Engineering of Cyborg Mammalian Cells as Therapeutic Materials

Introduction: Many advanced chronic or critical diseases lack effective long-term therapeutic options. This is particularly critical when the required stability and sustained activity of the treatment exceed the limits of materials-based approaches, cell therapies, and small molecules. Thus, there is a persistent need to develop new therapeutic methods that help address these unmet clinical needs. We have developed a new hybrid cellular platform combining principles of materials science, synthetic biology, and cell therapy that allows the creation of semi-synthetic cellular systems derived from mammalian cells. This new therapeutic paradigm, which we termed Cyborg Mammalian Cells, is created by assembling a synthetic polymer network inside each cell, rendering them incapable of dividing but preserving their core cellular capabilities, effectively transforming cells into microdevices with programmable functionality. Our system preserves the desired traits of the origin cell lines, including therapeutic molecule secretion, metabolism, and organelle integrity, while conferring tolerance to lethal stressors and showing increased stability. Moreover, we demonstrate the capabilities of the Cyborg Mammalian Cells as an enhanced therapeutic material by testing their angiogenesis potential and their stability in vivo using a hind limb ischemia mouse model. We envision our Cyborg Mammalian Cell platform as a novel therapeutic avenue that can be leveraged to overcome long-standing challenges in various chronic diseases.

Materials and

Methods: Production of Cyborg Mammalia Cells: Placental Mesenchymal Stem Cells (PMSCs) were incubated in a hydrogelation buffer. After incubation, cells were collected at the bottom of a conical tube. Supernatant was then discarded, and cells were washed once using DPBS. Following the washing step, cells were resuspended in DPBS and irradiated with UV light to crosslink the intracellular hydrogel. Cells were washed twice with DPBS before resuspending in growth media for further use. Assessing organelle integrity: The cytoskeleton, lysosome, Golgi apparatus, and mitochondria integrity were assessed using confocal or fluorescence microscopy and specific fluorescent stains to label each organelle. Assessing resistance of cyborg mammalian cells to extracellular stressors: Cyborg Mammalian Cells and untreated controls were incubated under osmotic stress conditions (solution with 3 mM ionic strength), high oxidative stress conditions (10% H2O2), and with 10 µM of the apoptotic inducer Staurosporine. Cell morphology, metabolic activity, and cell count after incubation with the stressors were analyzed using flow cytometry, fluorescence, and confocal microscopy. Secretome analysis: The secretome profile of the Cyborg Mammalian Cells and untreated controls was analyzed after incubation for 7 days in growth media using a 45plex Luminex Panel (ProcartaPlex Human Cytokine/Chemokine/Growth Factor Convenience Panel 1) and a Luminex 200 detection system.

Results, Conclusions, and Discussions: Results
We have created Cyborg Mammalian Cells derived from Placental Mesenchymal Stem Cells (PMSC) that are metabolically active and cannot divide (Fig 1a&b). The Cyborg Cells preserved their morphology, and organelle structure as observed through fluorescence and confocal microscopy by staining the cytoskeleton, lysosomes, and Golgi apparatus. Organelle integrity was preserved according to our microscopy analysis comparing controls and Cyborg Cells using organelle-specific fluorescent dyes. Cyborg Mammalian Cells also preserved their protein expression capacity as measured by quantifying fluorescent protein expression. Cyborg Mammalian Cells derived from stably transfected PMSCs preserved the same level of tdTomato expression as non-hydrogelated controls (Fig 1c). Moreover, the intracellular hydrogel conferred the Cyborg Cells resistance to osmotic and oxidative stress levels that otherwise kill their untreated counterparts (Fig 1d&e). Additionally, they tolerated lethal concentrations of Staurosporine (Fig 1f). Cyborg Cells preserved their cytokine, chemokine, and growth factor secretion capabilities as quantified by Luminex analysis. Some of the secreted molecules were detected above the untreated controls (Fig 1g), including the therapeutically relevant FGF-2 (involved in tissue repair and angiogenesis), IL-RA (anti-inflammatory protein), and IL-22 (involved in tissue repair and regeneration). Lastly, we demonstrate the application of the Cyborg Cells, showing sustained synthesis of therapeutic molecules and using a murine model of hind limb ischemia.

Conclusions
We demonstrate the creation of the novel Cyborg Mammalian Cell therapeutic paradigm. Our approach shows how cultured human stem cells can be enhanced using an intracellular synthetic hydrogel, thus, increasing their tolerance to extracellular stressors while preserving key characteristics such as cell morphology, metabolic activity, and therapeutic molecule secretion. We then leverage the increased stability and resistance of the Cyborg Mammalian Cells as a novel therapeutic biomaterial capable of dynamic and sustained immunomodulation, anti-inflammatory activity, tissue repair, and regeneration. This novel platform will enable the development of therapeutics for specific applications by varying the cell line used to create the Cyborg Cells.

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