# Improved cryopreservation of cardiomyocyte aggregates differentiated from GMP iPSC in a 3D culture format

## TL;DR

This paper presents a new method for cryopreserving cardiomyocyte aggregates derived from stem cells, maintaining their function and viability after freezing.

## Contribution

The study introduces an optimized cryopreservation protocol for cardiomyocyte aggregates using 10% human serum albumin as the best freezing medium.

## Key findings

- Cryopreservation using 10% human serum albumin maintained >90% cardiac marker expression after thawing.
- Spontaneous contraction activity and >80% cell recovery were observed in preserved cardiomyocyte aggregates.
- Optimized protocols successfully preserved complex cardiomyocyte aggregate structures.

## Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are in the focus of clinical research for cell-based therapies for heart failure patients. However, challenges include long-term storage of cardiomyocyte aggregates (CMAs). Current freeze/thawing protocols have been established for single cells, indicating a substantial gap for process development and optimization for cryopreservation. Here CMA differentiation was achieved by the combination of WNT-pathway modulation and TGF-β/SMAD- and FGF-pathway targeting. An efficient CMA differentiation with cardiac marker expression of ≥ 95% ACTN2/TNNT2 was established. For process optimization, different freezing media were combined with a pre-treatment strategy and comprehensive assessment of cell recovery, viability, physiology, and purity was performed before and after cryopreservation. Development and optimization of cryopreservation strategies for CMAs led to a freezing medium termed “10% human serum albumin (HSA)” representing the best option tested. Analysis at 5-days post thawing revealed maintenance of high cardiac marker expression (> 90%), spontaneous contraction activity, and overall recovery of > 80% vital cell counts compared to sample analysis before the freezing procedure. Extensive modification and optimization of established single cell CM cryopreservation protocols was achieved with successful recovery of CMs within the complex structure of aggregates.

The online version contains supplementary material available at 10.1038/s41598-025-32439-3.