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Bacteria can be genetically programmed to sense and report the presence of disease biomarkers in the gastrointestinal (GI) tract. However, diagnostic bacteria are typically delivered via oral administration of liquid cultures, resulting in poor survival and high dispersal in vivo. These limitations confound recovery and analysis of engineered bacteria from GI or stool samples. Here, we demonstrate that encapsulating bacteria inside of alginate core-shell particles enables robust survival, containment, and diagnostic function in vivo. We demonstrate these benefits by encapsulating a strain engineered to report the presence of the biomarker thiosulfate via fluorescent protein expression in order to diagnose dextran sodium sulfate-induced colitis in rats. Hydrogel-encapsulated bacteria engineered to sense and respond to physiological stimuli should enable minimally invasive monitoring of a wide range of diseases and have applications as next-generation smart therapeutics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Omid Veiseh and Jeffrey Tabor reports a relationship with Pana Bio that includes: board membership and equity or stocks. Samira Aghlara-Fotovat, Elena Musteata, Michael D. Doerfert, Moshe Baruch, Jeffrey J. Tabor, Omid Veiseh has patent pending to Pana Bio. Moshe Baruch is current employee of Pana Bio., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: While hydrogel encapsulation of cells has been developed to treat multiple diseases, methods to cryopreserve and maintain the composite function of therapeutic encapsulated cell products are still needed to facilitate their storage and distribution. While methods to preserve encapsulated cells, and post-synthesis have received recent attention, effective preservation mediums have not been fully defined., Methods: We employed a two-tiered screen of an initial library of 32 different cryopreservation agent (CPA) formulations composed of different cell-permeable and impermeable agents. Formulations were assayed using dark field microscopy to evaluate alginate hydrogel matrix integrity, followed by cell viability analyses and measurements of functional secretion activity., Results: The structural integrity of large > 1 mm alginate capsules were highly sensitive to freezing and thawing in media alone but could be recovered by a number of CPA formulations containing different cell-permeable and impermeable agents. Subsequent viability screens identified two top-performing CPA formulations that maximized capsule integrity and cell viability after storage at - 80 °C. The top formulation (10% Dimethyl sulfoxide (DMSO) and 0.3 M glucose) was demonstrated to preserve hydrogel integrity and retain cell viability beyond a critical USA FDA set 70% viability threshold while maintaining protein secretion and resultant cell potency., Conclusions: This prioritized screen identified a cryopreservation solution that maintains the integrity of large alginate capsules and yields high viabilities and potency. Importantly, this formulation is serum-free, non-toxic, and can support the development of clinically translatable encapsulated cell-based therapeutics., Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00739-7., (© The Author(s) under exclusive licence to Biomedical Engineering Society 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Proinflammatory cytokines have been approved by the Food and Drug Administration for the treatment of metastatic melanoma and renal carcinoma. However, effective cytokine therapy requires high-dose infusions that can result in antidrug antibodies and/or systemic side effects that limit long-term benefits. To overcome these limitations, we developed a clinically translatable cytokine delivery platform composed of polymer-encapsulated human ARPE-19 (RPE) cells that produce natural cytokines. Tumor-adjacent administration of these capsules demonstrated predictable dose modulation with spatial and temporal control and enabled peritoneal cancer immunotherapy without systemic toxicities. Interleukin-2 (IL2)-producing cytokine factory treatment eradicated peritoneal tumors in ovarian and colorectal mouse models. Furthermore, computational pharmacokinetic modeling predicts clinical translatability to humans. Notably, this platform elicited T cell responses in NHPs, consistent with reported biomarkers of treatment efficacy without toxicity. Combined, our findings demonstrate the safety and efficacy of IL2 cytokine factories in preclinical animal models and provide rationale for future clinical testing in humans.