1. Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures.
- Author
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Kraus A, Täger J, Kohler K, Manoli T, Haerle M, Werdin F, Hoffmann J, Schaller HE, and Sinis N
- Subjects
- Animals, Biomarkers analysis, Biomarkers metabolism, Cell Count, Cell Culture Techniques, Cells, Cultured, Fibroblasts cytology, Fibroblasts physiology, Immunohistochemistry, Median Nerve cytology, Median Nerve physiology, Nerve Growth Factors metabolism, Nerve Tissue Proteins, Organ Culture Techniques, Peripheral Nerves cytology, Rats, Rats, Inbred Lew, Receptors, Growth Factor, Receptors, Nerve Growth Factor metabolism, S100 Calcium Binding Protein beta Subunit, S100 Proteins metabolism, Schwann Cells cytology, Schwann Cells transplantation, Sciatic Nerve cytology, Sciatic Nerve physiology, Spinal Cord Injuries surgery, Time Factors, Cell Proliferation, Peripheral Nerves physiology, Schwann Cells physiology, Tissue Transplantation methods, Wallerian Degeneration physiopathology
- Abstract
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.
- Published
- 2010
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