Controlled-release Microbeads to Replace Growth Factors in Fetal Bovine Serum

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Project Summary / Abstract: Fetal bovine serum (FBS) contains a variety growth factors (GFs) and GF-stabilizing agents that have a potent influence on cell behavior. Lot-to-lot variability of FBS, however, results in fluctuation of initial GF levels. These fluctuations are further complicated by time-dependent decay at rates unique to each GF, resulting in changing GF ratios over time. These three FBS characteristics (variable initial GF levels, time-dependent decay, and varying GF ratios over time) make it difficult to know actual GF levels maintained during the culture period. Knowledge and control of GF levels, however, is needed to systematically formulate media to effectively replace the GFs provided by FBS We propose to manufacture controlled-release microspheres that maintain stable, defined GF levels to facilitate the development of an FBS-free medium. Replacing FBS GFs is a meaningful step toward addressing the FOA PA-18-816 goals of: ‘Better Defining Growth Medium to Improve Reproducibility of Cell Culture’ and ‘supporting technologies and products to expedite development of better serum substitutes’. A challenge to originating serum-free protocols is the rapid degradation of soluble GFs that are typically added to media at the time of use. StemCultures, LLC manufactures controlled-release poly lactic-co-glycolic acid (PLGA) microbeads that slowly release GFs to produce defined and stable GF levels in culture media. This flexible PLGA microbead platform will be used to generate a series of ‘CultureBeads’ that release key FBS GFs to maintain a stable media level over several days. Four FBS GFs will provide proof-of-concept for the controlled- release strategy. The initial four FBS GFs were selected based on: 1) presence in FBS, 2) lability and 3) bioactivity. Each CultureBead replaces one FBS GF, allowing CultureBeads to be combined to provide a stable GF mixture. The second aim advances automated microfluidic manufacturing to facilitate cost-effective current Good Manufacturing Process (cGMP) compliant CultureBeads production. The initial CultureBeads tool kit can be utilized for specific cell types dependent on the selected GFs, including retinal pigment epithelium (RPE) cells, certain mesenchymal stem cells and pluripotent stem cells where there is broad demand. CultureBeads manufacture of RPE has immediate application for an FDA-approved, NIH-sponsored clinical trial. In a future Phase 2 STTR application, the CultureBeads series will be expanded using multiplex analysis to identify additional factors to more completely replace FBS.