Feldman, S. A. | Search bioRxiv

bioRxiv2025

Robotic performance is comparable to human operators for cell therapy manufacturing tasks

Although numerous cell and gene therapies have received regulatory approval, their adoption has been hampered by high cost and challenges in scaling out manufacturing. Many autologous cell therapies are individually manufactured for each patient using traditional manual methods in high-cost environments. Therefore, robotics and automation offer a potential solution to meet the growing demand for such therapies. To ensure identical biological outcomes, automation must replicate validated manual workflows, a requirement that poses significant engineering challenges, especially for aseptic manipulation and compatibility with manual-centric instruments and consumables. Here we describe the design and performance of a modular robotic cluster consisting of specialized modules containing widely adopted equipment. The robotic arm uses custom end-effectors to handle standard consumables and instruments such as syringes, vials, bags, cell counters, bioreactors, incubators, and closed centrifuges. We compared the performance of skilled human operators against the robotic cluster across multiple tasks: transferring cells between sterile bags, cell counting, drawing volume from a vial to a syringe, and resuspension and sampling from both a bag and G-Rex 100M-CS bioreactor. The robotic system also executed high-complexity operations with industry-standard instruments: cell selection using a CytoSinct 1000 and wash/buffer exchange with a CTS Rotea Counterflow centrifugation system. Experimental results for each unit operation show that the robotic clusters performance is equivalent to manual operations on the selected key metrics. These data demonstrate that the robotic system can efficiently and robustly perform specific unit operations, which can be combined in any order for end-to-end cell therapy manufacturing processes. One Sentence summaryA modular robotic system can perform key unit operations in cell therapy manufacturing with accuracy comparable to human operators while ensuring throughput and flexibility.

bioengineering↗

bioRxiv2023

Inosine Induces Stemness Features in CAR T cells and Enhances Potency

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing. Statement of SignificanceAdenosine is well known to inhibit T cell function and substantial effort has focused on inhibiting adenosine generation and signaling. Here, we show that exhausted T cells are suppressed by adenosine, which is only modestly impacted by inhibiting adenosine generation or signaling. In contrast, metabolism of adenosine to inosine augmented T cell function and culture of T cells with inosine induced multi-level reprogramming leading to stemness and improved anti-tumor potency. We demonstrate the feasibility of introducing inosine during GMP cell manufacturing as a novel strategy to generate enhanced CAR-T cells.

immunology↗