title: A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves.
abstract: 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.
authors.affiliation: The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
authors.forename: Natalia D
authors.initials: ND
authors.lastname: Andersen
authors.affiliation: The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
authors.forename: Shruthi
authors.initials: S
authors.lastname: Srinivas
authors.affiliation: The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.|Universidad de Buenos Aires, CONICET, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Buenos Aires, Argentina.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Piñero
authors.affiliation: The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
authors.forename: Paula V
authors.initials: PV
authors.lastname: Monje
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation; D002470:Cell Survival; D015925:Cryopreservation; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009186:Myelin Sheath / Q000737:chemistry; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D012583:Schwann Cells / Q000166:cytology*; D012584:Sciatic Nerve / Q000166:cytology*
chemical_list: D005456:Fluorescent Dyes