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Engineering cells with intracellular agent–loaded microparticles to control cell phenotype

Abstract

Cell therapies enable unprecedented treatment options to replace tissues, destroy tumors and facilitate regeneration. The greatest challenge facing cell therapy is the inability to control the fate and function of cells after transplantation. We have developed an approach to control cell phenotype in vitro and after transplantation by engineering cells with intracellular depots that continuously release phenotype-altering agents for days to weeks. The platform enables control of cells' secretome, viability, proliferation and differentiation, and the platform can be used to deliver drugs or other factors (e.g., dexamethasone, rhodamine and iron oxide) to the cell's microenvironment. The preparation, efficient internalization and intracellular stabilization of 1-μm drug-loaded microparticles are critical for establishing sustained control of cell phenotype. Herein we provide a protocol to generate and characterize micrometer-sized agent-doped poly(lactic-co-glycolic) acid (PLGA) particles by using a single-emulsion evaporation technique (7 h), to uniformly engineer cultured cells (15 h), to confirm particle internalization and to troubleshoot commonly experienced obstacles.

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Figure 1: Flow diagram for the particle engineering protocol.
Figure 2: Generation of drug-loaded microparticles.
Figure 3: Surface modification of particles with PLL to enhance particle uptake.
Figure 4: Confirming cellular internalization of microparticles.
Figure 5: Troubleshooting particle aggregation to improve MSC uptake of particles.
Figure 6: Gating fluorescent particle–engineered MSCs in flow cytometry.
Figure 7: Potential for universal applicability of the particle-engineered cell platform.
Figure 8: Tailoring cells with intracellular depots for multiple applications.

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Acknowledgements

This work was supported by US National Institutes of Health grant no. HL095722 to J.M.K. and by a Movember-PCF Challenge Award to J.M.K. J.A.A. was supported by the Hugh Hampton Young Memorial Fellowship.

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Contributions

J.A.A., C.X., D.S., and J.M.K. conceived of the protocol and its initial applications. J.A.A., O.R.M. and K.S.N. optimized the protocol. J.A.A., O.R.M., K.S.N. and J.M.K. wrote the manuscript. J.A.A. designed and prepared all figures.

Corresponding author

Correspondence to Jeffrey M Karp.

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Competing interests

J.M.K. is a paid consultant of Sanofi and Stempeutics in the field of mesenchymal stem cell therapy.

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Ankrum, J., Miranda, O., Ng, K. et al. Engineering cells with intracellular agent–loaded microparticles to control cell phenotype. Nat Protoc 9, 233–245 (2014). https://doi.org/10.1038/nprot.2014.002

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