The ability to grow cells in culture i.e. in the lab, is essential for biotechnology and research
Applications of cell culture 1
RESEARCH (small scale usage)
growing bacterial cells for basic gene manipulation
culturing mammalian cells to observe the effects of drugs and hormones on the functioning of cells e.g. cancer studies
producing new plants
Applications of cell culture 2
BIOTECHNOLOGY (large scale usage)
agriculture e.g. silage production
pharmaceuticals e.g. genetically engineered bacteria to produce insulin
food production e.g. brewing and baking
biodegradation e.g. sewage treatment
Conditions needed for cell culture
In order for cells to grow, the conditions must be
just right for each cell type. The cytologist must
therefore consider the following:
Type of growth container or fermenter
Method for monitoring cell growth
Safety measures and implications
To avoid contamination of growth media and cultures
All inanimate and living objects, including the atmosphere carry large numbers of microorganisms.
A variety of techniques can be used to provide these conditions:
e.g. sterilisation of all utensils and media using heat . For example, using an autoclave (steam under pressure, necessary for bacterial spores) Growth of pure cultures
They are everywhere!
They highly adaptable to their surrounding environment
They are relatively easy to culture
They incredibly diverse and are able to colonise very extreme conditions e.g. salt pans, hydrothermal vents in the ocean floor
Classes of Microorganisms
There are 2 recognised categories of micro-organism:
1. Unicellular Algae / PHOTOTROPHS : use sunlight to make their own food 1. Bacteria & Yeasts (Fungi) / HETEROTROPHS : need more complex media containing an organic carbon source and other compounds e.g. amino acids
Culture & Uses
Food industry - cheese production, baking, wine & beer
Chemical production e.g. acetone
Bases of food chains
Commensal bacteria in digestive tract
Production of therapeutic compounds e.g. insulin
[See Scholar: Batch & Continuous culture]
Microbial Growth Culture Requirements
A few litres can be made in the lab
Thousands of litres can be made industrially
Micro-organisms are grown in a medium that supplies them with all nutrients necessary for growth.
This depends on …
the type of cell
the final purpose of the cell
Microbial Growth Culture Requirements
Important factors that must always be considered are:
the nutrient media
Unicellular algae , bacteria & yeast can be grown as batch cultures - no dilution is needed until max. density is reached. Growth can be limited by nutrient availability i.e. at the end of exponential growth
Chosen to imitate an organism’s natural environment
Generally supplies all the essential nutrients
A medium is classed as any solid or liquid preparation specifically for growth, storage or transport of micro-organisms
Must be at the correct pH and the correct gaseous concentration for the organisms to grow
There are 2 types of media commonly used:
1. Complex media - this has one or more crude sources of nutrients and their exact chemical composition and components are not known. Generally used for routine cultures 2. Defined media - otherwise known as synthetic media containing chemically known compounds and components which are in a relatively pure form REMEMBER: all media must be STERILE before use !!!
Mammalian Cell Culture
Many animal cells and tissues can be removed from an organism and cultured artifically. This allows the cell’s activities to be investigated e.g. control of the cell cycle
The process of culturing Mammalian Cells
Once the cells are obtained from animal tissues or other cell lines they are placed in a flat culture vessel that lies on its side
The cells stick or adhere to the inside of the vessel as they grow in the medium Most animal cells are ‘ANCHORAGE-DEPENDENT’ i.e. they need something to hold on to These cells usually form a monolayer that will eventually cover the entire surface of the medium
At this point, called confluence , it is necessary to subculture the cells into a fresh medium N.B. Cells that are associated with body fluids such as blood cells are NON-ANCHORAGE DEPENDENT and can be grown in suspension. Again, it is necessary to regularly subculture the cells into fresh medium N.B. All media and culture vessels are STERILISED to prevent the growth of micro-organisms
Mammalian Cell Growth Media
- mixture of glucose , amino acids , salts , water
- sometimes BASIC GROWTH SERUM is added
[This is animal serum prepared from blood and contains additional factors e.g. Platelet Derived Growth Factor,which enhances growth, 5-10% added or Fetal Bovine Serum (FBS) ]
- pH indicator e.g. phenol red: this shows
changes in pH due to waste production
(pH decreases red to yellow )
* Finally, the media must be incubated at the appropriate temperature for the chosen cells e.g. human cells - 37 o c *
Categories Of Mammalian Cell Cultures
There are 2 categories of animal cell cultures:
(1) Primary cultures:
These cells are taken directly from fresh tissue.
The disadvantage is that the cells have a limited lifespan; the cells only divide so many times in culture, so therefore long term culturing is difficult
Process of Cell Collection
The cells are treated with a proteolytic enzyme e.g. trypsin, to separate out the fragments into single cells.
The advantage of this process is that cells can be collected and cloned .
This is useful to isolate a mutant cell line i.e. deriving secondary cell cultures otherwise known as ...
(2) Continuous Cell Lines
These cells have an acquired capacity for infinite growth and division [they are immortal]
They are derived from tumours or the cells have been transplanted [neo-plastic - produce cancer if transplanted into animals] so they have lost their sensitivity to factors associated with growth control.
Generally, these cells will lose their anchorage dependence facility and so are often easier to culture
Continuous Cell Lines
The advantage of using continuous cell lines is that they can be cloned .
This allows easy :
isolation of mutant cells
investigation of cell growth
production of hybrid cells in biotechnology
This routine procedure is used to produce important pharmaceuticals e.g. vaccines and hormones
Bacterial & Fungal Cultures
Much easier to grow than mammalian cells !
Bacteria and Fungi require much simpler growth media requirements and culture conditions compared to animal cells.