FIG. 4.

Coating of peptide-conjugated microcarriers with poly-l-lysine (pLL) for hPSC culture in xeno-free medium. (A) Seeding efficiency of IMR90 and H9 cells on microcarriers featuring different surface treatments: CP+pLL: beads with conjugated peptide and pLL coating; CP: beads with conjugated peptide; FP+pLL: beads with free (unconjugated) peptide and pLL coating; pLL: beads with pLL coating only; N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS): beads subjected to the coupling reaction without peptide; Untreated: plain beads without any treatment. *p<0.05: CP+pLL versus pLL and CP+pLL versus FP+pLL, ^#p<0.05: CP versus EDC/NHS and CP versus untreated. Mean values are compared among different microcarriers for the same cell type (H9 or IMR90). (B) FDA-stained IMR90 cells on CP+pLL beads right after the seeding phase (left) and during spinner flask culture (right). (C) Time course of the concentration and viability of IMR90 human induced pluripotent stem cells (hiPSCs) cultured on CP+pLL beads in stirred suspension. (D) Relative expression of NANOG in IMR90 cells cultured for 6 days in a microcarrier suspension (bioreactor). The corresponding gene expression of IMR90 cells maintained in dishes is also shown. (E) Analysis of cultured cells (day 6) by flow cytometry for the expression of stem cell markers. Values are shown as mean±SD (n ≥3). (F) Immunostaining of cells after 6 days of expansion on CP+pLL beads in the bioreactor. Cells in (A–F) were cultured in TeSR2 medium. Scale bars in (B): 200 μm, (F): 50 μm. Color images available online at www.liebertpub.com/tea