BIOMADE: Development and Systems Characterization of Completely Synthetic Cell Culture Media enabled by VHH Growth Factor Alternatives

This project introduces novel nanobodies, or VHHs, as cost-effective, reproducible and tunable alternatives to traditional growth factors in cell culture media. Cell culture is fundamental to modern biotechnology, supporting applications from regenerative medicine to cultured meat production, yet current methods often rely on expensive and inconsistent animal-derived growth factors. VHHs, which are small antibody fragments, can be precisely designed to activate cell growth receptors, mimicking natural proteins at a significantly lower cost. Leveraging a novel microbial production system, we can drastically improve growth factor prototyping, reduce manufacturing costs and eliminate batch-to-batch variability common with conventional growth factors. Project outcomes will lead to advances in cell culture methods with broad impacts on human health, sustainable food production, and U.S. biomanufacturing competitiveness. This project aims to engineer novel VHH-peptide fusions as next-generation growth factor alternatives for synthetic cell culture media. Our core innovation involves creating bispecific molecules that combine a receptor-binding VHH with activating peptides. Specifically in this work we will target growth factor alternatives which activate the FGF receptor (FGFR) and the epidermal growth factor receptor (EGFR). These VHHs will be produced using a two-stage bacterial expression system in E. coli, which enables soluble expression, proper disulfide bond formation, and simplified purification, significantly reducing production costs (projected from $15,000/g to $100-500/g). Our approach is systematic, beginning with the design and validation of FGF receptor-activating VHH fusions using cell-based reporter assays and surface plasmon resonance to assess binding kinetics and activation. Subsequently, we will leverage Design of Experiments methodology to optimize complete synthetic media formulations for pluripotent stem cells and bovine satellite cells, ensuring robust proliferation and maintenance of cellular identity. A multi-omics strategy, encompassing RNA-seq transcriptomics, proteomics, phosphoproteomics, and targeted metabolomics, will provide a systems-level characterization of cellular responses to the VHH-fusions, comparing them to natural growth factors across multiple temporal points to capture signaling dynamics and validate functional equivalence. The platform will then be expanded to engineer EGFR-activating VHH-fusions for mesenchymal stem cell culture. This research will generate comprehensive molecular signatures and optimized media formulations, addressing critical challenges in cost, reproducibility, and scalability of cell culture. The integrated academic-industrial collaboration with Roke Biotechnologies provides a clear commercialization pathway, enhancing U.S. biomanufacturing competitiveness. This project is being jointly supported by the Division of Molecular and Cellular Biosciences at NSF and the BioMADE Manufacturing Innovation Institute. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.