Camilli, C. | Search bioRxiv

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↗

bioRxiv2020

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency

Vascular dysfunction contributes to the pro-oncogenic tumor microenvironment and impedes the delivery of therapeutics. Normalizing of the tumor vasculature has therefore become a potential therapeutic objective. We previously reported that the secreted glycoprotein, leucine-rich -2-glycoprotein 1 (LRG1), contributes to the formation of pathogenic neovascularization. Here we show that in mouse models of cancer, Lrg1 is induced in tumor endothelial cells. We demonstrate that the expression of LRG1 impacts on tumor progression as Lrg1 deletion or treatment with a LRG1 function-blocking antibody inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function resulting in significantly enhanced efficacy of cisplatin chemotherapy, adoptive T-cell therapy and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent (cold) to immune active (hot). LRG1 therefore drives vascular abnormalization and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics.

cancer biology↗