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, Cell culture
•Embryo
•Embryonated Eggs
because stage of differentiation)

Finely cut

Grow in media
-monolayer
Finely cut -suspension cells
tissue or explant

explant

organ
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 4°C for
up to 72hour.
 DISAGGREGATION
Enzymatic disaggregation
•Warm trypsin, 37˚C for 30 mins, cell damaged if too long
exposure.
•Cold pre exposure, soak at 4°C overnight and 37°C 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
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 (O2, CO2)
 a regulated physico-chemical environment (pH, osmotic
pressure, temperature)
MEDIA AND SERUM
 Common basal medium include
Eagle minimal effective
medium,Dulbecco’s 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 37°c 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 37°c 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 121°C 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-130°cis 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 BACTERIA YEAST FUNGI
FEATURE O0F
MICROIAL
CONTAMINATION

CHANGE IN PH PH DROP PH CHANGE WITH PH CHANGES

HEAVY SOME TIMES
INFECTIONS

CLOUDY MEDIUM SHIMMERING IN ROUND OR OVOID THIN

SPACES BETWEEN PARTICLES THAT FILAMENTOUS
CELLS;RODS OR BUDD OFF MYCELIA;SOMETI
COCCI MAY BE SMALLER MES CLUMPS OF
OBSERVED PARTICLES 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
Thank you