Cell Biology

This manuscript describes step-by-step procedures to establish and manage fresh and cryopreserved cultures of nerve-derived human Schwann cells (hSCs) at the desired scale. Adaptable protocols are provided to propagate hSC cultures through serial passaging and perform routine manipulations such as enzymatic dissociation, purification, cryogenic preservation, live-cell labeling, and gene delivery. Expanded hSCs cultures are metabolically active, proliferative, and phenotypically stable for at least three consecutive passages. Cell yields are expected to be variable as determined by the rate of growth of individual batches and the rounds of subculture. The purity, however, can be maintained high at >95% hSC regardless of passage. The cells obtained in this manner are suitable for various applications, including small drug screens, in vitro modeling of neurodevelopmental processes, and cell transplantation. One caveat of this protocol is that continued expansion of same-batch hSC populations is eventually restricted due to senescence-linked growth arrest.

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.

Primary neuronal culture from rodents is a key tool in neurobiology. However, the preparation of primary cultures requires precise planning, starting from animal mating. Furthermore, each preparation generates a high amount of cells that eventually go wasted. The possibility to cryopreserve primary neural cells represents a resource for in vitro studies and significantly reduces the sacrifice of animals. Here we describe that Neurostore buffer supports the cryopreservation of primary neurons.