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Derivation, expansion and cryopreservation of primary fetal organoids from second and third trimester human amniotic fluid cells

Nature Protocols (2025)Cite this article

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Abstract

Human primary fetal stem cell-derived organoids are used to model developing tissues in vitro. However, ethical and legislative constraints restrict fresh fetal tissue collection in several countries. Amniotic fluid (AF) is easily accessible with minimal ethical and regulatory constraints for collection. Our team recently showed that tissue-specific stem/progenitor cells can be isolated from fetal fluids collected during pregnancy through clinically indicated minimally invasive procedures conducted during the second and third trimesters. These samples consistently generate fetal lung, kidney tubule and gastrointestinal epithelial organoids autologous to the developing fetus. AF-derived organoids (AFOs) allow the investigation of fetal epithelia at developmentally relevant stages. Moreover, AFOs allow research to be conducted on late gestational stages, hardly accessible with other methods. Here, we provide a detailed protocol to establish, characterize and cryopreserve AFOs from viable AF cells. This includes the processing of patient-derived AF samples, viable cell sorting, seeding, establishment of clonal AFO lines, tissue phenotyping, expansion and cryopreservation. Additionally, we describe a straightforward immunofluorescence-based approach to pinpoint the tissue identity of the AFOs in a quick and cost-effective manner. In our hands, the protocol enabled the generation of primary fetal AFOs from 85.71% of samples (62.5% ascribed to the fetal lung, 59.4% to the kidney tubule and 6.2% to the small intestine). It takes 4–6 weeks to implement, requiring only standard equipment and expertise commonly available in cell biology laboratories.

Key points

  • This Protocol describes the derivation, expansion and cryopreservation of primary organoids from second and third trimester human amniotic fluid cells. These samples generate lung, kidney tubule and gastrointestinal epithelial organoids autologous to the developing fetus.

  • The generation of organoids from fetal tissue is often challenging owing to ethical and legal constraints. Generating fetal epithelial organoids from amniotic fluid cells enables overcoming some of these limitations.

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Fig. 1: Overview of the Protocol steps.
Fig. 2: Isolation of viable cells from human AF.
Fig. 3: Establishment of clonal AF-derived organoid lines.
Fig. 4: Passaging and clonal expansion of AF-derived organoids.
Fig. 5: Characterization of the organoids’ tissue identity.
Fig. 6: Cryopreservation and thawing of AF-derived organoids.

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Data availability

All data supporting the findings of this study are available within the paper and the supporting primary research paper9. Source data are provided with this paper.

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Acknowledgements

The work in M.F.M.G.’s laboratory is supported by an Academy of Medical Science Springboard Award (grant no. SBF009\1011). M.F.M.G. held a H2020 Marie Skłodowska-Curie Fellowship (grant no. 843265, AmnioticID) and received support from UCL Therapeutic Acceleration Support (TAS Call 10), the NIHR Great Ormond Street Hospital Biomedical Research Centre (NIHR GOSH BRC) and the Rosetrees Trust. G.D. is supported by an EMBO Scientific Exchange Grant (grant no. 11127). K.Y.S. is supported by a Kidney Research UK PhD Scholarship (grant no. Paed_ST_005_220221129). C.C. and G.D.’s work on this project was supported by the Italian Ministry of Health through the ‘cinque per mille’ research fund. A.L.D. is supported by the NIHR UCLH Biomedical Research Centre. A.F.P. and A.M. are supported by the European Research Council (ERC) under the European Union’s Horizon Europe research and innovation program (grant no. 101126209-3D.FETOPRINT). G.G.G. is supported by the UCL Therapeutic Acceleration Support (TAS) LifeArc Fund Rare Diseases Call (TRO award no.: 184646) and the NIHR GOSH BRC. P.D.C. is supported by the CDH-UK (grant no. 16ICH03) and the BREATH Consortium (grant no. 552269), by the National Institute for Health and Care Research (grant no. NIHR-RP-2014-04-046), H2020 (grant no. 668294, INTENS), OAK Foundation (grant no. W1095/OCAY-14-191), GOSH-CC (grant no. V5201) and NIHR GOSH BRC. Research at Great Ormond Street Hospital NHS Foundation Trust and UCL Great Ormond Street Institute of Child Health is made possible by the NIHR GOSH BRC. We are grateful to V. Karaluka, to A. Eddaoudi and the GOSICH Flow Cytometry facility, to the GOSICH Imaging facility and to UCL Genomics for the support provided. We are grateful to the patients and clinical teams who helped with collecting the AF samples: the UCL Hospital (UCLH) Fetal Medicine Unit (FMU) midwife and clinical team; the biobank staff at Ospedale Pediatrico Bambino Gesù, in particular, T. Franchin, G. Di Giovamberardino and V. Marcellini; and the King’s College Hospital staff, in particular, K. Nicolaides and the Fetal Medicine Foundation.

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Authors and Affiliations

  1. Department of Surgical Biotechnology, Division of Surgery and Interventional Science, University College London, London, UK

    Giuseppe Calà, Kylin Yunyan Sun & Mattia Francesco Maria Gerli

  2. Stem Cells and Regenerative Medicine Section, Great Ormond Street Institute of Child Health, University College London, London, UK

    Giuseppe Calà, Giorgia D’Ariano, Kylin Yunyan Sun, Gloria Ji Zhang, Giuseppe Matteo Carrino, Alessandro Mariani, Alessandro Filippo Pellegata, Marco Pellegrini, Giovanni Giuseppe Giobbe, Paolo De Coppi & Mattia Francesco Maria Gerli

  3. Department of Clinical Sciences and Translational Medicine, Università degli Studi di Roma Tor Vergata, Rome, Italy

    Giorgia D’Ariano & Roberto Bei

  4. Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Milan, Italy

    Giuseppe Matteo Carrino, Alessandro Mariani & Alessandro Filippo Pellegata

  5. Research Area of Fetal, Neonatal and Cardiological Science, Ospedale Pediatrico Bambino Gesù, Rome, Italy

    Carlotta Camilli, Isabella Fabietti, Marco Pellegrini & Paolo De Coppi

  6. Elizabeth Garrett Anderson Institute for Women’s Health, University College London, London, UK

    Anna L. David

  7. Department of Women and Children’s Health, School of Life Course Sciences, Faculty of Life Sciences and Medicine, King’s College London, London, UK

    Panicos Shangaris

  8. Specialist Neonatal and Paediatric Surgery Department, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK

    Paolo De Coppi

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  1. Giuseppe Calà
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Contributions

G.C., G.G.G., M.F.M.G. and P.D.C. conceived and developed the Protocol. G.C. performed the experiments and analyzed the data with the help of G.D. and K.Y.S. G.J.Z., G.M.C. and A.M. helped with the quantifications and supported the experimental work. G.C. and M.F.M.G. wrote the manuscript with the support of A.F.P., G.G.G. and P.D.C. A.L.D., C.C., I.F., R.B., P.S., M.P. and P.D.C. helped with fluid collection and data interpretation. M.F.M.G. and P.D.C. supervised the study and secured the necessary funding.

Corresponding authors

Correspondence to Paolo De Coppi or Mattia Francesco Maria Gerli.

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Competing interests

M.F.M.G., G.G.G., P.D.C. and G.C. are inventors of the patent ‘Derivation of Primary Organoids from the Fetal Fluids’ filed by UCL on 18 April 2023, with application number GB2305703.7.

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Nature Protocols thanks Yuanyuan Zhang and Bing Zhao for their contribution to the peer review of this work.

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Key reference

Gerli, M. F. M. et al. Nat. Med. 30, 875–887 (2024): https://doi.org/10.1038/s41591-024-02807-z

Extended data

Extended Data Fig. 1 Antibody panel validation.

a, Immunofluorescence images of a PAX8 positive KAFO displaying dim signal for NKX2-1. b, Immunofluorescence images of CDX2 staining on SiAFO, LAFO and KAFO. c, Immunofluorescence images of CDX2 staining on control fetal tissue-derived small intestinal (FSiO), lung (FLO) and kidney (FKO) organoids. d, Immunofluorescence images of NKX2-1/PAX8 co-staining on FLO, FKO, FSiO and SiAFO. e, Example of non-specific extracellular/cytoplasmic NKX2-1 or PAX8 signal in SiAFO (SiAFO_2, SiAFO_3). All scale bars, 50 µm.

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Source data

Source Data Fig. 2,3,6

Data analysis and quantifications.

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Calà, G., D’Ariano, G., Sun, K.Y. et al. Derivation, expansion and cryopreservation of primary fetal organoids from second and third trimester human amniotic fluid cells. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01227-8

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