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bioRxiv · 10.64898/2026.08.04.741960

Minimizing time in culture: A prototypic autologous manufacturing workflow for monoclonal iPSC lines within seven weeks

Haberhausen, D.·Woehle, C.·Raab, C.·Ludwig, C.·Kuchler, T.·Barth, S.·Wuellner, U.·Bosio, A.·Johannsen, H.·Knoebel, S.
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Abstract

Induced pluripotent stem cells (iPSCs) hold great promise for both allogeneic and autologous cellular therapies. However, broad application and clinical translation is hindered by fragmented, complex and time-intensive workflows, resulting in high manufacturing costs, poor standardization and increased risk of genomic aberrations in derived iPSCs. In this study we developed a standardizable, automatable and time-efficient process for the derivation of monoclonal iPSC lines straight from skin including a comprehensive and cascaded QC strategy. We generated monoclonal iPSC lines derived from human skin punch biopsies of ten donors (age 49-81) via mRNA-based reprogramming that subsequently underwent comprehensive and thorough characterization of phenotypic and genetic properties. The use of a combined mechanical and enzymatic fibroblast isolation protocol and a transient non-integrative reprogramming technology allowed us to obtain 78 monoclonal iPSC lines, ready for banking, molecular characterization and further differentiation within seven weeks from initial sample processing to passage four iPSC lines. The phenotypical characterization via flow cytometry-based pluripotency marker expression and 2D-directed differentiation into the three germ layers showed low intra- and inter-donor variability over all generated lines. A combination of SNP array based CNV analysis followed by whole exome sequencing proved to be the most efficient approach for assessment of genomic integrity. Proof-of-concept experiments for closed system processing revealed that a substantial part of the most error-prone and technically demanding steps can be transferred to semi-automated, closed systems. In conclusion, the described protocol allows for time-efficient, standardizable and automatable generation of high-quality monoclonal iPSC lines from human skin punch biopsies within seven weeks, thus moving the field of autologous iPSC manufacturing one step further towards cost-efficient clinical implementation.

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Haberhausen, D., Woehle, C., Raab, C., Ludwig, C., Kuchler, T., Barth, S., Wuellner, U., Bosio, A., Johannsen, H., Knoebel, S.. 2026-08-10. Minimizing time in culture: A prototypic autologous manufacturing workflow for monoclonal iPSC lines within seven weeks. https://doi.org/10.64898/2026.08.04.741960

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