Establishing Endometrial Assembloids to Model Human Embryo Implantation in vitro

Materials and reagents

1. Human endometrial biopsies (see Barros et al., 2016)
2. Collagenase from clostridium histolyticum type IA (Sigma-Aldrich: C9891)
3. Deoxyribonuclease I (DNase I; Worthington Biochemical Corporation, Lorne Laboratories)
4. RNAlater® (Sigma-Aldrich: R0901)
5. Dulbecco’s Modified Eagle Medium/ Ham’s F-12 (DMEM/F12) (1:1), HEPES with L-glutamine with phenol red (DMEM/F12 medium; Gibco^TM:31330038)
6. Dulbecco’s Modified Eagle Medium/ Ham's F-12 (DMEM/F12) (1:1), HEPES with L-glutamine, phenol free (Additive-free medium; Gibco^TM: 11039047)
7. Advanced Dulbecco’s Modified Eagle Medium/ Ham's F-12 (Advanced DMEM; Gibco^TM:12634010)
8. Charcoal (Sigma-Aldrich: C-9157)
9. Dextran (Sigma-Aldrich: D8821-10G)
10. Foetal bovine serum heat inactivated (FBS; Gibco^TM: 10500064)
11. Insulin, human recombinant (Sigma-Aldrich: 91077C)
12. Trypsin-EDTA solution (0.25%, sterile-filtered) (Gibco^TM: 25200)
13. L-Glutamine (Gibco^TM: 25030)
14. A83-01 Synthetic ≥98% (HPLC purified) (Sigma-Aldrich: SML0788)
15. Antibiotic-Antimycotic (100×) 10,000 units/ ml of penicillin, 10,000μg/mL of streptomycin, and 25μg/mL of Fungizone™ (Gibco^TM: 15240)
16. B-27 Supplement (50×), minus vitamin A, (50×) (Gibco^TM: A3353501)
17. Human epidermal growth factor (Animal free) (EGF; Peprotech: AF-100-15)
18. Human fibroblast growth factor 10 (FGF-10; Peprotech: 100-26)
19. Human hepatocyte growth factor (HGF; Peprotech: 100-39)
20. N-2 Supplement (100X) (Gibco^TM: 17502048)
21. Nicotinamide (Sigma-Aldrich: N0636)
22. Human NOGGIN (Mammalian) (Peprotech: 120-10C)
23. Human R-Spondin-1 (Peprotech: 120-38)
24. N-Acetyl-L-Cysteine (NAC; Sigma-Aldrich: A7250)
25. TrypLE Select Enzyme 10× (Gibco^TM: A1217701)
26. Insulin-Transferrin-Selenium-Ethanolamine (100×) (ITS -X; Gibco^TM: 51500-056)
27. MEM Non-Essential Amino Acids Solution (100×) (Gibco^TM: 11140-035)
28. MEM Amino Acids Solution (50×) (Gibco^TM: 11130-036)
29. Formaldehyde 4 % formalin 10 % buffered (VWR: 9713.1000)
30. 8-Bromoadenosine 3’,5’-cyclic monophosphate (cAMP) sodium salt (HPLC) (Sigma-Aldrich: B7880)
31. β-estradiol (Sigma-Aldrich: E2758)
32. Medroxyprogesterone 17-acetate (MPA) (Sigma-Aldrich: M1629)
33. Matrigel (Growth Factor Reduced) (Corning®: 356252)
34. PureCol® EZ Gel solution (Sigma-Aldrich: 5074)
35. Dasatinib (Cell Signaling Technology: 9052S)

Equipment

1. Costar 48 Well Clear TC-Treated Multiple Well Plates Individually Wrapped Sterile (Corning®: CLS3548)
2. 15 ml CELLSTAR® tubes (Greiner Bio-One: 188261)
3. 50 ml CELLSTAR® tubes (Greiner Bio-One: 227270)
4. 7 ml Bijoux tubes (Greiner Bio-One: 189176)
5. 1.5 ml centrifuge tubes (Sarstedt: 72.690)
6. FisherBrand^TM Sterile cell strainers, 40 µm (Fisher Scientific: 11587522)
7. 20 ml Luer syringe (BD: 300613)
8. 60 ml Luer syringe (BD: 309653)
9. Minisart™ NML Syringe Filters, Sterile (Sartorius: 16534-K)
10. FisherBrand^TM Soda lime glass Pasteur pipettes (Fisher Scientific: 1156-6963)
11. 25 cm² CELLSTAR® culture flasks (Greiner Bio One: 690175)
12. 75 cm² CELLSTAR® culture flasks (Greiner Bio One: 658175)
13. 5 ml serological pipettes (Greiner Bio One: 606180)
14. 10 ml serological pipettes (Greiner Bio One: 607180)
15. Pipette controller (Integra Biosciences^TM: 155015)
16. Vacuum-driven 0.22 μm filtration system (EMD Millipore: SCGPT05RE)
17. Walker Class II cell culture microbiological safety cabinet (Walkers Safety Cabinets, model: Class II MSC)
18. Fisherbrand^TM FB 70155 aspirator (Thermo Fisher Scientific:11533485)
19. Grant Instruments water bath (Grant Instruments, model: OLS200)
20. Thermo Scientific Heracell^TM 150i humidified tissue culture incubator (set at 37 °C and 5% CO2) (Thermo Fisher Scientific: 51026280)
21. Bright-field Leica DMIL microscope (Leica Microsystems, model: Leica DMIL)
22. Stereo Leica MZ12 microscope (Leica Microsystems, model: Leica MZ12)
23. EVOS® FL Auto time-lapse microscope and EVOS® Onstage Incubator (Thermo Fisher Scientific, models: AMAFD1000, AMC1000)
24. Eppendorf refrigerated micro-centrifuge (Eppendorf, model: Eppendorf 5424 R)
25. Eppendorf refrigerated bench top centrifuge (Eppendorf, model: Eppendorf 5810 R)

Procedure

Notes: 

1. Appropriate ethical and biosafety approvals should be obtained according to local rules.
2. Ensure sterility for all cell culture procedures. Work in a Class II microbiological safety cabinet and ensure full aseptic technique.
3. All centrifugation steps performed at 600 × g 4 °C for 6 minutes unless stated otherwise.

1. Processing of endometrial biopsies and primary cell cultures

Note: For full details on the isolation and primary culture of various cell types from whole human endometrial biopsies please refer to Barros et al, (2016). 

Passage of stromal cells

Notes: 

1. Before starting, pre-warm stromal cell growth medium and 0.25 % Trypsin-EDTA in the 37 °C water bath.
2. Inspect stromal culture to confirm confluency using a light microscope.

1. Aspirate medium from cell culture flasks using a vacuum aspiration line.
2. Add 5 ml of PBS to flask and wash cells by gently tilting the flask. Aspirate PBS.
3. Add 1 ml of 0.25 % Trypsin-EDTA to flask, tilting the flask to ensure it covers the entire surface. Incubate for 5 mins at 37 °C.
4. Dislodge any remaining attached cells in the flask by firmly tapping the side of the flask with the palm of your hand.
5. Observe under a light microscope to ensure all cells are free floating.
6. Add 9 ml of stromal cell growth medium to the flask and collect the cells in medium.
7. Transfer the cell suspension to a 14 ml tube and centrifuge for 5 mins at 300 × g.
8. While waiting for the centrifuge, prepare the required flasks for continuing the culture. For 1:2 passage, distribute 5 ml media into each flask and gently tilt/swirl to ensure it completely covers the culture surface of the flasks.
9. Aspirate supernatant and thoroughly re-suspend the cell pellet in 10 ml of media. Distribute equally between the pre-wetted flasks. Transfer new flasks to the cell culture incubator. Replace stromal cell growth medium every 2-3 days. Passage cells once they reach 80-90 % confluency. Assembloids should be established using 80-90% confluent P1 stromal cells.

Endometrial organoid culture

Notes: 

1. This procedure has been adapted from Turco et al, (2017);
2. Before starting, thaw Matrigel for at least 1 hour prior to organoid culture at 4 °C and keep on wet ice while in use in this procedure.
3. Pre-warm Expansion medium in the 37 °C water bath.

1. Following digestion of a fresh endometrial biopsy (refer to Barros et al., 2016), pass the digested sample through a 40 µm cell sieve into a labelled 50 ml tube, then wash 5 ml of stromal cell growth medium through the cell sieve. (This 50 ml tube contains everything except epithelial cell clumps)
2. Invert the cell sieve over a new labelled 50 ml tube and wash with 10 ml of additive-free DMEM/F12 medium through the strainer to collect epithelial cells.
3. Centrifuge epithelial tube(s) at 300 × g for 5 min.
4. Carefully remove supernatant and resuspend the epithelial pellet in 1 ml of additive-free DMEM/F12 medium. Gently pipette several times to dissociate the pellet.
5. Transfer this into a 1.5 ml microcentrifuge tube and pellet by centrifugation.
6. Remove supernatant, estimate volume of pellet, and add ice-cold Matrigel at a ratio of 1:20. Gently resuspend the pellet in the Matrigel to minimise the introduction of bubbles to the gel. Immediately place back on ice.
7. Aliquot 20 µl drops of the Matrigel-cell suspension into the centre of wells in a 48-well plate. Incubate at 37 °C in a cell culture incubator for 20 mins to cure.
8. Gently add 200 µl expansion medium. Return plates into the cell culture incubator.
9. Replace expansion medium every 2-3 days. Examine for the appearance of gland-like hollow structures using a light microscope. Passage organoids every 7-10 days for expansion.

Passage of Organoid cultures

Notes: 

1. This procedure is adapted from Turco et al (2017)
2. Before starting, thaw Matrigel for at least 1 hour prior to organoid culture at 4^oC and keep on wet ice while in use in this procedure.
3. Pre-cool additive-free DMEM/F12 medium on wet ice.
4. Pre-cool a bench-top microcentrifuge to 4 °C.
5. Pre-warm expansion medium in the 37 °C water bath.

1. Scrape each Matrigel organoid droplet using a 1000 µl pipettor with a pipette tip attached until dislodged and collect into 1.5 ml microcentrifuge tubes. Pool 6-8 droplets per tube and pellet by centrifugation.
2. Carefully remove supernatant and resuspend the epithelial pellet in 200 µl of additive-free DMEM/F12 medium. Keep tubes on wet ice. In turn, pipette each organoid suspension 300× to liberate the organoids from Matrigel.
3. Add 1 ml additive-free DMEM/F12 medium to each tube and pellet by centrifugation.
4. Carefully remove supernatant and resuspend the epithelial pellet in 200 µl of additive-free DMEM/F12 medium. Keep tubes on wet ice. In turn, vigorously pipette each organoid suspension 80× to break up the organoids.
5. Add 1 ml additive-free DMEM/F12 medium to each tube and centrifuge 600 × g for 6 minutes.
6. Remove supernatant, estimate volume of pellet and add ice-cold Matrigel at a ratio of 1:20. It is expected that the cells would have doubled in number therefore it is advised to use double the volume of Matrigel used in P0. Gently resuspend the pellet in the Matrigel to minimise the introduction of bubbles to the gel. Immediately place back on ice while obtaining and labelling a 48-well plate.
7. Aliquot 20 µl drops of the Matrigel-cell suspension into the centre of wells in a 48-well plate. Incubate at 37 °C in a cell culture incubator for 20 mins to cure.
8. Gently add 200 µl expansion medium. Place new plates in the cell culture incubator.
9. Replace expansion medium every 2-3 days. Examine for the appearance of gland-like hollow structures using a light microscope. Passage organoids every 7-10 days. Assembloids should be established using organoids when ready for passaging to P2.

1. Establishment of assembloid cultures

Notes: 

1. Before starting, pre-cool additive-free DMEM/F12 medium on wet ice.
2. Pre-cool a bench top micro-centrifuge to 4 °C. 
3. Pre-warm expansion medium, stromal cell growth medium and 0.25 % Trypsin-EDTA in the 37 °C water bath. 

1. Passage organoids:
   1. Scrape each Matrigel organoid droplet using a p1000 pipette with a pipette tip attached until dislodged and collect into 1.5 ml microcentrifuge tubes. Pool 6-8 droplets per tube and pellet by centrifugation
   2. Carefully remove supernatant and resuspend the epithelial pellet in 200 µl of additive-free DMEM/F12 medium. Keep tubes on wet ice. In turn, pipette each organoid suspension 300× to liberate the organoids from Matrigel.
   3. Add 1 ml additive-free DMEM/F12 medium to each tube and pellet by centrifugation.
   4. Carefully remove supernatant and resuspend the epithelial pellet in 200 µl of additive-free DMEM/F12 medium. Keep tubes on wet ice. In turn, vigorously pipette each organoid suspension 80× to dissociate the organoids into clumps.
   5. Add 1 ml additive-free DMEM/F12 medium to each tube and pellet by centrifugation.
   6. Place on wet ice
2. Passage stromal cells:
   1. Aspirate medium from cell culture flasks using vacuum.
   2. Add 5 ml of PBS to flask and wash cells by gently tilting the flask. Aspirate PBS.
   3. Add 1 ml of 0.25% Trypsin-EDTA to flask, tilting the flask to ensure it covers the entire surface. Incubate for 5 mins at 37 °C.
   4. Dislodge any remaining attached cells in the flask by firmly tapping the side of the flask with the palm of your hand.
   5. Observe under a light microscope to ensure all cells are free floating.
   6. Add 9 ml of stromal cell growth medium to the flask and pipette several times over the surface.
   7. Transfer the cell suspension to a 14 ml tube and centrifuge for 5 mins at 300 × g.
   8. Remove supernatant and thoroughly re-suspend the cell pellet in 1 ml of additive-free DMEM/F12 medium.
   9. Count stromal cells.
      1. Mix 10 µl stromal cell suspension with 10 µl Trypan Blue solution in a 0.5 ml microcentrifuge tube.
      2. Prepare a haemocytometer slide and add 10 µl to a chamber.
      3. Count the number of live cells in each of 4 outside squares.
      4. Average the counts from the 4 outside squares, multiply by 2 (dilution factor) and multiply by 10⁴.
3. Calculate the number of assembloid wells that can be prepared.
   1. EnSC and gland organoid pellets are mixed at an optimal ratio of approximately 1:1 (v/v). This equates to 5 × 10⁴ stromal cells and epithelial organoid fragments passaged at a ratio of 1:2-3 per assembloid culture. For example, 12 organoid Matrigel droplets and 1.5×10⁶ stromal cells would provide enough material for 24 assembloid droplets, with 3×10⁵ stromal cells remaining.
4. Mix the epithelial and stromal cells together as calculated and pellet by centrifugation.
5. Carefully remove the supernatant and add ice cold collagen hydrogel at a volume of 20 µl per assembloid to be plated. For example, for 24 assembloid droplets, a volume of 480 µl would be added.
6. Resuspend the cell pellet in the collagen hydrogel gently, being careful not to introduce any bubbles.
7. Aliquot in 20 µl volumes into a 48-well plate, one droplet per well, and allow the gel to cure in the cell culture incubator for 45 min.
8. Add 200 µl expansion medium supplemented with 10 nM E2 and place into the incubator.
9. Refresh the medium every 48 hours.
10. For decidualization experiments:
    1. Assembloid cultures are grown in expansion medium supplemented with E2 for 4 days to allow for growth and expansion.
    2. Assembloids are then either harvested or decidualized for a further 4 days in minimal differentiation medium. Again, the medium is refreshed every 48 hr and spent medium stored for further analysis.
    3. For tyrosine kinase inhibition, MDM is supplemented with 250 nM dasatinib.

1. Digestion of assembloids for downstream analysis

Notes: 

1. Before starting, pre-warm 5× TrypLE Select and 0.5 mg/ml collagenase I diluted in Additive-Free Medium, in the 37 °C water bath. 
2. Prior to this procedure you will need to grow stromal and organoid cultures to for 2 passages, as described above. Using a light microscope, inspect stromal culture confluency and confirm that organoids have formed gland-like hollow structures.

1. Collect medium from each assembloid droplet for further analysis.
2. Add 200 µl 0.5 mg/ml collagenase I diluted in additive-free DMEM/F12 phenol-free medium to each well and scrape each assembloid droplet using a 1000 µl pipettor with a pipette tip attached until dislodged and collect into 1.5 ml microcentrifuge tubes. Pool 6-8 droplets per tube.
3. Incubate assembloids in collagenase I in a 37 °C water bath for 10 min with regular vigorous shaking.
4. Centrifuge at 600 × g for 6 minutes.
5. Remove supernatant and incubate with 5× TrypLE Select diluted in additive-free DMEM/F12 phenol-free medium for 5 min in a 37 °C water bath.
6. Disrupt any remaining cell clumps by pipetting 300× manually.
7. Centrifuge at 600 × g for 6 minutes.
8. Remove supernatant and add 1 ml additive-free DMEM/F12 medium.
9. Centrifuge at 600 × g for 6 minutes.
10. Remove supernatant and resuspend assembloid cells in 0.1% bovine serum albumin (BSA) in PBS and pass the cells through a 35 µm cell sieve.
11. Harvest can be performed according to downstream processing required, for example digestion for single cell RNA sequencing or bulk RNA harvest or protein lysates.

1. Embryo co-culture 

Notes: 

1. The use of vitrified human blastocysts was carried out under a Human Fertilisation and Embryology Authority research licence (HFEA: R0155) with local National Health Service Research Ethics Committee approval (04/Q2802/26). Spare blastocysts were donated to research following informed consent by couples who had completed their fertility treatment at the Centre for Reproductive Medicine, University Hospitals Coventry and Warwickshire National Health Service Trust. 
2. Prior to their use in the co-culture, vitrified day 5 blastocysts are warmed using the Kitazato vitrification warming media (Dibimed, Spain) and undergo zona pellucida removal using a Saturn 5 Laser (CooperSurgical). Blastocysts are incubated for 1 hr under oil in 20 µl drops of culture media (ORIGIO Sequential Blast media, CooperSurgical) at 5 % O2, 6 % CO2, 89 % N2 at 37°C and allowed to re-expand. Pre-warm embryo medium in the 37 °C water bath. Pre-set the timelapse microscope incubator and stage to 37 ^oC, gassed with 5 % CO₂.
3. Prior to co-culture, replace the assembloid culture medium with 200 µl of embryo medium per well. 
4. Pull a Pasteur pipette prior to embryo co-culture and ensure that the pipette is bent to a 45° angle.

1. Using a pulled Pasteur pipette, gently poke the centre of the assembloid culture to create a small well in the centre of the droplet
2. Carefully collect a single blastocyst using a pulled Pasteur pipette and place into the pre-made well within the assembloid droplet under a stereo microscope.
3. Place well plate into a pre-heated gassed time-lapse microscope.
4. Set up a timelapse protocol to image the co-culture wells every 20 min for 96 hours. Samples are fixed upon completed of experiment using 10% formalin. Embryos must not be cultured beyond 14 days after fertilisation.

Troubleshooting Notes

1. Matrigel solidifies during plating of organoids
   1. Matrigel begins to cure quickly if allowed to reach room temperature and can cure in tubes and pipette tips. It is essential to keep all reagents and materials at 4°C during organoid droplet plating. Collagen hydrogel will also cure at room temperature if not kept at 4°C during assembloid droplet plating.
   2. Ensure that all samples containing Matrigel/collagen hydrogel are kept in microcentrifuge tubes on wet ice prior to plating.
   3. Pre-cool pipette tip boxes in the fridge prior to use.
2. Organoid or assembloid gel droplets are damaged
   1. Organoid/assembloid droplets may appear damaged or misshapen when observed under a light microscope.
   2. This is likely due to excessive force when adding or replacing medium: ensure the medium is added gently at the side of the well to minimise disruption to the gel droplets.
3. Bubbles in gels when plating
   1. Ensure to not introduce air when resuspending cells in either Matrigel or collagen hydrogel
   2. To remove bubbles, centrifuge the samples at 4°C at 600 × g for 6 minutes.
4. Gel droplet failed to cure
   1. Adding medium before a hydrogel has had the full time required to cure may lead to the gel melting.
   2. Matrigel requires 20 minutes to cure fully and the PureCol EZ collagen hydrogel requires 90 minutes to cure fully.

Recipes

     1.  Stromal cell growth medium (10 % DCC-FBS DMEM/F12) (Barros et al., 2016)

1. Add the following growth factors to 500 ml DMEM/F12 with phenol red:

         50 ml (10 % v/v) DCC-FBS

         1× Antibiotic-Antimycotic

         1× L-glutamine

         1 nM β-estradiol (prepare 100 µM stock in ethanol, store at -20°C, add 5 µl)

         2 μg/ ml recombinant human insulin (prepare 10 mg/ml stock solution in acidified water [1.5%, v/v, acetic acid in sterile water, add                100 µl])

     2.  Expansion medium (Turco et al., 2017)

Note: Prior to preparing ExM, it is essential to prepare stock solutions of each component and aliquot to minimise freeze-thaw events. 

1. Add the following growth factors to 20 ml Advanced DMEM/F-12:

         400 µl B27 supplement 50×

         200 µl N2 supplement 100×

         200 µl NAC 1.25 mM

         200 µl L-Glutamine 100×

         200 µl AB/AM 100×

         200 µl Nicotinamide 10 mM

         40 µl R-spondin-1 500 ng/ml

         20 µl Noggin 100 ng/ml

         20 µl FGF-10 100 ng/ml

         10 µl EGF 50 ng/ml

         10 µl HGF 50 ng/ml

         1.68 µl A83-01 500 nM

1. Filter into a 50 ml tube through a 20 ml syringe attached with a 0.2 µm syringe filter.
2. Store at 4 °C. Warm in 37 °C water bath before use.

    3.  Differentiation medium (Rawlings et al., 2021)

1. Add the following growth factors to 20 ml Advanced DMEM/F-12:

         400 µl B27 supplement 50×

         200 µl N2 supplement 100×

         200 µl NAC 1.25 mM

         200 µl L-Glutamine 100×

         200 µl AB/AM 100×

         E2 10 nM

         MPA 1 nM

         cAMP 0.5 mM

1. Filter into a 50 ml tube through a 20 ml syringe attached with a 0.2 µm syringe filter.
2. Store at 4 °C. Warm in 37 °C water bath before use.

    4.  Embryo medium (Rawlings et al., 2021)

1. Add the following growth factors to 20 ml Advanced DMEM/F-12:

         400 µl B27 supplement 50×

         200 µl N2 supplement 100×

         200 µl NAC 1.25 mM

         200 µl L-Glutamine 100×

         200 µl AB/AM 100×

         200 µl Nicotinamide 10 mM

         40 µl R-spondin-1 500 ng/ml

         20 µl Noggin 100 ng/ml

         20 µl FGF-10 100 ng/ml

         10 µl EGF 50 ng/ml

         10 µl HGF 50 ng/ml

         1.68 µl A83-01 500 nM

         Essential Amino acids 1×

         Non-essential amino acids 1×

         ITS 1×

         E2 10 nM

         MPA. 1 nM

         cAMP 0.5 mM

1. Filter into a 50 ml tube through a 20 ml syringe attached with a 0.2 µm syringe filter.
2. Store at 4 °C. Warm in 37 °C water bath before use.

References

BARROS, F. S. V., BROSENS, J. J. & BRIGHTON, P. J. 2016. Isolation and Primary Culture of Various Cell Types from Whole Human Endometrial Biopsies. Bio-protocol, 6, e2028.

RAWLINGS, T. M., MAKWANA, K., TAYLOR, D. M., MOLÈ, M. A., FISHWICK, K. J., TRYFONOS, M., ODENDAAL, J., HAWKES, A., ZERNICKA-GOETZ, M., HARTSHORNE, G. M., BROSENS, J. J. & LUCAS, E. S. 2021. Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids. eLife, 10, e69603.

TURCO, M. Y., GARDNER, L., HUGHES, J., CINDROVA-DAVIES, T., GOMEZ, M. J., FARRELL, L., HOLLINSHEAD, M., MARSH, S. G. E., BROSENS, J. J., CRITCHLEY, H. O., SIMONS, B. D., HEMBERGER, M., KOO, B. K., MOFFETT, A. & BURTON, G. J. 2017. Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium. Nat Cell Biol, 19, 568-577.