Scientific Abstract: The frequent use of fetal bovine serum (FBS) to support cell cultures is challenged by lot-to-lot variability, supply constraints, pathogen contaminants and ethical questions. These issues have increased significance due to the burgeoning use of FBS in cell research and therapy development. FBS has been used for over a century to culture cells, but with the need to define culture conditions for efficient manufacturing, along with modern approaches to control delivery of key molecules, many of the problems associated with FBS use can now be alleviated. The need to reduce animal products is critically important, as is the need to use human growth factors to optimize human cell growth. The company StemCultures specializes in controlling the growth factor (GF) component of cell culture media. Our goal is to replace FBS GFs with a bioengineered DISCTM (Defined Insert for Sustainable Culture) device providing controlled-release human recombinant GFs. The advanced cell manufacturing DISCTM device will address concerns about FBS and improve the reproducibility of cell production. In this application we will develop a DISCTM device for growth of human retinal pigment epithelial (RPE) cells. Human RPE was selected as a testbed for FBS replacement as these cells are a vanguard for stem cell replacement therapy in the US, Europe and Asia. A barrier to reducing FBS use is the lack of knowledge about the GF content of FBS that is critical for growth of the RPE cell product. We will measure levels of FBS GFs that stimulate human RPE cell proliferation. Selected recombinant human GFs will be encapsulated in controlled- release GF microbeads that provide a standardized growth signal over time, to replace the variable levels of bovine GFs provided by FBS with defined levels of native human recombinant GFs. The GF microbeads will be assessed in a factorial design experiment to identify microbead combinations that support RPE cell growth at a reduced level of FBS. The microbead combination that supports RPE cell growth without FBS or at the lowest level of FBS will be packaged in a Defined Insert for Sustainable Culture (DISCTM) device to simplify RPE cell manufacture. The DISCTM will be manufactured as a commercial product marketed to reduce FBS dependence of the commercially important RPE cells. Future work will apply the microbead/DISCTM platform strategy to reduce FBS dependence of other cell types. The company StemCultures (SCL) routinely manufactures microbeads and DISCsTM for sustained release of GFs in culture media and the co-located Neural Stem Cell Institute (NSCI) has extensive RPE cell culture expertise. The SCL-NSCI collaboration successfully completed Phase 1 aims, in preparation to make Phase 2 goals highly feasible.