(1243-D) Cell Growth and Maintenance of Undifferentiated State in Microcarrier Culture System for Human Induced Pluripotent Stem Cells using Laminin 511E8 Fragment

Abstract: Human pluripotent stem cells (hPSCs) hold a great promise for regenerative medicine. However, a large number of cells is required for a clinical study, where conventional monolayer and 3D suspension culture systems might pose problems in the scale-up and maintenance of critical cell functionality. The microcarrier culture system permits hPSC growth as monolayer on the microcarrier surface with the benefits of a homogeneous suspension culture. Nevertheless, this culture system with xeno-free matrix to support the strong hPSC adhesion and expansion has not yet been fully established.

Recombinant laminin (LM) 511E8 fragment (= iMatrixTM-511) is a xeno-free truncated form of laminin-511 containing the binding site for the integrin α6β1 predominantly expressed on hPSCs. The strong interaction of LM511E8 with integrin α6β1 enables hPSCs to preserve not only an undifferentiated state and pluripotency, but also long-term single cell passaging.

Previously, we introduced a series of workflows in a small-scale microcarrier culture system using LM511E8, which is scalable for therapeutic application. We presented the acquisition of multiple-layer human induced pluripotent stem cell (iPSC) expansion on microcarriers after 6 days in culture with the protocol. Moreover, we showed the dissociation of cells from microcarriers with 5 mM EDTA/D-PBS(-). In the current study, we continuously aim to establish the microcarrier culture system in the hiPSCs with LM511E8, particularly focusing on cell growth and maintenance of the undifferentiated state. We cultured hiPSCs with 11.2 mg of microcarriers and a seeding density of 3.0x105 cells with a working volume of 5 mL bioreactor stirring at 40 rpm for up to 6 days. We first quantified the number of cells at days 3 to 6 and found a linear correlation between cell expansion and culture period. The average growth rate at day 6 reached approximately 14-folds. We next investigated the preservation of undifferentiated state by performing flow cytometry and immunostaining analyses.

Both analyses revealed the expression of an undifferentiation marker, OCT3/4, in almost all cells up to day 4, while cells not expressing the marker started being observed at day 5 and longer. In conclusion, hiPSCs cultured with our current protocol expanded linearly and could maintain undifferentiated state up to day 4.