Biology subjects

Pellegata, A. F.

Publications and source records attributed to Pellegata, A. F..

4 recordsLinked to original sources

bioRxiv2026

Derivation of primary fetal epithelial organoids from cryopreserved human amniotic fluid cells.

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.

cell biology↗

bioRxiv2023

Lung viral infection modelling in a bioengineered whole-organ

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection with the engineered lung by infecting primary human airway epithelial cells. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral Remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.

bioengineering↗

bioRxiv2020

SARS-CoV-2 infection and replication in human fetal and pediatric gastric organoids

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global public health emergency. COVID-19 typically manifests as a respiratory illness but an increasing number of clinical reports describe gastrointestinal (GI) symptoms. This is particularly true in children in whom GI symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. By contrast, fetuses seem to be rarely affected by COVID-19, although the virus has been detected in placentas of affected women. These observations raise the question of whether the virus can infect and replicate within the stomach once ingested. Moreover, it is not yet clear whether active replication of SARS-CoV-2 is possible in the stomach of children or in fetuses at different developmental stages. Here we show the novel derivation of fetal gastric organoids from 8-21 post-conception week (PCW) fetuses, and from pediatric biopsies, to be used as an in vitro model for SARS-CoV-2 gastric infection. Gastric organoids recapitulate human stomach with linear increase of gastric mucin 5AC along developmental stages, and expression of gastric markers pepsinogen, somatostatin, gastrin and chromogranin A. In order to investigate SARS-CoV-2 infection with minimal perturbation and under steady-state conditions, we induced a reversed polarity in the gastric organoids (RP-GOs) in suspension. In this condition of exposed apical polarity, the virus can easily access viral receptor angiotensin-converting enzyme 2 (ACE2). The pediatric RP-GOs are fully susceptible to infection with SARS-CoV-2, where viral nucleoprotein is expressed in cells undergoing programmed cell death, while the efficiency of infection is significantly lower in fetal organoids. The RP-GOs derived from pediatric patients show sustained robust viral replication of SARS-CoV-2, compared with organoids derived from fetal stomachs. Transcriptomic analysis shows a moderate innate antiviral response and the lack of differentially expressed genes belonging to the interferon family. Collectively, we established the first expandable human gastric organoid culture across fetal developmental stages, and we support the hypothesis that fetal tissue seems to be less susceptible to SARS-CoV-2 infection, especially in early stages of development. However, the virus can efficiently infect gastric epithelium in pediatric patients, suggesting that the stomach might have an active role in fecal-oral transmission of SARS-CoV-2.

cell biology↗

bioRxiv2019

Engineering transplantable jejunal mucosal grafts using primary patient-derived organoids from children with intestinal failure

Intestinal failure (IF), following extensive anatomical or functional loss of small intestine (SI), has debilitating long-term effects on infants and children with this condition. Priority of care is to increase the childs length of functional intestine, jejunum in particular, to improve nutritional independence. Here we report a robust protocol for reconstruction of autologous intestinal mucosal grafts using primary IF patient materials. Human jejunal intestinal organoids derived from paediatric IF patients can be expanded efficiently in vitro with region-specific markers preserved after long-term culture. Decellularized human intestinal matrix with intact ultrastructure is used as biological scaffolds. Proteomic and Raman spectroscopic analyses reveal highly analogous biochemical composition of decellularized human SI and colon matrix, implying that they can both be utilised as scaffolds for jejunal graft reconstruction. Indeed, seeding of primary human jejunal organoids to either SI or colonic scaffolds in vitro can efficiently reconstruct functional jejunal grafts with persistent disaccharidase activity as early as 4 days after seeding, which can further survive and mature after transplantation in vivo. Our findings pave the way towards regenerative medicine for IF patients.

bioengineering↗