Cell Culture Methods Flashcards
Lecture 1 by Roisin (24 cards)
define in vitro cell culture
in glass
what is used to “feed” cells in culture?
originally Ross Granville Harrison used lymph, later blood plasma
today: cell culture media
what is the Hayflick limit?
cells grown in vitro could only divide for a limited number of times before becoming senescent
define tissue culture
the growth of cells in the lab
define microbiological safety cabinet
MSC
a hood used to maintain a sterile environment
define laminar flow hood
enclosed cabinet with a HEPA (high-efficiency particulate air) filtered laminar airflow system, run at positive pressure to ambient.
used to avoid bacterial contamination when experimenting with cell culture
tissue culture is maintained at a constant temp and pH of
37˚C (enzymes are optimised for this temp)
pH 7.2-7.4 similar to blood
overall mimics the in vivo environment
name six key reagents for cultivating cell culture.
Basal media (essential nutrients for cell growth)
Serum (growth factors/ hormones)
Glutamine (amino acid additive)
NEAA (non-essential amino acids)
PBS (phosphate buffered Saline)
Trypsin
what is the Basal Media used for? give an example
the basal media is the reagent added to cell culture that contains the ingredients required to sustain cell growth.
eg. DMEM, contains minimum necessary ingredients
what is the Serum used for as a reagent in cell culture?
Fetal Bovine Serum
supplies growth factors and hormones
what is glutamine used for as a reagent in cell culture?
an essential amino acid additive
(often unstable in sln so added separately)
what are NEAA? Reagent in cell culture
Non-essential amino acids
not produced by the cells themselves, must be added
what is PBS added for as a reagent in cell culture?
Phosphate Buffered Saline
basic salt solution with pH 7.4 (blood)
for washing cells
what is the purpose of trypsin as a reagent in cell culture?
trypsin is an enzyme which cleaves proteins – helps cells detach from surfaces
what is the Minimal Essential Medium (MEM) for cell culture?
developed by Eagle.
containing six salts + glucose + 13 amino acids + 8 vitamins
(later developed into DMEM)
how can an aseptic environment be maintained in tissue culture?
all solutions need to be autoclaved or filtered (0.02 µm)
all surfaces / bottles wiped with 70% ethanol
antibiotics used as an additive (although somewhat considered bad practice)
cells are typically grown on what material?
TCTP
Tissue Culture Treated Polystyrene
Cells grown on TCTP are maintained in incubators. what are the conditions in incubators?
humidified environment
5% CO2
18% O2 (note: has been criticised for inducing hypoxic conditions)
recall: TCTP is the Treated Polystyrene that Tissue Culture is grown on
what does it mean to say cells have reached “confluence” ?
Confluence: cells have covered the entire surface of the flask/dish
what is passaging?
detaching cells (using trypsin) from dishes/flasks, diluting them and placing in a new dish with fresh media.
(Hayflick’s limit actually referred to the max number of times that cells could be passaged)
what are some problems with modelling / predicting biological phenomena using tissue culture?
substrate stiffness - ignores mechanotransduction
2D layer (vs 3D in reality)
single cell type
cell culture media components (some poorly defined)
non-physiological oxygen and glucose levels
origin of cell – cancerous cell lines
antibiotics
SEE DIGRAM!!
Compare 3D in vitro model vs 2D monolayer models
2D: cells are grown on rigid/stiff substrate, cells lose their polarity and spread. submerged in liquid without oxygen/nutrient gradients. restricted interactions.
3D: less stiff hence more likely to mimic real tissue. Cell-cell and cell-ECM interactions. Less spreading. Gradients established.
what is Matrigel?
product derived from mouse cells, can be used to grow cells (in 3D). contains growth factors (analogous to Serum for 2D)
3D cell culture is preferred for…
disease modelling and drug discovery to better mimic diffusion in nutrients and gases / tissue structure
2D remains useful for processes requiring high throughput
