RESUMEN
Constructing three-dimensional (3D) tissues is an important process to improve cellular functions in tissue engineering. When transplanting artificially constructed tissues, a poor vascular network restricts oxygen and nutrient supplies to the tissue cells, which leads to cell death and reduced rates of tissue engraftment. Therefore, it is necessary to develop a system that builds a vascular network within 3D tissues. Here, we developed a hypoxia-responsive gene expression system for production of an angiogenic factor, vascular endothelial growth factor (VEGF), to improve hypoxia and nutrition deficiencies inside artificial 3D tissues. We demonstrated that cells into which the hypoxia-responsive VEGF gene expression system had been introduced autonomously controlled VEGF expression in a hypoxic stress-dependent manner. Next, we confirmed that VEGF expression within a 3D cell sheet was induced in response to a hypoxic environment in vitro. The genetically modified cell sheet was subcutaneously transplanted into mice to evaluate the feasibility of the hypoxia-responsive VEGF gene expression system in vivo. The results suggest that the hypoxia-responsive VEGF gene expression system is promising to prepare artificial 3D tissues in regenerative medicine.
Asunto(s)
Hipoxia , Factor A de Crecimiento Endotelial Vascular , Animales , Hipoxia de la Célula , Expresión Génica , Hipoxia/genética , Ratones , Factor A de Crecimiento Endotelial Vascular/genética , Factores de Crecimiento Endotelial VascularRESUMEN
Scale-up of production is needed for industrial applications and clinical translation of human induced pluripotent stem cells (hiPSCs). However, in cryopreservation of hiPSCs, successful rewarming of vitrified cells can only be achieved by convective warming of small volumes (generally 0.2 mL). Here, we present a scalable nano-warming technology for hiPSC cryopreservation employing inductive heating of magnetic nanoparticles under an alternating magnetic field. The conventional method by water bath heating at 37 °C resulted in a decrease of cell viability owing to devitrification caused by slow warming of samples with large volumes (≥ 20 mL). Nano-warming showed uniform and rapid rewarming of vitrified samples and improved viability of hiPSCs in the 20-mL system. In addition to single cells, hiPSC aggregates prepared using a bioreactor-based approach were successfully cryopreserved by the nano-warming technique. These results demonstrate that nano-warming is a promising methodology for cryopreservation in mass production of hiPSCs.
Asunto(s)
Criopreservación/métodos , Crioprotectores/farmacología , Calefacción/métodos , Células Madre Pluripotentes Inducidas/citología , Reactores Biológicos , Diferenciación Celular , Línea Celular , Supervivencia Celular , Convección , Humanos , Nanopartículas de Magnetita , VitrificaciónRESUMEN
To develop a remote control system of transgene expression through localized cellular heating of magnetic nanoparticles, a heat-inducible transgene expression system was introduced into mammalian cells. Cells were labeled with magnetic nanoparticles and exposed to an alternating magnetic field. The magnetically labeled cells expressed the transgene in a monolayer and multilayered cell sheets in which cells were heated around the magnetic nanoparticles without an apparent temperature increase in the culture medium. Magnetic cells were also generated by genetically engineering with a ferritin gene, and transgene expression could be induced by exposure to an alternating magnetic field. This approach may be applicable to the development of novel gene therapies in cell-based medicine.
