Project Summary/Abstract Preterm infants are at increased risk of health complications due to their under-developed organ systems at birth, and human milk receipt is recognized as an important intervention to promote infant health. Recent identification of cells in human milk which have similar characteristics to stem cells, as well as animal models which show integration of these cells into nursling's organs, indicate an important function of milk cells which is currently not understood. In the NICU, infants often receive human milk which has been refrigerated or frozen, and then warmed and/or thawed prior to feeding. The impact of these typical handling practices on the cellular components of milk that may provide an important mechanism of biologic protection in infant health has not been investigated. This work will determine how the real-life storage and handling of human milk impacts infant health and development, and is the first to evaluate protective mechanisms. There are currently two major knowledge gaps in the fields of human milk and lactation and neonatal care surrounding bioactive cellular components. The first is a lack of knowledge of how real-life storage and handling practices impact the protective ability of milk's cellular components for infants. Without this knowledge, infants who may benefit from milk with the most bioactivity (such as preterm infants) may not receive the properties of their mother's milk which provide important protection. The second major opportunity is the lack of knowledge on the mechanism(s) of protection in milk stem-like cells. Additionally, as research techniques advance our ability to investigate milk components, there is a great need to look at milk components in a biological systems perspective. The specific aims of the research project are to 1) examine how hospital storage practices (refrigeration and freezing) impact the protective mechanisms of human milk cells through the use of a tissue culture model of intestinal health, and 2) determine if milk cells are integrated as functioning cells specific to vital organs impacted by preterm birth (brain, heart, lungs, intestine) using a cross-foster mouse model. Upon completion of the specific aims of the K23 research strategy and training plans, the candidate will have advanced theoretical knowledge and technical skills to conduct human milk research with the ability to apply a biological systems approach to understand the complexity of the many components of milk which likely impact the function of each. The proposal research aims are supported by research training, didactic coursework, scientific meetings, and specific plans for dissemination and future growth. Ultimately, the short- and long-term goals of this research are to 1) improve infant health by optimizing the delivery of the most bioactive human milk components and 2) determine the mechanism(s) by which human milk cells protect infants and promotes growth and development.