The paper discusses the use of induced pluripotent stem cells (iPSCs) in various biomedical applications, particularly for toxicity testing. The authors outline their strategies for scalable production of iPSCs using the CERO 3D suspension culture platform and a benchtop bioreactor. They also report on recent efforts to establish robust, scalable differentiation protocols into cardiac, neural, and hepatic lineages to enhance the available research tools.  

“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.

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 showed a transcriptomic profile similar to adult human microglia, were immunopositive for microglial markers, and exhibited key functionalities such as cytokine secretion, phagocytosis, and ROS production, making them suitable for co-culture in neural 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

This study demonstrates the successful use of the CERO 3D system for the cultivation and neural differentiation of human iPSCs. 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 could

help accelerate the development of innovative and cost-effective stem cell-based treatments.

“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)