CELL CULTURING

Girija Maganti
M.Pharm
(pharmacology)
What is Cell Culture?

Cell culture refers to the removal of cells
from an animal or plant and their subsequent
growth in a favorable artificial environment.

Mouse, mammals,
Embryo
Embryonated Eggs
because stage of differentiation)
organ
explant
Grow in media
-monolayer
-suspension cells
Cell culture
Finely cut
Finely cut
tissue or explant
Enzymic digestion
STAGES OF CULTURE
Isolated tissue

(disaggregation)

Primary cell culture(limited lifespan after certain
proliferations undergo senescence)


Finite cell cultures


continous cell lines(immortalized cell line acquires ability to
proliferate indefinitely by transformation)


Growth Curve


ISOLATION OF TISSUES

Sterilize the site with 70% alcohol.
Remove tissue aseptically.
Transfer to the laboratory in transport medium
If delay in transporting to lab, keep at 4C for
up to 72hour.

Enzymatic disaggregation
Warm trypsin, 37C for 30 mins, cell damaged if too long
exposure.
Cold pre exposure, soak at 4C overnight and 37C for less 30
mins. Advantage: higher yield of viable cells, preserve
more cell types
Other enzyme
-collagenase benefit for connective tissues and muscle
(fibrous tissue)
- pronase, dipase, DNase, hyaluronidase
Mechanical disaggregation (prevent proteolytic
damage)
Scrapping or spillage
Sieving
Syringes
Trituration by pipette

DISAGGREGATION
CULTURE CONDITIONS
Culture conditions vary widely for each cell type.
a substrate or medium that supplies the essential
nutrients (amino acids, carbohydrates, vitamins,
minerals)
growth factors
hormones
gases (O
2
, CO
2
)
a regulated physico-chemical environment (pH, osmotic
pressure, temperature)

MEDIA AND SERUM
Common basal medium include
Eagle minimal effective
medium,Dulbeccos Modified
eagle medium,RPMI 1640,Ham
f10.
The above medium includes
aminoacids,glucose,salts,vitam
ins and nutrients.
L-glutamate,serum,antibiotics,
fungicides are added for complete
medium.

Serum may or may not be used
Most frequently used serum-fetal calf serum
Less expensive sera such as horse or calf sera
can be used.
Serum supports growth of cell.
Antibiotics and fungicides prevent microbial
contamination.
Some cell types like primary cell requrie
additional supliments(collagen
,fibronectin,epidermal growth factor)to attach
culture vessel and proliferate.
Sterility of the medium is tested by incubating
small aliquot at 37c for 48 hours,ifany microial
growth the medium should be discarded.
CULTURE VESSELS
MODES OF PROPAGATION
Depending on their origin, animal cells grow
either as adherent monolayers or in
suspension.
Adherent cells are anchorage dependent and
propagate as monolayer attached to the cell
culture vessel.
Eg:cells derived from tissues
Suspension cells:proliferate without being
attached to a substratum.
Eg:haemopoietic cell,cells derived from
tumours




PRIMARY CULTURE

Capable of only limited number of cell divisions
cells that are placed in culture directly from the
tissue of origin.
these are called primary cultures until the first
subculture


Epithelial cell

TYPES OF PRIMARY CELL CULTURE

Mouse embryos
Chick embryos
Human biopsy materials
Transplantable animal tumour
Chick embryo organ rudiments ( brain,
heart, lungs, liver, kidney, spinal cord, skin,)




SUBCULTURING
Subculturing or "splitting cells," is required to
periodically provide fresh nutrients and growing
space for continuously growing cell lines.
The frequency of subculture and the split ratio,
or density of cells plated depend on the
characteristics of each cell line being carried.
Sub culturing -
Adherent Cells
Suspension culture.

CONTINOUS CELL LINES

After the first subculture, primary culture may
be called secondary cultures, and thereafter, if
continued passage is possible, a continous cell
line are formed.
An established or immortalised cell line has
acquired the ability to proliferate indefinitely
either through random mutation or deliberate
modification, such as artificial expression of the
telomerase gene.

EXAMPLES OF ESTABLISHED CELL
LINES
May be derived from Normal or Tumor cells.

Cell line Organism Origin Tissue
HeLa Human Cervical cancer
293-T Human
Kidney (embryonic)
A-549
Human Lung carcinoma
ALC
Murine Bone marrow
CHO
Hamster Ovary
HB54
Hybridoma Hybridoma
FM3 Human
Metastatic lymph node
Mouse kidney cells
Secondary Hamster
kidney cells
Primary cell cultures
split several times:

INCUBATION CONDITIONS
Cell cultures should incubated in incubators
with tightly regulated temperatures(eg:water
jacketed incubator)and co2 con.
Most cell lines grows at 37c and 5%co2 with
saturating humidity.
STERILIZATION OF MEDIUM
The various media constituents and other reagents
used in cell cultures must be carefully sterilized
either by autoclaving or by filtration. Heat stable
constituents tike water, salts, supplements like
peptone or tryptase etc. are autoclaved at 121C for
20 min.

But heat labile constituents like serum, trypsin,
proteins, growth factors etc. must be sterilized by
filtration through a 0.2 mm porosity membrane
filter. Each filtrate should be tested for sterility to
avoid failure due to contamination.

.
CRYOPRESERVATION
Freeze preservation of animal cells
is now routine in all cell line banks.

A cryoprotective agent like DMSO
or glycerol (at-130cis generally
added to minimize injury to cells
during freezing and thawing.

Frozen ampoules are generally
stored in liquid nitrogen
refrigerators which are rather
convenient and quite safe.

CELL THAWING
preserved culture
collection will arrive
frozen and in order
to use the cells they
must be thawed and
put into culture
Contamination
Contamination with other cell lines
(cross contamination)
Yeast
Fungi
Viruses
Bacteria
mycoplasma


CHARACTERISTIC
FEATURE O0F
MICROIAL
CONTAMINATION
BACTERIA YEAST FUNGI
CHANGE IN PH PH DROP PH CHANGE WITH
HEAVY
INFECTIONS
PH CHANGES
SOME TIMES
CLOUDY MEDIUM SHIMMERING IN
SPACES BETWEEN
CELLS;RODS OR
COCCI MAY BE
OBSERVED
ROUND OR OVOID
PARTICLES THAT
BUDD OFF
SMALLER
PARTICLES
THIN
FILAMENTOUS
MYCELIA;SOMETI
MES CLUMPS OF
SPORES
Cross contamination
APPLICATIONS
Model Systems : Cell cultures provide a good model
system for studying 1) basic cell biology and
biochemistry, 2) the interactions between disease-
causing agents and cells, 3) the effects of drugs on
cells, 4) the process and triggers for aging, and 5)
nutritional studies
Drug screening and development
Toxicity testing
Cancer research
Virology
Largescale production of monoclonal
antibodies and vaccines
Genetic counselling
Gene therapy