Figure 6.

Cultispher-S microcarriers support expansion of undifferentiated SHEF3 hESCs. 3 × 10⁶ SHEF3 hESCs were seeded onto Cultispher-S microcarriers in KO-DMEM supplemented with 20% (v/v) KO-SR and 4 ng/mL FGF-2 in the presence (3D CM) or absence (3D Non-CM) of MEF conditioned medium (CM). Cells were cultured for 7 days, passaged and then cultured for a further 7 days. A: (i) Cell numbers plated and recovered at each passage for each condition are presented. (ii) The mean number of viable cells attached to microcarriers (±SD) are shown for each condition for the culture periods d0–d7 (upper panel) and d7–d14 (lower panel). B: Assessment of markers of pluripotency. (i) Expression of SSEA4, Tra-1–60 and Tra-1–85, were determined by flow cytometry. 2D indicates hESCs controls maintained in 2D culture on MEF plus FGF-2. RNA and protein were extracted from hESCs cultured in 2D or 3D conditions for the times indicated and expression of OCT-4 and NANOG determined by (ii) semi-quantitative RT-PCR; (iii) quantitative RT-PCR normalized to β-actin (±SD) or (iv) immunoblotting, normalized to SHP-2.