38 terms

Animal cell culture

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One square (a 9th) of haemocytometer
0.0001ml
Cell line
A cell culture after its first subculture.
Mechanical dispersion
By forcing tissue through syringe or sieve, or by dissecting it, collecting individual cells for primary cell culture.
Calculating cell density
Total cells / squares counted x 10^4 x DF
Cell counting technique
Count top and right.
Research and study
Cell culture reason. Studies cell biology, drug toxicity, aging and cancer.
Appearance of suspension cells
Cell looks round, nucleus and cytoplasm not easily differentiated.
Adherent cells
Cells that need to attach to surface in order to grow. Usually come from solid tissue
Observation of cells in cell culture
Check pH, density, shape
Serum (Fetal Bovine serum
Deactivates trypsin
Trypsin
Digest sticky proteins holding cells together. Used in cell separation and subculturing. Use at 37°C
Carbohydrates in culture medium
Glucose and fructose. Used as energy sources
Cell Culture
Growing animal cells taken from live tissues under laboratory conditions
Water for cells
Hydrates cells, used as solvent for nutrients, salts and oxygen.
Haemocytometer
Modified slide used for cell count
Appearance of adherent cells
Monolayer of cells, cells are flattened, spread out, cytoplasm and nucleus can be seen
Subculture of suspended cells
No need trypsin (already suspended), steps not involving trypsin are the same
Phenol red
Yellow<6.8Colour turns yellow: acid waste from bacteria. Color turns purple: alkaline waste from fungi
Glutamine
Precursor in biochemical reactions in cells that generate energy. Is an example of an essential amino acid
Serum in cell culture medium
Promotes cell growth. Has lipids, trace elements, hormones, growth and adherence factors, protease inhibitors
Reducing animal testing
Reason for cell culture.
Antibiotics
Reduce contamination frequency, but may also encourage growth of superbugs, poor aseptic techniques, and hide the presence of low level contaminants
Subculture of adherent cells:
examine culture for contamination, remove medium, rinse with PBS to remove serum, add trypsin, incubate at 37°C, add culture medium containing serum, centrifuge cells, pour away supernatant, add cell culture and mix for culture suspension. Count cells, determine cell density, seed cells, reincubate
Suspension cells
Suspended in culture medium, do not need to attach for growth. Come from blood or adherent cells adapted to suspension culture
Medical uses of animal testing
Test-tube babies, stem cell treatment
Subculture
Thin out cells ensuring sufficient space to grow, maintaining log phase of growth.
Primary cell culture
Cell culture derived from tissues originating from animal or human
Sodium bicarbonate
Used as buffering system to maintain pH around 7.2 to 7.4. Low cost, non toxic and provides nutritional benefits
Amino acids in culture medium
Used for protein synthesis. Usually essential amino acids added since cells cannot synthesise them.
Cell based manufacturing
Using animal culture to make vaccines, replacement tissues and organs.
Enucleated oocyte
Egg cell which has nucleus removed
Volume under haemocytometer
0.0001ml = 10^-4ml
The whole haemocytometer is 3mm3mm0.1mm.
Each small square is 1mm*1mm large.
Depth is 0.1mm.
Mechanism of action for sodium bicarbonate
NaHCO3 -> Na+ and OH- and CO2. OH- neutralises solution keeping pH at 6.9-7.0.
CO2 equilibrates dissociation.
HEPA filter
99.97% of all particles (>0.3micrometer) are removed
Cell culture incubator
37°C, 5%-10%, pH6.9-7.4
And also humidified atmosphere
Short term storage
-80°C
Long term storage
Liquid nitrogen at -196°C
Why cell separation (adherent)
Must be single cells in order to grow and spread out as a complete layer
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