Mammalian cell cryopreservation by using liquid marbles

Liquid marbles (LMs) are nonsticky droplets covered by micro- or nanometrically scaled particles and obtained by simply rolling small amounts of a liquid in a very hydrophobic powder. Since pioneer work by Aussillous and Quéré, a wide palette of hydrophobic materials for the preparation of LMs, as well as potential applications, have been reported. Because of the bioinspired origin of this concept, the applicability of LMs in biomedicine is gaining increasing attention, with remarkable advances in their use as microbioreactors for blood typing, drug screening, and tumor growth, among others. Herein, we explore the novel use of LMs as a biotechnological tool for the cryopreservation of mammalian cells as an alternative to conventional methods, which typically require the use of cryopreservant agents that commonly associate with some degree of cell toxicity. Murine L929 fibroblasts, a reference cell line for cytotoxicity studies, and poly(tetrafluoroethylene), a hydrophobic polymer widely used in cardiovascular surgery, were selected for the preparation of the cell-containing LMs. Our results reveal that there is a safe range of droplet volumes and cell densities that can be successfully used to cryopreserve mammalian cell lines and recover them after thawing without significantly affecting major cellular parameters such as adhesion, morphology, viability, proliferation, and cell cycle. We envision that progress in the exploration of cell-containing LMs could also open their impact as microreactors for the miniaturization of cytotoxicity procedures of drugs and materials in which powerful tools for cell evaluation such as flow cytometry could be used because of the elevated amount of cells handled