Preparation of Cilia from Human
Airway Epithelial Cells
For many years, the biochemical analysis of cilia,
and flagella has focused primarily on organisms that
are easy to grow and maintain in the laboratory and
from which axonemal structures can be isolated in
large quantities with a high degree of purity. For
example, the isolation of flagella from Chlamydomonas
in response to pH shock was described as early as 1972
(Witman et al., 1972) and cilia were isolated from Tetrahymena
using dibucaine in 1974 (Thompson et al.,
1974). While studies of these and other organisms have
provided a wealth of valuable information concerning
the structure and function of these fascinating
axonemal structures, it is clearly important to also
investigate the structure, function, and regulation of
mammalian, especially human, cilia and flagella.
While the axoneme of mammalian sperm has been isolated
by a number of techniques (e.g., San Agustin and
Witman, 1995), only a few reports detail isolations
of mammalian cilia from the airway. This probably
reflects in part the difficulty of obtaining sufficient
quantities of suitable starting material and the inherent
difficulties encountered when working with whole
In the last several years, the techniques used for
culturing airway epithelial cells from both animal and
human tissue have improved significantly. When cultured
at an air/liquid interface on a collagen matrix and
provided with suitable media, these cells have been
shown to undergo ciliogenesis in vitro
. In addition,
human cells can be expanded for one or two passages
and maintain their ability to differentiate, thus increasing
the number of ciliated cells obtained. By culturing
human airway epithelial cells under these conditions
and modifying a technique originally described by
Hastie et al.(1986) for isolating cilia from porcine
trachea, it is possible to routinely produce highly
enriched preparations of human ciliary axonemes.
Although these preparations are not completely free of
contamination by other cellular structures, they are
suitable for many biochemical studies (Zhang et al.,
2002; Reed et al., 2000; Ostrowski et al., 2002; Kultgen et
al., 2002). Because this procedure utilizes a detergent,
most of the ciliary membranes and soluble proteins are
removed during the isolation, and the recovered material
consists primarily of ciliary axonemes and their
attached structures (dynein arms, radial spokes, etc.).
Modifications of the procedure have been described
that allow for at least partial recovery of ciliary membranes
(Hastie et al., 1990; Salathe et al., 1993) from in
samples; these have not yet been tested extensively
in the in vitro
model. It should also be emphasized that
the starting material for this procedure is well-differentiated,
heavily ciliated airway cultures (Fig. 1A) grown
under the conditions described in detail by Bernacki
et al. (1999) and Randell et al. (2001) (and references
therein). If the cultures are not completely confluent
and heavily ciliated, the resulting preparation often
contains a significant amount of cellular debris. The use
of a nonionic detergent in the presence of calcium
causes the cilia to be removed from the cell at a point
just above the apical membrane, leaving the basal
bodies intact (Figs. 1B and 1C).
|FIGURE 1 Electron micrographs of ciliated cultures of human airway epithelial cells before (A) and after
(B and C) isolation of cilia. (A) A heavily ciliated culture after 6 weeks of culture. (B) A parallel culture fixed
immediately after removal of cilia. Note the intact basal bodies, which are shown at higher magnification in
C. Scale bar in A and B: 2.5 µm.
II. REAGENTS AND SOLUTIONS
Protease inhibitor cocktail (P8340), dithiothreitol
(DTT, D-9779), and Triton X-100 (T-8787) are from Sigma. β-Mercaptoethanol (0482) is from Amresco. The
protease inhibitor cocktail is thawed upon arrival and
frozen in small aliquots (100-200 µl). A 10% solution of
Triton X-100 is made in sterile distilled water and
stored in the refrigerator for no longer than 1 month.
DTT is conveniently frozen in small aliquots at
. All other chemicals are standard laboratory
reagents and can be obtained from most laboratory
- Deciliation buffer (DB): 10mM Tris-HCl (pH 7.5),
50 mM NaCl, 10 mM CaCl2, 1 mM EDTA, 0.1% Triton
X-100, 7mM β-mercaptoethanol, and 1% protease
inhibitor cocktail. Prepare a stock solution without
protease inhibitor, β-mercaptoethanol, or Triton X-100.
Sterile filter and store in the refrigerator. Before
starting the procedure, add to 4.9ml of the stock
solution 50µl of freshly thawed protease inhibitor
cocktail, 50 µl of 10% Triton X-100, and 2.45 µl of β-mercaptoethanol.
- Resuspension buffer (RB): 30mM HEPES (pH 7.3),
25mM NaCl, 5mM MgSO4, 1 mM EGTA, 0.1mM EDTA, 1 mM DTT, and 1% protease inhibitor cocktail.
Prepare a 10× stock solution without protease inhibitor
and DTT (300mM HEPES, 250mM NaCl, 50mM MgSO4,10mM EGTA, 1 mM EDTA), sterile filter, and
store in the refrigerator. Before starting the isolation
procedure, dilute an aliquot to 1X with sterile water.
To 4.9ml of diluted stock, add 50µl of freshly thawed
protease inhibitor cocktail and 50µl of freshly thawed
100 mM DTT.
As noted earlier, this procedure starts with heavily
ciliated cultures of human airway epithelial cells. Cilia can be isolated from four 30-mm Millicell-CM culture
inserts (PICM03050; Millipore) at one time. The inserts
are first transferred to a six-well plate for ease of
manipulation. Once started, the procedure should be
carried out quickly.
- Gently wash the mucus from the apical surface
of the culture with at least two rinses of 2 ml of chilled
phosphate-buffered saline (PBS). Add the PBS to the
surface of the culture, swirl gently, and aspirate. If the
cultures are producing large amounts of mucus,
perform additional washes until the visible mucus is
removed. After the last wash, carefully aspirate as
much fluid as possible from both apical and basolateral
sides of the insert.
- Add 300µl of cold DB to each insert.
- Cover the six-well plate and rock vigorously for
1 min. Tilt the plate from side to side so the solution
flows back and forth. Occasionally swirling the plate
so the solution washes around the edges helps remove
cilia from this area of the insert. Tilt the plate slightly
so that the solution collects on one edge and remove
with a pipetter. Pool the solution containing the cilia
from two inserts into one tube. Keep isolated cilia on
- Repeat steps 2 and 3. Pool the second collection
of cilia with the first. There should be 1.2 ml per microcentrifuge
- Pellet any cellular debris by centrifuging at 1000
RCF for 1 min at 4°C. Carry out the remaining steps in
a cold room.
- Collect the cilia containing supernatant into a
new tube. When starting with heavily ciliated cultures
that are washed well, there will be only a very small
pellet of debris, with some stringy material along the
sides of the tube. Try to remove the supernatant
without disturbing the pellet. We have found that spinning
at higher speeds or for longer times pellets cilia as well as debris. Depending on the goal of the experiment
(high purity or greatest recovery), the time of
this centrifugation may be adjusted.
- Pellet the ciliary axonemes by centrifugation at
12,000 RCF for 5 min at 4°C. After this centrifugation,
a compact white pellet should be obtained. Carefully
remove the supernatant and discard.
- Resuspend the ciliary axonemes in 0.5 ml of RB
buffer/insert (1.0ml per tube). The pellet does not go
into solution readily and must be pipetted gently to
disrupt. While a homogeneous solution is desired,
small clumps of material do not seem to affect the
- Optional: To remove ciliary membranes, add
50 µl of 10% Triton-X 100 (final concentration of 0.5%)
and incubate the solution on ice for 15min. Repeat
steps 7 and 8.
- Withdraw and spot 5µl of the solution onto
a slide, coverslip, and examine by phase-contrast
microscopy at 40× magnification. The axonemes
should be readily visible as individual strands, with
little cellular debris present (Fig. 2A).
- If a protein determination is desired, remove an
aliquot to a separate tube (typically 10%, or 100µl).
Pellet the cilia in both the sample and the protein
determination tube by centrifugation at 12,000 RCF for
5 min at 4°C.
- Remove as much of the supernatant as possible
and freeze the pellets at -80°C until needed.
|FIGURE 2 (A) Phase-contrast light micrograph of isolated cilia showing the appearance of the preparation
step 10 (original magnification, 400×). (B) Electron micrograph showing many intact ciliary
(C) Higher magnification of the same preparation as in B showing details of the isolated
Note the presence of radial spokes and dynein arms. Scale bars: 1.1 µm (B) and 0.15µm (C).
- Protein concentration is determined using the
BCA protein assay kit (Pierce, Rockford, IL). To solublize the axonemes, the pellet is dissolved in 0.5%
SDS before performing the assay. The protein values
obtained provide estimates of how much protein is
in the cilia pellet. Although the amount of ciliary
axonemes recovered varies with the degree of ciliated
cell differentiation, each insert typically yields about
50 µg of protein.
- The microscopic examination of the preparation
in step 10 provides a simple and rapid qualitative estimate
of the purity of the axonemes recovered.
- Cilia isolated by this procedure can be reactivated
by the addition of ATP to the cilia suspension
obtained after step 10. ATP is added to an aliquot of
the cilia and the reactivation is observed by phasecontrast
microscopy (Hastie et al., 1986).
- For detailed analysis, the pelleted axonemes
can be fixed in 2% formaldehyde/2% glutaraldehyde
with 0.5% tannic acid (Hayat, 1989) and examined by
transmission electron microscopy. An example of a
routine preparation of isolated axonemes examined by
electron microscopy is shown in Figs. 2B and 2C.
- A problem sometimes encountered has been the
fragmentation of the axoneme into individual microtubules.
While the cause of this phenomena has not
been identified, proteolytic degradation or the use of
old or improperly made solutions seems to be a likely
cause. When this is observed, fresh solutions should be
prepared and the procedure repeated to verify that
intact axonemes are obtained.
This work was funded in part by NIH Grant
HL63103 from the National Heart, Lung, and Blood
Institute. The author thanks Dr. Scott Randell and the members of the Cell and Tissue Core Facility for
helpful discussions and providing the human airway
epithelial cells, Kim Burns and the members of the Histology
Core Facility for providing electron microscopy
services, Kerri Kendrick for preparing the illustrations,
and Dr. William Reed for helpful suggestions concerning
the cilia isolation protocol.
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