Expansion of human induced pluripotent stem cells (hiPSCs) in a microcarrier bioreactor. Human iPSCs (B12-3, see Ref.⁶⁹) were seeded on Matrigel-coated beads and cultured in a spinner flask as described previously for the culture of hESCs on microcarriers.⁶⁵ (A) Temporal profiles of hiPSC concentration and viability for different seeding cell densities: solid line with circles: 0.25 × 10⁵ hiPSCs/mL; dashed line with squares: 0.5 × 10⁵ hiPSCs/mL; dotted line with triangles: 1 × 10⁵ hiPSCs/mL. The cells were cultured at 45 rpm. The efficiencies of initial attachment of hiPSCs on the beads for the above seeding densities are shown in the table as mean ± standard deviation. The upper arrow points indicates that cell viability values for the curve right under it should be read on the vertical axis on the right. Similarly, the lower arrow indicates that cell concentration values for the curve below this arrow are found on the vertical axis on the left. (B) hiPSCs on microcarriers on day 6. (C) hiPSCs dissociated from beads on day 8 were plated and stained for OCT3/4A (green) and SSEA4 (red), or (D) OCT3/4A (green) and TRA-1-60 (red). Scale bars: (B) 200 μm, (C, D) 20 μm. Color images available online at www.liebertonline.com/ten.

Human embryonic stem cells (hESCs) propagated as aggregates in stirred-suspension culture. (A) H1 hESC colonies were dissociated into single cells using accutase³¹ in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632. Single cells were incubated in mouse embryonic fibroblast (mEF)-conditioned medium with 10% Matrigel (to facilitate initial aggregation) and 10 μM Y-27632 for 30 min. Subsequently, the cell suspension was introduced into spinner flasks with mEF-conditioned medium and the agitation rate was set to 60 rpm. The cells formed stable aggregates and grew as shown in the micrographs from days 0, 2, 3, to 7. (B) Live cell concentration and viability of H1 hESCs cultured in a spinner flask with mEF-conditioned medium supplemented with Y-27632. The flask was seeded with 6 × 10⁴ hESCs/mL. The upper arrow points indicates that cell viability values for the curve right under it should be read on the vertical axis on the right. Similarly, the lower arrow indicates that cell concentration values for the curve below this arrow are found on the vertical axis on the left. (C) Quantitative polymerase chain reaction was employed to probe the expression of pluripotency genes by hESCs cultured in the bioreactor (dark bars). White bars represent the expression of the corresponding genes in hESCs maintained in Matrigel-coated dishes (control). Values are shown as mean ± standard deviation (n = 3). (D) Cells from the bioreactor were plated and immunostained for OCT4 (green) and SSEA-4 (red). Scale bars: (A) 200 μm, (D) 50 μm. Color images available online at www.liebertonline.com/ten.

Solution of a population balance equation for mouse ESCs (mESCs) cultured as aggregates in a 100 mL-spinner flask. Approximately 2 × 10⁴ mESCs/mL were seeded and cultured for 4 days at 60 rpm as described.¹⁷ Equation 12 was recast in terms of the logarithm of the mESC aggregate diameter d (i.e., ) instead of mass, assuming a spherical shape for single cells and aggregates. The von Smoluchowski kernel, , was used and the equation was solved as described before.¹⁷⁰ is an average shear rate (Eq. 3) in the culture vessel. The collision efficiency was estimated in 12-h intervals based on distributions of mESC aggregate sizes determined by image analysis of micrographs. (A) The first moment of the density function normalized to the total biomass (i.e., , where is the diameter of a single mESC) is shown for mESC aggregates. (B) Temporal profile of the total biomass. (C) Values for the average diameter of mESC aggregates at different times. Gray bars: simulation; black bars: experimental data. Color images available online at www.liebertonline.com/ten.