manner, the relative responsiveness of paired organoid and stroma cell cultures isolated from each woman could be assessed. In conclusion, we can isolate both epithelial and stromal cells from a single menstrual fluid sample, allowing us to establish organoids and cells in a paired manner. This protocol can greatly enhance our knowledge of the role of epithelial and stromal cells alone and in coordination.


Introduction
Diseases impacting the endometrium pose a critical health concern to women as well as to their children.Establishing an optimally receptive endometrium lays the groundwork for a healthy pregnancy, which can greatly impact the life-long health of an individual (1,2).Uterine development can be disrupted by exposures to substances in the environment or by disrupting normal endocrine signals including estrogen (3).Problems with implantation or placentation can lead to complications such as hemorrhage or pre-eclampsia (2,4) and the necessity of undesired interventions including bedrest or pharmacological treatments, which might inadvertently impact the developing fetus.In vitro models that recapitulate aspects of endometrial cell biology are crucial for understanding and testing the mechanisms regulating endometrial function and disease.Protocols for endometrial stromal cell culture are well established and can effectively model hormone-dependent processes in vitro (5).Processes intrinsic to epithelial cells of the endometrium, which are essential for embryo attachment and implantation, along with the secretion of key receptivity factors to support the pregnancy until the placental blood supply is fully developed (2,6), are not captured by stromal cultures.Until recently, growing primary endometrial epithelial cells in culture has not been very successful.Advances in endometrial organoid culture have paved the way to allowing scientists to study the roles of both epithelial and stromal cells in vitro (7,8).Due to variability between donors, primary cell cultures must be established from numerous individuals.Generally, endometrial epithelial and stromal cells can be isolated from an endometrial biopsy, however, this is collected in a clinical setting by an invasive transcervical procedure.Our goal was to develop a non-invasive method for the isolation of paired endometrial epithelial organoids and stromal cells from menstrual fluid collected in a reusable menstrual cup (9) from individual women, based on recent reports describing the isolation of endometrial epithelial organoids or endometrial stromal cells from menstrual fluid (10-12).
2 Reagents, materials, and equipment

• Instructions to participants
• Menstrual cups such as Diva cup brand and packaging with instructions • Specimen container for sample (such as Coviden 2210SA) • Water Bath • Cell culture incubator (37°C with 5% CO2) • Biological Safety Cabinet • Refrigerated Centrifuge with carriers for 15 and 50 ml conical tubes, capable of centrifugation at 600 RCF, with the ability to turn off the brake.

Tissue digestion solution
• Add the ingredients below then filter with a syringe filter (0.2 µm).Store on ice until digestion.
3 Methods 1.Under a NIEHS Institutional Review Board-approved protocol (000152) all donors must provide informed consent prior to specimen collection.Provide the participant with a menstrual cup, instructions, a specimen container, an ice pack, a biohazard bag, and a padded envelope.The participant is instructed to collect on their heaviest flow day for 6-12 hours and store it in a refrigerator.
2. Arrange drop off or courier transport of the collected sample in a specimen cup packaged in a biohazard bag inside a padded envelope with a frozen ice pack when it is available, ideally within 24-48 hours of collection.It is important to stress to the participants the importance of storing the sample at 4°C, as the integrity of the sample will diminish.4. Fill all tubes to 50 mL with cold PBS+p/s/f.Invert to mix.
5. Centrifuge sample at 600 RCF for 5-10 minutes at 4°C.Carefully remove the supernatant with a pipette, and avoid disrupting the pellet.Discard the supernatant.
6. Resuspend the pellet to 50 mL with cold PBS+p/s/f and centrifuge at 600 RCF for 5-10 minutes at 4°C, remove and discard the supernatant.Repeat resuspending in cold PBS +p/s/f and centrifuging until the supernatant is clear and colorless.Normally, this takes at least 4x.
7. Add cold PBS+p/s/f to pellets; if the sample was split into multiple tubes in step 3a, re-combine the material into one tube.Pass through a 100 µm cell strainer placed on top of a 50 mL tube.Avoid large fragments, clots, and mucus at first to facilitate flow through the strainer, but then pass all material through the strainer.If the strainer clogs, pass the remaining material through a new 100 µm cell strainer.Wash the strainer with more cold PBS+p/s/f.Wash until fragments remain in the strainer and most blood is rinsed off.
8. Invert the cell strainer over a 100 mm dish and backwash tissue fragments into a dish with cold PBS+p/s/f using a transfer pipette until all tissue is in the dish.
9. Chop tissue with scissors and forceps into small pieces (2-3 mm).Rinse with cold PBS+p/s/f to remove blood if needed.
10. Using forceps, transfer the tissue fragments to a 50 mL tube containing 20 mL tissue digestion solution.
11. Incubate in a water bath at 37°C for 20 minutes, inverting every 5 minutes to digest the tissue.
12. To stop the digestion, add 20 mL of a cold stop solution.
Mix by pipetting up and down 4+ times with a 25 mL pipette, and pass through a clean 100 µm cell strainer set on top of a clean 50 mL tube.
Cell strainer contains epithelial fragments (continue with Step 13).
Filtrate (in the 50 mL tube) contains stromal cells (go to Step 18).

Epithelial fragments/organoids
13. Invert the cell strainer over a 100 mm dish, and backwash retained epithelial fragments from the cell strainer with a cold stop solution into the dish using a transfer pipette until all fragments are in the dish.
14. Transfer the fragments from the 100 mm dish to a 15 mL tube and centrifuge for 10 minutes at 400 RCF at 4°C. 22. Resuspend the stromal cell pellet in 3 mL warm HESC culture medium and place cell suspension in a T25 flask.
23. Incubate at 37°C for 20 minutes and then transfer medium from this first flask into a second flask.Replace the medium in the first flask.Most stromal cells should be in the first flask and the second flask should contain stromal cells that did not adhere to the first flask as well as contaminating epithelial, blood, and immune cells.
24.After 60+ minutes, only stromal cells will have adhered to both flasks.Wash both flasks vigorously with room temperature PBS+p/s/f to remove other cell types and add 5 ml fresh warm HESC culture medium.

(Anticipated) Results
The method is summarized in the graphic in Figure 1.Images of organoids and stromal cells are shown in Figure 2. RT-PCR of RNA isolated from the cultures (steps 43-56 for stroma cells and 62-71 for organoid cultures) indicates the epithelial cellular marker CDH1 is enriched in organoids (Figure 2E) and the stroma cell marker HAND2 is enriched in stroma cells (Figure 2F).For the stromal cells, within 1-2 weeks colonies can be observed in the flasks.After 4-6 weeks, the first T25 flask of stroma cells will be confluent and can be passaged for expansion and cryopreservation.The passaged cells, seeded at 30-40% confluency, reach confluency within 1-2 weeks.We see variability in terms of organoid formation depending both on the amount of tissue in the menstrual fluid sample and individual donor variation in growth rates.From some samples, we obtain numerous organoids within 2-4 weeks, whereas few and more slowly growing organoids are formed from others.Often, initially, the organoid cultures will need to be replated/passaged despite the formation of only a few organoids due to the disintegration of Matrigel or to remove remaining non-epithelial cells (tissue debris, blood cells, and small presumed immune cells).One refinement to our method that would further enrich endometrial cells and decrease immune cells would be to employ the magnetic bead sorting described by Filby et al. (12) We have observed estrogen induction of IHH (Figure 3A), in organoid cultures (13), and indications of decidual response to estrogen, progesterone, and cAMP (EPC) treatment of stromal cells in terms of cell morphology (not shown) and induction of the decidual cell gene PRL (Figure 3B).Both of these responses are comparable to those observed in endometrial biopsy-derived cells.Other genes of interest that have been evaluated for estrogen response in organoids derived from endometrial biopsies include GREB1 and PGR (13).Similarly, IGFBP1 is another gene induced during decidualization (14).
All participants were between the ages of 18-35 years and had no known diagnoses of infertility, PCOS, or endometriosis.These menstrual fluid-derived cells exhibited inter-individual variability in hormonal responsiveness, as illustrated by the estrogen induction of IHH in organoids from three participants (Figure 3A) and the induction of the decidual marker PRL in stromal cells from the same three individuals (Figure 3B).This analysis illustrates that the relative responses between individuals differ in the two cell types.
For example, participant A showed the most robust PRL induction in stromal cells (Figure 3B), whereas participant A's IHH induction in organoids treated with estrogen was comparable to or less than that of the other two participants (Figure 3A).

Discussion
Model systems are crucial for experimental studies of human biology and disease.In the case of the human endometrium, well-  4. established stromal culture systems that recapitulated hormonedependent responses have been widely used (5).The culture of epithelial cells has proven more challenging, but the more recent development of organoid culture has led to an explosion of studies (7).Limitations of these systems include the wide variability in biological response characteristics between cultures derived from different individuals as well as the invasive procedure required to obtain endometrial samples for cell isolation.Utilizing menstrual fluid eliminates the need for endometrial biopsy, and greatly expands the breadth of potential donors, as the collection can be done without clinic visits.Ongoing studies will compare other characteristics, such as growth rates.Additionally, each participant can provide multiple samples, which will enable a longitudinal examination of the changes in the responsiveness of these tissues over time.Utilization of paired epithelial and stromal cultures from the same donor can be incorporated into studies as well.
The procedures employed to derive and maintain cultures are simple ones and can be done in any laboratory setting with access to basic mammalian cell culture equipment, supplies, and reagents.Therefore, we also think the procedure could be adapted for academic settings to serve the dual purpose of providing handson experience and training to life science undergraduate and graduate students while at the same time establishing cell lines with a broader representation of diverse communities.One limitation that could be a barrier for investigators is the costs of the reagents, especially for the Matrigel and for the additives that make up the organoid culture media.Recent work optimizing organoid culture media suggests the possibility of simplifying organoid ExM by omitting some of the additives or utilizing chemical inhibitors (15,16).Similarly, the development of extracellular matrix hydrogels used in place of or in combination with Matrigel is not only less expensive but can also result in organoids that more closely resemble uterine tissue (17).Menstrual fluid-derived cultures are a promising readily available source of biological samples for in vitro experimental studies.
Uterine responsiveness requires contributions of the signals from cells in both stroma and epithelium and the interaction between these signals.Current work is being undertaken to establish coculture conditions of epithelium and stroma to be more reflective of the in vivo condition (18).This approach will allow both homologous and heterologous coculture of epithelium and stroma to determine the factors regulating endometrial responsiveness.

A B
Relative responses of epithelial organoid and stromal cell cultures from three different individuals.(A) RT-PCR for IHH of RNA isolated from organoid cultures treated with vehicle (Veh) or estrogen (E2) for 6 days + 6 hours as described previously (13).Organoids were isolated from three different individuals (participants A, B, and C).(B) RT-PCR for the decidual cell marker PRL of RNA isolated from stromal cultures treated with vehicle (Veh) or 10 nM estradiol, 1 µM medroxyprogesterone acetate, and 100 µM cAMP (EPC) for 3 days to induce decidualization as described previously (14).Stromal cells isolated from the same three participants (A, B, and C) were used.Primer sequences are listed in Table 4. Significance was analyzed using paired t-tests in GraphPad Prism with *, p<0.05.

FIGURE 1
FIGURE 1 Isolation and culture of endometrial stromal cells and epithelial organoids from menstrual fluid samples.Participants collect menstrual fluid in a menstrual cup and provide it to the clinical center.Endometrial tissue fragments are digested with proteinases and stroma cells are separated from epithelial cell fragments.Stroma cells are separated from blood cells on a Ficoll gradient and cultured as adherent monolayers.Epithelial cells are cultured in Matrigel, forming organoids.

TABLE 1 Continued
: the remaining steps where the sample is exposed (open container) are performed in a biological safety cabinet 3. Transfer menstrual fluid from the specimen container into a 50 mL tube.Note the approximate volume of menstrual fluid.Rinse the container with cold PBS+p/s/f and transfer to a 50 mL tube.
*N2 and B27 are omitted from hormone treatments as they contain some progesterone.Note3a.If the original sample volume is greater than 15 mL, split the sample so that each 50 mL tube contains material from 5-7 mL of sample.
21. Collect cells from the interface, add a warm HESC culture medium, and centrifuge at 400 RCF for 10 minutes.