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Morphogenetic clonal growth of kidney epithelial cell line MDCK.

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THE ANATOMICAL RECORD 217:229-239 (1987)
Morphogenetic Clonal Growth of Kidney Epithelia1
Cell Line MDCK
JAMES A. McATEER, ANDREW P. EVAN, AND KENNETH D.GARDNER
Department of Anatomy, Indiana University School of Medicine, Indianapolis, IN 46223
(J.A.M., A.P.E.);Department of Medicine, University of New Mexico School of Medicine,
Albuquerque, NM 87106 (K.D.G.)
ABSTRACT
MDCK (Madin-Darby canine kidney) cells were cultured either 1)
dispersed within hydrated collagen gel (HCG) or 2) seeded atop a collagen substrate
and then immediately overlaid with HCG. Individual cells exhibited clonal growth
in three dimensions to form spherical cysts made up of a simple epithelium enclosing
a fluid-filled lumen. The cells of MDCK cysts were polarized with the basolateral
surface in contact with the collagen gel and the apical surface bordering the lumen.
The ultrastructure of MDCK cysts showed similarities to distal nephron. The cells
bore apical microvilli and solitary cilia and had occluding junctions and a simple
basolateral surface. MDCK cysts increased in size (> 800 pm diameter) with continued culture. MDCK cysts grown between layers of HCG were stripped free of the
overlying collagen to give direct access to basolateral surface membrane. Unlike
monolayer culture, morphogenetic clonal growth of cell line MDCK produces a
polarized cell population with a true lumenal and basolateral surface. Collagen-gelcultured MDCK cysts provide a n easily manipulable in uitro cell system that may
offer unique advantages for the study of renal cell structure and function.
The kidney epithelial cell line MDCK (Madin-Darby
canine kidney) (Madin and Darby, 1975) retains in culture many of the differentiated characteristics of a cell
of the distal nephron (Rindler et al., 1979; Taub and
Saier, 1979). The presumed distal nephron origin of the
MDCK cell is suggested by its ultrastructure (Valentich,
1981)and pattern of hormone responsiveness (Rindler et
al., 1979) and by the expression of a basolateral cell
surface antigen that is specific to thin ascending limb
and distal convoluted tubule in the dog (Herzlinger et
al., 1982). In addition, monoclonal antibodies raised
against MDCK cells have been localized to cortical and
medullary collecting duct of the dog (Garcia-Perez and
Smith, 1983).Although certain morphological characteristics of the MDCK cell have been shown to be dependent upon culture conditions (Valentich, 1981), in
monolayer, the cells are cuboidal, bear apical microvilli
and solitary cilia, produce basal lamina, and have tight
junctions (Cereijido et al., 1978; Valentich, 1981).
The physiology of the MDCK cell makes it a valuable
model for studies of vectorial fluid and ion transport
(Misfeldt et al., 1976). When grown to confluency on a
permeable substrate, the cells produce a transepithelial
electrical resistance (Cereijido, 1984). When raised on
a n impermeable substrate, MDCK cells develop multicellular domes (hemicysts or blisters) (Leighton et al.,
1969; Cereijido et al., 1981). Domes form when fluid
transported from apical to basolateral surface accumulates beneath the cell sheet, lifting region of the intact
cell layer away from the culture substrate (Leighton et
al., 1969).Domes are transient structures that may form
and then collapse (Leighton et al., 1969; Cereijido et al.,
1981).It has been shown that dome formation by MDCK
0 1987 ALAN R. LISS, INC.
cells is dependent upon active Naf transport (Abaza et
al., 1977; Lever, 1979a) and is influenced by a variety of
factors including CAMP(Lever, 1979a; Valentich et al.,
1979), various inducers of mammalian cell differentiation (Lever, 1979a,b), and cell-substrate interactions (Rabit0 et al., 1980).
Besides its role as a model for fluid transport by a
renal cell type, MDCK has also been used to study
aspects of cell-matrix interactions. Hall et al. (1982) observed that MDCK cells cultured in monolayer atop a
collagen membrane exhibit a “morphogenetic” response
when overlaid with collagen gel. When confronted with
collagen on both basolateral and apical surfaces the cell
sheet reorganizes to form a collection of tubulelike epithelial elements, each with the apical cell surface facing
a n enclosed lumen. This observation complements work
performed on nonrenal epithelial cell types in which
mouse mammary epithelium (Bennett, 1980; Bennett et
al., 1981; Yang et al., 1979, 1980) and hog thyroid cells
(Chambard et al., 1981)have also been shown to exhibit
histotypic reorganization and growth when cultured
within collagen gel. Such culture methods, which employ collagen gel a s the substrate, are a n alternative to
monolayer culture and provide a means to study the
effects of a three-dimensional extracellular matrix on
cell behavior.
In this report we describe the formation of polarized
epithelial cysts by MDCK cells suspended within collagen gel. Cyst formation under these culture conditions
occurs by the successive division (clonal growth) of isoReceived June 24, 1985; accepted August 12, 1986.
230
J.A. McATEER, A.P. EVAN, AND K.D. GARDNER
lated single cells. A distinct advantage of this system is
the nature of the collagen gel substrate, which is easily
manipulated to provide direct physical access to the
cultured cell population. Morphogenetic clonal growth
of MDCK cells within collagen gel may provide a novel
means to model the influence of the kidney interstitium
on the growth, structure, and function of the renal tubular epithelium.
MATERIALS AND METHODS
Culture of MDCK Cells Suspended Within Collagen Gel
MDCK cells (CCL 34, passage 53) obtained from the
American Type Culture Collection (Rockville, MD) were
subcultured two or more times at subconfluency in
serum-free medium patterned after medium K-1 of Taub
and Sat0 (Taub and Sato, 1979). Culture medium was a
1:l mixture of nutrient medium F12 and DMEM (KC
Biological, Lenexa, KS) containing 2.0 mg/ml sodium
bicarbonate, 10 mM HEPES, 10 nM sodium selenite,
0.1% BSA, and 100 U/ml penicillin-G and supplemented
with bovine insulin, 5 pg/ml, hydrocortisone 10-7 M,
PGE1, 25 ng/ml, and T3, 5 x 10-l2 M (all from Sigma
Chemical Co., St. Louis, MO). The cells were trypsinized
and then washed twice in medium containing 0.1% soybean trypsin inhibitor (Boehringer-Mannheim, Indianapolis, IN). Cells were resuspended in culture medium
to give 2.5 x 105 cells/ml and then gently triturated
through a n 18-gauge cannula and filtered (McAteer and
Cavanagh, 1983) through 20-pm Nitex screen (Tetko
Inc., Elmsford, NY). The dispersed cell suspension (4.3
ml) was added to a chilled collagen-medium solution.
The collagen-medium solution was prepared by combining 1 ml medium FlBK(1OX) (from CaCl2-deleted powder, special order, KC Biological, Lenexa, KS), 0.5 ml
5% NaHC03, 1ml 0.1 M HEPES, 100 p1 0.2 M CaC12,
and 100 pl 1 N NaOH with 3 ml of collagen stock,
prepared from rat tail tendon (McAteer and Cavanagh,
1982).Collagen gels were plated in 35-mm dishes (1ml),
placed in a n incubator (5%CO2 in air, 37°C) and allowed
to solidify (5 minutes). Each gel was then overlaid with
1.5 ml of culture medium that was replaced every other
day.
Cultures were also established from MDCK cells raised
in medium without supplemental hormones but containing 5%fetal bovine serum (KC Biological, Lenexa, KS).
Cultures Seeded Between Layers of Collagen Gel
Cultures were also established by seeding MDCK cells
between layers of collagen gel. Culture dishes were prepared with a solidified base layer of collagen gel. Cells
(2 x 105)were planted and allowed to settle (5 minutes);
then the medium was removed. The cells were overlaid
with collagen gel, which was allowed to solidify (as
above) before additional medium was added. This
method was used to prepare cultures to be viewed on a
daily basis for documentation of clonal growth. Cultures
were plated within Bionique chamber dishes (Corning,
Corning, NY) assembled with a glass coverslip. The
plastic lid of the chamber was replaced with a sterile
glass plate prior to photomicrography by using differential interference contrast (DIC) optics.
Preparation of Electron Microscopy
Cultures were fixed in a mixture containing 1.25%
glutaraldehyde and 1%OSOQin 0.1 M cacodylate-HC1
buffer (pH 7.41, en bloc stained in 0.25% uranyl acetate
in 0.1 M sodium acetate buffer (pH 6.3), and dehydrated
in a graded series of ethanols. Selected cultures were
fixed in the presence of ruthenium red (5 pg/ml). Specimens for transmission electron microscopy were embedded in Epon 812. Thin sections were double-stained with
uranyl acetate and lead citrate.
For scanning electron microscopy, intact cultures were
cryofractured from liquid nitrogen-frozen absolute
ethanol (Humphreys et al., 1974). Cultures established
by seeding cells between layers of HCG were fixed (as
above) after removing the upper collagen layer by using
fine forceps. This exposed MDCK cyst basolateral membranes for direct observation. MDCK cysts were also
isolated from living cultures by digestion of the collagen
matrix with 0.1% collagenase (type IV Worthington,
Millipore Corp., Freehold, NJ). Isolated MDCK cysts
were processed by using carriers fashioned from Nitex
(Tetko, Elmsford, NY) screen (McAteer et al., 1983).
Specimens for SEM were critical-point dried (Anderson,
19511, coated with gold-palladium, and viewed by using
a n AMR lOOOA scanning electron microscope.
Radioautography
MDCK cyst cultures at day 15 were incubated for 24
hours in medium containing 1 pCi/ml [meth~l-~H-]-thymidine (specific activity 20.0 Ci/mM) (New England Nuclear, Boston, MA). The cultures were fixed in 2.5%
glutaraldehyde in 0.1 M cacodylate/HCl, pH 7.4, dehydrated in graded ethanols, and embedded in JB-4 resin
(Polysciences, Warrington, PA). Sections were cut a t approximately 3 pm and placed on glass slides. Slides were
dipped in Kodak NTB-2 nuclear track emulsion (Kodak
Co., Rochester, NY) and then stored in light-tight boxes
containing CaS04. Slide boxes were placed in a refrigerator a t 8°C for exposure periods of 2, 4, or 6 weeks.
Following development in D-19, the tissue sections were
counterstained with 0.03% basic fuchsin. Nuclei were
considered to be positive for 3H-thymidine uptake when
they possessed 4~ the number of grains observed over
the background.
RESULTS
Clonal Growth Within HCG
MDCK cells seeded within collagen gel formed spherical, multicellular, cystlike structures (Figs. 1-4) in
which a simple cuboidal epithelium surrounded a central lumen (Figs. 2-4, 5). The epithelium was polarized,
with the cell apex bordering the lumen (Figs. 2,5).
MDCK cysts developed by 72-96 hours following seeding. Initially, cysts were small (< 20 pm) but increased
greatly in size ( > 800 pm) with continued culture. Cyst
formation was not synchronous and cysts did not appear
to increase in size a t the same rate.
During this study we recorded multiple selected fields,
using the inverted photomicroscope, at 12 hour intervals
throughout the first 10 days of culture. Cells within a
given field did not move from original position and prior
to cyst formation remained rounded in form. Only single
isolated cells appeared within the field (Fig. 3A,B; 2
days). We observed no cells with elongated or irregular
shape that would suggest movement through the collagen gel. Cells sumended within collagen pel did not
aggregate. MDCK kysts appeared, therifore: to form as
the result of the division (clonal growth) of isolated individual cells. The series of light micrographs displayed
v
v
MORPHOGENETIC CLONAL GROWTH OF MDCK CELLS
231
Fig. 1. Scanning electron micrograph of collagen-gelcultured MDCK cysts 12 days following
seeding. This culture was established by mixing a monodisperse cell suspension within fluid
collagen prior to plating. Numerous cysts (arrows) are exposed at the fractured surface of the
collagen gel (HCG). ~ 4 7 .
in Figure 3 shows two different fields (Fig. 3A, B) recorded at successive time points over a 10-day period.
The first frame of each set, recorded at day 2, shows
only single cells within the field. Subsequent frames
recorded at days 4 and 10 show the formation of a t least
one cyst per field derived from a single cell. These micrographs show the development of cysts from nonmigratory, isolated single cells, with no evidence of the recruitment of cells from the surrounding interstitium. In
addition, this series of photographs demonstrates that
not all isolated cells were capable of cyst formation.
Moreover, these frames show that MDCK cells initiated
cyst formation at different times following seeding and
that cysts grew in diameter at different rates.
The subsequent growth of MDCK cysts appeared to
occur by cell division and not by the addition of cells
from the interstitium. To test this, cysts were harvested
from the collagen substrate by treatment with collagenase. Cultures included those in the initial period of cyst
formation (72-96 hours) and older cultures of up to 1421 days. The population of cysts was washed free of
single cells; then individual cysts, some less than 20 pm
in diameter, were collected by using a micropipette.
These isolated cysts were replanted within collagen gel
that was free of dissociated cells. We observed that replanted cysts continued to grow in diameter and often
grew to be several hundred micrometers in diameter.
Such replanted cysts, especially those of larger diameter, often appeared to accumulate a modest number of
cells within the lumen. These cells were likely sloughed
from the cyst wall, possibly in response to treatment
with collagenase. The morphology of isolated-replanted
cysts was otherwise indistinguishable from cysts growing within the original cultures.
To further demonstrate that cyst growth involved mitotic activity, cultures were incubated with tritiated thy-
midine and processed for light microscopical radioautography. Figure 2B shows a n MDCK cyst bearing cells
with labeled nuclei. This indicates that cells are added
to the cyst wall by mitotic division.
Ultrastructure of Clonal MDCK Cysts
MDCK cysts were formed of a simple epithelium. The
cells bore abundant apical microvilli and commonly possessed a single cilium (Figs. 2, 5). Lateral cell surfaces
were usually simple, with few villi or infoldings (Figs.
2, 5). Desmosomes occurrred intermittently along the
intercellular interface. Occluding junctions were present adjacent to the lumen (Figs. 2, 5). Cells typically
possessed a well-developed complement of organelles,
including numerous mitochondria and a n elaborate
Golgi complex. Golgi stacks were commonly located on
the lumenal or lateral side of the nucleus (Figs. 2, 5).
The cytoplasm also contained smooth-surfaced vesicles
and appeared to have only a modest amount of rough
endoplasmic reticulum. Cells occasionally contained
large membrane-bound vacuoles containing material of
moderate density (Fig. 2).
The basal surface of MDCK cysts was relatively simple. Whereas cysts isolated by collagenase (Fig. 4) appeared to possess shallow clefts at intercellular margins,
specimens prepared by stripping away the overlying
collagen prior to fixation (Fig. 6) showed a smooth basal
surface. The uneven surface of enzyme-isolated cysts
was likely due to partial collapse of the cysts during the
isolation procedure. Indeed, many cysts were found to
tear and collapse completely during isolation with
collagenase.
MDCK cells appeared, by routine TEM (Figs. 2, 51, to
lack a well-organized, continuous basal lamina. Extracellular material occurred, instead, in irregular patches.
Fixation in the presence of ruthenium red, however,
Fig. 2. Structure of MDCK cysts cultured within collagen ge1.A
Light micrograph of living culture (10 days) showing numerous spherical cysts of various sizes. A large cyst (arrow) near the center of the
field measures approximately 800 pm in diameter. X59. B: Light
microscopy radioautograph of a plastic-embedded section from a 16day culture incubated with tritiated thymidine. Several cells (arrows)
show labeled nuclei. This suggests that mitotic activity was involved
in the growth of this cyst. ~ 8 9 6C:
. Transmission electron micrograph
of a solitary MDCK cyst a t 10 days following seeding. Cells are cuboidal, bear apical microvilli (mv), and are joined by tight junctions (circle)
and desmosomes (arrows). These contiguous cells form a polarized
epithelium with basal cell surface in contact with the surrounding
collagen matrix (HCG). X4,300. D: Scanning electron micrograph of a
cryofractured 12-day culture shows a spherical cyst surrounded by
collagen matrix (HCG). Apical cell surface is visible facing the lumen.
Each cell appears to possess a solitary cilium (arrows). ~ 8 9 6 .
MORPHOGENETIC CLONAL GROWTH OF MDCK CELLS
233
Fig. 3. MDCK cyst formation by clonal growth. These panels (A,B)
demonstrate the clonal growth of solitary MDCK cells to form polarized multicellular epithelia1 cysts. Two separate fields (3A, B) were
recorded by using differential interference contrast optics at days 2,4,
and 10 following seeding. Frames recorded at day 2 show only single
cells within the field. In both panels arrows mark the position of an
individual cell at day 2 that subsequently undergoes cyst formation. It
can be observed that not all single cells at day 2 underwent clonal
growth. ~ 2 6 2 .
demonstrated abundant flocculent electron-dense material associated with basolateral membranes (Fig. 5).
Cell shape within the population of cells composing
individual cysts was generally uniform. The great majority of cysts were made up of cells having cuboidal to
low-cuboidal morphology. Some cysts possessed moderately attenuated cells. Some degree of cell attenuation
appeared to accompany cyst growth; however, large cysts
rarely possessed highly attenuated cells.
Generally, cysts were spherical (Figs. 2,4). Cyst shape
was however, influenced by physical conditions of the
HCG matrix (see below) and by the cell population as
well. Cysts fused with one another if they came in contact during growth (Fig. 6).If contact and fusion occured
234
J.A. McATEER, A.P. EVAN, AND K.D. GARDNER
Fig. 4. Isolated MDCK cyst. Scanning electron micrograph of an intact single MDCK cyst
isolated from culture by digestion with collagenase. This cyst is a simple sphere. Basolateral
cell surface is visible. Cell boundaries appear demarcated (arrows) by shallow clefts induced
during isolation and preparation for microscopy. x 1,098.
between cysts of small to moderate size, the resultant
fused cyst usually grew to assume a spherical form. The
cysts of cultures in which cells were seeded between
HCG layers were not always spherical (Fig. 6) (see
below).
MDCK cysts were fluid-filled. Cysts never contained
collagen; however, lumina commonly contained a flocculant precipitate (Figs. 2, 5). Material within lumina
was preserved by routine processing for TEM but was
frequently displaced during cryofracture for SEM. Freefloating cells were often seen within cysts (Fig. 21, particularly in older cultures.
The ultrastructure of MDCK cysts cultured in serumfree media was indistinguishable from cysts grown in
serum-supplemented medium.
MDCK Cyst Formation Between HCG Layers
Cysts that developed in cultures in which cells were
planted at the interface between layers of collagen gel
(Fig. 6 ) often formed as moderately flattened sacs instead of spheres. Whereas many cysts appeared initially
to be spherical, nearly all developed flattened morphology with continued culture. The ultrastructure of cysts
in these cultures was indistinguishable from cysts raised
in collagen suspension. In layered gel cultures, growing
cysts often came into contact with one another. When
this occurred the cysts fused and their lumina coalesced
(Fig. 6). This pattern of development resulted in the
formation of large irregular channels with a common
interconnecting lumen and a n epithelia1 wall formed by
the recruitment of numerous individual cysts.
MORPHOGENETIC CLONAL GROWTH OF MDCK CELLS
Fig. 5. Ultrastructure of MDCK-cyst wall. A Transmission electron
micrograph shows portions of three cells. Apical surface bears microvilli (mv), while lateral and basal surfaces are relatively simple. Golgi
stacks and smooth-surfaced vesicles appear polarized toward the cell
apex. Basal lamina is not readily evident, although patches of apparent extracellular material are present (arrow). The lumen contains a
flocculent precipitate. Collagen gel (HCG). ~ 5 , 3 3 6 B:
. Scanning elec-
235
tron micrograph of cryofractured MDCK cyst shows abundant microvilli (mv) at the apical cell surface. Each cell also bears a long
filamentous single cilium (arrow). X3,955. C: Transmission electron
micrograph demonstrates ruthenium-red-positive extracellular material (arrows) along the basal surface and lateral interface between
adjacent cells. x 57,600.
236
J.A. McATEER, A.P. EVAN, AND K.D. GARDNER
Fig. 6. Cultures established between layers of collagen gel. A Scanning electron micrograph shows MDCK cysts at the interface between
the base and overlying layers of collagen gel. Cysts are not distributed
throughout the matrix but, instead, occupy the zone between collagen
gel layers. This specimen was prepared by stripping away the upper
collagen layer prior to fixation. X276. B: Light micrograph of a living
culture (10 days in vitro) demonstrates the irregular pattern of MDCK
cysts that develops when cells are seeded at the interface between
collagen layers. Cysts develop initially as solitary structures (arrowhead) but coalesce when they grow into contact (arrows) with one
another. Continued culture results in formation of large epithelia1
channels. ~ 6 3 C.: Scanning electron micrograph of a single MDCK
cyst from a specimen stripped free of its overlying collagen layer
immediately before fixation. The exposed basal surface of the epithelium is smooth and regular. Cell boundaries (arrows) are tightly apposed. Collagen gel (HCG). X812.
MORPHOGENETIC CLONAL GROWTH OF MDCK CELLS
DISCUSSION
We observe that the growth of MDCK cells dispersed
within collagen gel produces clonal colonies that display
the histotypic orientation of a kidney tubule. Apical cell
surfaces face a n enclosed lumen while basolateral surfaces border the interstitium. This morphogenetic form
of growth is likely due to multiple factors including both
the physical and biochemical composition of the collagen
gel substrate and intrinsic properties of the MDCK cell
type.
Our conclusion that the formation of MDCK cysts is
due to clonal growth of individual isolated cells is based
on frequent and repeated observations of living cultures
with light microscopy. It can be argued that intermittent observations might not disclose cell aggregation
involving rapid cell migration. However, the fact that
we have not observed any migratory cells at any time
during the period of cyst formation, in hundreds of cultures, indicates that cell movement does not occur, and
that cyst formation must, therefore, involve cell division. Greenburg and Hay (1982) have demonstrated that
certain embryonic and adult epithelia may lose polarity
and exhibit migratory behavior when cultured within
collagen gel. Such cells have a distinctive morphology.
They are bipolar or spindle shaped and possess elongated filopodia (Green and Hay, 1982).We do not observe
cells with this structure in our cultures. Instead, MDCK
cells within collagen gel remain rounded. Following division the newly formed cells have a cuboidal shape, as
part of the cyst wall epithelium. Thus our observations
indicate that MDCK cysts form due to mitotic activity
of individual cells (clonal growth). Likewise, we conclude
that subsequent growth in diameter of MDCK cysts
involves cell division and not cell aggregation. We observed that cysts isolated from culture and then replanted within fresh collagen gel continued to increase
in size and cell number. Growth under these conditions
could only be due to cell division. In addition, we demonstrated the nuclear incorporation of tritiated thymidine in cells of growing cysts. This indicates that mitotic
activity occurred during cyst growth.
Morphogenetic behavior is exhibited in a variety of
cell systems in which hydrated collagen gel is used as
the culture substrate. Mouse mammary epithelium
forms ductlike tubules within collagen gel (Yang et al.,
1979; Yang et al., 1980; Bennett, 1980; Bennett et al.,
1981). These structures form as outgrowths from mammary tumor explants embedded within collagen gel
(Bennett, 1980) or from cloned tumor cell lines cultured
atop a collagen substrate (Yang et al., 1979, 1980). In
addition, histotypic growth occurs when normal mouse
mammary cells are dispersed within collagen gel (Bennett, 1980). Tubule formation by mammary epithelial
cells has been shown to involve cell aggregation, reorganization, and subsequent growth. This seems to be the
case with other morphogenetic culture systems a s well,
including follicle formation by dispersed hog thyrocytes
(Chambard et al., 1981).Chambard et al. (1981)demonstrated that thyroid epithelial cells seeded atop a base
layer of collagen gel and then overlaid with collagen
will reorganize to form follicles in which the cells show
the apex-to-lumen polarity of the thyroid follicle in vivo.
Similar behavior is shown by MDCK cells cultured un-
237
der similar conditions. Hall et al. (1982)observed that a
confluent monolayer of MDCK cells cultured atop collagen will form multiple small cysts when overlaid with
collagen. In this case lumen formation is due to reorganization of the cell sheet, not clonal growth.
Cyst formation by MDCK cells suspended within a
three-dimensional matrix has been reported by others.
Leighton (1981) observed the development of polarized
epithelial cysts by MDCK cells seeded within a plasma
fibrin clot. Cell orientation of fibrin-cultured cysts was
similar to cysts we observed in collagen gels. Leighton’s
study (1981) did not attempt to document the growth
behavior of isolated cells; therefore, it is not known if
cyst formation within a fibrin matrix occurs by clonal
growth or, perhaps, involves cell aggregation.
The mechanism by which collagen gel promotes morphogenetic cell behavior is not known. Nor is it known
why certain cell types form histotypic structures within
collagen gels while others do not (Greenburg and Hay,
1982). Collagen clearly influences epithelial polarity,
but again, the mechanism of this interaction is not
known. Experimental evidence from several systems indicates that the polarity of a n epithelial cell is destabilized when collagen is applied to its apical surface. When
thyroid epithelial cells are cultured in fluid suspension
they form everted follicles in which the cell apex faces
the medium and basolateral surface encloses a lumen
(Mauchamp et al., 1979). These everted follicles reverse
polarity when collagen is added to the medium or when
the follicles are embedded in collagen (Chambard et al.,
1981).The cells, therefore, appear to reverse polarity in
order to avoid contact of the apical surface with the
surrounding collagen. Understandably, when a monolayer of MDCK cells grown on a collagen substrate is,
in turn, overlaid with collagen (Hall et al., 1982), polarity reversal will not free the apical surface from contact
with collagen. Instead, the cell sheet must reorganize to
create a lumenal space for apical surface to border.
We observe that when MDCK cells are dispersed
within collagen gel the cells remain rounded and do not
migrate through the matrix. Cyst formation in this system, therefore, does not involve reaggregation but is
dependent on cell division. The process of cyst formation
thus involves the development of a polarized epithelium
by the successive division (clonal growth) of a single cell.
Our observations suggest a series of events to describe
this clonal growth process. It is likely that MDCK cells
dissociated from monolayer culture lose polarity when
they are suspended within collagen. A cell fully surrounded by collagen is in contact over its entire surface
with substrate normally bordered by basolateral membrane. Because the isolated cell cannot reverse polarity
to avoid exposure of its apical surface to collagen it
probably eliminates apical surface altogether. The cell
must divide to reestablish polarity. We speculate that
when division occurs, daughter cells remain contiguous.
Their region of contact would create a collagen-free area
of the cell surface and provide a n interface that each cell
could polarize against. This would create a region of
membrane free to develop into apical surface. Thus,
with polarity established, all subsequent cell division
would occur within the plane of the epithelium. The
lumen would increase in diameter as cells are added by
division, to the cyst wall. Tight junctions would be estab-
238
J.A. McATEER, A.P. EVAN, AND K.D. GARDNER
lished between daughter cells at each division; thus, the
lumen would never be continous with the surrounding
interstitium.
A collagen matrix is not essential to support cyst formation by MDCK cells. Other three-dimensional matrices such a s soft agar can be used. The nature of the
substrate does, however, determine the polarity of the
cell population. We have grown MDCK cells suspended
within soft agar. As in collagen gel, the freshly isolated
cells remain rounded and do not migrate. Although cysts
do form in soft agar, cell polarity is opposite that of the
cysts that form in collagen gel. In soft agar, the apical
cell surface faces the agar medium while the basolateral
surface borders the enclosed lumen. Thus, the physical
support provided by a three-dimensional matrix does
not, in itself, determine cell polarity.
It is possible that fluid transport is involved in the
growth of MDCK cysts within collagen gel. We have
purposely ruptured isolated cysts before replanting them
within collagen. Collapsed cysts commonly reexpand to
original size within 48-72 hours, far faster than could
be attributed to growth by cell division. We have not,
however, determined if the reexpansion of a collapsed
cyst or the growth of a n intact cyst requires active fluid
transport. If unidirectional fluid transport does occur
during cyst growth the polarity of the cyst wall would
dictate that the direction of fluid flow must be opposite
that which occurs in vivo. This likewise would be opposite the direction of fluid transport that takes place
during dome formation in monolayer culture (Leighton
et al., 1969; Abaza et al., 1977; Lever, 1979a,b; Rabito et
al., 1980; Cereijido et al., 1981).
At present the literature describing cyst formation by
cells dispersed within a three-dimensional matrix is
somewhat limited (Leighton, 1981). Furthermore, the
process of morphogenetic clonal growth that we have
described for the MDCK cell has not been documented
for other cell types. This may suggest that morphogenetic clonal growth is a n uncommon cell behavior; however, it may be that other cell types are indeed capable
of this form of growth, but have not been studied under
similar culture conditions.
Not all cells of the parent MDCK cell population are
capable of morphogenetic clonal growth. We observe single cells within gels after even extended culture. These
cells can be harvested by filtration-separation following
digestion of the collagen matrix, and will seed, with
variable plating efficiency, to form a viable monolayer.
It therefore may be that clonal growth under these conditions is a population selection process.
Clonal growth in three dimensions offers potential
methodological advantages applicable to cell selection
and clonal analysis. We have demonstrated that MDCK
cysts can develop from single cells. Each confirmed (see
below) clonal cyst thus possesses a homogeneous cell
population in contrast to the recognized heterogeneity
of the parent line (Lewis and Spector, 1981; Richardson
et al., 1981; Valentich, 1981). Individual cysts can be
isolated and passaged to monolayer or collagen gel for
continued culture. This provides a unique method for
clonal selection of MDCK sublines, as in isolation of
agent-resistant or sensitive strains.
It must be noted that in this system MDCK cysts may
also develop from clusters of cells not fully dissociated
during preparation of the original cell suspension. In-
deed, the degree to which true clonal growth occurs is
influenced by the efficiency of preparation of a monodisperse cell population at the time of seeding. Assurance
that a n individual cyst is a true clonal cell population,
therefore, requires visual verification by sequential observations of the field.
Further value of the cellular homogeneity of MDCK
clonal cysts may become apparent should studies focus
on the biology of individual cysts. It seems reasonable to
suggest that investigation of a discrete clonally derived
cell population will allow a more precise assessment of
the functional properties of this kidney cell line.
The culture system we have described is a n alternative to monolayer culture that may provide the opportunity for novel experimental design in studies on the
biology of the MDCK cell. The physical characteristics
of the system are unique. We are aware of no other
kidney epithelial cell culture system that allows essentially unrestricted access to basolateral cell surface. Also,
unlike monolayer culture, the cells of MDCK cysts are
exposed to nutrient supply solely through their basolatera1 surface. This mimics the relationship of the vasculature to the basal cell surface in vivo and offers the
potential for selective manipulation of the “in vitro interstitial” compartment. In addition, it is possible to
alter the physical culture substrate of MDCK cysts. Not.
only can the composition of the collagen gel matrix be
tailored prior to seeding, but the substrate surrounding
growing cysts can be removed or replaced without disrupting cell-cell interactions. Thus, the system may
prove useful for study of the influence of extracellular
matrix on kidney epithelial cell structure and function.
ACKNOWLEDGMENTS
The authors wish to thank Philip Blomgren, Bret Connors, and Ellen Vance for their valuable technical assistance and Claudette Maurer and Mary Henderson for
typing the manuscript. This study was supported by
grant number AM3003 from the National Institutes of
Health and by the PKR Foundation (Kansas City).
LITERATURE CITED
Abaza, N.A., J. Leigbton, and S.G. Schultz (1977) Effects of Ouabain
on the function and structure of a cell line (MDCK) derived from
canine kidney. I. Light microscopic observations of monolayer
growth. In Vitro, 103172-183.
Anderson, T.F. (1951) Technique for the preservation of three dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci., 23:130-134.
Bennett, D.C. (1980) Morphogenesis of branching tubules in cultures
of cloned mammary epithelial cells. Nature, 285:657-659.
Bennett, D.C., B.L. Armstrong, and S.M. Okada (1981) Reconstitution
of branching tubules from two cloned mammary cell types in culture. Dev. Biol., 873193-199.
Cereijido, M. (1984) Electrical properties of Madin-Darby canine kidney cells. Fed. Proc., 43:2230-2235.
Cereijido, M., J. Ehrenfeld, S. Fernandez-Castelo, and I. Meza (1981)
F l u e s , junctions, and blisters in cultured monolayers of epithelioid
cells (MDCK). Ann. N.Y. Acad. Sci., 773422-441.
Cereijido, M., E.S. Robbins, W.J., Dolan, C.A. Rotunno, and D.D. Sabatini (1978) Polarized monolayers formed by epithelial cells on a
permeable and translucent support. J. Cell Biol., 773853-880.
Chambard, M., J. Gabrion, and J. Mauchamp (1981) Influence of collagen gel on the orientation of epithelial cell polarity: Follicle formation from isolated thryoid cells and from preformed monolayers.
J. Cell Biol., 91:157-166.
Garcia-Perez, A., and W.L. Smith (1983) Use of monoclonal antibodies
to isolate cortical collecting tubule cells: AVP induces PGE release.
Am. J. Physiol., 23:C211-c220.
MORPHOGENETIC CLONAL GROWTH OF MDCK CELLS
Greenburg, G., and E.D. Hay (1982) Epithelia suspended in collagen
gels can lose polarity and express characteristics of migrating
mesenchymal cells. J. Cell Biol., 95:333-339.
Hall, G.H., D.A. Farson, and M.J. Bissell (1982) Lumen formation by
epithelial cell lines in response to collagen overlay: A morphogenetic model in culture. Proc. Natl. Acad. Sci. USA, 79:4672-4676.
Herzlinger, D.A., T.G. Easton, and G.K. Ojakian (1982) The MDCK
epithelial cell line expresses a cell surface antigen of the kidney
distal tubule. J. Cell Biol., 93:269-277.
Humphreys, W.J., B.O. Spurlock, and J.S. Johnson (1974)Critical point
drying of ethanol-infiltrated cryofractured biological specimens for
scanning electron microscopy. In: Scanning Electron Microscopy.
Johari, 0. ed. IIT Research Institute, Chicago, pp. 275-282.
Leighton, J. (1981) Anatomic evidence of transport function by adenocarcinomas and a suggested role in transport in the spread of
cancer. Ann. N.Y. Acad. Sci., 372:455-464.
Leighton, J., 2. Brada, L.W. Estes, and G. Justh (1969) Secretory
activity and oncogenicity of a cell line (MDCK)derived from canine
kidney. Science, 163:472-473.
Lever, J.E. (1979a) Inducers of mammalian cell differentiation stimulate dome formation in a differentiated kidney epithelial cell line
(MDCK). Proc. Natl. Acad. Sci., USA 76:1323-1327.
Lever, J.E. (1979b)Cyclic AMP and inducers of mammalian cell differentiation stimulate dome formation in mammary and renal epithelial cell cultures. In: Hormones and Cell Culture. Cold Spring
Harbor Conference on Cell Proliferation. eds. G. Sat0 and R. Ross,
Cold Spring Harbor Press, New York, NY, pp. 727-738.
Lewis, M.G., and A.A. Spector (1981)Differences in types of prostaglandins produced by two MDCK canine kidney cell sublines. Prostaglandins, 21: 1025-1032.
.Madin, S.H., and N.B. Darby (1975) As catalogued (1958) in American
Type Culture Collection Catalogue of Strains, 2:47.
Mauchamp, J., A. Margotat, M. Chambard, B. Charrier, L. Remy, and
M. Michel-Bechet (1979) Polarity of three-dimensional structures
derived from isolated hog thyroid cells in primary culture. Cell
Tissue Res., 204:417-430.
McAteer, J.A., and T.J. Cavanagh (1982) Medium hydrated collagen
gel as an explant support in organ culture. J. Tissue Cult. Methods,
7: 117-122.
239
McAteer, J.A., and T.J. Cavanagh (1983) Nylon screen filtration devices for crude separation of dissociated cells for primary culture.
J. Tissue Cult. Methods, 8:45-47.
McAteer, J.A., A.P. Evan, and W.H.J. Douglas (1983) A method for
processing free-floating cultured cells for scanning electron microscopy. J. Tiss. Cult. Meth., 7:85-88.
Misfeldt, D.S., S.T. Hamamoto, and D.R. Pitelka (1976)Transepithelial
transport in cell culture. Proc. Natl. Acad. Sci., USA 73:1212-1216.
Rabito, C.A., R. Tchao, J. Valentich, and J. Leighton (1980) Effect of
cell-substratum interaction on hemicyst formation by MDCK cells.
In Vitro, 16:461-468.
Richardson, J.C., V. Scalera, and N.L. Simmons (1981)Identification of
two strains of MDCK cells which resemble separate nephron tubule segments. Biochem. Biophys. Acta, 673~26-36.
Rindler, M.J., L.M. Chuman, L. Shafter, and M.H. Saier (1979)Retention of differentiated properties in a n established dog kidney epithelial cell line (MDCK). J. Cell Biol., 81-635-647,
Taub, M., and M.H. Saier (1979) An established but differentiated
kidney epithelial cell line (MDCK), In: Methods in Enzymology,
Vol. LVIII. Cell Culture. W.B. Jakoby and I.H. Pastan, eds. Academic Press, New York, pp. 552-560.
Taub, M., and G.H. Sat0 (1979) Growth of kidney epithelial cells in
hormone-supplemented serum-free medium. J. Supramol. Struct.,
11:207-2
16.
.
Valentich, J.D. (1981) Morphological similarities between the dog kidney cell line MDCK and the mammalian cortical collecting tubule.
Ann. N.Y. Acad. Sci., 372.372-384.
Valentich, J.D., R. Tchao, and J. Leighton (1979) Hemicyst formation
stimulated by cyclic AMP in dog kidney cell line MDCK. J. Cell
Physiol., 100:291-304.
Yang, J., J. Richard, P. Bowman, R. Guzman, J. Enami, K. McCormick,
S. Hamamoto, D. Pitelka, and S. Nandi (1979) Sustained growth
and three-dimensional organization of primary mammary tumor
epithelial cells embedded in collagen gels. Proc. Natl. Acad. Sci.,
USA, 77:2088-2092.
Yang J., J. Richard, R. Guzman, W. Imagawa, and S. Nandi (1980)
Sustained growth in primary culture of normal mammary epithelial cells embedded in collagen gels. Proc. Natl. Acad. Sci. 76:34013405.
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