Derivation of primary fetal epithelial organoids from cryopreserved human amniotic fluid cells.
- 1Division of Surgery and Interventional Science, University College London, London, UK
Abstract
Primary fetal human epithelial organoids are canonically derived from tissue samples obtained after termination of pregnancy. Recently, we have demonstrated that these organoids can be consistently generated from amniotic fluid cells isolated prenatally during diagnostic and interventional procedures. Amniotic Fluid-derived Organoids (AFOs) are promising fetal epithelial lung, kidney and small intestine tissue models that bypass some of the ethical and legal constraints associated with obtaining primary fetal tissue. Despite this, the widespread adoption of this technology is limited by the lack of standardised and accessible pipelines for the biobanking, culturing and distribution of organoid-forming amniotic fluid cells. To enable broader AFO use for research and clinical purposes, this work investigates two cryopreservation strategies to optimise AF cell recovery for organoid derivation. In Strategy 1, viable AF cells were sorted before freezing, while in Strategy 2 unsorted AF was frozen, and viable AF cells were sorted for viability after thawing. We present here a comprehensive evaluation of 5 commercially available GMP-compliant freezing media (FM1-5) alongside a standard lab-grade control (FM CT). AFOs were assessed for formation efficiency, morphological characteristics, proliferation capacity, epithelial identity, and tissue type. Our results demonstrate that organoid-forming AF cells can be successfully cryopreserved both pre- and post-sorting. Strategy 1 yielded higher organoid formation efficiency, with AFOs derived from cryopreserved and fresh cells exhibiting comparable expansion potential. Finally, FM4 provided minimal to no decline in survival rate, making it the most effective GMP-grade freezing medium tested. In conclusion, we present two viable cryopreservation strategies adaptable to different laboratory settings and identify optimal GMP-compliant freezing media, to support safe distribution and centralised processing, thereby facilitating scalability and collaborative work on the AFO technology.
Related
- TLR2-dependent SOCS1 induction by Ureaplasma urealyticum attenuates IDO1 expression and amplifies chemokine production in human amniotic epithelial cells
- Asymmetric cortical mechanics shape epithelial cell geometry in organoids
- From Primary Cells to Organoids: Expanding the Biological Relevance of In Vitro Cellular Models
- WITHDRAWN: Evaluating phage-antibiotic synergy in differentiated primary airway epithelial cultures against Pseudomonas aeruginosa
- Genetically modified primary airway-epithelial cell culture models to study virus-host interactions of emerging respiratory viruses
BibTeX
@misc{dariano2026derivation,
title = {{Derivation of primary fetal epithelial organoids from cryopreserved human amniotic fluid cells.}},
author = {G. D'Ariano and G. J. Zhang and G. Cala and G. M. Carrino and A. Agarwal and A. Mariani and M. Marinaro and R. Matias de Sousa and G. Cenedese and C. Camilli and A. L. David and S. Eaton and A. F. Pellegata and M. Pellegrini and P. De Coppi and M. F. M. Gerli},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.07.749928},
url = {https://www.biorxiv.org/content/10.64898/2026.09.07.749928v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Derivation of primary fetal epithelial organoids from cryopreserved human amniotic fluid cells.
AU - G. D'Ariano
AU - G. J. Zhang
AU - G. Cala
AU - G. M. Carrino
AU - A. Agarwal
AU - A. Mariani
AU - M. Marinaro
AU - R. Matias de Sousa
AU - G. Cenedese
AU - C. Camilli
AU - A. L. David
AU - S. Eaton
AU - A. F. Pellegata
AU - M. Pellegrini
AU - P. De Coppi
AU - M. F. M. Gerli
PY - 2026
DA - 2026/09/07
PB - bioRxiv
DO - 10.64898/2026.09.07.749928
UR - https://www.biorxiv.org/content/10.64898/2026.09.07.749928v1
AB - Primary fetal human epithelial organoids are canonically derived from tissue samples obtained after termination of pregnancy. Recently, we have demonstrated that these organoids can be consistently generated from amniotic fluid cells isolated prenatally during diagnostic and interventional procedures. Amniotic Fluid-derived Organoids (AFOs) are promising fetal epithelial lung, kidney and small intestine tissue models that bypass some of the ethical and legal constraints associated with obtaining primary fetal tissue. Despite this, the widespread adoption of this technology is limited by the lack of standardised and accessible pipelines for the biobanking, culturing and distribution of organoid-forming amniotic fluid cells. To enable broader AFO use for research and clinical purposes, this work investigates two cryopreservation strategies to optimise AF cell recovery for organoid derivation. In Strategy 1, viable AF cells were sorted before freezing, while in Strategy 2 unsorted AF was frozen, and viable AF cells were sorted for viability after thawing. We present here a comprehensive evaluation of 5 commercially available GMP-compliant freezing media (FM1-5) alongside a standard lab-grade control (FM CT). AFOs were assessed for formation efficiency, morphological characteristics, proliferation capacity, epithelial identity, and tissue type. Our results demonstrate that organoid-forming AF cells can be successfully cryopreserved both pre- and post-sorting. Strategy 1 yielded higher organoid formation efficiency, with AFOs derived from cryopreserved and fresh cells exhibiting comparable expansion potential. Finally, FM4 provided minimal to no decline in survival rate, making it the most effective GMP-grade freezing medium tested. In conclusion, we present two viable cryopreservation strategies adaptable to different laboratory settings and identify optimal GMP-compliant freezing media, to support safe distribution and centralised processing, thereby facilitating scalability and collaborative work on the AFO technology.
ER -