Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, pro-angiogenic, and immune-modulatory effects in multiple disease models, such as skeletal muscle atrophy and Alport syndrome. A source of potential variability in EV biological functions is how EV are isolated from parent cells. Currently, a comparative study of different EV isolation strategies using conditioned medium from AFSCs is lacking. Herein, we examined different isolation strategies for AFSC-EVs, using common techniques based on differential sedimentation (ultracentrifugation), solubility (ExoQuick, Total Exosome Isolation Reagent, Exo-PREP), or size-exclusion chromatography (qEV). All techniques isolated AFSC-EVs with typical EV morphology and protein markers. In contrast, AFSC-EV size, protein content, and yield varied depending on the method of isolation. When equal volumes of the different AFSC-EV preparations were used as treatment in a model of lung epithelial injury, we observed a significant variation in how AFSC-EVs were able to protect against cell death. AFSC-EV enhancement of cell survival appeared to be dose dependent, and largely uninfluenced by variation in EV-size distributions, relative EV-purity, or their total protein content. The variation in EV-mediated cell survival obtained with different isolation strategies emphasizes the importance of testing alternative isolation techniques in order to maximize EV regenerative capacity.

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal surgical emergency in preterm neonates. Over the last five decades, a variety of experimental models have been developed to study the pathophysiology of this disease and to test the effectiveness of novel therapeutic strategies. Experimental NEC is mainly modeled in neonatal rats, mice and piglets. In this review, we focus on these experimental models and discuss the major advantages and disadvantages of each. We also briefly discuss other models that are not as widely used but have contributed to our current knowledge of NEC.