Project Summary/Abstract: Overview: Cultured cells form the basis of almost all TEMPs and Regenerative Medicine-based therapeutics. The long-term goal of this TR&D is to develop new technologies and strategies to achieve automated culture of cells that are phenotypically and functionally optimized for a particular TE application. Selecting cells with consistent attributes, including differentiation and expansion potential, is notoriously difficult because of variable donor genetics and a lack of understanding of cells responses to their environment. To overcome these limitations, we focus on (1) dynamic, inducible molecular reporters and non-invasive sensors to track, and control, cell and tissue manufacturing workflows in real time, and (2) targeted modifications of intracellular pathways and cell enrichment that result in more predictable and controllable cell phenotype and function. We will begin with human mesenchymal stem cells; but the strategies can easily be applied to, e.g., to tissue specific stem cells or iPS cells. This will be done following 3 Specific Aims: Aim 1: Generating genetically encoded cell sensors for dynamic and continuous sensing of cellular phenotype and function. We will generate fluorescent and/or bioluminescent (BLI) molecular reporters specific to the tissue types in development by our CPs, based on miRNA- and promoter-driven. These reporters will emit. BLI is useful to detect gene-expression activity in early differentiation events, as quantifiable data to track these events in real-time. High-resolution fluorescence imaging allows the spatio-temporal assessment of biomarker expression at a single-cell level. TR&D-1 will collaborate with TR&Ds-2, -3 and -4 to validate their technology, providing higher throughput than commonly used end-point destructive assessments. Aim 2: Developing molecular sensors for ultra-sensitive, non-invasive monitoring of cell function. We will develop molecular sensors and reporters to sense, at very low concentration, specific biological molecules within- or produced by cells in real-time, through fluorescence and electrochemical sensing systems. Our validated universal electrochemical biosensor will be adapted to any molecule of interest using aptamers and the high specificity of the CRISPR-Cas12(13) platforms. These non-invasive measurements will be designed for integration into the Tissue Foundry automation backbone as described in TR&D-4. Aim 3: Achieving dynamic control of cell phenotype and function. After assessing the spatio-temporal dynamics of differentiation using real-time molecular reporters (SA-1 & -2), inducible genetic control and/or enrichment and pre-conditioning of cells will be implemented. This will be accomplished using inducible systems such as CRISPR-activation, Tet-inducible systems and miRNA mimicking/antagonist. Alternately cell populations can be enriched for desirable biological features through novel sorting techniques (e.g., microfluidic immunomagnetic sor...