Human whole‐blood culture system for ex vivo characterization of designer‐cell function

Abstract

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.

Programming gene and engineered-cell therapies with synthetic biology

“…Such organoids may one day facilitate testing of therapeutic synthetic gene circuits in a setting that closely mimics relevant disease pathologies. Another approach might be to characterize genetic circuits embedded in engineered "designer cells" by using human wholeblood culture systems (52,85). These systems simulate the environment that the engineered cells will be exposed to in a patient, helping to more accurately predict their performance in vivo.…”

“…These systems simulate the environment that the engineered cells will be exposed to in a patient, helping to more accurately predict their performance in vivo. For example, engineered HEK-293T cells encapsulated in alginate and cocultured with whole blood were able to respond to the TNF-a produced by primary immune cells stimulated with bacterial lipopolysaccharides (85). The continuing development of such platforms will enable high-throughput circuit characterization and optimization in more physiologically relevant settings.…”

Influence of K+ and Na+ ions on the degradation of wet‐spun alginate fibers for tissue engineering

“…However, biomaterials applied to tissue engineering often involves the culture of cells on a scaffold being played the role of an artificial extracellular matrix, have traditionally been designed to be inert and not to interact with biological systems in the host. [1][2][3][4] With the progress of technology, recent evolution in the tissue engineering field has now led to the definition of a biomaterial as a material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body, and boundaries for the use of biomaterials are still expanding. [5][6][7] Nowadays, the design of novel biomaterials is focused on mimicking the extracellular matrices of body tissues, as these can regulate host responses in a well-defined manner, and naturally derived materials have recently been obtained much attention owing to their inherent biocompatibility.…”

“…Therefore, there is an increasing need for tissue engineered grafts to replace damaged or diseased tissue. However, biomaterials applied to tissue engineering often involves the culture of cells on a scaffold being played the role of an artificial extracellular matrix, have traditionally been designed to be inert and not to interact with biological systems in the host . With the progress of technology, recent evolution in the tissue engineering field has now led to the definition of a biomaterial as a material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body, and boundaries for the use of biomaterials are still expanding .…”

Engineering of synthetic gene circuits for (re‐)balancing physiological processes in chronic diseases

“…Although in its infancy, synthetic biology has produced advances that have evolved into promising therapeutic modalities for the treatment of a wide range of chronic conditions with recurrent dynamics (e.g., metabolic diseases, cancers, and autoimmune diseases) . Engineered synthetic prosthetic networks within living cells are especially suited for such purposes because the autonomous detection and monitoring of changes in the levels of endogenous disease‐associated biomarkers can be directly linked to the expression of the therapeutic output (Table ).…”