Facile engineering of xeno-free microcarriers for the scalable cultivation of human pluripotent stem cells in stirred suspension

Abstract

A prerequisite for the realization of human pluripotent stem cell (hPSC) therapies is the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable expansion and directed differentiation of hPSCs. Comparative analyses of commercially available microcarriers clearly show the need for developing synthetic substrates supporting the adhesion and growth of hPSCs in three-dimensional cultures under agitation-induced shear. Moreover, the low seeding efficiencies during microcarrier loading with hPSC clusters poses a significant process bottleneck. To that end, a novel protocol was developed increasing hPSC seeding efficiency from 30% to over 80% and substantially shortening the duration of microcarrier loading. Importantly, this method was combined with the engineering of polystyrene microcarriers by surface conjugation of a vitronectin-derived peptide, which was previously shown to support the growth of human embryonic stem cells. Cells proliferated on peptide-conjugated beads in static culture but widespread detachment was observed after exposure to stirring. This prompted additional treatment of the microcarriers with a synthetic polymer commonly used to enhance cell adhesion. hPSCs were successfully cultivated on these microcarriers in stirred suspension vessels for multiple consecutive passages with attachment efficiencies close to 40%. Cultured cells exhibited on average a 24-fold increase in concentration per 6-day passage, over 85% viability, and maintained a normal karyotype and the expression of pluripotency markers such as Nanog, Oct4, and SSEA4. When subjected to spontaneous differentiation in embryoid body cultures or directed differentiation to the three embryonic germ layers, the cells adopted respective fates displaying relevant markers. Lastly, engineered microcarriers were successfully utilized for the expansion and differentiation of hPSCs to mesoderm progeny in stirred suspension vessels. Hence, we demonstrate a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable, xeno-free propagation and differentiation of human stem cells intended for therapies.

FIG. 1.

Seeding of human pluripotent stem cells (hPSCs) on Matrigel-coated microcarriers. (A) Efficiencies as fractions of seeded cells attached on the microcarrier surface are shown for seeded dispersed hPSCs or clusters. (B) Fraction of beads colonized after being seeded with dispersed cells or clusters. H9 human embryonic stem cells (hESCs) on Matrigel-coated beads stained with FDA after being seeded as (C) single cells or (D) small clusters. Microcarriers loaded with dispersed cells are shown at days 0, 3, and 6. Stars mark microcarriers with only a few or no cells. Scale bars: 200 μm. (E) Expansion of hESCs in spinner flasks after their seeding on Matrigel-coated microcarriers and 4 h (dashed curve) or overnight (solid curve) treatment with ROCK inhibitor. [^#p<0.05 in (A, B).] Color images available online at www.liebertpub.com/tea

FIG. 2.

Multi-passage expansion of H9 hESCs on Matrigel-coated microcarriers. (A) Growth profile and viability of microcarrier-attached cells cultured for five passages. (B) Corresponding cumulative lactate dehydrogenase (LDH) activity. Cells were probed for the expression of pluripotency markers by (C) quantitative polymerase chain reaction (qPCR), (D) flow cytometry, and (E) immunostaining (scale bars: 50 μm). Color images available online at www.liebertpub.com/tea

FIG. 3.

Peptide surface density of microcarriers and seeding efficiency of hPSCs. (A) Surface peptide density versus the total amount of peptide used in the amidation reaction. (B) Seeding efficiency of IMR90 cells on microcarriers conjugated with different amount of peptides. Cells were cultured in TeSR2 medium. (C) IMR90 cells seeded on CP beads and cultured in spinner flasks failed to spread on the beads and formed aggregates. Scale bar: 200 μm. Color images available online at www.liebertpub.com/tea

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

FIG. 5.

Human PSCs cultured for multiple passages on CP+pLL microcarriers in stirred-suspension vessels. (A) Growth profile, viability, and (B) cumulative LDH activity of IMR90 hiPSCs cultured for five passages on microcarriers in spinner flasks. After each passage, the expression of pluripotency markers was characterized by (C) qPCR and (D) flow cytometry. (E) After the last passage, cells were plated and stained for pluripotency markers (scale bars: 50 μm). (F) Karyotyping results for IMR90 cells after their culture for five passages on CP+pLL beads in spinner flasks. Color images available online at www.liebertpub.com/tea

FIG. 6.

Differentiation potential of hPSCs after their propagation on CP+pLL microcarriers for five passages. (A) IMR90 hiPSCs were subsequently cultured as embryoid bodies and expression of markers of the three embryonic germ layers was assessed. The expression of undifferentiated IMR90 cells was used for normalization. Alternatively, propagated cells were plated and subjected to directed differentiation toward (B, E) definitive endoderm (DE), (C, F) mesoderm, and (D, G) neuroectoderm. The expression was evaluated by qPCR (B–D) and immunostaining (E–G). Gene expression was normalized to IMR90 cells differentiated in basal medium without differentiation factors. Scale bars in (E–G): 50 μm. Color images available online at www.liebertpub.com/tea

FIG. 7.

Directed differentiation of hPSCs cultured on CP+pLL microcarriers in a stirred-suspension bioreactor. (A) Expression of MS genes after IMR90 hiPSC differentiation in the bioreactor. Gene expression was normalized to hiPSCs differentiated in basal medium without factors. (B) Immunostaining of differentiated cells on beads. Stars denote the position of microcarriers. Scale bars: 100 μm. (C) Flow cytometric analysis of KDR expression. A flow cytometry graph is shown from a representative experiment. Curves correspond to samples of negative control (black), cells in dishes incubated in basal media without differentiation factors (green; dish control), cells differentiated in dishes (red) and cells differentiated in microcarrier suspension culture (blue). The gate excludes 99% of the negative control cells. Results are summarized in the bar graph (n=3) as mean±SD.