A simple and efficient cryopreservation method for mouse small intestinal and colon organoids for regenerative medicine

Organoid cryopreservation method is one of key step in the organoid culture. We aimed to establish a simple and efficient cryopreservation method for mouse small intestinal organoids (MIOs) and colon organoids (MCOs) using various concentrations of cryoprotectant. Based on the theoretical simulation, we optimized the dimethyl sulfoxide (DMSO) concentration by pretreating the organoids with 5, 7.5, and 10% DMSO for 30 min at 4 °C to allow penetration into the organoids and evaluated their viability, proliferation, and function after cryopreservation. Gene expression in the MIOs and staining of lineage markers were examined real-time PCR. The organoids in the DMSO-treated groups as well as the control, expressed ChrgA, Ecad, Muc2, Lyz, villin, and Lgr5, and there are no significant. A forskolin-induced swelling assay for MIOs was performed to confirm normal cystic fibrosis transmembrane conductance regulator (CFTR) activity. Similar forskolin-induced swelling was observed in the DMSO-treated groups and the control. In addition, MCOs were transplanted into mouse colon for confirmation of regeneration therapy efficacy. Thawing organoids were cultured for two and four sequential passages after cryopreservation with 5% DMSO to confirm any changes in the gene expression of lineage markers after subculture. We developed a simple and efficient organoid freezing method using 5% DMSO with low potential toxicity and validated our findings with theoretical simulation.