Efficient Cryopreservation of Human Midbrain Organoids Using Conventional DMSO Protocols with Ice Recrystallization Inhibitors

Human midbrain organoids (hMOs) are a powerful stem cell-derived model for studying human neurodevelopment and neurological disease in vitro; however, their broad adoption is limited by long culture times, high cost, and inter-batch variability. Cryopreservation offers a strategy to address these limitations by enabling long-term storage and reducing experimental heterogeneity, but its effectiveness depends on robust cryoprotective strategies. Here, we evaluated hMO cryopreservation using a standard 10% dimethyl sulfoxide (DMSO) protocol, alone and in combination with a panel of five ice recrystallization inhibitors (IRIs), including betaine-derived ammonium salts and N-aryl-d-gluconamides. Across all conditions, hMOs were successfully recovered following freeze–thawing, with preservation of overall structural integrity, including maintained morphology and reduced cellular debris. Importantly, post-thaw viability and cell recovery in the 10% DMSO condition were comparable to those of fresh controls, demonstrating that conventional DMSO-based cryopreservation is sufficient to maintain hMO survival and gross structural preservation. While supplementation with select IRIs, particularly N-ethyl betaine ethyl ester iodide (NEBEE-I), yielded modest improvements in viability relative to DMSO alone and a non-significant trend toward enhanced electrophysiological activity in multielectrode array recordings, these effects were secondary to the robust performance of the baseline DMSO protocol. Collectively, these findings establish standard 10% DMSO cryopreservation as an effective and broadly applicable method for hMO preservation, while suggesting that small-molecule IRIs may provide incremental benefits in selected outcome measures.