Pluripotent Stem Cells
Scalable Expansion of iPSCs and Their Multilineage Derivatives
This paper explores the use of induced pluripotent stem cells (iPSCs) in biomedical applications, with a focus on toxicity testing.he authors detail strategies for scalable iPSC production using the CERO 3D suspension culture platform and a benchtop bioreactor. Additionally, they highlight recent advancements in scalable differentiation protocols to generate cardiac, neural, and hepatic lineages, expanding the toolkit for research and therapeutic development.
“Cultivation and expansion of iPSCs in 3D culture conditions. A: Workflow options of the 3D suspension cultivation. A1: iPSC clumps are seeded on Matrigel™- coated alginate microcarriers. A2: iPSC single cells are seeded on Matrigel™-coated alginate microcarriers in medium supplemented with 10 µM Y-27632. A3: iPSC single cell suspension forms aggregates with ROCK-inhibitor Y-27632 support. B: Morphology of the cells cultured as clumps and single cells on Matrigel™-coated alginate microcarriers (MC) and as aggregates. The cell line UKBi005-A was used for both microcarriers approaches and the cell line BIONi010-C for the aggregates approach. Scale bar = 500 µm.“
Reference: Keong Kwok et. al. (2022) Scalable expansion of iPSC and their derivatives across multiple lineages, Reprod Toxicol. 2022 May 17;S0890-6238(22)00068-5.
Scalable Production of Microglia
The CERO 3D Incubator & Bioreactor was employed for the large-scale production of embryoid bodies from human iPSCs and for co-culturing cortical spheroids containing iPSC-derived microglia (iPSdMiG) over periods of 1 to 4 weeks. This research highlights an in vitro differentiation method for producing and cryopreserving iPSdMiG at scale. These microglia displayed a transcriptomic profile similar to adult human microglia, expressed key microglial markers, and demonstrated essential functions, including cytokine secretion, phagocytosis, and ROS production, making them ideal for integration in neural co-culture models.
“Schematic illustration depicting the generation of iPSdMiG via scalable production of EBs that are fated to exhibit both primitive hematopoiesis and early neural precursors and are subsequently propagated on mesh macrocarriers. Microglia can be harvested continuously from week 6 up to week 12 of differentiation.”

“C Representative immunofuorescence images demonstrating that iPSdMiG (IBA1, red) migrated into and evenly distribute across 3D cortical spheroids (TUBB3, green); nuclei stained with DAPI. Scale bars=500 m. D High magnifcation images revealing that integrated iPSdMiG (IBA1, red) exhibit a ramifed morphology after 4 weeks of co-culture with 3D cortical spheroids (TUBB3, green). Scale bar=100 m”
Reference: Mathews et. al. (2022) Reenacting Neuroectodermal Exposure of Hematopoietic Progenitors Enables Scalable Production of Cryopreservable iPSC-Derived Human Microglia, Stem Cell Reviews and Reports
Enabling Efficient and Scalable Stem Cell Therapies
This study demonstrates the successful use of the CERO 3D system cultivating and differentiating human iPSCs into neural lineages. Bulk cryopreservation of iPSCs, followed by expansion and differentiation in CERO 3D bioreactors, offers a promising approach for large-scale production of stem cells for therapeutic applications. This technology has the potential to accelerate the development of innovative, cost-effective stem cell-based therapies.

“Morphology of UKKi011-A and BIONi010-C-41 cells before and after inoculation in scalable bioreactors. (A) Condensed schematic illustration of the workflow. The hiPSC lines UKKi011-A and BIONi010-C-41 were cryopreserved at a density of 2 × 107 cells/mL using slow-rate freezing in cryo vials (1 mL, 2 × 107 cells in total) and cryo bags (50 mL, 1 × 109 cells in total) and directly inoculated in a CERO 3D-suspension bioreactor after thawing ( = stemness maintenance). (B) Phase contrast images showing the morphology of both cell lines before cryopreservation in 2D. Scale bars indicate 200 m. (C) Representative phase contrast images of UKKi-011-A spheroids generated in a suspension-based bioreactor on day 1 and day 3 for the non-frozen samples, and the cryopreserved samples in cryo vials and cryo bags on day 1 and 3 after thawing. Scale bars indicate 500 m.”
Reference: Meiser et. al. (2023) Application-Oriented Bulk Cryopreservation of Human iPSCs in Cryo Bags Followed by Direct Inoculation in Scalable Suspension Bioreactors for Expansion and Neural Differentiation, Cells. 2023 Jul; 12(14)
Learn More
The CERO 3D Incubator & Bioreactor provides the solution for scale-up and automation platforms, simplification and cost reduction of stem cell expansion projects in biobanks, cell-based drug discovery, toxicity testing and regenerative medicine. The CERO 3D Incubator & Bioreactor is a specialized incubator that facilitates 3D cell cultivation, allowing cells to grow in a more physiologically relevant environment compared to traditional 2D cultures.
- Microcarrier-free
- Stable pluripotency over many passages
- Easy to set-up and simple workflow
- Free-floating 3D aggregates
- Able to differentiate in 3 germ layers
- Homogeneous iPSC and ESC aggregates


Pluripotent stem cells are inoculated as single cells directly into the CEROtube, where they undergo self-aggregation to form uniform cell clusters that can be expanded across multiple passages. This process achieves significant biomass increase with just ~2 minutes of hands-on time per day.
The resulting 3D stem cell aggregates are ready for direct processing in differentiation applications, such as organoid formation, or in 3D and 2D assay development for various downstream applications.

Human iPSC after expansion in CERO 3D Incubator & Bioreactor tested for pluripotency.

Human iPSC after expansion in CERO 3D Incubator & Bioreactor tested for differentiation in three germ layers.
Pluripotent stem cells expanded in CERO 3D (former name “BioLevitator”) will maintain pluripotency and can be differentiated into all 3 germ layers, as described by Elanzev et. al. 2015; Biotechnol. J. 2015, 10, 1589–1599:
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