Prolonged continuous in vitro human platelet production using three-dimensional scaffolds.

Methods producing human platelets using growth on plastic, on feeder layers, or in suspension have been described. We hypothesized that growth of hematopoietic progenitors in a three-dimensional (3D) scaffold would enhance platelet production sans feeder layer. We grew CD34 positively selected human cord blood cells in surgical-grade woven polyester fabric or purpose-built hydrogel scaffolds using a cocktail of cytokines. We found production of functional platelets over 10 days with two-dimensional (2D), 24 days with 3D scaffolds in wells, and more than 32 days in a single-pass 3D perfusion bioreactor system. Platelet numbers produced daily were higher in 3D than 2D, and much higher in the 3D perfusion bioreactor system. Platelet output increased in hydrogel scaffolds coated with thrombopoietin and/or fibronectin, although this effect was largely obviated with markedly increased production caused by changes in added cytokines. In response to thrombin, the platelets produced aggregated and displayed increased surface CD62 and CD63. Use of 3D scaffolds, especially in a bioreactor-maintained milieu, may allow construction of devices for clinical platelet production without cellular feeder layers.