Probing MSC and Tumor Cell Secretome Locally via Dynamic Sampling Platform (DSP)

Background & Aim Within any cell culture, especially on the larger scale of commercial bioreactors, there is considerable heterogeneity that directly impacts the final cell product. This variation results in part from the local shear forces experienced by cells as well as the biochemical microenvironment, e.g., metabolic products and secreted protein factors that can act in a paracrine fashion to modify nearby cells. The ability to measure these critical quality attribute (CQA) biomarkers with high spatial and temporal resolution is foundational to the development of real-time quality monitoring for cell manufacturing. Standard analytical methods quantify neither the local heterogeneity nor the transient nature of cell growth. The Dynamic Sampling Platform (DSP, figure 1) overcomes both barriers by enabling local sampling of the secretome for rapid, high fidelity, and label free CQA identification via inline electrospray ionization mass spectrometry (ESI-MS). Real-time monitoring of the microenvironment with DSP will help predict cell health, viability, and potency, thus enabling the Chemistry, Manufacturing and Controls (CMC) standards for the scale-up and scale-out of cell-based therapies. Methods, Results & Conclusion DSP with ESI-MS sensing enables continuous collection, treatment, and direct infusion of ultra-small samples taken directly from cell culture bioreactors for ESI-MS detection of the cell secretome. DSP-nanoESI-MS is comprised of three elements: 1) a non-invasive sampling interface, which has demonstrated the ability to continuously sample from 2D cell cultures aseptically; 2) a microfabricated mass exchanger for sample treatment which simultaneously removes compounds detrimental to MS analysis, e.g., inorganic salts, and introduces compounds that enhance MS sensitivity; and 3) a spray port for direct infusion nanoESI-MS analysis resulting in identification/fingerprinting of biomolecules in cell cultures. DSP has been applied to multiple live cell cultures to identify differences in the secretome associated with cell state. Analysis of wild type versus interferon gamma primed mesenchymal stromal cells (MSCs) as well as OS-17 and 143-B tumor cell types reveal an enriched ESI-MS signature associated with the different cell types. Localized sampling by DSP is shown to be critical in order to capture higher concentration biomarkers near the cell membrane vs bulk samples that are masked by excessive abundance of biochemical background within the media. The Dynamic Sampling Platform (DSP) samples directly from a cell culture with high spatial resolution to capture critical quality attribute (CQA) biomolecules which indicate the instantaneous cell state and growth trajectory. Rapid, inline sample treatment using the microfabricated mass exchanger removes contaminants, such as inorganic salts, and infuses chemicals that enhance the sensitivity of ESI-MS analysis. Larger CQA biomarkers are selectively retained in the sample channel by the size selective nanomembrane, while smaller contaminants and enhancers transfer across the membrane freely. Real-time electrospray ionization mass spectrometry (ESI-MS) provides detection and identification of broad range of CQA biomolecules with no a priori knowledge of chemical composition.