Abstract 12: Cord Blood Banking, Technical and Clinical grade GMP- Development of Advanced Therapy Products as HLA-Homozygous iPSC-derived Cardiomyocytes

Abstract Introduction The Cord Blood Bank (CBB) in Düsseldorf stores 21477 active cryopreserved cord blood units (CBUs) licensed by the Paul-Ehrlich Institute, with 1517 transplants delivered worldwide. We have previously documented a stable storage time of 29 years in liquid nitrogen (Liedtke et al. 2024). Since establishing confirmatory typing and high-resolution HLA sequencing for CBUs and segments in 2016, the CBB progressed into advanced cellular therapies with high-qualified source material and applicable grants (BMBF-161B0760B and HEAL-101056712). Production permission for isolation and short-term expansion of CD34+ cells from licensed CBUs was granted, allowing the GMP reprogramming of 17 HLA-homozygous lines in collaboration with CATALENT (Terheyden-Keighley et al. 2024, accepted in SCTM). Objectives As part of the HEAL project, CD34+ HSC-derived iPSCs were differentiated into cardiomyocytes (CM) using suspension cultures to produce CM-aggregates instead of single cells, as studies in non-human primates indicate better engraftment of aggregates than single cells (Gruh et al. 2024). An optimized cryopreservation process for CM-aggregates is under development applying techniques available to CBU banking. Methods iPSCs were differentiated into CM-aggregates via an improved WNT pathway modulation protocol in suspension culture. In addition to the WNT inhibitor CHIR99021, BMP, Activin A and FGF-2 were added to target accessory pathways and achieve robust scalability across different platforms. Following differentiation, the CM-aggregates were cryopreserved in 10% DMSO with HSA or KO-SR supplementation using a controlled-rate freezer adapted to tissue freezing to ensure uniform freezing processes. Results The improved protocol allowed for straightforward upscaling of CM-aggregate differentiation from 6-well plates to Erlenmeyer flasks of different volumes, leading to a 1-2 fold yield of CM-aggregates with a purity of up to 95 % cardiac troponin T (cTnT) and sarcomeric actinin (sAct). Post-cryopreservation recovery of aggregates was between 60-90% with a viability of ~90%. Although CM culture after thawing remains problematic (<60% CM recovery after 5 days), the aggregates restored their morphology and function. Discussion The CBU banking, processing and storage should meet quality and release criteria, including genetic testing, allowing both allogeneic and related CBU selection to provide the best possible starting material. These advanced therapy products can be utilized e.g. for cardiomyocyte production for potential clinical applications. Overview of a GMP-process development for advanced therapy products from selected HLA-homozygous Cord Blood Units