Simple and Rapid Model to Generate Differentiated Endometrial Floating Organoids

Materials and reagents

Biological materials

1. Human endometrial tissues (0.5 × 1.0 cm) (generated in patients who underwent assisted reproductive technologies at the Reproductive Medicine Unit, Gynecology and Obstetrics Department of the International Evangelical Hospital, Genova, Italy); samples were collected during the secretory phase of the cycle, without hormonal stimulation, with a Pipelle catheter.

Reagents

1. Collagenase Type V (Sigma-Aldrich, catalog number: C9263-100MG)

2. Dispase II (Sigma-Aldrich, catalog number: D4693-1G)

3. DMEM/F12 (Euroclone, catalog number: ECM0095L)

4. Fetal bovine serum (FBS) (Gibco, catalog number: 10270106)

5. DNase I (Sigma-Aldrich, catalog number: DN25-100MG)

6. PBS 1× (Euroclone, catalog number: ECB4004L)

7. Antibiotics Pen/Strep 100× (Euroclone, catalog number: ECB 3001D)

8. L-Glutamine 200 mM (Euroclone, catalog number: ECB3000D)

9. Ethanol (Sigma-Aldrich, CAS number: 64-17-5)

10. Parafilm (AMCOR, catalog number: PM-996)

11. Matrigel^TM growth factor reduced (GFR) (Corning, catalog number: 354230)

12. 16% formaldehyde solution (Thermo Scientific, catalog number: 28908)

13. 0.1 M glycine (Sigma-Aldrich, CAS number: 56-40-6)

14. Normal goat serum (Sigma-Aldrich, catalog number: G9023)

15. Triton X-100 (Sigma-Aldrich, CAS number, 9036-19-5)

16. Rabbit anti-vimentin (EPR3776) antibody (Abcam, catalog number: ab92547)

17. Mouse anti-cadherin (HECD-1) antibody (Abcam, catalog number: ab1416)

18. Mouse anti-acetyl-α-tubulin (Lys40) (6-11B-1) antibody (Cell Signaling, catalog number: 12152)

19. Goat anti-mouse IgG (H-L) Alexa Fluor^TM 568 antibody (Invitrogen, catalog number: A-11004)

20. Goat anti-rabbit IgG (H+L) Alexa Fluor^TM 647 antibody (Invitrogen, catalog number: A21245)

21. Sytox Blue nucleic acid stain (Invitrogen, catalog number: S11348)

22. Cell recovery solution (Corning, catalog number: 354253)

23. Aurum Total RNA mini kit (Bio-Rad, catalog number: 7326820)

24. Sodium acetate (Sigma-Aldrich, CAS number, 127-09-3)

25. Calcium acetate (Sigma-Aldrich, CAS number, 62-54-4)

26. Edu DetectPro Cell Proliferation Imaging kit (Baseclick GmbH, catalog number BCK-EduPro-IM488)

Solutions

1. Collagenase V stock solution 100× (see Recipes)

2. Dispase II stock solution 40× (see Recipes)

3. DNase I stock solution 100× (see Recipes)

4. DMEM/F12 basal medium (see Recipes)

5. 10% FBS-DMEM/F12 complete medium (see Recipes)

6. Enzyme digestion solution (see Recipes)

7. 4% Formaldehyde stock solution (see Recipes)

8. 1% Formaldehyde working solution (see Recipes)

9. Blocking-permeabilizing solution 1× (see Recipes)

Recipes

1. Collagenase V stock solution 100×

Dissolve 100 mg of collagenase type V powder in 2.5 mL of PBS w/o Ca²⁺ and Mg²⁺ and sterilize through a 0.22 μm filter. Store aliquots at -20 °C.

2. Dispase II stock solution 40×

Prepare a 20 mL solution of 10 mM NaAc (pH 7.5) with 5 mM CaAc and sterilize by filtration with a 0.22 μm filter. Dissolve 1 g of Dispase II in 10 mL of the filtered NaAc + CaAc solution.

3. DNase I stock solution 100×

4. DMEM/F12 basal medium

5. 10% FBS-DMEM/F12 complete medium

6. Enzyme digestion solution

7. 4% formaldehyde stock solution

8. 1% formaldehyde working solution

9. Blocking-permeabilizing solution 1×

Laboratory supplies

1. 1.5 mL microcentrifuge tubes (Eppendorf, catalog number: 0030120086)

2. 15 and 50 mL conical tubes (Euroclone, catalog numbers: ET5015B, ET5050B)

3. 5 and 10 mL sterile pipettes (Euroclone, catalog numbers: EPS05N, EPS10N)

4. 10, 200, and 1,000 μL sterile tips (Euroclone, catalog numbers: ECTD00010, ECTD00200, ECTD01005)

5. 100 μm cell sieves (100 μm cell strainer) (Corning, catalog number: 431752)

6. 35, 60, and 100 mm Petri dishes (Euroclone, catalog numbers: ET2035, ET2060, ET2100)

7. Disposable stainless steel blade scalpels No. 23 (GIMA, catalog number: 27067)

8. Sterile tweezers

9. 3 mL plastic Pasteur pipette (Copan, catalog number: 200)

10. Empty 96-well rack of 10 μL tips (Eppendorf ep Dualfilter T.I.P.S., catalog number: 0030078500)

11. Nunc non-treated multidish 4 wells (Thermo Scientific, catalog number: 179820)

Equipment

1. Refrigerator 2–8 °C (Liebherr, model: SRFvg 3501-20A-001)

2. Freezer -20 °C (Liebherr, model: GP 2733 Index 20D/001) and -80 °C (Forma Scientific, model: 925)

3. Water bath (GFL)

4. Biological safety cabinet (BioAir Top-Safe, model: 1.8)

5. Laboratory centrifuge with rotor for 10- and 50-mL conical tubes (Eppendorf, model: 5810 R)

6. Laboratory centrifuge with rotor for 1.5- and 2.0-mL tubes (Eppendorf, model: 5415 R)

7. Water jacketed CO₂ incubator (Forma Scientific Instruments, model: 3111)

8. Automated cell counter (Bio-Rad, model: TC20)

9. Digital inverted microscope (Leica Microsystems, model: DMIL) equipped with camera (Leica Microsystems, model: ICC50HD)

10. Confocal microscope (Leica Microsystems, model: Stellaris 8 STED)

Procedure

A. Isolation of endometrial gland elements

1. Perform all procedures in a biosafety cabinet to preserve sterility.

2. Prepare Recipes 1, 2, and 3; sterilize all solutions by filtration through a 0.2 μm filter and store the aliquots at -20 °C.

3. Prepare a culture Petri dish (100 mm) with 10 mL of sterile PBS solution supplemented with 0.1 mL of 100× Pen/Strep (final concentration: penicillin 100 U/mL, streptomycin 0.1 mg/mL) to clean the endometrial tissue collected by Pipelle.

Note: Pipelle biopsies should preferably be processed on the day of collection. If immediate processing is not possible, store the sample at 4 °C to preserve vitality.

4. Transfer the sample tissue to the Petri dish and gently agitate to remove clots and debris (Figure 1A).

Figure 1. Dissociation of an endometrial Pipelle biopsy. (A) Petri dish containing an endometrial Pipelle biopsy after cleaning (step A4). (B) Brightfield representative image of gland tubular elements after dissociation (step A11); white arrows mark the optimal size of the glandular fragments, and black arrows mark too-long glandular elements. (C) Magnification of gland elements.

5. Transfer the washed tissue into a new Petri dish containing 10% FBS-DMEM/F12 complete medium (see Recipe 5) and cut the tissue with a sterile steel blade scalpel into small fragments (0.1 × 0.1 cm).

6. Transfer the dissected tissue into a 50 mL sterile centrifuge tube.

7. Centrifuge at 146 rcf for 8 min.

8. Prepare enzyme digestion solution following Recipe 6. Recalibrate the volume of enzyme digestion in relation to the size of the biopsy (larger tissue fragments may require a larger digestion volume).

9. Carefully remove the supernatant without disturbing the tissue pellet using a pipette.

10. Add 4 mL of enzymatic digestion solution to the tissue pellet and incubate at 37 °C with gentle shaking to ensure uniform exposure of the tissue to the enzyme solution.

11. After 20 min, check that digestion has taken place by observing the presence of free glandular elements. Take up 20 μL of the sample, place it on a 35 mm Petri dish, and observe under the inverted microscope. Continue incubation at 10-min intervals until digestion is complete (Figure 1B).

Caution: The time of incubation is a critical step. Monitoring the digestion is necessary since the enzyme can cause cell death, but insufficient digestion limits the release of glandular elements.

12. Terminate incubation by diluting the sample with 4 mL of 10% FBS-DMEM/F12 complete medium to minimize further disaggregation.

13. Leave the tube to stand for 2 min to allow undigested fragments to sediment.

14. Filter the supernatant through a 100 μm cell sieve and wash with 5 mL of DMEM/F12 basal medium (see Recipe 4).

15. Invert the sieve over a 60 mm Petri dish and backwash the glandular elements from the membrane by strongly pipetting 5 mL of DMEM/F12 basal medium using a plastic Pasteur pipette. Transfer the backwashed glandular elements to a 15 mL tube and pellet by centrifuging at 500× g for 5 min.

16. Resuspend the pellet in 1 mL of DMEM/F12 basal medium and gently pipette up and down several times to partially dissociate the glands.

17. Test the gland fragmentation by observing a test droplet under the microscope; if long tubular fragments are still evident, continue to dissociate. In Figure 1B, the optimal (white arrow) dimensions of the gland fragments are indicated.

18. Transfer to a 1.5 mL microcentrifuge tube and pellet by centrifugation at 500× g for 8 min.

19. Remove the supernatant and estimate the volume of the pellet.

20. Flick the tube to loosen the pellet and place it on ice for 2–5 min.

B. Preparation of Parafilm mold

1. Cut 3 × 4 cm Parafilm strips and store them at -20 °C (low temperature increases parafilm stiffness, favoring the subsequent steps, but it is not required).

2. Place the Parafilm strip on the top of an empty rack of a 96-well box of 10 μL tips (Figure 2B).

Figure 2. Preparation of the Parafilm mold. (A) Material needed to prepare the Parafilm molds. (B) Formation of round depressions on the Parafilm strip (steps B2–3). (C) Cutting of the Parafilm strip (step B4).

3. Press the Parafilm strip into the rack’s holes using the bottom of a 1.5 mL tube to create round depressions (Figure 2B).

4. Cut the Parafilm strip to obtain 2–4 depression spots (Figure 2C).

5. Sterilize the Parafilm strip by immersion in 100% ethanol for 30 min.

6. Dry the Parafilm strips by leaving them in a new Petri dish in a biosafety cabinet.

7. Use the Parafilm molds directly for the experiment or store them at 4 °C for further use.

C. Floating organoid seeding

1. Put a Parafilm strip into a sterile 60 mm Petri dish at room temperature.

2. Thaw a Matrigel^TM aliquot at 4 °C and then put it on ice.

3. Resuspend the pellet (from step A18) in DMEM/F12 basal medium 1:1 (v/v) on ice (i.e., 20 μL of gland fragments + 20 μL of DMEM/F12 basal medium = 40 μL of suspension).

4. Mix the resuspended pellet with ice-cold Matrigel^TM by adding 10× volume to the suspension (i.e., 40 μL of suspension + 360 μL Matrigel^TM).

Notes:

1. This step is critical and not always standardizable. The size of the gland fragments may show differences between samples and can be affected by enzymatic digestion; moreover, the quantification and the resuspension of the pellet (step C3) are critical and sometimes difficult to estimate. It is recommended to place a test droplet (suspension/Matrigel^TM) into a Petri dish and observe it under an inverted microscope to optimize sample concentration (Figure 3A).

2. If the tissue fragment concentration is too dense, further dilute the tissue sample with media and Matrigel^TM in a 1:9 ratio. In our experience, we also diluted the sample only with DMEM/F12 basal medium, modifying the ratio of suspension/Matrigel^TM and therefore the stiffness of droplets. The minimum ratio we tested was 20:80 of diluted sample to Matrigel^TM. In any case, floating organoids also developed properly in a 1:4 ratio of sample to Matrigel^TM.

Figure 3. Floating organoid development in Matrigel^TM droplets. (A) Representative Matrigel^TM droplet within gland tubular fragments at the time of seeding (day 0). The yellow dotted line marks the edge of the droplet. The green line marks the optimal density of the glandular fragments into Matrigel^TM droplets. The red line marks an area with an excessive density of glandular elements. (B–D) Representative images of floating organoids grown after 3 (B), 15 (C), and 25 (D) days of culture seeded at high gland fragment density. (B’–D’) Representative images of floating organoids originated by a culture seeded at low gland fragment density after 3 (B’), 13 (C’), and 25 (D’) days in vitro. Scale bars: 100 μm.

5. Dispense 30 μL of droplets of gland fragments (suspension)/Matrigel^TM into each depression of the Parafilm mold, avoiding generating bubbles.

6. Incubate the plate at 37 °C with 5% CO₂ for 30 min.

7. Remove the Matrigel^TM droplets from the Parafilm mold: using a 5 mL pipette, gently spurt 1–2 mL of 10% FBS DMEM/F12 complete medium sideways on the droplets.

8. Remove and throw away the Parafilm support and add 10 mL of 10% FBS DMEM/F12 complete medium per dish.

9. Check the floating droplets under a microscope and place the Petri dishes in the incubator at 37 °C with 5% CO₂ (see Figure 3).

10. Change the medium every 3–4 days; carefully remove 8–9 mL of the old medium with a 5 mL pipette, making sure not to aspirate the floating droplets, and replace with the same volume of fresh 10% FBS DMEM/F12 complete medium.

11. After 20–40 days in culture, organoids are mature enough for subsequent analysis (Figure 3D, D’ and Figure 4A, B).

Figure 4. Floating organoid development in Matrigel^TM droplets. Representative images of floating organoids (low-density) grown for 34 (A) and 45 (B) days in culture; magnification of organoid structures delimited by the boxes. Scale bar: 100 μm.

D. Immunofluorescence of 3D floating organoids

1. Perform all procedures in a biosafety cabinet to preserve sterility.

2. Put 400 μL of PBS 1× per well of a new 4-well multidish.

3. Transfer floating organoids into a 4-well multidish, with one Matrigel^TM droplet per well, using a plastic Pasteur pipette.

4. Wash floating organoids with PBS 1×.

Note: To wash floating organoids, use a 1 mL blue tip. Tilt the plate toward you and gently remove the media, always monitoring the movement of the organoid, which will move downward following the reduction of the liquid (like a fish in a pool). Discard the medium into a 2 mL tube; this way, you can eventually recover the sample accidentally sucked by the tip.

5. Fix with 1% formaldehyde solution (see Recipe 8) for 15 min.

6. Carefully remove formaldehyde solution.

7. Wash with PBS 1×.

Pause point: Fixed and unstained samples can be stored for months at 4 °C in sterile PBS. Ensure that the PBS does not evaporate too much; in case of long storage, rehydrate the sample.

8. Incubate with 200 μL of 0.1 M glycine for 10 min.

9. Remove glycine.

10. Add 200 μL of blocking-permeabilizing solution (see Recipe 9), at room temperature for at least 2.5 h.

11. Remove the blocking-permeabilizing solution.

12. Incubate overnight at 4 °C in 100 μL of blocking-permeabilizing solution with primary antibody [anti-vimentin (EPR3776), anti-cadherin (HECD-1), and anti-acetyl-α-tubulin (Lys40) (6-11B-1)] diluted 1/100. Tilt the dish by placing it on a support (i.e., the cap of a 50 mL tube).

13. Remove the solution and wash the floating organoids with PBS 1× for 15 min.

14. Repeat step D13 three times.

15. Incubate overnight at 4 °C in 100 μL of blocking-permeabilizing solution with the appropriate fluorescent conjugated secondary antibodies (AF568 anti-mouse or AF647 anti-rabbit antibody) and Sytox nuclear dye, all diluted 1/200.

16. Remove the solution and wash the floating organoids with PBS 1× for 15 min.

17. Repeat step D16 three times.

18. Store your sample at 4 °C in PBS in a 4-well multidish sealed with Parafilm until analysis by confocal immunofluorescence microscopy.

Caution: Immunofluorescence staining is degraded by long storage. We recommend performing confocal acquisition within one week of sample staining.

E. Extraction of total RNA from floating organoids

1. Put 400 μL of PBS 1× per well of a new 4-well multidish.

2. Transfer floating organoids into a 4-well multidish, with one floating organoid per well, using a plastic Pasteur pipette.

3. Wash floating organoids with PBS 1×.

4. Transfer three floating organoids into a 1.5 mL microcentrifuge tube.

5. Remove PBS 1× and add 250 μL of cell recovery solution and place on ice for 60 min.

6. Mix with pipette and spin at 600× g for 6 min at 4 °C.

7. Remove the medium and wash the pellet with 1 mL of PBS 1×.

8. Spin at 600× g for 6 min at 4 °C.

9. Add 350 μL of lysis buffer from the Aurum Total RNA mini kit.

10. Proceed with the RNA extraction following the kit instructions.

Note: RNA extraction yield is generally low; in our experience, by pulling three floating organoids, we obtained approximately 2.5 μg of mRNA.

Validation of protocol

The expression of both epithelial and stromal markers in floating organoids was established through immunofluorescence staining and qRT-PCR analysis.

Organoids developed into floating Matrigel^TM droplets form round or elongated empty structures composed of monolayers of epithelial cells, integrated with stromal cells, distributed between or into the epithelial structures. This was demonstrated by the presence of cells expressing the epithelial markers acetyl-α-tubulin and E-cadherin and the stromal marker vimentin (Figures 5 and 6). For further immunofluorescence analysis, please refer to Figures 3 and 4, Videos, and Supplementary figures of [14].

Figure 5. Immunofluorescence staining of epithelial and stromal cells in floating organoids. (A) Whole-mount immunostaining for acetyl-α-tubulin (red) and vimentin (cyan) expression in floating organoids. Scale bar: 100 μm. (B–D) Magnifications of the selected zones delimited by the boxes. Scale bar: 10 μm.

Figure 6. Immunofluorescence staining of epithelial and stromal cells in floating organoids. (A) Whole-mount immunofluorescence representative image of a floating organoid pulsed overnight with 5-EdU stained for the epithelial marker E-cadherin (red), the stromal marker vimentin (cyan), the proliferative EdU-positive cells (green), and Sytox (blue) nuclei. Scale bar: 10 μm. (B) Magnification of stromal and epithelial interaction in a floating organoid. Scale bar: 10 μm.

Expression of endometrial and decidual genes was analyzed by qRT-PCR in comparison with conventional organoids before and after in vitro hormonal differentiation. Results support that endometrial organoids grown by following the protocol described here increase the differentiated gene expression profile (progesterone receptor, osteopontin, IGF1-binding protein 1, and FOXO1, among others). For detailed results of qRT-PCR experiments, please refer to Figures 6 and 7 and Supplementary figures of [14].

This protocol (or parts of it) has been used and validated in the following research article: Bajetto et al. (2025) Floating endometrial organoid model recapitulates epithelial-stromal cell interactions in vitro [14].

General notes and troubleshooting

General notes

1. Matrigel^TM should always be handled on ice since it can degrade and solidify at room temperature. Thaw a Matrigel^TM unopened bottle in an ice bucket stored overnight at 4 °C. Make single-use aliquots of Matrigel^TM and store them at -20 °C.

2. Floating organoids are almost transparent, breakable structures. All steps must be performed carefully, always monitoring the number of organoids present in the Petri dishes. Do not use the vacuum pump, only pipette tips. Keeping a test tube for media changes and washes will be useful for recovering any accidentally aspirated sample. Place a black sheet or tray under the dishes to form a black background to improve organoid visibility.

Troubleshooting

Problem 1: Fixation with formaldehyde solution influences the Matrigel^TM droplet solidity.

Possible cause: Formaldehyde solution and long-term culture damage the Matrigel^TM structure, while organoids grown into the droplets strengthen the structure. After fixation, floating droplets can break into several units, each of which contains some organoids.

Solution: The broken units can be stained by immunofluorescence and included in paraffin or cryostat embedding medium. If the organoids are well developed, you can use 4% formaldehyde solution to fix them.

Problem 2: Concentration of gland fragments in the Matrigel^TM droplet is too high.

Possible cause 1: Sample is not properly mixed with the Matrigel^TM droplets.

Solution 1: Mix the tissue fragments several times with the Matrigel^TM on ice. Avoid the formation of bubbles by using a pipette of a smaller volume than that of the sample.

Possible cause 2: Incorrect estimate of pellet volume.

Solution 2: Spot a test Matrigel^TM/gland fragment droplet on a Petri dish and observe it under an inverted microscope. Optimize the sample concentration by adding medium and Matrigel^TM to reach a suspension/Matrigel^TM ratio of 1:9.

Problem 3: Restricted ability to sustain long-term in vitro propagation.

Possible cause: The acquisition of the differentiated status reduces the stemness and limits organoid propagation to one or two passages (when disassembled, they are no longer able to grow).

Solution: Gland fragments isolated from the biopsy can be cryo-stored for further experiments.

Acknowledgments

Authors’ contributions: A.B., T.F.: Conceptualization. A.B., A.P., A.C., B.F.T., S.T., F.B.: Investigation. A.B., T.F.: Writing—Original Draft. All authors: Writing—Review & Editing. A.B., T.F.: Supervision.

The authors are grateful to all women who donated tissue for research, to Monica Gatti, Valerio Pisaturo, Denise Colia, Elena Pastine, and Mauro Costa, International Evangelical Hospital (Genova, Italy) for providing Pipelle biopsies, and to Dr. E. Gatta and DIFILAB laboratory (Univ. of Genova) for the Leica Stellaris 8 STED confocal microscope use.

The protocol described here was based on the previous original research paper: Bajetto et al. (2025), Exp Cell Res. 452(1): 114749. Doi:10.1016/j.yexcr.2025.114749 [14].

Competing interests

The authors declare that they have no competing interests.

Ethical considerations

All tissue samples used in this study were obtained with written informed consent from all participants, in accordance with the Declaration of Helsinki 2000 and with institutional Liguria Ethical Committee approval (approval n° P.R. 406REG2017).

References

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