Cell Culture Flashcards
What does recombinant DNA technology make?
Enzymes, synthetic hormones,monoclonal antibodies, anticancer agents
What do biotechnology produce?
Manufacture of viral vaccines
What is transfection?
Introduction of foreign DNA
Express a protein of interest
RNAi constructs: suppress expression of a gene/protein
What is cell culture?
Removal of cells from animal /plant to be grown in a favourable artificial environment
What is primary culture?
Cells isolated from tissue & proliferated until they occupy all available substrate
What is a cell line?
1st subculture (subclone)
What is a cell strain?
Select a subpopulation (e.g. Cloning)
What is a finite cell line?
Normally cells only divide a limited number of times, then stop proliferating (senescence)
What is a continuous cell line?
Virally induced transformation to become “immortal”
What is the media for cell culture?
Dulbeccos modified eagle medium
What basic equipment is needed?
Cell culture good Incubator Water bath Centrifuge Refrigerator and freezer (-20oC) Automated cell counter Inverted microscope Liquid nitrogen for storage Sterilizer (autoclave)
What is used to make benches aseptic?
70% ethanol spraying
What do the hood do?
Protect working environment from dust & airborne contaminants
What is immortalized cell line?
Acquired ability to proliferate indefinitely
What does the medium contain?
Essential nutrients: amino acids, carbohydrates, vitamins,minerals
Growth factors & hormones
Gases: oxygen & carbon dioxide
Right pH, osmotic pressure & temperature
How can you tell if bacteria is been contaminated into your culture?
Culture cloudy (turbid) with thin film on surface. Drop in pH, tiny moving granules among cells
How can you tell if your culture is contaminated with yeast?
Turbid, pH rises when heavy, ovoid/spherical particles
How can you tell if contained with molds?
Hyphae: connected filaments
How can tell if viruses in it?
v. difficult to detect in culture
How are infections prevented in the cell culture?
Antibodies or antifungals are added to media
How do you prevent cryopreservation?
Treat with protective agent (DMSO or glycerol)
Stored at -130oC: liquid nitrogen
Name some cell types?
Fibroblasts
Epithelial
Lymphoblast
What is plating density?
No. cells per volume of culture medium
Suspension
Adherent: extracellular matrix (collagen/laminin)
What is confluence?
No. Of adherent cells in culture dish
What are some technical considerations?
Nutrient depletion/changes in pH in the media
Accumulation of dead cells
Contact inhibition m: cell to cell contact stimulates cell arrest
Cellular differentiation stimulated by cell to cell contact
Adherent:extracellular matrix
What is passinging?
Transferring a small number of cells into a new vessel
How do you avoid technical considerations?
Passaging of cells
What is trypsin-EDTS used for ?
Passinging of adherent cells
