Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Oct;5(10):1289-1301.
doi: 10.5966/sctm.2015-0253. Epub 2016 Jul 1.

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors

Affiliations
Free PMC article

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors

Christina Kropp et al. Stem Cells Transl Med. .
Free PMC article

Abstract

: The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 106 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse-transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism, suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture.

Significance: Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine.

Keywords: Human pluripotent stem cell expansion; Induced pluripotent stem cells; Metabolism; Perfusion; Single-use stirred tank bioreactors; Three-dimensional suspension culture.

Figures

Figure 1.
Figure 1.
Impact of feeding strategies on aggregate formation and size distribution. (A): Human induced pluripotent stem cells (hCBiPS2) were detached from monolayer cultures and seeded as single-cell suspensions on day 0 to stirred tank bioreactors. During the first 48 h cultures were maintained without any medium exchange. On culture day 2 the first complete manual medium exchange was performed for the repeated batch cultures (repeated the following days [days 3–6]), and in parallel automated continuous medium change was started for the perfusion cultures (4.2 ml/hour), resulting in equal medium throughput for both feeding strategies. On culture day 7 cells from both processes were harvested and analyzed. (B): On process days 1 and 2 (prior to perfusion start and first manual medium change in repeated batch bioreactors) as well as days 3–7 aggregates were assessed by light microscopy as shown (scale bars = 200 µm). (C): Applying AxioVision LE (Zeiss) and GraphPad prism software, between 240 and 1,480 aggregates from four independent experiments were analyzed (supplemental online Fig. 1C) from each time point and visualized as single squares. (D): The average aggregate volume calculated from the mean aggregate volume of the individual runs is depicted as columns whereby each column represents the mean of four independent bioreactor runs for each feeding strategy. Results are reported as mean ± SEM. Differences were considered statistically significant at ∗, p < .05, ∗∗, p < .01, and ∗∗∗, p < .001. Abbreviation: d, day.
Figure 2.
Figure 2.
Comparison of growth kinetics and viability during repeated batch and perfusion cultures, using hCBiPS2 cells. (A): Cells were seeded at 0.5 × 106 cells per milliliter on day 0. Cell densities (columns) and viability (solid circles) were determined daily (n = 4). Up to 4.6-fold increase in repeated batch cultures and up to 6.7-fold increase with perfusion could be achieved. Results are reported as mean ± SEM. Differences were considered statistically significant at ∗∗, p < .01 and ∗∗∗, p < .001. (B): Highest specific growth rates µ were observed on day 2 of cultivation (between 24 and 48 hours after inoculation). Abbreviation: d, day.
Figure 3.
Figure 3.
Metabolic activity of hCBiPS2 cells in repeated batch and perfusion cultures. Perfusion was initiated after 48 hours of culture. In parallel entire medium was replaced daily from day 2 onward in repeated batch cultures, keeping the daily and overall medium throughput equal. (A, B): Feeding strategies result in respective concentration pattern for glucose (A) and lactate (B) (both n = 4). (C): The specific yield coefficient of lactate from glucose Y(qLac/qGlc) is highest in the earlier process phase and decreases for both feeding strategies from process day 4 onward. (D–F): In response to repeated batch, heterogeneous “zigzag-like” patterns for pH (D) and dissolved oxygen (DO) (E) as well as culture osmolarity (n = 3) (F) in contrast to homogeneous progression at perfusion were observed. To ensure clarity of illustration, pH levels and dissolved oxygen are depicted for one bioreactor run of each feeding strategy. Samples for glucose, lactate, and osmolarity analysis were taken every 24 hours of culture and additionally after medium exchange for repeated batch cultures.
Figure 4.
Figure 4.
Pluripotency marker expression of hCBiPS2 cells after expansion with different feeding strategies. (A): Immunofluorescence of cryosections of aggregates harvested at process day 7 showed positive staining for the pluripotency-associated markers TRA1-60, SSEA3, OCT4, and NANOG as well as the proliferation-associated marker Ki-67. Respective isotype controls confirmed specificity of staining (data not shown). Scale bars = 100 µm. (B): Flow cytometry revealed that the majority of repeated batch- and perfusion-expanded cells expressed pluripotency-associated surface markers SSEA4 and SSEA3 as well as the transcription factors NANOG and OCT4 comparable to monolayer precultures (isotype control shown in gray). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.
Figure 5.
Figure 5.
Repeated batch and perfusion bioreactor-expanded hCBiPS2 cells differentiate into derivatives of all three germ layers. (A): Immunofluorescence analysis of day 7 bioreactor-derived aggregates from repeated batch as well as perfusion cultures induced for spontaneous differentiation revealed expression of marker proteins representative of all three germ layers (positive staining in red or green): sarcomeric actinin, cardiac Troponin T (mesoderm), α-fetoprotein (AFP) (endoderm), and β3 Tubulin (ectoderm). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Respective isotype controls confirmed specificity of stainings (data not shown). Scale bars = 100 µm. (B): Furthermore, modulation of the Wnt pathway by small molecules resulted in cardiac differentiation of cells from repeated batch as well as perfusion cultures. Expression of cardiac-specific markers sarcomeric actinin and αMHC as well as βMHC could be detected by flow cytometry. Abbreviations: MHC, major histocompatibility complex; sarc., sarcomeric.
Figure 6.
Figure 6.
Expression analysis revealed high similarity between hCBiPS2 cells from 2D precultures and 3D suspension culture. (A): Quantitative real-time analysis for transcription factors OCT4 and NANOG showed no significant deviation in expression levels of expanded cells either under repeated batch or under perfusion conditions and furthermore compared with 2D precultures (n = 3). Primers were defined to detect endogenous variant of the factor to exclude false positive results in induced pluripotent stem cells. (B): Expression levels of genes associated with pluripotency and early differentiation for repeated batch and perfusion-based cultures displayed as absolute processed fluorescence intensity (top) and compared with 2D precultures (bottom, n = 3). (C): Principle component analysis on global gene expression of hiPSC 2D precultures on day 3 (gray), static suspension cultures on day 3 (blue), dynamic suspension cultures with either perfusion or repeated batch on day 3 (perfusion, light green; repeated batch, orange), or process endpoint day 7 (perfusion, dark green; repeated batch, red). Visualization comprises >100 genes with σ/σmax < 0.255 and p < .001 in a group comparison. See also supplemental online Figure 4A for details of the experimental setup. (D): The hierarchically clustered heat map displays differentially expressed genes between 2D precultures, repeated batch (day 3 and day 7), and perfusion (day 3 and day 7) samples of genes allocated to the GO categories “glycolytic process,” “regulation of glycolytic process,” “oxidative phosphorylation,” and “mitochondrial electron transport” (multigroup comparison; p < .1). The microarray dataset presented in this figure was deposited in Array Express with accession no. E-MTAB-3898 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3898). Abbreviation: d, day.
Figure 7.
Figure 7.
Characterization of perfusion cultures using hHSC_F1285T_iPS2 cells confirmed the robustness of the expansion process. (A): An average viable cell density of 3.01 × 106 cells per milliliter was obtained in 7-day lasting perfusion cultures representing an approximately sixfold hPSC expansion. Cell densities (columns) and viability (symbols) were determined daily (n = 3). (B): Representative microscopic images of aggregate formation and progression (scale bars = 200 µm). (C, D): Specific growth rate (C) and yield coefficient of lactate from glucose Y(qLac/qGlc) (D) were highest in earlier process phase for the independent second cell line as well (both n = 3). Results are reported as mean ± SEM. Differences were considered statistically significant at ∗, p < .05. (E): Flow cytometry analysis revealed that the majority of cells harvested on day 7 were positive for pluripotency markers OCT4, NANOG, SSEA4, and TRA 1-60 (red; isotype in gray). (F): Quantitative real-time analysis for pluripotency transcription factors OCT4 and NANOG revealed no significant deviation in expression levels for cells harvested on day 7 compared with 2D monolayer precultures (n = 3). Primers were defined to detect endogenous variant of the gene only. (G): Hierarchically clustered heatmap displaying differentially expressed genes between 2D preculture and perfusion (day 3 and day 7) samples allocated to the GO categories “glycolytic process,” “regulation of glycolytic process,” “oxidative phosphorylation,” and “mitochondrial electron transport” (multigroup comparison: p < .1). The microarray dataset presented in this figure was deposited in Array Express with accession no. E-MTAB-4149 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4149). Abbreviation: d, day.

Similar articles

See all similar articles

Cited by 24 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback