Selective culture of mitotically active human Schwann cells from adult sural nerves.
We devised a simple method to isolate mitotically active human Schwann cells from sural nerve biopsy specimens and expand the population in culture. Nerve fascicles were treated with cholera toxin for 7 days in culture before dissociation, which increased the cell yield at least twenty-five-fold over immediated tissue dissociation. Digesting the tissue completely with enzymes in serum-containing medium resulted in the highest cell viability, and released 2 to 6 x 10(4) cells/mg of tissue. Seeding the cells on a poly-L-lysine substrate in a small volume of serum-free medium optimized the plating efficiency. Although Schwann cells comprised 90% of the initial culture population, their numbers declined over time due to a faster mitotic rate of the fibroblasts in the presence of cholera toxin alone. However, treating the cultures with a combination of cholera toxin and forskolin, which act synergistically to elevate cyclic AMP levels, inhibited fibroblast growth without causing Schwann cell toxicity. Adding glial growth factor to the adenyl cyclase activators maximized Schwann cell proliferation, and the population rapidly and selectively expanded. Therefore, it should be possible to generate large numbers of Schwann cells from diseased nerves to study defects in cell function or from normal nerves to study the effects of Schwann cell grafts on neuronal regeneration.
Rutkowski
J L
JL
Department of Pediatrics, University of Michigan, Ann Arbor 48109-0570.
Tennekoon
G I
GI
McGillicuddy
J E
JE
D000262:Adenylyl Cyclases / Q000378:metabolism; D000328:Adult; D001369:Axons / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D046508:Culture Techniques / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects; D020904:Glia Maturation Factor; D006801:Humans; D009186:Myelin Sheath / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D016196:S Phase; D012583:Schwann Cells / Q000166:cytology*; D013497:Sural Nerve / Q000166:cytology / Q000187:drug effects
D016895:Culture Media, Serum-Free; D020904:Glia Maturation Factor; D009419:Nerve Tissue Proteins; D005576:Colforsin; D002772:Cholera Toxin; D000262:Adenylyl Cyclases