(229x) Production of Self-Assembled Aggregates of Human Mesenchymal Stem Cell in the Wave bioreactorTM for Cell Therapy

Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and regenerative medicine, and have been tested in clinical trials for a wide range of diseases. Recent studies showed that hMSC have natural ability to self-assemble into three-dimensional (3D) aggregates that enhance their therapeutic functions with augmented multi-lineage differentiation potential, migration ability, secretions of anti-inflammatory and angiogenic factors, and resistance to ischemic conditions post-transplantation. Presently, various laboratory methods have been developed for hMSC aggregates production, including manual hanging drops, centrifugation with microfabricated surface, low attachment surface treatment, thermal lifting, and microfluidic technologies. However, these method have limited scalability and/or poor control in aggregate size, and can not meet the required production in clinical trials. This study is to produce size-controlled aggregates of hMSCs in a scalable bioreactor for cell therapy.

The WAVE bioreactor^TM provides a repeated back and forth gentle wave motion that accelerates cell-cell collisions and boosts hMSC aggregation at low shear stress. We first investigated the influence of rocking angle (3Ë?, 6Ë?, and 9Ë?) and rocking speed (10, 15, 20 rpm) on aggregation kinetics and size distribution. hMSC aggregates were formed in an ultra-low attachment 6-well plate on a WAVE bioreactorTM base at a certain rocking condition for 18 h to 72 h. Aggregate size was converted by the projected area of aggregates based on the light microscope images with an assumption that the aggregates are spherical in shape. Since the fluidic shear stress increases the cell-cell collision but decreases the binding kinetics upon cell-cell contract, the shear stress significantly impacts the aggregation process and aggregate size distribution. Computational fluid dynamics analysis using COMSOL Multiphysics indicates that the maximum shear stress was proportional to the rocking angle and speed under a two-variable linear regression, and the rocking angle had a more significant effect than the rocking speed. Aggregate size distribution was also found to be significantly dependent on the cell-cell collision frequency as alteration in cell seeding density and incubation time influence aggregate size distribution. Therapeutic functional tests supported that hMSCs derived from engineered aggregates in WAVE bioreactor^TM have significantly higher expression of stem cell genes and secretion of anti-inflammatory factors compared with those from monolayer culture.