Human induced pluripotent stem cells (hiPSCs) have a number of potential applications in stem cell biology and regenerative medicine, including precision medicine. However, their potential clinical application is hampered by the low efficiency, high costs, and heavy workload of the reprogramming process. Here we describe a protocol to reprogram human somatic cells to hiPSCs with high efficiency in 15 d using microfluidics. We successfully downscaled an 8-d protocol based on daily transfections of mRNA encoding for reprogramming factors and immune evasion proteins. Using this protocol, we obtain hiPSC colonies (up to 160 ± 20 mean ± s.d (n = 48)) in a single 27-mm 2 microfluidic chamber) 15 d after seeding ~1,500 cells per independent chamber and under xeno-free defined conditions. Only ~20 µL of medium is required per day. The hiPSC colonies extracted from the microfluidic chamber do not require further stabilization because of the short lifetime of mRNA. The high success rate of reprogramming in microfluidics, under completely defined conditions, enables hundreds of cells to be simultaneously reprogrammed, with an ~100-fold reduction in costs of raw materials compared to those for standard multiwell culture conditions. This system also enables the generation of hiPSCs suitable for clinical translation or further research into the reprogramming process.

Microfluidic reprogramming to pluripotency of human somatic cells

Gagliano O.;Luni C.;Qin W.;Bertin E.;Torchio E.;Galvanin S.;Urciuolo A.;Elvassore N.
2019

Abstract

Human induced pluripotent stem cells (hiPSCs) have a number of potential applications in stem cell biology and regenerative medicine, including precision medicine. However, their potential clinical application is hampered by the low efficiency, high costs, and heavy workload of the reprogramming process. Here we describe a protocol to reprogram human somatic cells to hiPSCs with high efficiency in 15 d using microfluidics. We successfully downscaled an 8-d protocol based on daily transfections of mRNA encoding for reprogramming factors and immune evasion proteins. Using this protocol, we obtain hiPSC colonies (up to 160 ± 20 mean ± s.d (n = 48)) in a single 27-mm 2 microfluidic chamber) 15 d after seeding ~1,500 cells per independent chamber and under xeno-free defined conditions. Only ~20 µL of medium is required per day. The hiPSC colonies extracted from the microfluidic chamber do not require further stabilization because of the short lifetime of mRNA. The high success rate of reprogramming in microfluidics, under completely defined conditions, enables hundreds of cells to be simultaneously reprogrammed, with an ~100-fold reduction in costs of raw materials compared to those for standard multiwell culture conditions. This system also enables the generation of hiPSCs suitable for clinical translation or further research into the reprogramming process.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3412563
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