Stirred Suspension Bioreactor Culture of Porcine Induced Pluripotent Stem Cells

Abstract

Induced pluripotent stem cells (iPSCs) are an attractive cell source for regenerative medicine and the development of therapies, as they can proliferate indefinitely under defined conditions and differentiate into any cell type in the body. Large-scale expansion of cells is limited in adherent culture, making it difficult to obtain adequate cell numbers for research. It has been previously shown that stirred suspension bioreactors (SSBs) can be used to culture mouse and human stem cells. Pigs are important preclinical models for stem cell research. Therefore, this study investigated the use of SSBs as an alternative culture method for the expansion of iPSCs. Using an established porcine iPSC (piPSC) line as well as a new cell line derived and characterized in the current study, we report that piPSCs can grow in SSB while maintaining characteristics of pluripotency and karyotypic stability similar to cells grown in traditional two-dimensional static culture. This culture method provides a suitable platform for scale-up of cell culture to provide adequate cell numbers for future research applications involving piPSCs.

Keywords: bioreactor; iPS cells; porcine.

FIG. 1.

Experimental design. Schematic representation of SSB, where Wi, Di, Dt represents an impeller width of 0.95 cm, an impeller diameter of 3.5 cm, and a vessel diameter of 3.88 cm, respectively (A). Schematic representation of comparative culture (B). SSB, stirred suspension bioreactor. Color images are available online.

FIG. 2.

Apparent doubling time, viability, and cytogenetic analysis. Apparent doubling (A) and cell viability (B) comparing SSB to static cultures over eight passages. Cells were passaged every 3 days, and cells were collected at day 3 for analysis n = 5. Error bars = SD. Cytogenetic analysis of piPSCs (C; image shows piPSCs from SSB culture). Cytogenetic analysis was performed on preculture piPSCs, P8 SSB piPSCs, and P8 static piPSCs; n = 5. piPSCs, porcine induced pluripotent stem cells. Color images are available online.

FIG. 3.

Appearance of piPSCs in static and SSB culture. piPSCs (constitutively expressing GFP) grown in static (A, A^II) and SSB (B^I, B^II, C^I, C^II). Aggregates formed in SSB culture are of relatively uniform size (61.8 ± 1.6 μm in diameter at P6 and 87.5 ± 14.2 μm at P8, n = 462 aggregates) and shape; scale bar = 200 μm. Color images are available online.

FIG. 4.

Protein and gene expression of pluripotency-associated and proliferation markers. (A) qPCR analysis assessing relative gene expression levels of pluripotency-associated markers; n = 4. (B) Flow cytometry illustrating percentage of pluripotency-associated (OCT4, SOX2, SSEA-4) and proliferative (Ki67)-positive piPSCs; n = 3. (C, D) Immunocytochemistry of piPSCs cultured in SSBs (C) and piPSCs cultured statically (D) for pluripotency-associated (OCT4; purple, SOX2; red, SSEA-4; purple) and proliferation (Ki67; red) markers; scale bar = 50 μm. qPCR, quantitative polymerase chain reaction. Color images are available online.

FIG. 5.

Gene expression analysis. Upregulation of genes indicative of differentiation (A–C) and the subsequent downregulation of pluripotency-associated gene expression (D–F) on day 3 of spontaneous embryoid body differentiation. Differentiation medium with or without DOX was used to determine the differentiation capacity of the piPSCs. Significant differences in gene expression were observed for (A) ACTA2 (^a,bP < 0.05); (B) GATA6 (^a,bP < 0.05); (C) NESTIN (^a,bP < 0.05; ^a,cP < 0.05; ^b,cP < 0.05); (D) cMYC (^a,bP < 0.05); (E) KLF4 (^a,bP < 0.05); and (F) SOX2 (^a,bP < 0.05). Gene expression was normalized to GAPDH and represented in log2 scale; n = 5. Error bars = SD. DOX, doxycycline. Color images are available online.

FIG. 6.

Differentiation-associated protein expression in day 3 spontaneously formed embryoid bodies from piPSCs. Intact embryoid bodies were collected and stained for ectoderm (NESTIN; purple) and mesoderm (α-SMA; purple) proteins to illustrate differentiation into the respective germ lineages. Cells are constitutively expressing GFP (green). Scale bar 50 μm. Color images are available online.

FIG. 7.

Apparent doubling time, viability, and cytogenetic analysis. Apparent doubling (A) and cell viability (B) comparing SSB to static cultures over eight passages. Cells were passaged every 3 days, and cells were collected at day 3 for analysis n = 3. Error bars = SD. Cytogenetic analysis of piPSC (C) image shows piPSCs from SSB culture. Cytogenetic analysis was performed on preculture piPSCs, P8 SSB piPSCs, and P8 static piPSCs. Color images are available online.