Cell Culture Flashcards
Organ culture
The entire embryo or organ is excised from the body and culture
Advantages and Disadvantages of organ culture
Advantages
- Normal physiological functions are maintained
-Cells remain fully differentiated
Disadvantages
-Scale-up is not recommeneded
-Difficult maintanence
-Fresh explantation is required for every experiment
Tissue culture
Fragments of excised tissue are grown in culture media
Ad and Dis of Tissue Culture
Advantages
-Some normal functions may be maintained
-Better than organ culture for scale-up but still not ideal.
-Can prolong tissues indefinitely by submerging them in liquid Nitrogen
Disadvantages
-Original organization of tissue is lost.
Cell culture
Tissue from an explant is dispersed, mostly enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture.
What is celll culture used for
-model systems for studying basic cell biology and interactions between disease- causing agents and cells
-Toxicity testing: new drugs
-Cancer research-How cells turn cancerous
-Virology: vaccine
-Genetic engineering: Large scale production of proteins or viruses
-Gene Therapy: Replace non-functional genes in cell
Ad and Dis of cell culture
advantages
-absolute control of physical environment
-Homogeneity of sample
-Less compounds needed than in animal models
-Large upscaling possible
Disadvantages
-Hard to maintain
-Not cheap
-Dedifferentiation (loss of specialized function)
-Instability and aneuploidy after accumulating many mutations after many replications
Major developments in cell culture technology
-antibiotics inhibit the growth of contaminants ( penicillin and streptomycin)
-Trypsin removes adherent cells to subculture further from the culture vessel
-Culture medium which uses complex chemicals to provide nutrition to cells
Primary culture
these are cells directly taken from tissue but their disadvanatge is they can only replicate a set number of times
contact inhibition
It is a growth mechanism which functions to keep cells growing into a o layer one cell thcik
Secondary cultures
They are derived from primary cultures and exhibit contact inhibiton and anchorage dependance
Immortalised cell line
Cells will keep replicating but are not cancerous.
To prevent mutations, you must stop replicating cells after a certain point
Transferd cell line
This occurs when a cell turns cancerous
Qualities of cancer cells
- Growth factor independency
-No response to growth factors
-Evasion of apoptosis
-Can promote angiogenesis
-Unlimted proliferation
-Invasive
What are the 4 cell types
-Fibroblasts
-Epithelial
-Lymphoblastic
-Neuronal
Epithelial cells
They are attached to substrate and appear flat and polygonal
Lymphoblast cells
Cells remain in suspension with a spherical shape
Fibroblast cells
Cells are attached to substrate and appear elongated and bipolar
Anchorage dependance
cells that will grow only on suitable substrates. Most cells are like this except for suspension cells
What is Mr Frosty
It is a piece of equipment used to freeze cells to eventually reach -178 degrees when cooled in liquid nitrogen. However this device cools the cell down progressively to prevent formation of water crystals
Confluency
It is how much of the available space is taken up by cells.
What is normal confluency
It is anything between 70%-80% as if they are at 100%, then the cells are in high competition and will be continuously stressed.
Adherent cells
These are cells that are anchorage dependant
What does EDTA do
It binds to Mg and CA in a cell dish and removes them from the medium. It is added in addition to Trypsin in order to deattach cells
Human breast adenocarcinoma
They are cancer cells that do not have hormone receptors and so is hard to develop drugs for.
-mainly affects women of colour
Human embryonic kidney cells (HEK)
Epithelial morphology
-used to determine if drug kills healthy cells
suspension cells
are not anchorage dependent
-They are easier to culture but harder to assay
Why sub culture
In order for cells to be healthy and in a growing stage they must be sub cultured
this usually occurs at 90% confluency
Therefore sub culturing reduces confluency
What do cells need to grow?
Substrate or liquid
-For cells to bind to or float in
Nutrients
Environment:
-CO2, temperature at 37, humidity so that they don’t dry out and that the osmolarity of the plate remains constant
Sterility: aseptic technique and antibiotics
Why is pH control important
Physiological pH is at 7
pH can affect:
-Cell metabolism
- Growth rate
-Protein synthesis
-Availability of nutrients
CO2 acts as a buffering agent to maintain the same pH.
Types of contaminants of cell cultures
Chemical- difficult to detect but could be caused by metal ions or disinfectants
Biological- Cause visible effects on culture and could be yeast, bacteria or fungus
MTT assay
-It is used to determine the percentage cell survival in response to a drug, antibody, etc.
-MTT is a yellow salt that becomes converted into purple formazan crystals in the mitochondria of viable cells.
-The absorbance of formazan is directly proportional to cell viability and it is read at 570nm.
-Since formazan is only produced in viable cells, the higher the absorbance of formazan ( the darker the color), the higher the number of viable cells
enzyme in MTT
Mitochondrial Succinate Dehydrogenase converts MTT into Formazan
How to passage cells
Why passage cells
-To maintain cell culture, so that they don’t overgrow and become stressed
-to increase cell number for experiments/storage
Factors affecting cell behaviour in Vivo
