Development of a novel feeding regime for large scale production of human umbilical cord mesenchymal stem/stromal cells

Human umbilical cord mesenchymal stem/stromal cells (hUC-MSCs) have attracted significant research interests in regenerative medicine and cell-based therapies due to their minimally invasive isolation procedure, abundant availability, allogenic nature, improved proliferation capacity and tri-lineage differentiation potential. However, the challenge in harvesting a sufficient number of hUC-MSCs through conventional static culture for downstream application hinders the downstream clinical translation of hUC-MSCs. Hence, an alternative culture method that can facilitate large-scale expansion is highly desirable. Herein, we developed a microcarrier-based dynamic culture system to culture hUC-MSCs combined fed-batch mode with medium refreshment to decrease concentrations of metabolic wastes, improve nutrient supplement and reduce the amount of medium used for cell culture. Instead of refreshing medium based on the pre-determined frequency, the replacement and feeding of medium using the novel feeding regime were carried out based on consumption of nutrients (glucose and glutamine) and production of metabolic waste (lactate and ammonia) to maintain a balanced and benign culture microenvironment. The optimal process allowed over 20 folds increase of cell with a maximum cell density at (24.13 ± 0.59) × 105 cells/mL. In addition, no significant alteration of cell surface markers of hUC-MSCs derived from dynamic conditions was observed compared to static conditions. Impressively, over 99.8% of the cellular population showed the desired positive expression of CD73, CD90 and CD105, while less than 0.2% of the population showed undesired negative expression of CD34, CD45 and HLA-DR. More importantly, hUC-MSCs derived from our dynamic culture condition still maintained their trilineage differentiation capacity.