Isolation, Culture, and Cryopreservation of Adult Rodent Schwann Cells Derived from Immediately Dissociated Teased Fibers.

Adult Schwann cell (SC) cultures are usually derived from nerves subjected to a lengthy step of pre-degeneration to facilitate enzymatic digestion and recovery of viable cells. To overcome the need for pre-degeneration, we developed a method that allows the isolation of adult rat sciatic nerve SCs immediately after tissue harvesting. This method combines the advantages of implementing a rapid enzymatic dissociation of the nerve fibers and a straightforward separation of cells versus myelin that improves both cell yield and viability. Essentially, the method consists of (1) acute dissociation with collagenase and dispase immediately after removal of the epineurium layer and extensive teasing of the nerve fibers, (2) removal of myelin debris by selective attachment of the cells to a highly adhesive poly-L-lysine/laminin substrate, (3) expansion of the initial SC population in medium containing chemical mitogens, and (4) preparation of cryogenic stocks for transfer or delayed experimentation. This protocol allows for the procurement of homogeneous SC cultures deprived of myelin and fibroblast growth as soon as 3-4 days after nerve tissue dissection. SC cultures can be used as such for experimentation or subjected to consecutive rounds of expansion prior to use, purification, or cryopreservation.

Magnetic-Activated Cell Sorting for the Fast and Efficient Separation of Human and Rodent Schwann Cells from Mixed Cell Populations.

To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75NGFR, O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations. A simple protocol for the growth of hybridoma cell cultures as a source of monoclonal antibodies for cell surface immunolabeling of SCs and fibroblasts is provided as a cost-effective alternative for commercially available products. These steps allow for the timely and efficient recovery of purified SC populations without compromising the viability and biological activity of the cells.

A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves.

We herein developed a protocol for the rapid procurement of adult nerve-derived Schwann cells (SCs) that was optimized to implement an immediate enzymatic dissociation of fresh nerve tissue while maintaining high cell viability, improving yields and minimizing fibroblast and myelin contamination. This protocol introduces: (1) an efficient method for enzymatic cell release immediately after removal of the epineurium and extensive teasing of the nerve fibers; (2) an adaptable drop-plating method for selective cell attachment, removal of myelin debris, and expansion of the initial SC population in chemically defined medium; (3) a magnetic-activated cell sorting purification protocol for rapid and effective fibroblast elimination; and (4) an optional step of cryopreservation for the storage of the excess of cells. Highly proliferative SC cultures devoid of myelin and fibroblast growth were obtained within three days of nerve processing. Characterization of the initial, expanded, and cryopreserved cell products confirmed maintenance of SC identity, viability and growth rates throughout the process. Most importantly, SCs retained their sensitivity to mitogens and potential for differentiation even after cryopreservation. To conclude, this easy-to-implement and clinically relevant protocol allows for the preparation of expandable homogeneous SC cultures while minimizing time, manipulation of the cells, and exposure to culture variables.

Exploring the positive allosteric modulation of human α7 nicotinic receptors from a single-channel perspective.

Enhancement of α7 nicotinic receptor (nAChR) function by positive allosteric modulators (PAMs) is a promising therapeutic strategy to improve cognitive deficits. PAMs have been classified only on the basis of their macroscopic effects as type I, which only enhance agonist-induced currents, and type II, which also decrease desensitization and reactivate desensitized nAChRs. To decipher the molecular basis underlying these distinct activities, we explored the effects on single-α7 channel currents of representative members of each type and of less characterized compounds. Our results reveal that all PAMs enhance open-channel lifetime and produce episodes of successive openings, thus indicating that both types affect α7 kinetics. Different PAM types show different sensitivity to temperature, suggesting different mechanisms of potentiation. By using a mutant α7 receptor that is insensitive to the prototype type II PAM (PNU-120596), we show that some though not all type I PAMs share the structural determinants of potentiation. Overall, our study provides novel information on α7 potentiation, which is key to the ongoing development of therapeutic compounds.