Chapter 3 - Characteristics of Cells in Culture

Question 1

What is primary culture?

Answer 1

A culture started from cells, tissues or organs taken directly from the animal

Question 2

What is explant outgrowth?

Answer 2

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

Question 3

What is physical dissociation?

Answer 3

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)

Question 4

What enzymes and agents are used with enzymatic or chemical dissociation?

Answer 4

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)

Question 5

What methods are used in cell selection?

Answer 5

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)

Question 6

What is subculturing?

Answer 6

Subculturing or passaging is the transfer or transplantation of cells from one culture vessel to another; in order to increase the cell numbers

Question 7

Explain the two passaging methods.

Answer 7

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.

Question 8

What are the two types of cell lines?

Answer 8

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.

Question 9

How can a cell culture be immortalized?

Answer 9

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)

Question 10

What is differentiation?

Answer 10

The process leading to fully mature cells in vivo expressing specific phenotypic properties

Question 11

What are the two pathways to differentiation?

Answer 11

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

Question 12

What are the two ways of judging the state of differentiation of the cells?

Answer 12

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)

Question 13

What are reasons for cells to lose differentiated functions?

Answer 13

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)

Question 14

What can be done to induce differentiation?

Answer 14

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.

Question 15

How is differentiation affected based on cell density?

Answer 15

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.

Question 16

What are the two types of neoplastic transformation?

Answer 16

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)

Question 17

What is the in vivo criteria to identify neoplastically transformed cells?

Answer 17

Cells will produce tumours in suitable host organisms (ie. nude mice, nude mice or other immunosuppressed animals)

Question 18

What is the in vitro criteria to identify neoplastically transformed cells?

Answer 18

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

Question 19

What is anoikis?

Answer 19

Apoptosis caused by loss of adhesion to a substrate; normal cells placed in dishes 0.5% agarose will show anoikis.

Question 20

List 2 cryoprotectants

Answer 20

Dimethyl sulfoxide (DMSO) or Glycerol

Question 21

What sorts of damages can be caused to cells during cryoprotectants?

Answer 21

Mechanical injury by ice crystals
Concentration of electrolytes
Dehydration
pH changes
Denaturation of proteins
Other factors

Question 22

How can lethal effects of cryopreservation be minimized?

Answer 22

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

Question 23

What is mycoplasma?

Answer 23

A pleuropneumonia-like organism (PPLO) with a 0.1 um diameter. The have no cell walls so antibiotics are ineffective.

Question 24

What are the sources of mycoplasma contamination?

Answer 24

Human - oral source from mouth pipetting

Contaminated commercial bovine sera

Contaminated commercial porcine trypsin

Contamination of the original tissue or organ

Question 25

How is testing for mycoplasma done?

Answer 25

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

Question 26

How can you avoid cross contamination?

Answer 26

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

Question 27

How do you test for cross contamination?

Answer 27

Chromosome examination or karyotyping

DNA finger printing

Isozyme analysis

Species specific antigen-antibody reactions