Amniotic fluid plays a crucial role in fetal development, yet a comprehensive understanding of its composition, temporal changes, and mechanisms of action remains elusive. The increased accessibility and abundance of third-trimester amniotic fluid from Cesarean sections provide an opportune moment to explore its therapeutic potential. Necrotizing Enterocolitis (NEC) stands as the leading cause of gastrointestinal-related mortality in premature infants within the Neonatal Intensive Care Unit (NICU). Affecting 1-5% of neonatal intensive care admissions and 5-10% of very low birth weight infants, NEC's pathophysiology involves inflammation, bacterial invasion, cellular damage, and necrosis, often leading to fatal outcomes. Surgery is required in 20-30% of NEC cases, with high fatality rates, contributing to substantial NICU costs. Exclusive human milk- based diets (EHMD) have shown promise in reducing NEC risk, mortality, and hospitalization costs. However, the expense associated with EHMD, particularly using Prolacta Bioscience Inc.'s exclusive fortifier, poses a significant financial challenge for hospitals. Non-privately-owned hospitals, with limited budgets, may benefit from a lower- cost preventative therapy compatible with bovine fortifiers while still reducing the risk of NEC, mortality, and overall expenses. The proposed research aims to explore the potential of amniotic fluid, a vital in utero growth medium containing cellular and non-cellular elements, as a therapeutic product to mitigate the risk of NEC in the NICU. After isolating amniotic fluid cells, their characterization included assessing mesenchymal stem cell qualities through plastic adherence, stem cell marker expression, and differentiation. Similarly, extracellular vesicles isolated from amniotic fluid were characterized as exosomes based on size, morphology, and positive exosome protein identification. Subsequently, an in vitro NEC model utilizing T84 intestinal epithelial cells and Lipopolysaccharides (LPS) was employed to examine the impact of these components on proliferation, cell viability, intestinal barrier function, and tight junction gene regulation. Notably, isolated exosomes from amniotic fluid demonstrated a positive effect on intestinal epithelial cell proliferation and barrier function, effectively preventing LPS-induced intestinal barrier injury. Importantly, this preventative effect cannot be solely attributed to increased proliferation or changes in mRNA expression of tight junction proteins, necessitating further exploration of other mechanisms of action. Despite extensive research, NEC incidence rates and mortality have only marginally decreased by 5% over the past two decades. The potential of third trimester human amniotic exosome exposure to prevent NEC suggests a promising avenue for further therapeutic development.