Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells

Abstract

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

Figure 1

Schematic illustration of the core-shell microfiber culture system for human induced pluripotent stem (iPS) cells. (A) An illustration of the double co-axial laminar flow microfluidic device used for the formation of the human iPS cell-laden core-shell hydrogel microfiber. (B) The cells encapsulated within the core-shell microfibers proliferate and form cell aggregations with uniform thickness by expanding along the microfiber.

Figure 2

The thickness and the viability of human iPS cell aggregations in the core-shell microfiber culture system. (A) Microscopic images showing the morphologies of human iPS cell aggregations in the core-shell microfiber culture and the suspension culture systems on day 4 and 8. Scale bar: 500 μm. (B) Thickness distributions of cell aggregations in the core-shell microfiber culture and the suspension culture systems on days 4 and 8. The thickness was calculated by measuring the minor axis of cell aggregation regarded as an ellipse. (C) The cell viabilities in the core-shell microfiber culture and the suspension culture systems after the retrieval of cell aggregations from the microfibers and dissociation of cell aggregations into single cells on days 4 and 8 (N ≥ 3). *P < 0.05.

Figure 3

Human iPS cells cultured in various types of core-shell microfibers. (A) Phase-contrast images showing the different types of human iPS cell-laden core-shell microfibers immediately after fabrication (day 0). Scale bar: 500 μm. (B) Merged phase-contrast and fluorescence images of the six types of core-shell microfibers cultured by day 4. Live cells were stained with calcein AM (green) and dead cells were stained with ethidium homodimer-1 (red). Scale bar: 500 μm. (C) Viability of the cells cultured in the six types of microfibers after the retrieval of cell aggregations from the microfibers and dissociation into single cells on day 4 (N ≥ 3).

Figure 4

Optimization of the initial cell densities and the extracellular matrix (ECM) components in the core-shell microfibers. (A) Expansion rates of human iPS cells in the core-shell microfiber culture systems. The cells were retrieved on day 4, and their expansion rates were calculated (N ≥ 3). *P < 0.05. (B) Reverse transcription polymerase chain reaction (RT-PCR) analysis of the pluripotency-associated marker genes expression in cells cultured in six types of human iPS cell-laden core-shell microfibers for 4 days. 2D, cells cultured on a Matrigel-coated culture plate (positive control) and EB, embryoid bodies cultured in differentiation medium (negative control). Full-length gels are presented in Supplementary Figure S9. (C) Flow cytometry analysis of OCT3/4 expression in cells cultured with collagen at initial cell density of 1.0 × 10⁷ or 1.0 × 10⁸ cells/mL for 4 days (N = 3). The black solid line and gray dashed line indicate stained cells and the negative control, respectively.

Figure 5

Passaging of human iPS cells in the core-shell microfiber culture system. (A) Expansion rates of human iPS cells cultured in the microfibers accompanied with collagen at low initial cell density (1.0 × 10⁷ cells/mL) before and after passage (N ≥ 3). The cells were passaged on day 4, 6, or 8. *P < 0.05. (B) Expansion rates of the cells cultured in the microfibers by serial passaging (N ≥ 3). Cells were passaged every 4 days and cultured until 8 passages (32 days).

Figure 6

Characterization of the human iPS cells cultured in the core-shell microfiber culture system. (A) RT-PCR analysis of pluripotency-associated marker genes. 2D, cells cultured on a Matrigel-coated culture plate (positive control); Fiber, cells after culture for 44 days (11 passages) in the core-shell microfiber culture system; and HDF, human dermal fibroblasts (negative control). Raw data of gels are presented in Supplementary Figure S10. (B) Fluorescence images of cell aggregations stained for OCT3/4 (green) or NANOG (red) after culture for 44 days (11 passages). Nuclei were stained with Hoechst 33342 (blue). Scale bar: 100 μm. (C) RT-PCR analysis of differentiation marker genes. 2D, cells cultured on a Matrigel-coated culture plate (positive control); Fiber, cells after culture for 44 days (11 passages) in the core-shell microfiber culture system; and Differentiated fiber, cells cultured in the core-shell microfiber culture system with differentiation-induction medium after maintaining the culture for 48 days (12 passages) in the culture system. Full-length gels are presented in Supplementary Figure S11. (D) Fluorescence images of the in vitro differentiated fibers and teratoma using cells cultured for more than 32 days (8 passages) in the core-shell microfiber culture. The cells and tissues were stained with an ectoderm marker (TUJ1: green), a mesoderm marker (αSMA: red), an endoderm marker (AFP: red), and Hoechst 33342 (nuclei: blue). Scale bar: 100 μm.