Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures

J Neurotrauma. 2010 Jan;27(1):197-203. doi: 10.1089/neu.2009.0995.

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.

MeSH terms

  • Animals
  • Biomarkers / analysis
  • Biomarkers / metabolism
  • Cell Count
  • Cell Culture Techniques
  • Cell Proliferation*
  • 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
  • Peripheral Nerves / physiology*
  • 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 / physiology*
  • Schwann Cells / transplantation
  • Sciatic Nerve / cytology
  • Sciatic Nerve / physiology
  • Spinal Cord Injuries / surgery
  • Time Factors
  • Tissue Transplantation / methods*
  • Wallerian Degeneration / physiopathology*

Substances

  • Biomarkers
  • Nerve Growth Factors
  • Nerve Tissue Proteins
  • Receptors, Growth Factor
  • Receptors, Nerve Growth Factor
  • S100 Calcium Binding Protein beta Subunit
  • S100 Proteins
  • Ngfr protein, rat