Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures.
The efficacy of Schwann-cell cultivation can be enhanced by in vitro predegeneration of the harvested cells compared to immediate culture. The aim of this study was to improve Schwann-cell culture efficacy by comparing three different durations of predegeneration. The sciatic and median nerves of 6-8-week-old Lewis rats were harvested and subjected to either 2-day, 7-day, or 14-day predegeneration in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin. Afterward, tissue was enzymatically dissociated and placed in a modified melanocyte growth medium. The cell count was determined immediately after dissociation while the cell purity was determined one subculture/trypsinization cycle later after cell attachment to the culture plate by means of optical microscopy and immunocytochemistry. Particular attention was then paid to the Schwann-cell-to-fibroblast relation. The cumulative cell count in the culture was 5.8 x 10(5) for 2-day, 1.12 x 10(6) for 7-day, and 1.48 x 10(6) for 14-day predegeneration. The culture purity was approximately equal for 2- and 7-day predegeneration (88% Schwann cells, 12% fibroblasts after 2 days; 85% Schwann cells, 15% fibroblasts after 7 days). After 14 days, however, cell cultures were significantly debased by fibroblast proliferation (57% Schwann cells, 43% fibroblasts). In vitro predegeneration is a particularly suitable procedural method to increase the cultural Schwann-cell yield. The number of cultivated rat Schwann cells is doubled by 7-day in vitro predegeneration in comparison to 2-day predegeneration. After 14-day predegeneration, however, the culture is significantly debased by fibroblasts. Therefore, 7-day in vitro predegeneration is an advisable predegeneration period.
Kraus
Armin
A
Department of Hand, Plastic, Reconstructive and Burn Surgery, BG-Trauma Center, Eberhard Karls University, Tüebingen, Germany.
Täger
Joachim
J
Kohler
Konrad
K
Manoli
Theodora
T
Haerle
Max
M
Werdin
Frank
F
Hoffmann
Jürgen
J
Schaller
Hans-Eberhard
HE
Sinis
Nektarios
N
D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D007150:Immunohistochemistry; D008475:Median Nerve / Q000166:cytology / Q000502:physiology; D009414:Nerve Growth Factors / Q000378:metabolism; D009419:Nerve Tissue Proteins; D009924:Organ Culture Techniques; D010525:Peripheral Nerves / Q000166:cytology / Q000502:physiology*; D051381:Rats; D011917:Rats, Inbred Lew; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000378:metabolism; D012583:Schwann Cells / Q000166:cytology / Q000502:physiology* / Q000637:transplantation; D012584:Sciatic Nerve / Q000166:cytology / Q000502:physiology; D013119:Spinal Cord Injuries / Q000601:surgery; D013997:Time Factors; D016378:Tissue Transplantation / Q000379:methods*; D014855:Wallerian Degeneration / Q000503:physiopathology*
D015415:Biomarkers; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C068313:Ngfr protein, rat