PARUL INSTITUTE OF PHARMACY
cell culture is the complex process by which
cells are grown under controlled conditions,
generally outside of their natural
By this process prokaryotic,eukaryotic or
plant cells are grown under controlled
condition but in practice it refers to the
culturing of cells derived from animal cells.
Cell culture was first successfully undertaken
by Ross harrison in 1907
In 1885 Roux first time maintained
embryonic chick cells in cell culture.
1911: Lewis and Lewis made the first liquid
media consisted of sea water, serum, embryo
extract, salts and peptones. They observed
limited monolayer growth
1913: Carrel introduced strict aseptic
techniques so that cells could be cultured
for long periods.
1952: Gey established a continuous cell line
from a human cervical carcinoma known as
HeLa (Helen Lane) cells.
1927: Carrel and Rivera produced the first
viral vaccine - Vaccinia.
1933: Gey developed the roller tube
1982: Human insulin became the first
recombinant protein to be licensed as a
1985: Human growth hormone produced
from recombinant bacteria was accepted for
1987: Tissue-type plasminogen activator
(tPA) from recombinant animal cells became
First development was use of antibiotics
which inhibits the growth of contaminants.
Second was the use of trypsin to remove
adherent cells to subculture further from the
Third was the use of chemically defined
MODEL SYSTEM: for studying basic cell
biology, interaction between disease causing
agent and cells, effect of drugs on cells,
process and triggering of aging & nutritional
TOXICITY TESTING: in study of effect of new
CANCER RESEARCH: study function of various
chemical, virus & radiation to convert normal
culture cell to cancerous cells.
VIROLOGY: cultivation of virus for vaccine
GENETIC ENGGINERING: production of
commercial protein, large scale production of
viruses for use in vaccine production.
GENE THERAPY: cells having a functional gene
Can be replaced to cell which are having
3 Types of cell cultures
1)Primary cell culture: This is the maintenance of
growth of cells dissociated from the parental tissue
(such as kidney or liver) using mechanical or
enzymatic methods, in culture medium using
suitable glass or plastic containers. The primary
cell culture could be of two types depending upon
the kind of cells in culture.
Adherent cells - Cells shown to require attachment
for growth are said to be anchorage dependent
cells. The adherent cells are usually derived from
tissues of organs such as kidney where they are
immobile and embedded in connective tissue.
Suspension cells - Cells which do not require attachment for
growth or do not attach to the surface of the culture vessels
are anchorage independent cells/suspension cells. All
suspension cultures are derived from cells of the blood
system because these cells are also suspended in plasma in
vitro e.g. lymphocytes.
2)Secondary cell cultures: When a primary culture is subcultured, it becomes known as secondary culture or cell line.
Subculture (or passage) refers to the transfer of cells from
one culture vessel to another culture vessel. This is
periodically required to provide fresh nutrients and growing
space for continuously growing cell lines. The process
involves removing the growth media and disassociating the
adhered cells (usually enzymatically). Such cultures may be
called secondary cultures.
3)Cell Line: A cell line or cell strain may be finite or
continuous depending upon whether it has limited culture life
span or it is immortal in culture. On the basis of the life span
of culture, the cell lines are categorized into two types:
Finite cell lines - The cell lines which have a limited life
span and go through a limited number of cell generations
(usually 20-80 population doublings) are known as finite cell
lines. These cell lines exhibit the property of contact
inhibition, density limitation and anchorage dependence. The
growth rate is slow and doubling time is around 24-96 hours.
Continuous cell lines - Cell lines transformed under
laboratory conditions or in vitro culture conditions give rise to
continuous cell lines. These cell lines show the property of
ploidy (aneupliody or heteroploidy), absence of contact
inhibition and anchorage dependence. They grow either in a
monolayer or in suspension (see below). The growth rate is
rapid and doubling time can be 12-24 hours.
Monolayer cultures - When the bottom of the culture
vessel is covered with a continuous layer of cells, usually one
cell in thickness, they are referred to as monolayer cultures.
Suspension cultures - Majority of continuous
cell lines grow as monolayers. Some of the
cells which are non-adhesive e.g. cells of
leukemia or certain cells which can be
mechanically kept in suspension, can be
propagated in suspension.
HUMAN CELL LINES:
DU145 (prostate cancer)
THP-1 (acute myeloid leukemia)
MCF-7 (breast cancer)
Saos-2 cells (bone cancer)
PC3 (prostate cancer)
o PLANT CELL LINES:
Tobacco BY-2 cell
o Rat tumor cell lines:
GH3 (pituitary tumor)
o Mouse cell lines:
MC3T3 (embryonic calvarium)
Choice of media depends on the type of cell
Commonly used Medium are GMEM,
Media is supplemented with antibiotics viz.
penicillin, streptomycin etc.
Prepared media is filtered and incubated at 4
Laminar cabinet-Vertical are preferable
Incubation facilities- Temperature of 25-30 C
for insect & 37 C for mammalian cells, co2 25% & 95% air at 99% relative humidity. To
prevent cell death incubators set to cut out at
approx. 38.5 C
Refrigerators- Liquid media kept at 4 C,
enzymes (e.g. trypsin) & media components
(e.g. glutamine & serum) at -20 C
Microscope- An inverted microscope with
10x to 100x magnification
Tissue culture ware- Culture plastic ware
treated by polystyrene
Possibly keep cultures free of antibiotics in order
to be able to recognize the contamination
Never use the same media bottle for different cell
lines. If caps are dropped or bottles touched
unconditionally touched, replace them with new
Necks of glass bottles prefer heat at least for 60
secs at a temperature of 200 C
Switch on the laminar flow cabinet 20 mts prior
to start working
Cell cultures which are frequently used should be
subcultered & stored as duplicate strains.
Use actively growing cells that are in their log
phase of growth, which are 80-90% viable
Keep exposure to trypsin at a minimum
Handle the cells gently. Do not centrifuge
cells at high speed or roughly re-suspend the
Feeding & sub culturing the cells at more
frequent intervals then used with serum
containing conditions may be necessary
A lower concentration of 104cells/ml to
initiate subculture of rapidly growing cells & a
higher concentration of 105cells/mlfor
slowing growing cells
Several cultured epithelial cell lines of human or animal
origin can be used as in vitro drug absorption models to
predict the absorption of compounds across intestinal
tissue in vivo.
The Caco-2 human colonic cell line is perhaps the most
widely used cell line for this purpose. .
Caco-2 cells adopt a polarized morphology and express
many intestinal transport proteins and other enzymes
characteristic of epithelial cells of the small intestine.
Absorption Systems offers:
1)In vitro assays for absorption across human or animal
2)Oral bioavailability studies in rodents, dogs, pigs and
3)Intestinal loop perfusion studies in rats
The Caco-2 cell line is an immortalized line of
heterogeneous human epithelial colorectal
Caco-2 cell monolayers spontaneously
differentiate to express morphological and
functional characteristics of mature smallintestinal enterocytes. The differentiated
monolayers are polarized, with microvilli on the
it is developed by the Sloan-Kettering Institute
Spontaneously differentiate to express
morphological (polarized columnar cells)
and functional characteristics of mature
Four times higher in transepithelial
resistance compared to HT 29-cell
It expresses various drug metabolizing
enzymes like aminopeptidase, esterase, and
Tissue in the villus contains more than one
Dose not produce the mucus and unstirred
water Observed in the intestine
No P-450 drug metabolizing enzyme activity
has been reported
Time consuming as 21 days required for full
The necessity of LC / MS or HPLC for
Influence of P-gp is difficult to estimate.
A. Trans Epithelial Electrical Resistance (TEER)
Measurement of the integrity of the caco-2 cell
After washing the cell monolayer with 37°C
tempered D-PBS (with Ca2+, Mg2+) (Dulbecco's
Phosphate Buffered Saline), 1600 μl transport
medium was added into the apical and 2800 μl
transport medium was added into the basal
Allow for equilibrium for 60 min in the cell culture
4. The measurement chamber was tempered to
37°C with transport medium before the
measurement. For the post-experimental TEER
measurement, the withdrawn volume in the apical
compartment was replaced with transport medium
before TEER was measured.
5.Caco-2 monolayer with TEER values exceeding
250 Ωcm2 were used for transport experiments.
6. Background TEER may be recorded in wells
without cell monolayers, and can be subtracted
from the raw TEER values with cells.
Rinse the monolayer three times with 300 µL HBSS ( Hank’s
buffer saline solution) in the apical wells and 28 mL in the
Add 300 µL of LY solution to each well in the filter plate
Add 600 µL HBSS to each well of a 24-well receiver tray
Assemble the filter plate and 24-well receiver plate and
incubate for 1–2 hours at 37°C.
Remove the filter plate from the receiver plate and place the
receiver plate into a fluorescent plate reader. Determine the LY
fluorescence using an excitation wavelength of 425-430 nm
and an emission wavelength of 515-520 nm, 540 nm.
Calculate the percent of LY rejection across the cell
monolayer by measuring fluorescence in the receiver plate as
compared to an ‘equilibrium’ standard.
The standard plate should consist of 4 wells with 600 µl HBSS
(blank) and 4 wells with 200 µl LY (100 µg/ml) + 400 µl
HBSS (equilibrium samples)
calculate the LY rejection using the following equation:
LY rejection=100%-%ly passage
Caco-2 human colon adenocarcinoma cells are grown to confluence and
differentiated for three weeks on filters. Test agent is added to one side of
the monolayer, and permeability is assessed by analyzing the concentration
of the test agent on the other side of the monolayer using LC-MS/MS.
The following Caco-2 assays are available:
Monodirectional: High permeability predicts good human oral
Bidirectional: High efflux ratio indicates possible P-gp or other transporter
Bi-directional (P-gp substrate determination): Test agent is incubated on
either side of the monolayer in the presence and absence of a known P-gp
Bi-directional (P-gp inhibitor determination): Test agent is incubated on
either side of the monolayer in the presence and absence of a known P-gp
Bi-directional (Other transporters): it require regarding the testing of your
test agents to be substrates or inhibitors of other efflux transporters (e.g.
BCRP, MRP1, MRP2, etc.).
After the desired cell growth period, remove the plate from the
incubator and determine the electrical resistance for each well
(as described above). Wash the monolayer, exchanging the
volume three times using sterile HBSS, pH 7.4. After washing,
remove the buffer from the filter plate and feeder tray.
Transfer the filter plate to a 24-well transport analysis plate.
To determine the rate of drug transport in the apical to
basolateral direction, add 300 µL of the test compounds to the
filter well. Drug concentrations typically ranging from 10 µm
to 200 µm may be used (achieve desired concentration using
HBSS, pH 7.4 or an alternative buffer of desired pH).
Fill the wells of the 24-well receiver plate with 600 µL buffer.
To determine the rate of drug transport in the basolateral to
apical direction, add 600 µL of the test compounds to the 24well receiver plate.
Fill the filter wells (apical compartment) with 300 µL of
Join the filter and receiver plates once all drugs and buffer
have been added. Begin timing the experiment.
Incubate at 37°C shaking at 60 rpm on a rotary shaker. Typical
incubation times are 1 to 2 hours.
CALCULATION OF APPEARENT
Area * time
VA= volume in ml in the acceptor well
area =the surface area of the membrane
time= total transport time in seconds
1) In Drug Discovery: To test the absorption profiles of
the new molecular entities in the lead optimization state.
2) In pre-clinical drug development:
US FDA recognizes Caco-2 to measure permeability
as part of the bioequivalence waiver process.
3) To evaluate effect of pharmaceutical excipients.
4) To study transport mechanism from many compounds
5) In drug metabolism & toxicity effects.
6) Others like study of CFTR, regulation of protein
expression; genetics study.
PAMPA & caco-2 should not be considered as
competing permeability methods.
Good correlation between PAMPA & caco-2 data for
a compound indicates a predominance of passive
diffusion in its permeation.
Lack of correlation indicates
absorptive (active, paracellular ,gradient effect for
ecretarys (efflux, gradient effect for bases)
one of the commonly used cell monolayer systems to assess
the human intestine barrier.
MDCK cell lines can reach full differentiation in 3-7days and
are therefore relatively easy for cell culturing and assay
MDCK cell lines originate from dog kidney.
The expression of transporters is quite different from human