This protocol describes a simple method to cryopreserve mammalian cells within filter papers as an alternative to conventional slow-freezing approach. The method involves treating paper fibers with fibronectin, using low concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), and slow freezing cells to -80 °C at a 1 °C min
-1 rate. In our method, the biocompatibility, large surface area, 3D porosity and fiber flexibility of the paper, in combination with the fibronectin treatment, yield recovery of cells comparable to conventional approaches, with no additional fine-tuning to freezing and thawing procedures. We expect that the paper-based cryopreservation method will bring several advantages to the field of preserving mammalian cells, including accommodation of a higher number of cells within a unit volume and no cell loss after release. The method requires a minimal storage space, where paper platforms with large areas can be rolled and/or folded and stored in stocks, and allows for efficient transportation/distribution of cells in an on-demand manner. Moreover, an additional feature of this method includes the formation and cryopreservation of cellular spheroids and 3D cell cultures.