Growing Madin-Darby Canine
Kidney Cells for Studying
Epithelial Cell Biology
Epithelial cells display a structural and functional
polar organization (Simons and Fuller, 1985). In these
cells, the plasma membrane can be divided into two
distinct domains, the apical membrane and the basolateral
membrane, each containing different sets of
proteins. The apical membrane facing a secretory or
an absorptive lumen is delimited by a junctional
complex from the basolateral membrane. The tight
junction (zonula occludens) is the most apical member
of the complex. It is found at the intersection between
the apical and the lateral plasma membranes and
joins each cell to its neighbors, thus limiting the
diffusion of molecules between the luminal and the
serosal compartments (Gumbiner, 1987). This junction
also prevents the lateral diffusion of membrane proteins
from one domain to another, thus maintaining
their unique composition. Immediately basal to the
tight junctions is the intermediate junction (zonula
adherens or belt desmosomes). The other more
basal junctional elements are desmosomes (maculae
adherentes) and gap junctions, which attach the
lateral membranes of adjacent cells to each other.
The junctional complex is involved in sealing the
epithelium; it prevents molecules from diffusing
between adjacent cells. The basolateral membrane
faces the bloodstream and is involved in cellcell
contact and cell adhesion to the basement
For most studies on epithelial cell polarity, cultured
cells have been used. These cells are superior to cells obtained from tissues because they can be grown
under carefully controlled conditions and are easily
manipulated. The cell population is homogeneous.
Biosynthetic experiments using pulse-chase techniques
with radioactive precursors can be accomplished
at an analytical level with a short time
resolution. Endocytosis and transcytosis can also be
The most well-studied epithelial cell is the
Madin-Darby canine kidney (MDCK) cell. This cell
line is derived from normal dog kidney (McRoberts et
al., 1981). An unusual feature of these cells is that while
in culture they retain many differentiated properties
characteristic of kidney epithelial cells. Among these
are an asymmetric distribution of enzymes and vectorial
transport of sodium and water from the apical to
the basolateral faces. The latter gives rise to "domes"
or "blisters" in confluent cultures, which are transient
areas where collected fluid has forced the monolayer
to separate from the substratum. Morphologically,
the cells resemble a typical cuboidal epithelium with
microvilli on the apical side of the cells. Two different
strains of the MDCK cell are known (Richardson et al.,
1981; Balcarova-St/inder et al., 1984). Strain I cells are
derived from a low-passage MDCK cell stock
and these cells form a tight epithelium with transepithelial
resistance above 2000Ω.cm2
. Strain II cells
form a monolayer of lower resistance of 100-200Ω.
. MDCK strain II cells have been used primarily for
studies of the cell biology of epithelial cells. Transcytosis
is, however, studied more conveniently in
MDCK strain I cells because of their high electrical
Several factors are important for optimal expression
of the epithelial phenotype in vitro
(Simons and Fuller,
1985). A primary consideration is the polarity of nutrient
uptake. In vivo
, many nutrients reach the epithelial
sheet from the basolateral side, which faces the blood
supply; however, when epithelial cells are cultured on
glass or plastic, they are forced to feed from the
apical surface, which faces the culture medium. Hence,
the basolateral surface becomes isolated from the
growth medium as the monolayer is sealed by the formation
of tight junctions. To grow properly, the epithelial
sheet must remain somewhat leaky or expose
basolateral proteins responsible for the uptake of
nutrients and binding of growth factors on the apical
These problems can be overcome simply by
growing the epithelial cells on permeable supports,
such as polycarbonate and nitrocellulose filters.
Epithelial cells form monolayers with a higher degree
of differentiation when the basolateral surface is
directly accessible to the growth medium. This is
evident from the morphology of the cells, their
increased responsiveness to hormones, and the
exclusion of basolateral proteins from their apical
II. MATERIALS AND
Minimal essential medium with Earle's salt (MEM)
is purchased as a powder (Cat. No. 11700-077) from
Biochrom, mixed with Milli-Q-filtered H2
0, and sterile
filtered. Glutamine (200mM
, Cat. No. 25030-024),
penicillin (10,000 IU / ml)-streptomycin (10,000 mg/ml)
(Cat. No. 15140-122), trypsin (0.05%)-EDTA (0.02%)
(Cat. No. 25300-054), and phosphate-buffered saline
(PBS, Cat. No. 041-04040H) are from GIBCO-BRL. The
Transwell polycarbonate filters (2.45 cm, Cat. No. 3412,
and 10cm, Cat. No. 3419) are from Costar. Tissue culture
, Cat. No. 156499) are from Nunc. The
glass petri dishes (140mm diameter and 30mm high)
for holding six 2.4-cm Transwell filters are from Schott
Glasware. The laminar flow hood (Steril-Gard Hood
Model VMB-600) is from Baker. The CO2
(Model 3330) is from Forma Scientific. The inverted
Diavert microscope is from Leitz. The electrical resistance
measuring device (EVOM) is from World Precision
Instruments. The centrifuge (Type 440) is from
A. Growing Madin-Darby Canine Kidney
Cells on Plastic
MDCK I and II cells are passaged every 3-4 days up
to 25 passages. One flask is usually split into five new
flasks. MDCK II cells usually form domes within 2
days of splitting, whereas MDCK I cells do not blister.
- MEM growth medium:
|5% fetal calf serum
|10% fetal calf serum
|2 mM glutamine
|100 IU / ml penicillin
|100 / µg/ml streptomycin
- Phosphate-buffered saline
- 0.05% trypsin/O.02% EDTA
- Wash hands and wipe laminar flow hood with
70% ethanol. Warm all solutions to 37°C. All manipulations
are done in the laminar flow hood. When splitting
the cells, remove the growth medium from the
75-cm2 flasks containing the confluent layer of MDCK
cells and add 10ml PBS. Rinse and discard wash
- Add 5 ml trypsin-EDTA solution, seal flask, and
incubate for 10-15min at room temperature (until
small patches of cells are rounded up but not yet
detached from the flask).
- Remove the trypsin-EDTA solution and add
1.5ml of fresh trypsin-EDTA. Reseal the flask and
incubate at 37°C for 10-15 min (MDCK II) or 25-30 min
(MDCK I). At this point the cells should flow down to
the bottom of the flask when the flask is turned up. Hit
the flask hard against the palm of your hand.
- Add 10ml prewarmed MEM growth medium
and resuspend the cells with a sterile 10-ml pipette
(at least five times up and down). Using the inverted
microscope, check that the cells are not sticking to each
- Plate 2 ml of the cell suspension in a new 75-cm2 flask containing 20 ml of MEM growth medium.
B. Seeding MDCK Cells on Polycarbonate
- MEM growth medium containing 10% fetal calf serum,
penicillin-streptomycin, and 2 mM glutamine (see solution
- Phosphate-buffered saline
- 0.05% trypsin/O.02% EDTA
|FIGURE 1 (A) The glass petri dish contains one filter holder for
one Transwell 2419 filter. (B) The glass petri dish contains six filter
holders for Transwell 3412 filters.
C. Transepithelial Resistance Measurement
- Seed the cells on the filters at high density, higher
than that achieved by confluent cells on plastic. The
cells form tight junctions within 24h and reach
maximum tightness on the filters in 4 days. During this
time cell density increases to more than five times that
achieved on plastic. We place the filters in the petri
dishes containing growth medium. For seeding we use
one 75-ml flask, containing a confluent layer of MDCK
I and II cells, for 2.4-cm-diameter filters (Transwell
3412). If you use large 10-cm-diameter filters (Transwell
3419), seed one 75-cm2 culture flask of MDCK
cells into each large filter.
- Pour off medium from the culture flask and rinse
cells with 10 ml of warm PBS. Pour off PBS.
- Add 5 ml of warm trypsin-EDTA to cells. Leave
in laminar flow hood.
- After 15min remove the trypsin-EDTA with a
pipette, add 1.5 ml of trypsin-EDTA, and put the flask
into a CO2 incubator (37°C) for 10-15min (MDCK II)
or 25-30 min (MDCK I).
- Remove flask (cells should be loose). Hit the flask
hard against your palm. Add 10ml of warm growth
medium and suspend cells by pipetting up and down
with a 10-ml pipette. Put suspension into a 50-ml
Falcon tube and centrifuge for 5 min at 1000rpm in a
- Remove supernatant and suspend cells in 9.5 ml
of growth medium.
- Pour 90 ml medium into glass petri dish containing
six Transwell 3412 filters. Use 140ml for one Transwell
3419 filter. The petri dishes contain filter holders
specially made to fit either 3412 or 3419 filters (Fig. 1).
Autoclave these units before use. Place filters into filter
holders and allow filters to get wet from the bottom
with medium. This should be done while the cells are
in the centrifuge.
- Add 1.5 ml of cell suspension to each filter in its
holder. Use six Transwell 3412 filters or one Transwell
3419 filter per petri dish. Be careful not to spill cells
over the edge of the filter holder.
- Swirl petri dish gently to remove any trapped air
from beneath filters.
- Place the petri dish with the filters in the CO2 incubator.
- Leave for 3-4 days in the incubator. No medium
change is required during this time.
- Transepithelial resistance of filter-grown MDCK
cells is measured with EVOM "chop-stick" electrodes.
Each leaf has an outer and an inner electrode. The
outside electrodes are small silver pads for passing
current through the membrane sample. Inside the electrodes
are small Ag/AgC1 voltage sensors.
- To test the instrument, switch the mode switch to
R and turn the power on. Push the test R button. With
the range switch in the 2000-V position, the meter will
read 1000 (±1 digit). In the 20-k range, the meter will
read 1.00. The meter is now ready for use.
- To test the electrodes, insert the small telephonetype
plug at the end of the chopsticks electrode cable
into the jack on the front panel of the EVOM. Place the tips of the electrodes into 0.1M KCl. Switch the mode
switch to Volts. Turn the power switch on. The digital
panel meter may read I or 2 mV due to the asymmetry
of the voltage sensor pair. After 15min, adjust
this voltage to 0mV with the screwdriver adjustment
labeled "Zero V."
- Measure resistance. The electrode set is designed
to facilitate measurements of membrane voltage and
resistance of cultured epithelia in culture cups by
dipping one stick electrode inside the cup on top of the
cell layer and the second stick electrode in the external
bathing solution. To measure resistance, immerse the
electrode pair again into the electrolyte and set the
mode switch to "Ohm." The display should read zero;
if not, adjust the display to zero with the Ohms
Zero screwdriver adjustment. Push the measure R
button. A steady ohm reading of the resistance should
|a. Measure resistance
R from solution + sample
|b. Measure resistance R
from solution + membrane
support + tissue
|c. Subtract (a) from (b)
||189- 109- 80, R (tissue) - 80 V
|d. Calculate resistance
× area product
||resistance × area
= 1.2 cm × p × r2
= 80V × 3.14 × (1.2cm)2
= 361.9 V cm2
- When moving the electrodes from one dish to
another it is best not to rinse the electrodes with distilled
water. If it is necessary to wash the electrodes
between measurements, they should be rinsed with
the membrane perfusate (e.g., PBS). Do not touch the
cell layer with the internal electrode when making a
measurement. Small differences in the apparent fluid
resistance may occur if the depth to which the electrodes'
tips are immersed varies. If the tips are unusually
dirty, a light and very brief sanding with a fine
nonmetallic abrasive paper will clean the sensor tip.
For sterilization the electrodes may be soaked in
alcohol or bactericides. After sterilization, the electrodes
should be rinsed extensively with sterile perfusing
solution or 1 M KCl.
The cell layer on the filter cannot be observed in the
inverted microscope because the filters are not transparent.
Transparent filters are also available commercially'
but they are more expensive than the
polycarbonate ones; however, either the cells in one
filter can be stained or the transepithelial resistance
can be measured to ensure that the layer is intact. Our
experience is that when one filter in the petri dish
checks out, the other filters will also be fine.
It is recommended that MDCK cells not be used for
more than 20-25 passages. New stock cells should then
be thawed from liquid nitrogen storage.
We use filter holders for growing MDCK cells on
either 2.4- or 10-cm polycarbonate filters. It is possible
to grow cells in either the six-well plate for the Transwell
3412 filter or in the petri dish supplied with the
Transwell 3419 filters. Under these latter culture conditions,
growth media have to be changed every day,
as the cells do not get enough nutrients and do not
grow to optimal density. The problems with changing
the medium every day are (1) the extra work involved
and (2) the considerably increased risk of contamination.
Therefore, we prefer to place filter holders in petri
dishes into which one can add enough growth
medium to last 4 days.
Balcarova-Stander, J., Pfeiffer, S. E., Fuller, S. D., and Simons, K.
(1984). Development of cell surface polarity in the epithelial
Madin-Darby canine kidney (MDCK) cell line. EMBO J. 3
Gumbiner, B. (1987). Structure, biochemistry and assembly of epithelial
tight junctions. Am. J. Physiol. 253
McRoberts, J. A., Taub, M., and Saier, M. H., Jr. (1981). The Madin-
Darby canine kidney (MDCK) cell line. In "Functionally Differentiated
(G. Sato, ed.), pp. 117-139. A. R. Liss, New York.
Richardson, J. C. W., Scalera, V., and Simmons, N. L. (1981). Identification
of two strains of MDCK cells which resemble separate
nephron tubule segments. Biochim. Biophys. Acta 673
Simons, K., and Fuller, S. D. (1985). Cell surface polarity in epithelia.
Annu. Rev. Cell. Biol. 1