The loss of wild biodiversity has prompted the development of cryobanks, such as those of somatic cells. This is the reality of Pumas, wild felids of ecological importance that suffer from anthropogenic actions, population decline, and subsequent loss of genetic diversity. Somatic cell banks are a strategy for conserving population diversity. We compared different concentrations and types of intracellular cryoprotectants (dimethyl sulfoxide, DMSO; ethylene glycol, EG) associated with 0.2 M of sucrose (SUC) in the cryopreservation of the somatic cells of captive Pumas. The cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum and varying concentrations of DMSO and EG in the absence or presence of SUC. The cells were analyzed for morphological characteristics, viability, proliferative activity, metabolic activity, and apoptosis levels. Cells maintained similar fusiform morphology before and after cryopreservation. There was no difference in viability, regardless of the reduction in the concentration and type of intracellular cryoprotectants and sucrose. Similarly, proliferative activity, metabolic activity, and apoptosis levels were not altered by the composition of the cryoprotectants. In summary, we demonstrate that reducing the concentration of DMSO or EG ensures adequate cryopreservation of Puma somatic cells, regardless of the presence of SUC.