PROJECT SUMMARY Human pluripotent stem cells (hPSCs) and their derived cardiomyocytes (hPSC-CMCs) are valuable for studying and treating human diseases. However, their widespread use and further advancement to clinics are currently limited by the difficulty of manufacturing these cells at large scales. The current cell culture methods have low efficiency, are not scalable, and can only produce cells at small scales with high cost. For instance, making>10^10 hPSC-CMCs per batch is still very challenging. Additionally, there are significant variations in the yield and properties of cells from different batches, labs, and companies, leading to inconsistent research and clinic results. Further, the produced hPSC-CMCs have immature phenotypes that limit their use both in vivo and in vitro. The overall goal of this project is to develop a novel cell culture technology to address this challenge. Toward this goal, the team has developed a scalable, dissolvable, and cell-friendly 3D microbioreactor in the preliminary studies. The microbioreactor provides cells a uniform, reproducible, well-controlled, and friendly microenvironment, resulting in extremely high culture efficiency and consistency. The team has demonstrated expanding hPSCs and differentiating hPSCs into cardiomyocytes in the microbioreactor. The viability, growth rate, yield, and quality of cells in the microbioreactor offer large advances over current methods. The volumetric yield is 250 times of current state-of-the-art. The new technology has all the needed features to address the large-scale hPSCs and hPSC-CMCs manufacturing challenge. The proposed research will further develop and vigorously validate the technology and the associated methods to make it robust and ready for hPSCs and hPSC- CMCs manufacturing. Leveraging sound preliminary studies and a diverse team of experts, the specific aims are to (1) validate the technology for culturing hPSCs from diverse backgrounds and (2) further develop and validate the technology for preparing high-purity hPSC-CMCs. Completing this project, a scalable, clinically compatible, and low-cost cell culture technology for culturing hPSCs and hPSC-CMCs will be available. The technology can also be used for culturing other cells (such as platelets and red blood cells from hPSCs) related to National Heart, Lung, and Blood Institute’s mission. The technology is enabling and will significantly advance medicine for heart disease.