Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors

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Adv Healthc Mater. 2022 Oct 31;e2201138

Debbie L L Ho ¹, Stacey Lee ¹, Jianyi Du ¹, Jonathan D Weiss ¹, Tony Tam ¹, Soham Sinha ¹, Danielle Klinger ¹, Sean Devine ², Art Hamfeldt ², Hope T Leng ¹, Jessica E Herrmann ^1 3, Mengdi He ⁴, Lee G Fradkin ¹, Tze Kai Tan ^5 6 7, David Standish ², Peter Tomasello ², Donald Traul ², Noushin Dianat ⁸, Rukmini Ladi ², Quentin Vicard ⁸, Kishore Katikireddy ², Mark A Skylar-Scott ^1 9 10

• PMID: 36314397
• DOI: 10.1002/adhm.202201138

Abstract

Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering.

Keywords: 3D bioprinting; cell manufacturing; organoids; pluripotent stem cells; suspension culture.

Ho DLL, Lee S, Du J, Weiss JD, Tam T, Sinha S, Klinger D, Devine S, Hamfeldt A, Leng HT, Herrmann JE, He M, Fradkin LG, Tan TK, Standish D, Tomasello P, Traul D, Dianat N, Ladi R, Vicard Q, Katikireddy K, Skylar-Scott MA. Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors. Adv Healthc Mater. 2022 Oct 31:e2201138. doi: 10.1002/adhm.202201138. Epub ahead of print. PMID: 36314397.