WEEK 4 Flashcards
Tissue culture
refers to the cultivation of eukaryotic tissues or dissociated cells 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
the culturing of dissociated cells rather than pieces of tissues
Tissue culture: clinical applications
- diagnosis of chromosomal disorders from the culture of blood or 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 of the early embryo
Roux (1885)
demonstrated that neural tissue from the chick embryos can be maintained for a short period of time in a saline solution. however, this is considered to be temporary survival rather than true tissue culture as the media did not support long-term growth
Harrison (1907)
first demonstration of maintaining animal cells in a substrate supporting growth and long-term survival. he removed small sections of frog embryos and embedded them in blood clots. he was able to observe the outgrowth of nerve cells over a period of weeks.
Carrel and Burrows (1991)
established much longer term cultures by refining the growth media and by employing careful aseptic technique. they were able to generate the first indefinitely growing cell line from embryonic chicken heart.
Lewis & Lewis (1911)
demonstrated that they were able to grow embryonic chic tissue in a simple defined liquid media, rather than in blood products.
Rous and Jones (1916)
demonstrated the use of the proteolytic enzyme trypsin to dissociate tissues into individual cells for culture.
“Passaging”
- cell adhesion molecules on the surface of many cells bind strongly to components of the ECM. these contacts have to be destructed to detach cells without causing cell death.
- the use of trypsin allowed for the replating of cells grown attached to a substrate. Passaging is essential when culturing dividing celles which will eventually grow to occupy all the space in a cell culture vessel.
Extracellular matrix (ECM)
most cell types, with the exception of blood cells, grow attached to an extracellular matrix. the ECM is composed of a mix of polysaccharides and proteins such as collagens and laminin.
- tissue culture vessels coated with purified or unpurified components of the ECM help to support attachment and normal functioning of many types of adherent cells.
Contact inhibition
when cells no longer have the space to grow, they undergo contact inhibition, which stops the cell from dividing further and can alter the characteristics of the cells under investigation.
Trypsin alternatives for cell passaging
- trypsin is still in use to enzymatically dissociate tissues into single cells, however for passaging cells, trypsin can cause a degree of cell death.
- gentler enzymes such as accutase and non-enzymatic methods such as EDTA are increasingly used.
- non-enzymatic methods chelate ions such as calcium that are essential for the function of the cell adhesion molecules. Disruption of these contacts allows the cells to be rinsed from the flask with much lower levels of cell death.
Carrel and Baker (1923)
- Harrison’s “hanging drop technique” posed problems, including the fact that the cells in their clot were difficult to view under a microscope.
- Carrel and Baker developed the Carrel flask, which is manufactured with an angled neck to prevent airborne particles from setting into the flask when open. The next design also allowed for sterilisation with a flame, reducing the risk of airborne contaminants affecting the culture.
Modern tissue culture vessels
- plastic, sterile, single use.
- flasks have vented lids to prevent airborne particles entering, whilst allowing for the free exchange of gases
Mycoplasma
- smallest known bacterial cells, making them difficult to observe under conventional microscopes
- a significant problem in long-term cultures, affecting the growth and normal function of cells.
- there are some antibiotics which are effective against mycoplasma, however best practice is to prevent contamination in the first place by employing good aseptic technique.
Class I cabinets
- designed to protect both the user and the environment from hazardous microbes
- dates back to 1909, and was a simple ventilated hood developed for tuberculosis
- the sample is not protected from airborne particles in the environment
Class II cabinets
- developed in the 1960s
- rely on a continuous uniform flow of clean filtered air traveling down over the sample. the airflow then splits and flows through grills at the front and the back of the cabinet.
- unfiltered air entering the front is drawn directly into the grill and out through a filtered exhaust without passing over the sample
- this protects the sample from outside contamination
Class III cabinets
- used for samples which require an even greater level of containment
- these completely enclose the sample and can only be accessed through gloves integrated into the cabinet
Tissue culture incubators
- developed to maintain a number of critical parameters to allow optimal growth and survival. these include constant levels of temperature, humidity, CO2, and oxygen.
- most media currently in use for tissue culture use buffers that require an atmosphere of 5% CO2 to maintain a physiological pH.
- iPSCs grow better under low oxygen conditions
Hayflick and Moorhead (1961)
derived the first strains of human fibroblasts (WI-38)
Primary cells, cell lines, and cell strains
- primary cells: derived from normal tissue and grown without passaging
- cell strains: derived from primary cells which have a limited capacity for growth and division but retain a normal karyotype
- cell lines: capacity to grow indefinitely and invariably, and have abnormal karyotypes
Gey (1951)
cultured cells from Henrietta Lacks who had cervical cancer. he found that cells derived from the tumor could grow and divide indefinitely, and generated a cell line from a single cell he named “HeLa cells”
- HeLa cells have aided in the development of the first polio vaccine
Evans (1982): embryonic stem cells
established cultures of cells derived from mouse blastocysts. these can generate any cell type of the body in a cell culture dish
Thomson (1998): human embryonic stem cells
used human embryonic stem cells to generate inaccessible cell types, such as neurons, in large numbers for the first time.
- there are ethical issues surrounding the generation of human embryonic stem cells as they require the use and destruction of human embryos
Yamanaka (2006/2007)
directly generated embryonic stem cells by manipulating fibroblasts in a process called reprogramming.
- iPSCs have become a powerful tool to study genetic diseases and inaccessible cell types such as neurons, without the ethical issues of embryonic stem cells
Personalized regenerative medicine
- an individual who is suffering from Parkinson’s can give a small sample of blood, which can be reprogrammed to iPSCs and then differentiated to dopaminergic neurons. these neurons can then be transplanted back into the individual to replace the ones that have been lost
- the advantage is that the transplanted cells will be genetically identical to the individual, eliminating risk of rejection and use of immunosuppressant drugs
