https://orcid.org/0000-0002-1707-6461
Figures & data
Table 1 Primer Sequences for Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
Figure 1 Summary of the experimental scheme.
Notes: Undifferentiated human induced pluripotent stem cells (IPSCs) (IMR90-1) were incubated with fresh mTeSR™1 and 10 µM Y-27632. They were then resuspended in 1.1% (w/v) low-viscosity alginic acid and 0.2% (v/v) bovine gelatin at room temperature (20‒25°C). Using a peristaltic pump and a drop height of 3 cm, the cell-gel solution was passed through a 25-gauge needle into a sterile 100 mM CaCl2 solution (Sigma‑Aldrich) at pH 7.4. Gelation was instant upon contact with the CaCl2 solution, resulting in the formation of spherical beads (approximately 2.5 mm in diameter after swelling). Several conditions were tested to optimize the viability/proliferation of cells within the three-dimensional (3D) environment: static versus dynamic culture; addition of adhesion protein to alginate (Matrigel™; the Matrigel™ matrix (BD) versus gelatin); and addition of Y-27632 to the culture.
Figure 2 Characterization of human-induced pluripotent stem cells (IPSCs) before and after encapsulation.
Notes: (a) Image from day 5 showing large and tightly packed human IPSCs in multicellular colonies with distinct borders; this represented the optimal time for passaging and was used as a control. (b) Passaging of human IPSCs as single cells after incubation with fresh mTeSR™1 and 10 µM Y-27632 for 2 h, resuspended in 1.1% (w/v) low-viscosity alginic acid for encapsulation. (c and d) Hematoxylin and eosin staining to visualize cellular detection and overall cell distribution within the hydrogel. (e) 3D static culture from day 5. Red arrow revealed that aggregated cells and debris might derive from necrotic or dead cells. a: Scale = 200 μm. b, c, d, e, and f: scales = 20 μm. (f) Day 5 from 3D dynamic culture. Red arrow revealed highly proliferated and packed viable cells without debris.
Figure 3 Comparison of proliferation between two-dimensional (2D), static, and dynamic cultures of human induced pluripotent stem cells.
Notes: (a) Images of live/dead staining show cell viability at day 15. Red arrows show the necrotic cores that occur under static conditions. Scale bar, 2 mm; (b) proliferation was determined using MTS assays. Cell growth was evaluated at an absorbance of 570 nm. Cells encapsulated in 1.1% alginic acid and 0.2% gelatin under dynamic conditions presented significant differences in proliferation on different time point with both 2D and static conditions. All data represent the mean of three independent experiments. P-values obtained by two-way analysis of variance **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Figure 4 Proliferation of human induced pluripotent stem cells encapsulated within alginate hydrogel red gelatin (3DD G) or alginate hydrogel+ Matrigel™ (3DD M) in 3D dynamic culture.
Notes: Proliferation was determined using MTS assays. Proliferation differed significantly between cells encapsulated in 1.1% alginic acid and 0.2% gelatin under dynamic motion and between those encapsulated in 1.1% alginic acid and Matrigel™ under dynamic motion on day 10. All data represent the mean of three independent experiments. P-values obtained by two-way analysis of variance ****p < 0.0001.
Figure 5 Proliferation of human induced pluripotent stem cells encapsulated within alginate hydrogel + gelatin and cultured with mTeSR™ medium with ROCK inhibitor 3DD (RI) or without ROCK inhibitor 3DD (W/O RI) in dynamic three-dimensional (3D) culture.
Notes: Proliferation plot based on the results of an MTS assay. Cellular proliferation was significantly higher on days 5 and 20 in 3D dynamic culture in the presence of 10 µM ROCK inhibitor than in the absence of ROCK inhibitor. All data represent the mean of three independent experiments. P-values obtained by two-way analysis of variance ****p < 0.0001.
Figure 6 Comparison of the pluripotency and differentiation markers between the static and dynamic conditions.
Notes: (a) Expression of pluripotency markers OCT3/4 under static and dynamic conditions on days 5 and 20. (b) Expression of pluripotency marker Utf1 under dynamic conditions on days 5 and 20. (c) Expression of pluripotency markers Sox2 under static conditions on day 5 versus day 20. (d) Expression of pluripotency markers Nanog under static conditions on day 5 versus day 20. (e) Expression of pluripotency markers Rex1 under static conditions on day 5 versus day 20. (f) Expression of the spontaneous differentiation marker PAX6 under static and dynamic culture conditions on days 5 and 20. Real-time quantitative polymerase-chain reaction (RT-qPCR) for the spontaneous differentiation marker PAX6. Bars show the mean fold change on days 5 and 20; error bars denote one standard deviation; n = 3 biological replicates; data are normalized to 2D culture on day 5 (2−ΔΔCt). P-values obtained by two-way analysis of variance *p ≤ 0.5, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure 7 Effect of hypoxia on encapsulated cells during prolonged adaptation under both static and dynamic conditions.
Notes: (a) Expression of the acute hypoxia marker HIFα1 under three-dimensional (3D) culture conditions at different time points. Real-time quantitative polymerase-chain reaction (RT-qPCR) for HIF1α expression. (b) Expression of the chronic hypoxia adaption marker HIFα2 under 3D culture conditions on days 5 and 20. RT-qPCR for HIFα2 expression. Bars show the mean fold change on days 5 and 20; error bars denote one standard deviation; n = 3 biological replicates; data normalized to 2D culture on day 5 (2−ΔΔCt). P-values obtained by one-way analysis of variance *p ≤ 0.5 and ****p ≤ 0.0001.
Figure 8 Extracellular matrix (ECM) secretion by human induced pluripotent stem cells (IPSCs) cultured in a three-dimensional (3D) environment.
Notes: (a) Expression of LAMA5 under static and dynamic culture conditions on days 5 and 20. Real-time quantitative polymerase-chain reaction (RT-qPCR) for ECM markers. (b) Expression of FN1 under dynamic culture conditions on days 5 and 20. qRT-PCR for ECM markers LAMA5 and FN1. Bars show the mean fold change on days 5 and 20; error bars denote one standard deviation; n = 3 biological replicates; data are normalized to 2D culture on day 5 (2−ΔΔCt). P-values obtained by one-way analysis of variance *p ≤ 0.5, ****p ≤ 0.0001 and ***p ≤ 0.001.
