Facile Bead-to-Bead Cell-Transfer Method for Serial Subculture and Large-Scale Expansion of Human Mesenchymal Stem Cells in Bioreactors

Abstract

The conventional planar culture of adherent cells is inefficient for large-scale manufacturing of cell and gene therapy products. We developed a facile and efficient bead-to-bead cell-transfer method for serial subculture and large-scale expansion of human mesenchymal stem cells (hMSCs) with microcarriers in bioreactors. We first compared culture medium with and without nucleosides and found the former maintained the expression of surface markers of hMSCs during their prolonged culture and enabled faster cell proliferation. Subsequently, we developed our bead-to-bead cell transfer method to subculture hMSCs and found that intermittent agitation after adding fresh microcarriers to cellpopulated microcarriers could promote spontaneous cell migration to fresh microcarriers, reduce microcarrier aggregation, and improve cell yield. This method enabled serial subculture of hMSCs in spinner flasks from passage 4 to passage 9 without using proteolytic enzymes, which showed faster cell proliferation than the serial planar cultures undergoing multiple enzyme treatment. Finally, we used the medium containing nucleosides and our bead-to-bead cell transfer method for cell culture scale-up from 4-to 50-L cultures in single-use bioreactors. We achieved a 242-fold increase in the number of cells to 1.45 Â 10 10 after 27-day culture and found that the cells harvested from the bioreactors maintained proliferation ability, expression of their surface markers, tri-lineage differentiation potential and immunomodulatory property. This study shows the promotive effect of nucleosides on hMSC expansion and the potential of using our bead-to-bead transfer method for larger-scale manufacturing of hMSCs for cell therapy.

Seed Train Optimization in Microcarrier-Based Cell Culture Post In Situ Cell Detachment through Scale-Down Hybrid Modeling

“…Using the method of cell detachment from MCs is crucial for optimal seed training. Some studies suggest the possibility of cell migration from confluent MCs to newly added bare MCs through bridging mode as a method of seed training and scaling up [ 42 , 43 , 44 , 45 ]. This non-enzymatic bridging mode prevents any potential physiological damage to the cells [ 45 ].…”

“…Some studies suggest the possibility of cell migration from confluent MCs to newly added bare MCs through bridging mode as a method of seed training and scaling up [ 42 , 43 , 44 , 45 ]. This non-enzymatic bridging mode prevents any potential physiological damage to the cells [ 45 ]. However, the reported recovery of this method is lower compared to the detachment and re-attachment approaches, 59 ± 19% compared to 85 ± 4 [ 24 ].…”

A controllable gelatin-based microcarriers fabrication system for the whole procedures of MSCs amplification and tissue engineering

“…In contrast, for microcarriers composed of indissolvable polymers such as dextran and polystyrene, harvest programs typically involve detaching cells from the microcarriers and separating the microcarriers from the cell suspension. The former step often involves treatment with trypsin for more than 12 min [ 35 ] while the latter step often employs filtration methods. However, achieving sufficient harvest using these methods can be challenging and may limit the final yield.…”

Current Advances in 3D Dynamic Cell Culture Systems

“…In addition, differences in morphology, surface markers, gene expression profiles, and secretome are observed between the microcarrier and conventional 2D systems in the stem cell culture [ 116 , 120 ]. The other characteristic of microcarrier cell culture is that cells can go through a bead-to-bead transfer [ 121 ], which simplifies the subculture process in the large-scale production of stem cells. In addition to large-scale production, microcarriers can also serve as a vehicle for cell delivery in tissue regeneration, especially in bone and cartilage tissue engineering.…”