Influence of supramammary lymph node extract on cell growth in vitro [electronic resource] /
Abstract (Summary)Abstract: The bovine supramammary lymph node, located on the dorso-caudal surface of the udder, is a protein rich organ that is discarded on a regular basis during slaughter. Experiments were conducted to evaluate whether or not the lymph node extract could be used as a substitute for bovine growth serum in cell media. Two different preparations of lymph node extract were made and tested. Three different cell lines (MDA-MB-435, MAC-T, and 1C6) were used in assays to evaluate the lymph node extract as a supplement. The first extract yielded a protein concentration of 3.0 mg/ml (heat inactivated) and the second extract contained 27 mg/ml (heat inactivated). MTT and CyQuant assays were used to determine cell viability and proliferation, respectively, in the presence of lymph node extract. Both heat inactivated preparations supported cell growth as well as or better than BGS following a two day serum starvation phase in culture. The second lymph node preparation provided a stimulatory effect following serum starvation on all three cell lines at 10 and 20% supplementation. Direct culture of cells into 10% lymph node extract did not support cell growth as expected. However, gradual adaptation process with lymph node supplementation into media maintained cell growth up to a full 10% extract. Once cells were trypsinized and re-seeded with the 10% lymph node extract they were unable to re-adhere, leaving them detached, and eventually appeared dead. Analysis of DNA revealed any apparent cell death visually observed was not due to apoptosis. The stimulatory effect at 10 and 20%, illustrated by the CyQuant assays, may provide explanation as to why adherence was not achieved in culture. These higher protein concentrations may present an over abundance of mitogenic protein/factors resulting in over-stimulation of cells. Lowering the protein concentration, or fractionating the extract, could possibly provide a better environment for cells to grow and adhere in culture. In conclusion, substitution of BGS at 10% dramatically stimulates cells but does not maintain cell growth in vitro. Further research may prove lower concentrations can sustain cells in culture by lowering mitogenic protein/factors to sub-stimulatory concentrations.
School Location:USA - South Carolina
Source Type:Master's Thesis
Date of Publication: