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Mammalian Cell Culture. Majeed Arsheed Sabbah , phD Biotechnology research center, Alnahrain university. What is cell culture. Current work in Iraq. Cells isolation. Ficoll. Immunomagnetic separation. Cell viability. Trypan Blue exclusion.
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Mammalian Cell Culture MajeedArsheedSabbah, phD Biotechnology research center, Alnahrain university
Cells isolation Ficoll Immunomagnetic separation
Cell viability Trypan Blue exclusion live cells exclude the dye as the membrane is impermeable, but dead cells uptake the dye due to the lack of an intact cell membrane. Viability is then determinedby manually counting the stained sample in a hemacytometer, a time consuming and labor intensive process. More importantly, trypan blue stained samples have to be analyzed within 3-5 minutes, as longer incubation will increase blue colored cells. Trypan blue staining may result in an overestimate of viable cells especially for low viability samples.
Cytotoxicity assays 1-Neutral red uptake 2- MTT 3- Crystal violet Neutral red uptake
Principle: Formazan crystals are formed by mitochondrial succinate dehydrogenase in living and early apoptotic cells, but not in dead cells. MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) MTT cytotoxicity test in cell culture http://www.ib.amwaw.edu.pl/home/dslado/video/mtt.html
Cell Identification Flowcytometry Immunohistochemistry
Improvement cell culture research Establish suitable laboratories for cell culture. Supply continuous electricity.
Importing cells, reagents and growth factors in suitable cooled containers. Establish cell bank for continuous cell supply.
Future work Production of monoclonal antibodies Tissue Engineering
Growing cells in 3D Forming Tissue in Bioreactor stem cell-mediated therapy