Microfibrous carriers for integrated expansion and neural differentiation of embryonic stem cells in suspension bioreactor

Abstract

A scalable suspension culture process for integrated expansion and neural differentiation of murine embryonic stem cells (mESCs) was developed using polyethylene terephthalate (PET) microfibrous carriers, which can provide large surface areas and an in vivo-like three-dimensional (3-D) environment to facilitate high density ESC growth and lineage-specific differentiation. Cells inoculated within microfibrous carriers in a 125-mL bioreactor were studied at two different agitation speeds, 60rpm and 90rpm. Agitation at 60rpm supported faster and more uniform cell seeding, and led to a higher cell expansion fold compared to agitation at 90rpm. For integrated expansion and neural differentiation, cells in the microfibrous carriers were cultured in the expansion medium for 4 days and then in the astrocyte-conditioned medium to induce neural differentiation for 10 days. In this integrated process, mESCs were expanded 22-fold with 60–67% of cells exhibiting neural markers (i.e., nestin, neurofilament) as analyzed by flow cytometry. The neural differentiation was further verified with reverse transcriptase polymerase chain reaction (RT-PCR), which showed up-regulation of neural genes nestin, Nurr1, and NF-68 (a light chain of neurofilament), and down-regulation of pluripotent genes Oct-4, Rex-1 and Nanog. The microfibrous carrier-based suspension culture system is simple and easy to scale up, and can be used to produce large amounts of ESC-derived cells for clinical applications.