Fundamentals of tissue culture Flashcards
What is a tissue culture?
Cultivation of eukaryotic tissues outside of the organism
- in a growth media with the necessary nutrients
Which nutrients are required in a tissue culture to function in a physiologically normal manner?
- Inorganic salts
- pH (potential of hydrogen, acid/basic)
What is a ‘cell culture’?
Culturing of dissociated cells rather than pieces of tissue (tissue culture)
Why is tissue culture useful?
> Model system for studying basic processes of cell biology
> Clinical applications
How did Kohler and Milstein (1975) clinically apply tissue culture?
Generation of monoclonal antibodies
- production of vaccines as a result of the development of hybridoma cells
How did Steptoe and Edwards (1977) clinically apply tissue culture?
In vitro fertilisation
- through techniques developed for culture of early embryo
Who achieved short term maintenance of tissue outside an organism in 1885?
Wilhelm Roux:
- short term maintenance of neural folds from early chick embryos in a saline solution
- BUT media did not support long term growth
Who achieved the first maintenance of animal cells in a substrate supporting growth AND long-term survival in 1907?
How?
Ross Granville Harrison:
- removed small section of frog embryos and embedded them in blood clots on underside of coverslips to allow microscopic evaluation
- good aseptic technique
-> he observed the outgrowth of nerve cells over several weeks
Who achieved the first long-term cell cultures in 1911?
Carrel and Burrows:
- generated the first ‘cell line’ from embryonic chicken heart
How can cell lines grow indefinitely in culture?
Through genetic mutations and chromosomal abnormalities
Which problems rose from media based upon blood products?
Why?
Problems with reproducibility of results
- due to its poorly defined nature
Who made the first defined liquid media to overcome the problems of blood-based media in 1911?
How?
Margaret Reed Lewis and Warren H. Lewis:
- cultivation of tissues from chick embryos in simple defined liquid media
- solutions of NaCI, CaCI2, KC1 and NaHCO3
In what kind of serum are cell types typically grown with nowadays?
Media containing serum
- e.g. human fibroblasts
Who first dissociated tissues into individual cells for culture in 1916?
How?
Francis Peyton Rous and F.S. Jones:
- enzymatic dissociation
- use of proteolytic enzyme trypsin
How do most cell types - with the exception of blood cells - grow?
Attached to an extracellular matrix (ECM)
What is an extracellular matrix (ECM) composed of?
A complex mixture of polysaccharides and proteins
- e.g. collagens and laminin
How do tissue cultures support attachment and normal functioning of many types of adherent cells?
> Tissue culture vessels are coated with purified or unpurified components of the ECM
> Binding of cell adhesion molecules to components of the ECM needs to be disrupted to detach cells without causing cell death
> Use of trypsin allows the passaging (re-plating) of cells grown attached to a substrate
- essential for dividing cells to grow and occupy all space in a cell culture vessel
What happens to dividing cells when they no longer have room to grow in a cell culture vessel?
They undergo contact inhibition:
- stops the cells from dividing further
- can alter characteristics of the examined cells
What is the current use of trypsin?
Still used to enzymatically dissociate tissues into single cells
What is the limitation of trypsin?
For the passaging of cells, trypsin can cause a degree of cell death
What are the alternative methods to trypsin?
What do they tend to result in?
> Gentler enzymes such as Accutase
Non-enzymatic methods such as EDTA solutions (ethylenediamine tetraacetic acid)
=> reduced cell death
How do non-enzymatic methods work for the passaging of cells?
They chelate ions (e.g. Ca2+) that are essential for the function of cell adhesion molecules
What are chelating agents?
Organic compounds capable of linking together metal ions to form complex ring-like structures called chelates