Effect of inoculum density on human-induced pluripotent stem cell expansion in 3D bioreactors

Abstract

Objective: For optimized expansion of human-induced pluripotent stem cells (hiPSCs) with regards to clinical applications, we investigated the influence of the inoculum density on the expansion procedure in 3D hollow-fibre bioreactors.

Materials and methods: Analytical-scale bioreactors with a cell compartment volume of 3 mL or a large-scale bioreactor with a cell compartment volume of 17 mL were used and inoculated with either 10 × 10⁶ or 50 × 10⁶ hiPSCs. Cells were cultured in bioreactors over 15 days; daily measurements of biochemical parameters were performed. At the end of the experiment, the CellTiter-Blue^® Assay was used for culture activity evaluation and cell quantification. Also, cell compartment sections were removed for gene expression and immunohistochemistry analysis.

Results: The results revealed significantly higher values for cell metabolism, cell activity and cell yields when using the higher inoculation number, but also a more distinct differentiation. As large inoculation numbers require cost and time-extensive pre-expansion, low inoculation numbers may be used preferably for long-term expansion of hiPSCs. Expansion of hiPSCs in the large-scale bioreactor led to a successful production of 5.4 × 10⁹ hiPSCs, thereby achieving sufficient cell amounts for clinical applications.

Conclusions: In conclusion, the results show a significant effect of the inoculum density on cell expansion, differentiation and production of hiPSCs, emphasizing the importance of the inoculum density for downstream applications of hiPSCs. Furthermore, the bioreactor technology was successfully applied for controlled and scalable production of hiPSCs for clinical use.

Keywords: 3D culture; bioreactor culture; cell expansion; human-induced pluripotent stem cells; inoculum density.

Figure 1

Bioreactor types used for the expansion of hiPSCs with their capillary structure. The picture shows the analytical‐scale bioreactor (A) with a cell compartment volume of 3 mL and the large‐scale bioreactor (B) with a cell compartment volume of 17 mL. The schematic image on top shows a section of the capillary structure inside the bioreactor, consisting of the following four compartments: medium capillaries I (red) and II (blue) for countercurrent medium perfusion, gas capillaries (yellow) and the space surrounding the capillaries, which serves as the cell compartment (white). The scale bars correspond to 2 cm

Figure 2

Comparison of clinical chemistry parameters during the culture of human‐induced pluripotent stem cells over 15 days in analytical‐scale bioreactors inoculated with either 10 × 10⁶ (AS 10, n = 4) or 50 × 10⁶ (AS 50, n = 3) cells, or the large‐scale bioreactor inoculated with 50 × 10⁶ (LS 50, n = 1) cells. Values are presented as mean ± SEM (AS 10 and AS 50) or single values (LS 50). The two analytical‐scale bioreactors were compared by means of the areas under the curves (AUCs) and the tipping points (TP). Differences were considered significant at P < 0.05

Figure 3

Gene expression analysis after 15 days of hiPSC culture in analytical‐scale bioreactors inoculated with 10 × 10⁶ (AS 10) or 50 × 10⁶ (AS 50) cells, in the large‐scale bioreactor inoculated with 50 × 10⁶ cells (LS 50), on 2D culture plates, or after formation of embryoid bodies. The figure displays gene expression data of POU Class 5 Homeobox 1 (POU5F1, [A]), Nanog Homeobox (NANOG, [B]), Alpha‐Fetoprotein (AFP, [C]), SRY‐Box 17 (SOX17, [D]), C‐X‐C Motif Chemokine Receptor 4 (CXCR4, [E]), Paired Box 6 (PAX6, [F]), Neurofilament Light (NEFL, [G]), GATA Binding Protein 2 (GATA2, [H]) and T‐Box Transcription Factor T (T, [I]). Expression data were normalized to the housekeeping gene Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH), and n‐fold expression values were calculated relative to undifferentiated hiPSCs before inoculation on d0 using the ΔΔC _t method. Data are presented as mean ± SEM (AS 10 n = 3; AS 50 n = 3; LS 50 n = 1; 2D d15 n = 5; EB's d15 n = 3). Differences between AS 10, AS 50 as well as 2D cultures and embryoid bodies were detected using the one‐way ANOVA; calculated values were considered significant at *P < 0.05, **P < 0.01 and ***P < 0.001

Figure 4

Comparison of CellTiter‐Blue^® fluorescence values over a time period of up to 60 minutes on the final day of the experiment (day 15). The figure shows measurements performed in analytical‐scale bioreactors (AS) inoculated with 10 × 10⁶ (AS 10, n = 2) or 50 × 10⁶ (AS 50, n = 3) cells, the large‐scale bioreactor (LS) inoculated with 50 × 10⁶ cells (LS 50, n = 1) and 2D cultures (2D d15, n = 4). Differences in the gradients of the corresponding linear correlation were detected using the unpaired, two‐tailed Student's t test and considered statistically significant at *P < 0.05 and **P < 0.01

Figure 5

Immunohistochemical staining of undifferentiated human‐induced pluripotent stem cells (hiPSCs) and hiPSCs after culture in analytical‐scale bioreactors (AS) inoculated with 10 × 10⁶ cells (AS 10) or 50 × 10⁶ cells (AS 50), in the large‐scale bioreactor (LS) inoculated with 50 × 10⁶ cells (LS 50) and in embryoid bodies (EB's d 15). The figure shows staining of POU Class 5 Homeobox 1 (POU5F1, A‐E), marker of proliferation (MKI67, F‐J), α‐smooth muscle actin (α‐SMA, K‐O) and vimentin (VIM, P‐T), alpha‐fetoprotein (AFP, U‐Y) and nestin (NES, Z‐AD). Nuclei were counterstained with Dapi (blue). Scale bars correspond to 100 µm