Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells

Abstract

Efficient translation of human induced pluripotent stem cells (hiPSCs) requires scalable cell manufacturing strategies for optimal self-renewal and functional differentiation. Traditional manual cell culture is variable and labor intensive, posing challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient- and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation and generated functional neurons, cardiomyocytes, and hepatocytes. To validate our approach, we compared robotic and manual cell culture operations and performed comprehensive molecular and cellular characterizations (e.g., single-cell transcriptomics, mass cytometry, metabolism, electrophysiology) to benchmark industrial-scale cell culture operations toward building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy.

Keywords: CEPT cocktail; biomanufacturing; cell differentiation; high-throughput; iPS cell; mass cytometry; robotic cell culture; single-cell transcriptomics; standardization.

Graphical abstract

Figure 1

Overview of the automated CTST system Features and components of CTST, including flask incubator, plate incubator, storage of large volumes of medium, cell counting, viability analysis, microscopic imaging, and a sterile HEPA-filtered cabinet housing a robotic arm, various pipettes, and a chilling unit to store temperature-sensitive reagents such as recombinant proteins.

Figure 2

Characterization of hiPSCs (LiPSC-GR1.1) cultured by CTST (A) Characteristics and advantages of automated cell culture. (B) Representative hiPSCs growing in densely packed colonies at 3 days post-passaging. Scale bar, 500 μm. (C) Colony of hiPSCs showing typical morphological features of human pluripotent cells at 3 days post-passaging. Scale bar, 250 μm. (D) hiPSCs immunostained for pluripotency-associated markers OCT4 and NANOG. Scale bar, 100 μm. (E) Long-term robotically cultured hiPSCs maintain a normal karyotype (passage 40). (F) Seahorse XF glycolysis stress test profile comparison of glycolytic function in hiPSCs maintained by automated or manual cell culture. Cells were treated with serial injections of metabolic modulators (glucose, oligomycin, 2-deoxyglucose [2-DG]). (G) Seahorse XF mitochondrial stress test profile comparison of mitochondrial function in hiPSCs maintained by automated or manual cell culture. Cells were treated with serial injections of metabolic modulators (oligomycin, FCCP, and rotenone/antimycin A [Rot/AA]). (H) Comparison of medium change intervals during automated and manual cell culture of hiPSCs. (I–O) Supernatants of cultures maintained manually or robotically were measured daily (Vi-Cell MetaFLEX Bioanalyte Analyzer). Boxplots show the variation of spent medium from hiPSC cultures. (I) pO₂, (J) pH, (K) cLac, (L) cGlu, (M) cCa⁺, (N) cNa⁺, (O) cK⁺. (P) Image-based analysis comparing cell growth in hiPSC cultures expanded manually and robotically. (Q) Automated cell expansion strategy showing massive scale-up in only 12 days. Data expressed as mean ± SD, n > 3 biological replicates using two independent cell lines (B–P). p = 0.0001 in (I), unpaired t test.

Figure 3

Single-cell RNA-seq and comparison of manual and automated cell culture (A and C) t-SNE plots illustrating (A) hiPSCs (LiPSC-GR1.1) and (C) hESCs (WA09) maintained either manually or robotically show a high degree of transcriptomic similarity. (B and D) Venn diagrams showing overlap of expressed genes in (B) hiPSCs (LiPSC GR1.1) and (D) hESCs (WA09). (E) Direct comparison of transcriptomes of hiPSCs (LiPSC-GR1.1) and hESCs (WA09) cultured manually and robotically. Data from n = 5,573, 4,835, 4,485, and 3,922 single cells obtained from n = 4 independent experiments using two independent cell lines for hESC manual, hiPSC manual, hESC auto, and hiPSC auto, respectively (A–E). Single-cell RNA-seq data were analyzed in the Seurat R package.

Figure 4

Mass cytometry of hiPSCs and hESCs and comparison of manual and automated cell culture (A) Uniform manifold approximation and projection (UMAP) plots showing subpopulations of cells within each group organized into eight clusters identified by FlowSOM and ConsensusClusterPlus algorithms. Cluster 6 was prominent in hiPSCs (LiPSC-GR1.1) when cultured manually and its representation was mitigated by automated culture. Core pluripotency markers OCT4, NANOG, and SOX2 were expressed at similar levels across clusters. Surface-antigen CD24 was expressed at a considerably higher level in cluster 6 in hiPSCs cultured manually (red arrow). (B) Heatmaps comparing protein expression levels for each analyzed marker in individual clusters and the abundance of the clusters within the hiPSC populations (LiPSC-GR1.1) cultured manually or by automation. Manual culture led to a large proportion of CD24-negative cells, 66% versus 11% in automated culture. (C) Heatmaps of protein expression levels and cluster abundances in hESCs (WA09) after manual and automated cell culture. The abundance of the major cluster 3 was similar in both culture conditions, and CD24-negative cluster 6 was represented at a negligible level. UMAP plots were constructed from 8,000 single cells per sample (n = 4 independent experiments) obtained from two independent cell lines (A–C). CyTOF data were analyzed using a modified CyTOF workflow (Robinson et al., 2017).

Figure 5

Controlled multi-lineage differentiation of hPSCs by using CTST (A) Western blot of hiPSCs (LiPSC-GR1.1) and hESCs (WA09) before (OCT4) and after differentiation into ectoderm (PAX6) at day 7, mesoderm (Brachyury) at day 5, and endoderm (SOX17) at day 5. Tubulin was used as loading control. (B) Immunocytochemical analysis of hiPSC (LiPSC-GR1.1)-derived ectoderm (PAX6) at day 7, endoderm (SOX17) at day 5, and mesoderm (Brachyury) at day 5. Cultures were differentiated by CTST. Scale bar, 200 μm. (C) Single-cell RNA-seq of pluripotent and differentiated cultures (LiPSC-GR1.1). (D) Heatmap showing the highly expressed genes for pluripotent cells (LiPSC-GR1.1) and differentiated cultures representing ectoderm (day 7), endoderm (day 5), and mesoderm (day 5). (E) Comparison of undifferentiated and differentiated hESCs (WA09) and hiPSCs (LiPSC-GR1.1) shows that gene expression signatures are similar. Data are from n = 4 biological replicates using two independent cell lines (A and B). Data are from n = 19,759 or 16,582 single cells obtained from n = 4 independent experiments using two independent cell lines (C–E). Cell counts for hiPSCs: 4,772 pluripotent, 6,457 ectoderm, 4,160 endoderm, and 4,370 mesoderm. Cell counts for hESCs: 3,627 pluripotent, 5,062 ectoderm, 4,267 endoderm, and 3,626 mesoderm. Single-cell RNA-seq data were analyzed in the Seurat R package.

Figure 6

Robotic scalable production of hiPSC (LiPSC-GR1.1)-derived human neurons (A) Neuronal differentiation strategy established for automated cell culture. (B) Phase-contrast image showing a typical neuronal culture (day 30). Scale bar, 200 μm. (C) Neurons develop a dense network of neurites upon maturation (day 50). Scale bar, 200 μm. (D–F) hiPSC-derived cortical neurons (day 40) immunostained for (D) TUJ1 and CUX1, (E) MAP2 and CTIP2, and (F) vGLUT. Scale bar, 50 μm. (G) hiPSC-derived neuronal cells (day 40) showing immunoreactivity for inhibitory neurotransmitter GABA. Scale bar, 20 μm. (H) Robotic MEA platform used for high-throughput electrophysiology and functional cell characterization. (I and J) Comparison of (I) spontaneous neuronal spikes and (J) spike amplitudes in hiPSC-derived cultures after 6 weeks of manual or robotic cell differentiation as measured by MEA. Representative data are expressed as mean ± SD, n > 3 biological replicates (B–G, I, J). p > 0.5, unpaired t test.

Figure 7

Characterization of cardiomyocytes and hepatocytes derived by automated cell culture (A) Overview of cardiomyocyte differentiation protocol. (B) Western blot showing induction of cardiac troponin and NKX2.5 in undifferentiated (abbreviated as U) and differentiated (abbreviated as D) hESCs (WA09) and hiPSCs (LiPSC-GR1.1) at day 24. GAPDH was used as a loading control. (C and D) (C) Immunocytochemistry and (D) quantification shows that hiPSC-derived cardiomyocytes express cardiac troponin (day 24). Scale bar, 75 μm. (E) Comparison of spontaneous spike amplitudes in hiPSC-derived cardiomyocytes differentiated manually or robotically (day 24). (F and G) Comparison of (F) beat periods and (G) field potential duration in cardiomyocyte cultures (LiPSC-GR1.1) differentiated manually or robotically and measured by MEA (day 24). (H) Overview of hepatocyte differentiation protocol. (I) Immunocytochemistry at day 10 shows most hiPSC (LiPSC-GR1.1)-derived cells express FOXA2 and HNF4A. Scale bar, 200 μm. (J) hiPSCs (LiPSC-GR1.1) differentiated into hepatocytes express α-fetoprotein (AFP) and HNF4A (day 20). Scale bar, 200 μm. (K) Immunocytochemistry showing albumin-expressing hepatocytes robotically differentiated in a 384-well plate. Representative overview of 18 whole wells containing hepatocytes. Scale bar, 2 mm. Representative data are expressed as mean ± SD, n > 3 biological replicates. p > 0.5, unpaired t test.