Cryopreservation of human lung adenocarcinoma spheroids using MMC based cryomixtures

Cryopreservation remains crucial bottleneck for storing and transporting bioengineered 3D cell models, vital for preclinical drug development and cancer research. Conventional cryoprotectants like fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) present cytotoxicity challenges and lack efficacy in maintaining structural integrity and viability in complex 3D culture models. This study investigates the efficacy of two carbohydrate-based macromolecular crowders (MMCs), polydextrose III (PD) and resistant maltodextrin (rMD), in cryopreserving human lung adenocarcinoma spheroids as alternatives to FBS. Spheroids were cryopreserved at −80 and −196 °C using MMC-based cryomixtures, with subsequent evaluation of cell viability, structural integrity, and proliferation markers post-thaw. Results indicate that MMC-based cryomixtures, particularly PD, provide superior cryoprotection, preserving the structural and functional integrity of A549 spheroids over a 60-day storage period at −196 °C. Immunocytochemistry of vimentin and Ki67 biomarkers demonstrated that PD-cryopreserved spheroids exhibited consistent structural stability and retained proliferative capacity, contrasting with those stored in conventional FBS-based cryomixtures, which showed marked deterioration in cellular morphology and viability. Apoptosis profiling revealed a lower incidence of cell death in MMC-preserved spheroids, with live cell percentages stabilizing around 50% at −80 °C and approximately 54% at −196 °C over the extended storage period. Further characterization revealed protection of the necrotic core and cellular junctions PD-cryopreserved spheroids. These findings suggest that MMC-based cryomixtures, especially PD, are effective alternatives for cryopreservation of tumor spheroids. The increased cellular viability and structural preservation provided by MMCs could advance their application in 3D culture preservation, addressing limitations of conventional cryopreservation in drug testing, regenerative medicine, and cancer research.