Optimization of Slow Freeze Protocol for 3D Cell Structures Using Brain Organoids and Chondrospheres as Models

BACKGROUND: Cryopreservation is a valuable tool for long-term preservation of viability of cell cultures or complex cellular structures, including organoids. Currently, cryopreservation protocols have been developed for some types of organoids, which have not yet been optimized for three-dimensional structures of the brain and cartilage tissue. Selection of the optimal composition of the cryoprotective solution and the stage at which it is preferable to perform the cryopreservation procedure will help to increase the viability of the preserved material. Main relevance is the search for a suitable protocol for cryopreservation of organoids based on differentiated derivatives of induced pluripotent stem cells (iPSCs), due to the great prospect of using such cells in various fields of biology and medicine. AIM: Our aim was to optimize protocols for slow cryopreservation of iPSC-derived neural organoids and chondrospheres to minimize the negative impact of the procedure on morphofunctional characteristics. METHODS: Neural organoids obtained from iPSCs differentiated in the neuronal direction were cryopreserved in 92% FBS + 8% DMSO or 82% DMEM + 10% FBS + 8% DMSO solutions on days 9, 14, 22, 29 and 43. After thawing, the integrity and size of the organoids, real-time PCR for neural markers MAP2 and NESTIN, as well as immunohistochemical (IHC) staining for TUBB3, MAP2, SOX2 and PCNA were assessed. Chondrospheres were obtained from human chondrocytes and cryopreserved 29 days after transfer to 3D culture conditions in DMEM+10%FBS+8%DMSO solution. After thawing, IHC analysis was performed for the expression of chondrogenic marker proteins aggrecan, type II collagen and SOX9, as well as PCNA. RESULTS: RT-PCR, as well as IHC staining, showed the preservation of the neuronal phenotype by the cells of neuronal organoids 2 weeks after defrosting, despite the lag phase and size changes. Chondrospheres retained their integrity and phenotype after thawing. CONCLUSION: According to the results of our study, the optimal stage for cryopreservation of neural organoids based on iPSCs can be considered the 3rd week from the beginning of differentiation, and the cryoprotective solution 82% DMEM + 10% FBS + 8% DMSO is the most suitable for freezing.