Drying of Functional Hydrogels: Development of a Workflow for Bioreactor-Integrated Functional Freeze-Drying of Protein-Coated Alginate Microcarriers for iPS Cell-Based Screenings

Protein-coated ultra-high viscosity (UHV)-alginate hydrogels are essential to mimic the physiological in vivo environment for several in vitro applications. This work presents an optimized bioreactor-integrated freeze-drying process of protein-coated UHV-alginate microcarrier in the context of iPSC expansion. The impact of freeze-drying on the UHV-alginate microcarriers using trehalose 100 mg/ml in 0.9% NaCl as a lyoprotective agent, as well as the stem cell response using hiPSCs was analyzed by microscopy-based screenings. First observations of the process showed that the integrity of the cake was preserved in the samples with maximum vapor exchanging rate. After rehydration of the samples, there were no morphological changes of the UHV-alginate microcarriers observed. By addition of Poloxamer 188, the stickiness and bubble formation were reduced. The expansion of iPSC resulted in a 5-7-fold change with a total cell count of at least 1,3x107 cells with viabilities over 80% after seven days cultivation in a suspension bioreactor. The pluripotency factors OCT3/4 and SSEA4 in flow cytometry analysis resulted in positive signals over 98% and less than 10% positive cells for differentiation factor SSEA1. This study presents, for the first time and supported by preceding in silico predictions of drying time, basic steps towards a “ready-to-use” bioreactor-integrated freeze-drying process for UHV-alginate microcarriers in iPSC context.