Optimization of slow freeze protocol for 3D cell structures using brain organoids and chondrospheres as models

BACKGROUND: Cryopreservation is a widely used method for long-term viability preservation of cultured cells and complex cellular structures, including organoids, which serve both scientific and clinical purposes, as well as for screening applications. However, data on cryopreservation of organoids derived from differentiated induced pluripotent stem cells (iPSCs) remain limited. AIM: The work aimed to optimize slow freeze protocols for neural organoids derived from iPSC-differentiated cells and chondrospheres. METHODS: Neural organoids differentiated from iPSCs were cryopreserved on days 9, 14, 22, 29, and 43 using four different cryopreservation media. Post-thaw assessment included organoid integrity and size measurements, quantitative polymerase chain reaction for MAP2 and NES markers, and immunohistochemical (IHC) staining for βIII-tubulin, MAP2, SOX2, and proliferating cell nuclear antigen (PCNA). Chondrospheres were derived from human chondrocytes and cryopreserved 29 days after transition to 3D culture conditions in a solution of 82% DMEM + 10% FBS + 8% DMSO + 10 µM ROCK inhibitor Y27632 (Ri, Rho-associated protein kinase inhibitor). Post-thaw IHC analysis was performed to assess the expression of chondrogenic marker proteins, including aggrecan, collagen type II, and SOX9, as well as PCNA. RESULTS: Neural organoids cryopreserved between weeks 3 and 6 of differentiation retained the highest post-thaw structural integrity. Although organoid diameter decreased by 14.5% on average 1 week after thawing, size was nearly restored to the values observed on the corresponding day of differentiation for organoids not subjected to cryopreservation by week 2. Quantitative polymerase chain reaction and IHC staining confirmed maintenance of neuronal phenotype 2 weeks post-thaw. Chondrospheres showed no change in diameter over 2 weeks post-thaw and exhibited 100% preservation of organoid integrity. IHC analysis confirmed the presence of chondrocytic proteins in chondrospheres 2 weeks after thawing. CONCLUSION: Based on the study findings, the third week of differentiation was identified as the optimal stage for cryopreservation of iPSC-derived neural organoids. The cryopreservation medium composed of 82% DMEM, 10% FBS, and 8% DMSO was optimal for both neural organoids and chondrospheres.