Animal Cell Culture
Animal Cell Culture (ACC)
Types of Animal Cell Culture
cell culture
removal of cell from an animal or plant and their subsequent growth in a favourable artificial environment
cell obtainment
removal from tissue directly
derivation from a cell line or cell strain
in vivo → inside a living organism
in vitro → outside the organism
why use ACC?
discontinuation of the usage of animal testing
research and study
cell biology
biochemistry
drug toxicity
aging
cancer
cell-based manufacturing
vaccines
organ and tissue replacement
primary cell culture
originates from an animal or human
separates individual cells by:
enzymes
trypsin
digests away sticky proteins that hold the cells together
mechanical dispersion
forcing tissue fragments through a syringe
collecting cells when a fragment of tissue is dissected
pressing fragments of tissue through a series of sieves
grown from fragments of tissues
a culture is considered a primary culture up to its first subculture; after that it is a cell line
subculture of adherent cells
why?
thin out the cells so that they have sufficient space and nutrients to grow
maintain the cells at the log phase of cell growth
adherent cells
cells that need to be attached to a surface in order to grow are adherent cells
monolayer & attached to each other
cells look flattened and spread out
nucleus and cytoplasm can be seen
cells usually come from solid tissue
suspension cells
cells are suspended in the culture medium
cells look round
nucleus and cytoplasm not easily differentiated
come from blood forming tissues or from adherent cells adapted to suspension culture
Types of Lab Equipment & Materials
biological safety cabinet (BSC)
provides a working environment that is protected from dust and contamination by a constant flow of filtered air passing over the work surface
classes of BSCs
class I
non-toxic, non-infectious agents
class II
low-to-moderate toxic or infectious agents
class III
totally enclosed system to handle high risk pathogens
air in a BSC is kept sterile by a high-efficiency particulate air (HEPA) filter
a HEPA filter removes 99.97% of all particles >0.3 μm from the air that passes through
air flow in a biological safety cabinet
cell culture incubator
controls temperature, carbon dioxide levels and humidity
temperature maintained at 37ºC for mammalian cells
humidified atmosphere prevents evaporation of culture medium
5% - 10% carbon dioxide to equilibrate the bicarbonate in the culture medium to maintain the culture at pH 6.9-7.4
animal cells need carbon dioxide to simulate conditions in the body
inverted microscope
important in observing cell cultures
cell culture is placed on the stage
observe cell cultures for changes in cell growth, cell shape and signs of microbiological infection
haemocytometer
modified slide used to count cells
the haemocytometer grid helps us count the number of cells
liquid nitrogen storage materials and facilities
cells are stored in liquid nitrogen until they are needed
cells are suspended in cryoprotectant so as no ice crystals are formed at freezing temperature
this storage in frozen form in liquid nitrogen is called cryopreservation
ppe when handling liquid nitrogen
safety goggles
lab coat
cryogloves
insulated, waterproof gloves
jeans
boots
non-slip mat
cryovial
tube made of a special material to withstand freezing at very low temperatures
cryocooler
specially constructed box that controls freezing such that the temperature of the vials drop slowly at 1ºC or less per minute
put in lab freezers to cool to -20ºC
moved to -70ºC freezer
cell culture disposables
serological pipettes
measuring volumes and handling liquids
multidishes, plates, and flasks
sterilized, plasticware made of polystyrene
sometimes treated to increase attachment by adherent cells
treatment with poly-L-lysine or collagen
electrically charged
Cell Culture Medium
components in cell culture medium
water
keep cells hydrated
water is the solvent for the nutrients, salts, and oxygen that the living cells need
the medium is isotonic (0.9% sodium chloride) such that no osmosis or intercellular / extracellular shift occurs
amino acids
protein synthesis
essential amino acids must be added as cells are not able to synthesise them on their own
glutamine
acts as a precursor for the Krebs’ cycle
carbohydrates
glucose and fructose as energy sources
serum
contains:
lipids
trace elements (iron, iodine)
hormones and growth factors (insulin)
adherence factors that help in cell adhesion
protease inhibitors
plasma (from blood) without clotting factors to prevent coagulation
antibiotics
reducing frequency of contamination
penicillin and streptomycin commonly used
antibiotics may:
encourage the development of antibiotic-resistant microorganisms
hide the presence of low-level contaminants that grow when the antibiotics are removed
encourage poor aseptic technique
sodium bicarbonate
most cells require a pH of 7.2-7.4
sodium bicarbonate is used as a buffering system
used due as it is low cost, non-toxic, and provides additional nutritional benefits to the cells
$NaHCO_3⇌Na^++HCO_3$$^-⇌Na^++CO_2+OH^-$
during cell culture, incubator is set to 5% - 10% carbon dioxide level.
sodium bicarbonate also dissociates to release carbon dioxide into the atmosphere and hydroxide ions into the medium
in a freshly prepared medium, the enriched carbon dioxide atmosphere will help to equilibrate the association of ions to maintain the pH at 6.9-7.0
carbon dioxide partially dissociates into carbonic acid
hydroxide ions neutralise the carbonic acid in the medium
pH indicator
phenol red is added so the pH status of the medium is constantly indicated by the colour
the culture medium should be changed or replenished if the colour turns yellow (acid) or pinkish purple (alkali)
nutrients
lipids
fatty acids and cholesterol
salts involved in cell function and metabolism
nucleic acids
vitamins, hormones, growth and adherent factors
preparation of cell culture medium
contains various ingredients to mimic physiological fluids (blood, lymph) so as to provide optimal conditions for cell growth
all ingredients must be added in the right quantity
mole concept is your best friend here !!
v/v
e.g. 7% pipagao solution (v/v)
7 ml of pipagao + 100 ml of water
w/v
20% milo solution (w/v)
20 grams of milo powder & top up until 100 ml solution volume reached
dilution of stock solution
using $C_1V_1=C_2V_2$ and solve (that’s literally it)
From Tissue to Cell Line
trypsin
a type of protease
cells secrete sticky proteins which let the cells stick to the flask and each other
trypsin digests the proteins to break up the cell clumps
can affect cell viability if incubated for too long
deactivated by rinsing fragments of tissue with culture medium that contains serum
during subculturing of suspension cells
examine the cell suspension for signs of contamination
pipette a sample and count the cells from the cell suspension
pipette a portion of the cells into a new culture vessel
add in culture medium and incubate
during subculturing of adherent cells
remove the cell culture medium
detach the cells from the culture vessel by using trypsin
transfer a portion of the cells to the new culture vessels so that they have enough space to grow
steps in subculturing adherent cells
Examine the culture for contamination
To subculture, remove the medium
Rinse the adherent cells with phosphate buffered saline (PBS) to remove traces of serum in the culture medium
Add trypsin solution and incubate at 37°C until cells rounded off (5-15 min)
Add culture medium containing serum and mix with the detached cells to deactivate the trypsin
Transfer the cells to a centrifuge tube and centrifuge to pellet the cells
Pour away the supernatant
Add in culture medium to mix with the cells to create a cell suspension
Count a sample of the cells from the cell suspension
Calculate the cell density and determine how many cells to put into a new culture vessel (a process also known as seeding)
Seed the cells into the new culture vessel
Incubate the culture vessel to grow the cells
cell counting using a haemocytometer
count grid by grid
include cells on the top and left side of the grid
exclude cells on the bottom and right of the grid
Animal Models
what are animal models?
non-human species used in medical research
mimics aspects of a disease found in humans
used to obtain information about a disease and its prevention, diagnosis, and treatment
researchers use animals instead of humans due to ethical issues
common animal models
pigs
useful in cardiology research
share same heart size and blood supply system as humans
useful in burn would healing and plastic surgery
skin similar to humans
used in crash testing
big size
rabbits
suitable for cardiovascular and immune system research
useful in eye experiments
each eye only has one tear duct, so chemicals are not washed away easily
draize rabbit eye test
acute toxicity test
assessment of the effects of chemicals, substances, and mixtures that may cause eye irritation
mild temper makes them easy to handle
dogs
beagles and puppies under 1 year of age are most commonly used as they are small and docile
hearts and lungs similar to that of humans
suitable for transplantation experiments
birds
domestic fowl is the most common bird used
farmed poultry is one of the world’s biggest industries
experiments are mainly research into new feeding materials, diseases, and vaccines against them
agricultural chemicals and growth hormones
sheep
used to research human pregnancy
pregnancies only last 5 months
newborn lambs and babies weigh about the same
ideal for genetic engineering and cloning research
mice
more than 10 million mice are used in the US each year
more than 95% of animals used in the lab
most commonly used is teh albino house mice
used in studying human evolution
human genome studies
to mimic the human biological response
highly available, small size, low cost, ease of handling, fast reproduction rate
genome fully sequenced
more than 80% of their DNA is similar to humasn
good model to study gene functions by using gene knock-in or knock-out technology
widely used in studying diseases like diabetes, obesity, cancer, AIDS, alzheimer’s disease etc.
zebrafish
genome fully sequenced
lower cost and easier to handle than mice
high reproductive rate
short life-cycle
transparent embryo
used in regenerative medicine
they can regenerate their fins, skin, and even heart
used in genetic study
zebrafish have a short lifecycle of 90 days
their transparent embryos can easily be genetically engineered
effects of addition and deletion of genes can be observed