Week 4: Tissue Culture: Growing and Studying Neural Cells in a Dish
ONE: Appreciate major developments in the history of tissue culture
TWO: Understand the main challenges for successfully maintaining cells in tissue culture
THREE: Discuss, with examples, the major applications of tissue culture
FOUR: Critically evaluate published research that has employed these techniques
What is tissue culture?
Cultivation of eukaryotic tissues outside of the organism, in a growth media with the necessary nutrients, inorganic salts and pH required to function in a physiologically normal manner.
Cell Culture:
culturing of dissociated cells rather than pieces of tissue
Why is it useful?
Useful as a model system for studying the basic processes of cell biology
Clinical Applications:
Diagnosis of chromosomal disorders from the culture of blood oir amniotic fluid samples
Generation of monoclonal antibodies for the production of vaccines as a result of the development of hybridoma cell lines
In vitro fertilization through techniques developed for the culture odf the early embryo
Willhelm Roux maintained that neural folds from early chick embryos in a saline solution
Ross Granville Harrison removed small section of frog embryos and embedded them in blood clots on the underside of coverslips to allow microscopic evaluation
Alex Carrell and Montrose Thomas Burrows generated the first cell line from embryonic chicken heart
Development of growth media:
Media based upon blood products gave rise to problems with reproducibility of results due to its poorly defined nature
Made first defined liquid media to try to overcome this problem:
Grew embryonic chick tissue in a relatively simple defined liquid media
Nowadays, cell types are still typically grown with media containing serum
Many reseachers looked to try and make fully chemical growth media. Lewis and Lewis were able to grow chick embryonic in a relatively simply defined media
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Enzymatic dissociation of tissue into cells and their "passaging"
Francis Peyton Rous and F.S. Jones
Most Cell types. with the exception of blood cells grow attached to an extracellular matrix (ECM):
The extracellular matrix is composed of a complex mixture of polysacchrinides and proteins such as collagens and laminin
Tissue culture vessels coated with purified of unpurified components of the ECM help to support attachement and normal functioning of many types in adherent cells
Cell adhesion molecules on the surface of many cells bind strongly to the components of the ECM
The use of trypsin also allowed for the re-plating of cells grown attached to a substrate
Still used todaty to enzymatically dissociate tissues into single cells but can kill cells so gentler enzymes such as EDTA soutions are increasingly used and some non-enzymatic methods as they result in lower cell death.
Freezing Cells:
Dissociated cells can be frozen indefinitely in liquid nitrogen
In order to freeze and thaw cells they are A) detached using the same methods used for passaging cells and re-suspen ded in a solution with a cryo-protectant such as DMSO
Cells are frozen down in a freezer with a vessel filled with isopropanol
Cells are then transferred to to a liquid nitrogen vessel and then revived by rapidly thawing.
Culture Vessels
Alexis Carrel developed a new vessel for tissue culture—the carrel flask.
The angled neck prevents airborne particles from settling into the flask when open
Allows for sterilisation with a flame further reducing the risk of airborne contaminants infecting the culture.
Introduction
This is a reductionist approach to studying the physiology of cells. There are many clinical applications for this too.
Cells may behave differently when cultured
Trypsin, one of our digestive enzymes, can be used to break down tissue into dissociated cells
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