Chapter 3 - Characteristics of Cells in Culture Flashcards
What is primary culture?
A culture started from cells, tissues or organs taken directly from the animal
What is explant outgrowth?
Fragmenting the tissue through mechanical dissociation. The explants are placed in culture dish with proper medium. Cells grow out from the explants.
Draw the process
What is physical dissociation?
Tissue is passed through a series fo sieves to break apart the cells; only suitable for cells that are loosely bound to each other (ie. nervous tissue)
What enzymes and agents are used with enzymatic or chemical dissociation?
EDTA - chelating agent which binds to calcium
Trypsin - breaks down proteins
Collagenase - breaks down collagen
Hyaluronidase - breaks down proteoglycans
DNase - used because some cells may be killed from trypsinizing so DNase breaks down DNA (which has a sticky texture)
What methods are used in cell selection?
Physical method - separate cells based on density gradient centrifugation on Ficoll (polysaccharides solution)
Selection during Culturing - During cell culturing, certain cells attach to growth substrates more rapidly than others (ie. fibroblasts attach to substrates first so they are easier to remove from culture)
What is subculturing?
Subculturing or passaging is the transfer or transplantation of cells from one culture vessel to another; in order to increase the cell numbers
Explain the two passaging methods.
Physical - Using a cell scraper to remove cells from surface of the culture vessel. Cells have to be robust otherwise the membrane will break, killing the cells.
Enzymes - Trypsin is used to separate cells from surface of culture vessel and each other. The time in trypsin solution is very important; too much will kill cells and too little cells wont separate. Serum is used to neutralize trypsin, centrifuge to separate trypsin before passaging.
What are the two types of cell lines?
Finite - These cells have limited doubling potential and maintain diploid status.
Continuous - These cells have unlimited doubling potential and are not diploid/have random additions in the chromosome.
How can a cell culture be immortalized?
Spontaneous - Without the use of physical or chemical agents; only typical cell culturing methods. Very species specific.
Directed or experimental - A result of a specific experimental treatment (ie. cell fusion or introduction of specific genes)
What is differentiation?
The process leading to fully mature cells in vivo expressing specific phenotypic properties
What are the two pathways to differentiation?
Tissue undergoing continuous renewal - pluripotent stem cells give rise to committed precursor cells; (ie. haemotopoietic system, skin and intestinal epidermis)
Tissues that renew under stress - Cells respond by losing some of their differentiated properties, divide and re-differentiate to renew cell population
What are the two ways of judging the state of differentiation of the cells?
Lineage Markers - characteristics of the cells within the lineage (ie. intermediate filament proteins such as cytokeratins for epithelium)
Terminal Differentiation Markers - represent mature phenotype of the cell (ie. haemoglobin in RBC)
What are reasons for cells to lose differentiated functions?
Dedifferentiation - the specialized properties of the cell are lost irreversibly (conversion to more primitive phenotype)
Deadaptation - the synthesis of specific products are lost but can be reacquired if correct culture conditions can be recreated
Selection - continuous proliferation may select undifferentiated precursors (in absence of correct culture conditions the cells do not differentiate)
What can be done to induce differentiation?
Medium - addition of soluble factors to the medium (ie. hormones or growth factors)
ECM Interactions - commercial components of the matrix can influence cell differentiation
Cell-to-cell Interactions - placing different cell types together may induce change
Polarity and shape - hepatocytes grown on collagen gel, shrink gel; get a change in the hepatocytes shape into fully matured hepatocytes.
How is differentiation affected based on cell density?
When cell densities are low (through serial passage/subculturing) cell proliferation occurs; little differentiation.
High cell densities (in low serum and appropriate hormones) promotes differentiation and inhibits cell proliferation.
What are the two types of neoplastic transformation?
Spontaneous - normal cells in culture dishes transform into neoplastic cells
Directed - normal cells in culture dishes transform into neoplastic cells through treatment methods (ie. oncogenic viruses, oncogenes, chemical carcinogens and irradiation)
What is the in vivo criteria to identify neoplastically transformed cells?
Cells will produce tumours in suitable host organisms (ie. nude mice, nude mice or other immunosuppressed animals)
What is the in vitro criteria to identify neoplastically transformed cells?
Growth characteristics - the cell lines are immortal or continuous, loss of contact inhibition of growth, lose substrate dependent growth
Genetic properties - heteroploid karyotype , spontaneous mutation rate, overexpressed oncogenes, deleted suppressor genes
Structural alterations - modified actin cytoskeleton, loss of cell surface-associated fibronectin
Neoplastic/malignant/invasive properties - angiogenic (induces blood vessel proliferation), enhanced protease secretion (able to digest ECM)
**Invasiveness can be tested using the Matrigel assay or chicken heart invasion assay
What is anoikis?
Apoptosis caused by loss of adhesion to a substrate; normal cells placed in dishes 0.5% agarose will show anoikis.
List 2 cryoprotectants
Dimethyl sulfoxide (DMSO) or Glycerol
What sorts of damages can be caused to cells during cryoprotectants?
Mechanical injury by ice crystals Concentration of electrolytes Dehydration pH changes Denaturation of proteins Other factors
How can lethal effects of cryopreservation be minimized?
Slowing the cooling rate to allow water to move out of cells (one degree decrease per minute)
Store below -130C to slow down crystal growth (usually liquid nitrogen @ -196C)
Rapid warming from -50C to 0C
Addition of cryoprotectants
What is mycoplasma?
A pleuropneumonia-like organism (PPLO) with a 0.1 um diameter. The have no cell walls so antibiotics are ineffective.
What are the sources of mycoplasma contamination?
Human - oral source from mouth pipetting
Contaminated commercial bovine sera
Contaminated commercial porcine trypsin
Contamination of the original tissue or organ
How is testing for mycoplasma done?
Microbiological culturing
Immunofluorescent staining with antisera specific to mycoplasma
Fluorescent DNA staining - Hoescht
Biochemical tests - testing for adenosine phosphorylase and other enzymes found in mycoplasma and not in mammalian cells
Electron microscopy
How can you avoid cross contamination?
Never simultaneously culture more than one cell line at a time
Keep separate media, trypsin, versene (EDTA) for each different cell line
Only manipulate one culture at one time and clean flow food thoroughly between uses
How do you test for cross contamination?
Chromosome examination or karyotyping
DNA finger printing
Isozyme analysis
Species specific antigen-antibody reactions
