Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred-Suspension Culture

Abstract

Human induced pluripotent stem cells (hiPSCs) hold great hopes for application in regenerative medicine due to their inherent capacity to self-renew and differentiate into cells from the three embryonic germ layers. For clinical applications, a large quantity of hiPSCs produced in standardized and scalable culture processes is required. Several groups, including ours, have developed methodologies for scaled-up hiPSC production in stirred bioreactors in chemically defined medium. In this study, we optimized the critical steps and factors that affect hiPSC expansion and yield in stirred-suspension cultures, including inoculation conditions, seeding density, aggregate size, agitation rate, and cell passaging method. After multiple passages in stirred-suspension bioreactors, hiPSCs remained pluripotent, karyotypically normal, and capable of differentiating into all three germ layers.

Large‐scale expansion of feeder‐free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation

“…Our future studies will focus on translating these protocols to hESC and hiPSC lines. Although human pluripotent cells are more challenging to work with, numerous authors have shown expansion and chromosomal normality following single‐cell inoculation (Abbasalizadeh, Larijani, Samadian, & Baharvand, 2012; Abecasis et al, 2017; Haraguchi, Matsuura, Shimizu, Yamato, & Okano, 2015; Kwok et al, 2018) and aggregate preformation (Hunt, Meng, Rancourt, Gates, & Kallos, 2014; Meng, Liu, Poon, & Rancourt, 2017) into stirred suspension bioreactors. As our lab has had success with translating bioprocess protocols from mouse to human stem cells in the past (Baghbaderani et al, 2008; Gilbertson, Sen, Behie, & Kallos, 2006) and are hopeful this can be replicated with our PSC work.…”

Bioengineering Considerations for a Nurturing Way to Enhance Scalable Expansion of Human Pluripotent Stem Cells

“…The change in size distribution of aggregate is affected directly by cell division, or indirectly by joining of new cells from outside and the association of smaller aggregates to form larger ones, the breakage of aggregates to form smaller ones, cell growth, and death owing to the stress caused by fluid motion . The bioreactor's operating parameters determine the distribution of aggregates in a culture fluid, size of the aggregates, and expansion capacity through controlling amount of shear force applied on the cells . Agitation and shear stress, which are concurrently making and breaking cell–cell and cell–substrate interactions within cell aggregates, may have adverse effects on aggregate morphology and growth kinetics.…”

“…Once aggregates have formed, they gather at the bottom of the culture vessel due to the lack of mixing and generate larger aggregates owing to fusion of aggregates, leading to cell death at the central region of the aggregates. Many studies have been carried out to gain knowledge on shear stress fluid shear stress caused by mixing in the bioreactor as a function of cell types . Cormier et al reported that mouse ESCs (mESCs) successfully expanded as aggregates in spinner flasks at an agitation of 80–100 rpm which corresponds to a shear stress in the range from 4.5 to 6.1 dyn cm −2 .…”

“…However, mESCs exhibit minimal growth and extensive damage, when agitation was increased to 120 rpm or 7.8 dyn cm −2 . The boundary conditions for aggregate size and culture time in suspension culture depends on numerous parameters, in particular the cell types, the seeding density, culture media, culture platform, and the stirring device . This will have a negative effect on their culture quality, which partly explains why this type of culture is associated with lower efficiency during large‐scale production compared to that of static suspension culture.…”

“…Suspension culture based on matrix-free cell-only aggregates solely relies on cell adhesion properties, in which cells require time to control the size of aggregates, while maintaining high cell viability during/after aggregate formation and optimal biological characteristics of hPSCs. [62][63][64][65][66][67][68] Figure 4 shows a schematic illustration of aggregate morphology and growth kinetics of hPSCs in static-and dynamic-suspension culture systems. The kinetics of hPSC aggregation can be predicted by modeling the steps that lead to aggregate growth; common model assumes that cellular aggregation determines the rate of collisions with other cells (or aggregates) and the rate of cell division, which is a prerequisite for aggregate growth.…”