RESUMEN
Induced pluripotent stem cells (iPS cells) represent a particularly versatile stem cell type for a large array of applications in biology and medicine. Taking full advantage of iPS cell technology requires high throughput and automated iPS cell culture and differentiation. We present an automated platform for efficient and robust iPS cell culture and differentiation into blood cells. We implemented cell cluster sorting for analysis and sorting of iPS cell clusters in order to establish clonal iPS cell lines with high reproducibility and efficacy. Patient-specific iPS cells were induced to differentiate towards hematopoietic cells via embryoid body (EB) formation. EB size impacts on iPS cell differentiation and we applied cell cluster sorting to obtain EB of defined size for efficient blood cell differentiation. In summary, implementing cell cluster sorting into the workflow of iPS cell cloning, growth and differentiation represent a valuable add-on for standard and automated iPS cell handling.
RESUMEN
In vitro tissue engineering of vascular grafts requires dynamic conditioning in a bioreactor system for in vitro tissue maturation and remodeling to receive a mechanically adequate and hemocompatible implant. The goal of the current work was to develop a bioreactor system for the conditioning of vascular grafts which is (i) able to create a wide range of flow, pressure and frequency conditions, including physiological ones; (ii) compact and easy to assemble; (iii) transportable; (iv) disposable. The system is driven by a small centrifugal pump controlled via a custom-made control unit, which can also be operated on batteries to allow for autonomous transportation. To show the potential of the newly developed bioreactor system small-caliber vascular composite grafts (n = 5, internal diameter = 3 mm, length = 12.5 cm) were fabricated using a fibrin scaffold embedding human umbilical artery smooth muscle cells and a polyvinylidene fluoride warp-knitted macroporous mesh. Subsequently, the vascular grafts were endothelialized and mounted in the bioreactor system for conditioning. The conditioning parameters remained within the predefined range over the complete conditioning period and during operation on batteries as tested for up to 25 h. Fabrication and pre-conditioning under arterial pressure and shear stress conditions resulted in robust and hemocompatible tissue-engineered vascular grafts. Analysis of immunohistochemical stainings against extracellular matrix and cell-specific proteins revealed collagen I and collagen III deposition. The luminal surface was confluently covered with endothelial cells. The developed bioreactor system showed cytocompatibility and pH, pO2, pCO2, glucose and lactate stayed constant. Sterility was maintained during the complete fabrication process of the vascular grafts. The potential of a versatile and mobile system and its functionality by conditioning tissue-engineered vascular grafts under physiological pressure and flow conditions could be demonstrated.