Encapsulated designer cells implanted into mice are currently used to validate the efficacy of therapeutic gene networks for the diagnosis and treatment of various human diseases in preclinical research. Because many human conditions cannot be adequately replicated by animal models, complementary and alternative procedures to test future treatment strategies are required. Here we describe a novel approach utilizing an ex vivo human whole-blood culture system to validate synthetic biology-inspired designer cell-based treatment strategies. The viability and functionality of transgenic mammalian designer cells co-cultured with primary human immune cells were characterized. We demonstrated that transgenic mammalian designer cells required adequate insulation from the human blood microenvironment to maintain viability and functionality. The biomaterial alginate-(poly-l-lysine)-alginate used to encapsulate the transgenic designer cells did neither affect the viability of primary granulocytes and lymphocytes nor the functionality of lymphocytes. Additionally, alginate-encapsulated transgenic designer cells remained responsive to the release of the pro-inflammatory cytokine tumor necrosis factor (TNF) from the whole-blood culture upon exposure to bacterial lipopolysaccharide (LPS). TNF diffused into the alginate capsules, bound to the specific TNF receptors on the transgenic designer cells' surface and triggered the expression of the reporter gene SEAP (human placental secreted alkaline phosphatase) that was rewired to the TNF-specific signaling cascade. Human whole-blood culture systems can therefore be considered as valuable complementary assays to animal models for the validation of synthetic circuits in genetically modified mammalian cells and may speed up preclinical research in a world of personalized medicine. Biotechnol. Bioeng. 2016;113: 588-597. (c) 2015 Wiley Periodicals, Inc.