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MICROSCOPY RESEARCH AND TECHNIQUE 39:71–84 (1997)
Monolayer and Three-Dimensional Cell Culture and Living
Tissue Culture of Gallbladder Epithelium
YASUNI NAKANUMA,* KAZUYOSHI KATAYANAGI, YASUHITO KAWAMURA, AND KAZUYOSHI YOSHIDA
Second Department of Pathology, Kanazawa University School of Medicine, Kanazawa 920, Japan
KEY WORDS
gallbladder; monolayer cell culture; three-dimensional cell culture; explant;
tissue culture; collagen gel
ABSTRACT
Several models for preparing and isolating human and animal gallbladder epithelial cells, including low-grade gallbladder carcinoma cells, as well as proposed systems for culturing
these isolated epithelial cells are reviewed here. Several reports concerning tissue culture of the
gallbladder are also reviewed. The cell culture systems are divided into monolayer cell culture on
collagen-coated or uncoated culture dishes or other culture substrate and three-dimensional cell
culture in collagen gel. To prepare and isolate gallbladder epithelial cells, digestion of the
gallbladder mucosa, abrasion of the mucosal epithelial cells, and excision of epithelial outgrowth of
mucosal explants are applied. In monolayer cell culture, most of the specific biological features of
isolated and cultured cells characteristic to the gallbladder are gradually lost after several passages,
though quantitative and objective analyses of the pathophysiology of cultured cells and their
secretory substances can be performed. Tissue culture using explants of the gallbladder has mainly
been used for physiological studies of the gallbladder, such as investigating the transport of water
and electrolytes. In this tissue culture system, quantitative assessment is difficult, though the
original and specific biological and histological characteristics of the gallbladder are retained.
Three-dimensional collagen gel culture could be an ideal model combining monolayer cell culture
and tissue culture systems, and create controllable conditions or environments when several
biologically active substances, such as growth factors, proinflammatory cytokines and adhesion
molecules, are added to the culture medium. Advantages and shortcomings of individual cultivation
models are discussed, and selecting the culture model most appropriate to the purpose of the study
will facilitate investigations of the biology and pathogenetic mechanisms of gallbladder diseases
such as cholelithiasis. Microsc. Res. Tech. 39:71–84, 1997. r 1997 Wiley-Liss, Inc.
INTRODUCTION
The gallbladder performs many physiologic functions, namely absorption and secretion of water and
electrolytes, control of the content and concentration of
bile (Carey and Cahalane, 1988; Hoerl et al., 1992;
Sherlock and Dooley, 1992; Tilvis et al., 1982). The
characteristic morphologic and phenotypic properties
and developmental processes of the gallbladder are also
known (MacSween and Scothorne, 1994; Rhodin, 1974).
The mucus coat on the luminal surface is thought to be
a barrier protecting the mucosa; but it is also providing
an environment for microcaliculi formation in cholelithiasis (Carey and Cahalane, 1988). Mucosubstances
are secreted from the surface epithelial cells and to a
lesser degree the cervical glands. This organ also has a
role related to local mucosal immunity mediated by
secretion of immunoglobulins, particularly Ig A and
other nonspecific bactericidal substances like lactoferrin and lysozyme (Duitton et al., 1985; Nagura et al.,
1987).
In addition, the gallbladder is affected by a number of
diseases. In particular, gallstone disease remains a
leading cause of human morbidity and mortality in
developed countries including Japan. Gallstone pathogenesis is supposed to involve alterations in the gallbladder wall (Carey and Cahalane, 1988; Conter et al.,
r 1997 WILEY-LISS, INC.
1992; Moore, 1990), affecting local circulation and
mucus secretion, motor functions, and bile composition
(Purdum et al., 1993). Yet, despite extensive research
into the physiology and pathophysiology of the gallbladder, little is known about mucosal modifications that
precede and contribute to gallstone disease.
In vitro techniques are required for further investigations on the pathologic and physiologic properties of the
gallbladder wall and of its epithelium. Epithelial cell
cultures represent one of the adequate in vitro approaches. Several methods of monolayer and threedimensional cell culture after preparation and isolation
of biliary epithelial cells from normal or pathologic
biliary tract are available (Gall and Bathal, 1987; Hoerl
et al., 1992; Joplin et al., 1990; Kawamura et al., 1989).
Tissue culture using explants of the gallbladder mucosa
is an interesting alternative to cell culture (Elhamady
et al., 1983).
In this review, several representative models for the
preparation and isolation of gallbladder epithelial cells
and for their use in monolayer and three-dimensional
cell culture are examined. Finally, tissue culture of the
*Correspondence to: Yasuni Nakanuma, MD, Second Department of Pathology,
Kanazawa University School of Medicine, Kanazawa 920, Japan.
Accepted in revised form 5 June 1995.
72
Y. NAKANUMA ET AL.
biliary tract, particularly the gallbladder, will be described. The respective advantages, shortcomings and
applications of these cultivation methods will also be
considered.
IDENTIFICATION OF GALLBLADDER
EPITHELIAL CELLS FOR IN VITRO CULTURE
Several approaches are available for identifying cells
isolated from the gallbladder which are proliferating
and growing in the culture system. The morphologic
features or phenotypic markers described in this chapter are most frequently used for recognizing a biliary
epithelial lineage.
It is, however, noteworthy that the expression and
intracellular distribution of the phenotypic characters
described here are dependent upon the degree of in vivo
differentiation and the anatomical location or level of
the biliary epithelial cells that have been isolated and
cultured (Berdichevsky et al., 1994; Nakanuma et al.,
1997). The methods of cell isolation and of culture used,
specifically the use of supporting or of coated matrices,
whether growth factors and serum are added to the
medium or not, and the duration and number of passages of cultures, are also important factors. Therefore,
the morphologic and phenotypic characteristics described in this paragraph are not constantly expressed
in the cultured biliary epithelial cells, while they are in
vivo. However, when positive, the cells are most probably originating from biliary epithelium.
Morphologic Characteristics
Several morphologic characteristics, particularly ultrastructural ones, can be used to distinguish biliary
epithelial cells from other cell lineages (Fig.1). In
addition to the usual cytologic appearance of epithelial
cells themselves, the presence of dense microvilli and
cilia on the luminal surface, of complicated intercellular digitation or infoldings, of basally or centrally
located nuclei and mucus droplets are in favor of a
biliary epithelial lineage. Basement membrane is specific for biliary epithelial cells though its presence
depends on the culture model.
Mucin Histochemistry
Cytoplasmic mucin droplets and a layer of mucinous
substance on the luminal surface including a glycocarix
can be demonstrated by several histochemical stainings. For example, alcian blue (AB) at pH 1.0 and 2.5
(Fig. 2A), mucicarmine stain, colloidal iron, high iron
diamine (HID) and diastase-digested periodic acid Schiff
(d-PAS) stainings (Sano, 1976) are routinely used for
this purpose. Ruthenium red staining is another method
to identify this luminal acid glycoprotein, which represents a surface marker (Nagase, 1988) of biliary epithelial cells.
Other Phenotypic Markers of Gallbladder
Epithelial Cells
Expression of the phenotypic markers described here
is highly suggestive of a biliary epithelial cell lineage.
However, their expression largely depends on the animal species, the ontogeny and the degree of maturation
of the biliary epithelial cells, as well as on the material
and methods used for cultures. These makers are
Fig. 1. Ultrastructure of the gallbladder epithelium. There are
dense microvilli on the luminal surface and basally and centrally
located nuclei. Note developed intercellular infoldings (asterisks). L,
lumen of the gallbladder; N, nucleus. Normal rabbit gallbladder
epithelium, 32,000.
detectable by using immunoperoxidase, immunofluorescence, and lectin histochemistry with avidin-biotinperoxidase complex, or by using enzyme histochemistry
(Hsu et al.,1981; Nagase, 1988).
Cytokeratin (CK). Biliary type CK such as CK 7, 9
and 19 is known to be expressed on human biliary
epithelial cells. Particularly, CK 19 is useful in the
identification of biliary epithelial cells. These CKs are
detected by using commercially available monoclonal
antibodies such as AE1, AE3, KL1, Ks19.1 and CAM5.2
(Auth et al., 1993; Van Eyken and Desmet, 1993). CK
detected by polyclonal anti-CK antibodies may also be
used as a marker of biliary epithelial cells (Nakanuma
and Ohta, 1986).
Lectin Binding Properties. Concanavalin A (Con
A), Pisum sativum agglutinin (PSA), wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), and
soy bean agglutinin (SBA) bind to the apical surface
and/or cytoplasm of gallbladder epithelial cells in vivo
(Fig. 2B). These lectins are known to bind to the specific
carbohydrate residues of glycoproteins (Terada and
Nakanuma, 1994; Yoshida et al., 1996). Therefore, it
seems reasonable to propose that such carbohydrate
residues which are detectable by lectin histochemistry
CULTURE OF GALLBLADDER EPITHELIAL CELLS
73
Fig. 2. A: Normal gallbladder. There is a layer of mucinous
substance (arrows) on the luminal surface. L, gallbladder lumen.
Normal rabbit gallbladder. Alcian blue staining at pH 2.5. B: Normal
gallbladder. Dolichos biflorus agglutinin is bound to the luminal
surface of the normal rabbit gallbladder (arrows). L, lumen of the
gallbladder. Lectin immunohistochemistry using avidin-biotin-peroxidase complex and Dolichos biflorus agglutinin and hematoxylin.
be used as a phenotypic marker of the glycoprotein of
the gallbladder epithelium in cultures.
Mucin Core Protein. Recently, mucus core proteins
(MUC 1-7) have been described as the backbone of
glycoprotein, and antibodies to these core proteins are
becoming available. Among these MUC proteins, MUC
1 is now available commercially (Sasaki and Nakanuma, 1994), and the expression of this protein can be
used as a marker of the glycoprotein in biliary epithelial cells.
Enzyme Histochemistry. Gamma-glutamyltranspeptidase is a well known as a marker of biliary
epithelial cells in man and in experimental animals
(Hoerl et al., 1992; Yoshida et al., 1995).
Other Phenotypic Markers. Epithelial membrane
antigen and a specific glycoprotein which are both
constantly expressed in the biliary epithelial cells in
vivo including the gallbladder (Momburg et al., 1987)
may be used as markers of cultured biliary epithelial
cells.
Hepatocytes. Expression of albumin or glucose-6phosphatase and the presence of abundant glycogen
and canalicular structures (Rhodin, 1974) are in favor
of hepatocyte lineages.
Cellular Markers to Detect Contaminated
Mesenchymal Cells and Hepatocytes
Mesenchymal Cells. Vimentin, an intermediate filament found in mesenchymal cells, and factor VIII–
related antigens are constantly negative in biliary
epithelial cells but positive in mesenchymal cells and in
endothelial cells of blood vessels, respectively. So, they
are available for identification of these contaminated
cells, though vimentin is also expressed on secondary
damaged bile ducts (Nakanuma and Kono, 1992) and
possibly cultured biliary epithelial cells.
Evaluation of Dynamic Changes
of Cultured Epithelial Cells
Autoradiographic analysis of [3H] thymidine incorporation into DNA (Hoerl et al., 1992) and immunostaining of proliferating cell nuclear antigen and BrdU may
be used for investigating the proliferative activities.
Flow cytometric analysis is also an interesting method
for this purpose (Oda et al., 1991). Uptake of [3H]
uridine is used for demonstrating RNA synthesis. Glycoprotein synthesis and secretion can be observed by
following the incorporation of [14C] glycosamine into
tissue glycoprotein and its subsequent secretion into
the culture medium (Oda et al., 1991).
MONOLAYER AND THREE-DIMENSIONAL
CELL CULTURE
Monolayer cell culture is the most usual method for
culturing epithelial cells from the biliary tract, namely
the gallbladder of humans and experimental animals
(Joplin et al., 1990; Parola et al., 1988). Threedimensional cell culture is also challenging in the
analysis of biological and pathological characteristics of
the gallbladder, particularly with respect to stereotypical analysis (Kawamura et al., 1989).
As for the human gallbladder and biliary tract,
autopsy materials are not suitable for cultivation of
epithelial cells because of prominent and rapid autoly-
74
Y. NAKANUMA ET AL.
sis postmortem. Gallbladders without or with only
minimal necrotic or inflammatory changes resected by
the surgeon for gallstones or for other reasons like
prophylactic resection or liver transplantation are recommended for cultivation (Katayanagi et al., submitted; Yoshitomi et al., 1987). For optimal results it is
important that the surgically resected gallbladder specimens should be cultured within the first 2 hours (Auth
et al., 1993).
In general, both monolayer and three-dimensional
cell culture systems involve two successive stages,
namely the preparation and isolation of the biliary
epithelial cells and the cultivation process(es). These
stages will be successively described. Viability of isolated epithelial cells is usually tested by trypan blue
staining.
Preparation and Isolation of Normal and
Malignant Gallbladder Epithelial Cells
In addition to adequate culture conditions, cell purity
is mandatory for a reliable in vitro culture study. There
have been numerous reports on obtaining viable and
purified gallbladder epithelial cells. Techniques can be
subdivided into the following three categories.
Preparation of Desquamated Epithelial Cells
from the Mucosa Using a Whole Gallbladder or
Parts of a Gallbladder. There are several approaches to this procedure (Auth et al., 1993; Conter et
al., 1992; Hoerl et al., 1992; Kawamura et al., 1989; Oda
et al., 1991; Yoshitomi et al., 1987). Our method using
rabbit gallbladder (Kawamura et al., 1989; Yoshida et
al., 1996) will be presented first. After the gallbadder
was resected in aseptical conditions, the bile was gently
aspirated from the lumen with a syringe. T/E mixed
medium containing 0.02% ethylenediaminetetraacetic
acid (EDTA) and 0.25% trypsin was injected into the
empty gallbladder. The organ was then immersed in
Hank’s culture medium in a moist chamber at 37oC for
50–70 minutes. Thereafter, T/E medium was recovered
and centrifuged. Approximately 80% of all cells in
smears from the pellets were isolated epithelial cells
but there were also several epithelial cell clumps (12%
of clumps consisted of 6 to 10 cells and 4% of clumps
consisted of more than 11 cells, respectively). Usually,
almost all isolated and clumped cells were viable as
shown by trypan blue staining, while contamination by
mesenchymal cells was minimal. After the pellets were
dispersed and resuspended in culture medium with
15% fetal calf serum, penicillin G, streptomycin, and
epidermal growth factor, the cells could be used in
either monolayer or three-dimensional cell culture.
This procedure appeared to be a simple and reproducible way to prepare singly isolated mucosal cells. However, separation of single cells from small clumps of
epithelial cells or from some contaminating mesenchymal cells is not possible with this method. Recently, Oda
et al. (1991) have reproduced this procedure using
canine gallbladders.
Yoshitomi et al. (1987) and Auth et al. (1993) developed other but similar methods for isolation of gallbladder epithelial cells. The former used functional gallbladders of patients undergoing cholecystectomy for
gallstones and incubation of the specimens with protease of dispase, while the latter used gallbladders taken
at the time of a prophylactic cholecystectomies and
have proposed a 0.125% collagenase IV incubation. The
mucosa was rinsed with culture medium through a
small incision in the gallbladder wall and wiped several
times with gauze in order to remove adherent bile. After
prolonged incubation with protease or collagenase,
careful mechanical abrasion of the gallbladder mucosa
was carried out. After the abrasion the same process
was repeated several times, the culture fluid containing
epithelial cell suspensions being centrifuged twice at
85g for 5 minutes each time. This appeared to be the
most efficient way to remove bile sludge and peripheral
blood cells while preparing adequate cell pellets. According to Auth et al. (1993), freshly isolated gallbladder
epithelial cells form characteristic clusters of 10 to 150
cells (organoid bodies).
Hoerl et al. (1992) also developed a method using
fragments or explants (approximately 1 cm2) of human
surgical specimens with minimal pathological alterations. They removed as much as possible muscle and
perimuscular connective tissue from the gallbladder
fragments in the culture medium. The roughly denuded
mucosal fragments of the gallbladder were floated in
0.17% trypsin in phosphate buffer saline at 4oC overnight and the epithelial cell layer was then separated
from the mucosa using curved forceps. The isolated
epithelial cells were then processed for cultivation.
Several modifications of this method have been proposed. According to our most recent method, samples
from which the major part of the muscle layer and
connective tissue is removed are immersed in a solution
of T/E mixed medium (see above). This fluid containing
the fragments of gallbladder mucosa is then gently
stirred for 24 hours at 37oC and the supernatant is
centrifuged. The obtained cell pellets contain many
isolated and clumped gallbladder epithelial cells as well
as a considerable number of mesenchymal cells. Kaminsky et al. (1985) described another method using a 25
gauge needle to inject the submucosa of the gallbladder
with iced 0.25% NaCl and 2.5% EDTA pH 7.5. at 4oC.
The gallbladder was then shaken in the same ice-cold
solution for 5 minutes. While shaking, the mucosal cells
became separated from the muscle layer. The solutions
were pooled and mucosal cells collected by centrifugation at 2,500g for 5 minutes. The pellets were washed
twice with iced Krebs-Ringer phosphate solution pH
7.5, and recentrifuged to remove blood cells. This
separation technique yielded approximately 500 mg of
mucosal cells per gallbladder.
Preparation and Isolation of Outgrowing Epithelial Cells from Explant of the Gallbladder on
Collagen Gel. This procedure is applicable to human
and animal gallbladders (Katayanagi et al., submitted).
After removing the gallbladder aseptically, the organ
was subdivided into several small pieces in culture
fluid. These small explants were incubated in a culture
fluid containing 0.05% collagenase and dispase at 37oC
for 30 minutes and then thoroughly rinsed in culture
fluid. The explants were then placed on collagen gel and
cultured in an incubator for 1 or 2 weeks. Using
repeated observations under phase contrast microscopy, it was observed that epithelial cells of the small
explants were proliferating and were spreading like a
sheet on the collagen gel surface (Fig.3A). Although
mesenchymal cells were actively growing and spreading simultaneously in the collagen gel or under the
CULTURE OF GALLBLADDER EPITHELIAL CELLS
75
Fig. 3. A: Explant of the mouse gallbladder. Phase contrast microscopy of explant (E)
of the mouse gallbladder cultured on collagen
gel for 1 week and epithelial sheet (asterisks)
growing out from the explant. The edge of
spreading epithelial sheet is shown by arrowheads. B: Explant of the mouse gallbladder
cultured on collagen gel for 5 days. The
mucosal epithelium of the explant (E) is viable. There is outgrowing epithelial sheet
from the explant. Epithelial cells of the sheet
is columnar (arrow) near the explant, while
they become flat (arrowhead) at the front of
this sheet. Mesenchymal cells are also growing into the collagen gel (asterisks). H&E
staining.
superficially spreading epithelia cells (Fig.3B), some
sites of the culture, particularly at the periphery, were
predominantly or purely composed of epithelial cells.
Such areas of collagen gel on which a pure epithelial
cell sheet was being observed were cut into fragments
which were then plated onto other collagen gel surfaces.
The latter secondary explants were cultured in a similar way for up to 1 to 2 weeks. From these secondary
microexplants, actively proliferating gallbladder epithelial cells without mesenchymal contamination grew
and spread on the collagen gel. Up to seven passages of
cultured epithelial cells using subsequent explants
which form such epithelial cell sheets are possible.
Preparation of Carcinoma-Derived Gallbladder Epithelial Cells. Several cell lines have been
derived from human gallbladder carcinomas (Johzaki
et al., 1989; Koyama et al., 1980; Morgan et al, 1981;
Purdum et al., 1993). Because of their adequate of
differentiation and because they share many properties
with normal gallbladder epithelial cells they have been
proposed as a substitute for non-malignant cells in
several studies.
Confluent Monolayer Cell Culture
and Supporting Systems
There have been several reports describing methods
of monolayer cell culture. This procedure offers a reliable and convenient source of experimentation and has
been widely used in research. Hoerl et al. (1992) and
Oda et al. (1991) developed and partially characterized
a confluent monolayer of cultured canine gallbladder
epithelial cells. Kawamura et al. (1989) reported monolayer cell culture of rabbit gallbladder in tissue culture
flasks (Fig.4). Monolayer cell cultures can be passaged
76
Y. NAKANUMA ET AL.
Fig. 4. A: Monolayer culture of rabbit gallbladder epithelial cells
on collagen coated plastic dish for 1 week. There is a monolayer of
epithelial cells with occasional vacuoles, suggesting mucus droplets.
Phase contrast microscopy. B: Ultrastructure of monolayer culture of
mouse gallbladder epithelial cells on collagen gel for 5 days. The
epithelial cells are of low columnar epithelial cells resembling the in
vivo gallbladder epithelial cells. There are dense microvilli on the
luminal surface (arrowheads) and well-developed intercellular lateral
infoldings. N, nucleus. L, lumen facing the culture medium, 35,000.
Fig. 5. Three-dimensional culture of rabbit gallbladder epithelial cells in collagen gel under phase
contrast microscopy. A: One day after being cultured, a small epithelial clump seems to contain small cyst
(arrow). B: Three days after being cultured, epithelial cell clump increases in size and seems to be a large
cyst (arrow). A and B, 3100.
CULTURE OF GALLBLADDER EPITHELIAL CELLS
several times (primary, secondary, tertiary cell culture,
and so on). These cultured cells after several passages
under all conditions eventually enlarge, show vacuolization, and demonstrate irreversible growth arrest.
Methods of Monolayer Cell Culture. Monolayer
cultivation of isolated gallbladder epithelial cells consists of the following several components and steps.
Culture supporting systems including culture vessels
and substrate. Several supporting systems are now
being applied to monolayer cell culture. It is well known
that extracellular matrix component(s) are necessary
for the culture of gallbladder epithelial cells, particularly long-term ones requiring successive replications
of epithelial cells. For example, collagen, a major constituent of the extracellular matrix in vivo, enhances
the growth as well as differentiation of many epithelial
cells (Kawamura et al., 1989; Yang et al., 1979, 1980),
and has been used for decades as a culture substrate
(Elhamady et al., 1983; Kumar and Jordan, 1986).
These extracellular matrix components are also important as an attachment factor in cultured epithelial cells.
Therefore, it is recommended to coat culture vessels
like plastic dishes, flasks or plates with the following
factors or substances: collagen type I, fibronectin, Matrigel (containing laminin, type IV collagen, and other
basement membrane components), poly-L-lysine, vitrogen, or other components of basement membrane,
polycarbon supporting matrix, or Kaiser’s glycerol gelatin. Culture vessels with a feeder layer of irradiated
contact-inhibited Swiss albino 3T3 mouse fibroblasts
are also preferable for long-term culture up to 6 weeks
and provide optimal culture rates, differentiation and
proliferation. Collagen gel matrix (see below) itself is
another example of culture substrate.
Uncoated plastic dishes in serum-free cell culture
may also be used for short-term culture of gallbladder
epithelial cells for special experimental study such as
immunological study, though proliferation and differentiation of cultured cells is limited (Auth et al., 1993).
Culture medium. A variety of culture media are now
being used for the culture of gallbladder epithelial cells
according to the aim of the study. Dulbecco’s modified
Eagle’s medium with Ham’s F12 in a 1:1 mixture,
William’s medium E, Eagle’s minimum essential medium, MCDB 153 medium, and serum-free keratinocyte growth medium are examples (Auth et al., 1993;
Hoerl et al., 1992; Kawamura et al., 1989; Oda et al.,
1991; Yoshitomi et al, 1987). The first four are usually
supplemented with 10% fetal calf serum with or without growth factors. Several antibiotics such as penicillin G and streptomycin are added in the culture media.
These cultures are usually incubated in a 5% CO2
atmosphere at 37oC. Culture media are usually changed
every 2 to 3 days, though the frequency depends on the
aim of the study.
Procedures. Singly isolated or clumped gallbladder
epithelial cells are suspended or dispersed in the culture medium and plated on coated or uncoated culture
vessels supplemented with an appropriate culture medium. They are cultured for up to 2 weeks (primary
culture). Subsequent monolayer cell culture of the primary
culture (secondary culture) and that of secondary culture
(tertiary culture) and so on are also frequently done.
Observations. Attachment, growth and spread of cultured epithelial cells on coated or uncoated culture
77
vessels are usually surveyed and confirmed periodically
and routinely by phase contrast microscopy. Morphologic observations by light microscopy, histochemistry,
immunohistochemistry, and electron microscopy are
also available, when necessary, and proliferative and
other dynamic activities are also evaluable by several
methods (see above).
Growth and Morphologic and Phenotypic Differentiation of Monolayer Cultured Cells. Epithelial
cells which adhere to the surface of the culture dish,
culture flask or collagen gel within 24 hours or between
24 and 72 hours after being plated immediately start to
proliferate and spread. Attached cells have oval nuclei
with one or more nucleoli and polygon-shaped cytoplasm with some granules around the nucleus. After 2
to 5 days, they show a cobble stone morphology and
gradually form a confluent monolayer after approximately 1 week. Under phase-contrast microscopy, the
proliferating cells appear flattened and show a sheetlike growth pattern (Fig.4A). There are occasional
vacuoles (cytoplasmic mucous vacuoles) among the cell
sheets. These growth patterns and the occurrence of
vacuoles become clearer in the secondary culture. Histologically, the outgrowing epithelial cells in culture
vessels are largely spindle-shaped or elliptical, and
their borders indistinct. The cytoplasm is slightly eosinophilic, and the nuclei are ovoid or elliptical. Occasional
multinucleation and mitoses suggest active proliferation. Variably sized vacuoles in the cultured epithelial
cytoplasm are positive for PAS, AB at pH 1.0 and 2.5,
colloidal iron, HID, and mucicarmine stainings. Such
cultured epithelial cells exhibit biliary-type CK, namely
CK 7 and CK 19, consistent with their gallbladder
epithelial origin; they secrete mucosubstances into the
culture medium. Carbohydrate residues detectable by
immunohistochemistry and lectin histochemistry are
also detected in the cytoplasm of cultured biliary epithelial cells. Transmission electron microscopy shows that
the growth and spread of epithelia on the supporting
matrices predominantly form a single layer of cuboidal
or low columnar cell epithelial cells (Fig.4B). This layer
gradually acquires cell polarity towards the culture
medium and towards the culture dish, respectively.
These patterns are similar to those of the in vivo
gallbladder epithelial layer.
By transmission and scanning electron microscopy,
cultured epithelial cells show variable amounts of
surface microvilli, and a few organelles such as mitochondria and Golgi apparatus are seen in cytoplasm.
There are variably developed intercellular adhesive
devices and intercellular digitations between neighboring cells. These findings suggest the development of cell
polarity.
Occasionally it has been noted that monolayer cell
cultures of normal human gallbladder do not reach
confluency (Kumar and Jordan 1986; Purdum et al.,
1993). Interestingly, Purdum et al. (1993) observed that
well-differentiated carcinoma-derived gallbladder epithelial cells easily formed a confluent monolayer and
acquired cellular characteristics and functions similar
to those of the normal cells, namely apical orientation
and predominantly monolayer growth with cellular
interdigitations. However, they noted that timing was
critical for obtaining preparations composed mainly of
monolayers suitable for variable experimentation. If
78
Y. NAKANUMA ET AL.
overgrowth is allowed to develop, the cells tend to
head-up or pile up, presumably reflecting their carcinomatous origin.
Advantages, and Shortcomings and Applications. Monolayer cell culture is an easy cultivation
method for examining the characteristics of biliary
epithelium.
Quantitative assessment of the characteristics of
cultured cells and their alterations with the in vivo
pathology is possible. In addition, measuring the membrane permeability to water and electrolytes, secretory
activities and the amount of substances secreted in the
culture media is also possible (Lu et al, 1988; Okumura
et al., 1988; Purdum et al., 1993; Tilvis et al., 1982).
Furthermore, in vitro studies of immunological and
biological characteristics, such as expression of adhesion molecules and their ligands or receptors, can be
performed by adding chemical mediators and proinflammatory cytokines, like interferon or interleukin-2 to the
medium (Auth et al., 1933; Joplin et al., 1990). The
direct or indirect effects of carcinogenic substances on
the biliary epithelium can also be evaluated using this
system (Lu et al., 1989).
It is also possible to multiply the number of pure
gallbladder epithelial cells for biochemical and immunological studies of adequate samples. For example, several passages of explant culture on collagen gel forming
pure epithelial sheets treated with collagenase can
provide a great number of isolated cells (McAteer et al.,
1988). Other approaches with monolayer cell culture
yielding pure epithelial cells are also possible.
Finally, gallbladder mucosal epithelial cells offer a
unique model for the study of epithelial cells in general
(Oda et al., 1991).
As to the shortcomings, cultured biliary epithelial
cells tend to become detached after long-term culture,
particularly after more than seven passages. However,
this problem depends on the conditions of the cell
culture, particularly on the substances used to coat the
culture vessels and on the culture medium. In addition,
the biological and morphologic features characteristic
of the gallbladder epithelium are gradually vanishing
with the increased number of passages. Therefore there
are only few applications of monolayer cell cultures for
the study of the biological characteristics of gallbladder
epithelium. However, addition of biologically active
substances to the culture medium and the coating of
culture vessels with several extracellular substances
can improve the culture conditions so that the cells at
least partly regain their original characteristics.
Three-Dimensional Cell Culture
in Collagen Gel Matrix
This culture system was first established by Yang et
al. (1979, 1980) using mammary carcinoma tissue. This
method is needed for analyzing three-dimensional
growth of cultured cells and stereotypical changes,
namely of cell polarity, under several culture conditions
(Berdichevsky et al., 1994). It has been applied to
several other organs such as pulmonary alveolar epithelium, thyroid follicular epithelium and adipose tissue
(Sugihara et al., 1993; Toda et al., 1990). Our group also
applied this method to the cultivation of isolated rabbit
gallbladder epithelial cells for up to 6 weeks (Kawamura
et al., 1989; Yoshida et al., 1995). In this system,
individually isolated epithelial cells or cell clumps start
to proliferate while elongating and branching eventually forming multicellular cysts, ramified tubes or other
organoid structures covered by one or more cuboidal or
low columnar epithelial cells layers. Secretion of several substances into the cystic or tubular lumens by the
epithelial cells is observed. This methodology may
provide important information on physiological and
pathological conditions in the gallbladder.
Methods. The collagen gel matrix is usually supplemented with a modified Eagle’s minimum medium,
fetal calf serum, hormones, growth factors, and antibiotics, in order to promote sustained growth of normal or
cancerous gallbladder epithelial cells in three-dimensional culture.
In our studies, the isolated epithelial cells or epithelial cell clumps were dispersed and immersed in a fluid
collagen gel matrix (0.3% acid soluble collagen gel (type
I collagen:fetal calf serum:concentrated William’s medium (7.5:1.5:1) containing EGF). This collagen fluid in
which cells were embedded was gently planted on
plastic culture bottles which had been previously coated
with solidified collagen gel. This fluid soon became
gelatinous. Thereafter the cultures were covered with
William’s medium E supplemented with fetal calf serum, penicillin G, streptomycin, and EGF, which was
changed every 2 or 3 days according to the experimental design. Culture was maintained at 37oC in humid
air containing CO2 for 6 weeks or more. The growth and
spread or cyst formation of the cultured epithelial cells
in collagen gel was repeatedly observed by phasecontrast microscopy (Fig. 5). Their morphologic changes
can be followed by repeated examination as in the
monolayer cell culture method.
Growth and Phenotypic and Morphologic Differentiation. Cultured cells in collagen gel show increasing changes between the initial stage of culture and the
formation of multicellular cysts lined by biliary epithelial cells with gradual acquisition of many in vivo
characteristics.
Time sequential morphological changes of isolated
and cultured epithelial cells in collagen gel can be
comprehensively categorized into three phases: loss of
cell polarity (up to 12 hours), early reestablishment of
cell polarity with proliferation and/or conglomeration
of epithelial cells (12–24 hours), and reestablishment of
cell polarity with formation of multicellular cysts (after
24 hours)(Kawamura et al., 1989; Yoshida et al., 1995).
The loss of cell polarity was characterized by the loss
of distribution of organelles and by the transient appearance of autophagic vacuoles. While many round epithelial cells were present singly in collagen gel, there were
also considerable numbers of clumps of two to several
epithelial cells. These cells contained cytoplasmic vacuoles of various sizes, which were negative for PAS,
mucicarmine, HID, and AB (pH 2.5). They were autophagic vacuoles (Gautam et al., 1987) and they failed to
bind to the five lectins noted above and disappear by 24
hours.
Ultrastructurally, the cell volume of the cultured
cells and the numbers of cellular organelles were
considerably reduced. The cellular adhesion apparatus
was not clearly detected in the singly isolated cells;
however, clumps composed of several cells retained this
apparatus. A few mucin droplets in the cytoplasm, the
CULTURE OF GALLBLADDER EPITHELIAL CELLS
foci of microvilli, and several infoldings on the cell
surfaces were occasionally identifiable. In most cells,
the remaining cell organelles were poorly polarized,
and the lateral infoldings were also considerably reduced. Instead, autophagic vacuoles of various sizes
which were positive for acid phosphatase (autophagic
vacuoles) were frequently found. These vacuoles were
positive for acid phosphatase.
The early reestablishment of cell polarity with an
increased number of clumps of more than four cells was
characterized by the appearance of mucin dots (Fig.6A).
One or more small dots positive for PAS, AB (pH 2.5),
mucicarmine and HID were present in these cell clumps.
Some of these dots showed variable binding to Con A,
WGA, and DBA after 24 hours, and binding to PSA and
SBA was shown after 24 hours. Ultrastructurally, the
mucin dots contained small lumens sealed by a few
epithelial cells or by cytoplasmic inclusions, both being
covered with microvilli and containing an amorphous
substance (Fig.7A). Sometimes, a few mucin droplets
and a centriole were seen in the vicinity of the small
lumens or cytoplasmic inclusions. Some cytoplasmic
inclusions were found to be continuous with the small
lumens, appearing as diverticula. These inclusions or
lumens increased in size, and small lumens consisting
of several cells became enlarged, forming multicellular
cysts (see below). The microvilli of these inclusions and
microcysts were ultrastructurally positive for DBA,
WGA, SBA, and ruthenium red.
In the late reestablishment of cell polarity, the multicellular clumps began to organize into distinct multicellular cysts (Figs.5A and 6B,C). Their luminal borders
and contents were positive for PAS, AB (pH 2.5), HID,
and mucicarmine. The lining epithelia of these cysts
were composed of one or two layers of cuboidal epithelial cells, while there were secondary mucin dots within
the cytoplasm of the epithelial cell layers. The luminal
borders of almost all the multicellular cysts showed
strong binding to Con A, PSA, WGA, DBA, and SBA.
Mucin ‘‘vacuoles’’ in the epithelial layer of the cysts and
in the epithelial cell clumps showed similar staining
and lectin binding patterns.
Ultrastructurally, epithelial cells of the multicellular
cysts began to show many features of epithelial cell
polarity: dense luminal microvilli, mucin droplets in
the subluminal cytoplasm, Golgi apparatus, and abundant mitochondria and rough endoplasmic reticulum in
the supranuclear cytoplasm (Fig.7B). The formation of
junctional complexes between neighboring cells in these
cysts was clear. The microvilli of these epithelial cells
were positive for DBA, WGA, SBA, and ruthenium red.
After 72 hours, the multicellular cysts were still
enlarging with mucinous substances filling the lumen.
The structure of the cells in these cysts became more
and more similar to those of in vivo gallbladder epithelial cells. The phenotypic and structural features of the
cells in these multicellular cysts were active production
and secretion of mucinous substances via the apical
surfaces into their cystic lumens. No basal lamina was
found around the basal side of the multicellular cysts of
pure gallbladder epithelial cells.
Advantages, Shortcomings and Applications. As
to the advantages, three-dimensional outgrowth and
morphologic changes of isolated cells and of their
polarity (loss and restoration) could be observed and
79
followed sequentially. Moreover, this method is reproducible and is relatively easy to perform.
Addition of biological active substances such as growth
factors or proinflammatory cytokines produces morphological and phenotypical changes in these cells.
Co-culture with other mesenchymal cells or immunocompetent cells from patients or addition of proinflammatory cytokines such as interleukin-2 or interferon-g
to the culture media may be applied to investigate the
pathogenesis of immune-mediated biliary tract diseases (Auth et al., 1993).
Such cultured gallbladder mucosal epithelial cells
exhibit parameters similar to those of native gallbladder epithelium and may offer a convenient new research tool for the study of the pathophysiology of
gallstone formation and of other gallbladder disease.
Loss and restoration of epithelial cell polarity are
evaluable using this model (Yoshida et al., 1996).
As to the shortcomings, quantitative assessment of
cultured cells and biochemical analysis of secreted
substances from cultured cells are rather difficult or
impossible in this system.
Modifications of Monolayer and
Three-Dimensional Cell Culture of Gallbladder
Epithelial Cells by Addition of Biologically
Active Substances
Recent studies indicate that many different agents
are involved in the stimulation or inhibition of in vitro
growth and morphogenesis of normal epithelial cells.
Evidence is accumulating that agents such as fetal calf
serum, insulin, cortisol, prostaglandin E1 and growth
factors also act by regulating the increased rate of
initiation of DNA synthesis (Berdichevsky et al., 1994).
It is possible to clarify which substance(s) or factor(s)
influence the outgrowth and morphogenesis of cultured
gallbladder epithelial cells. Receptor(s) on the cultured
cells for these agents and morphogens could determine
their effects.
Effects of Growth Factors as a Morphogen. Yet
there are a number of growth factors, like transforming
growth factor b1 (TGF-b1) and hepatocyte growth
factor (HGF), and of adhesion molecules like a2b1,
a3b1, and a6b4 integrins, that act as morphogens
(Berdichevsky et al., 1994; Yoshida et al., 1996). For
example, the addition of TGF-b1, which is able to
influence the proliferation and maturation of epithelial
cells, caused the epithelial cells in the multicellular
cysts cultured in the collagen gel to become taller and
more multilayered (Fig.8A). Their nuclei became more
or less hyperchromatic and their cytoplasm was more
acidophilic, whereas the mucin dots or vacuoles in the
epithelial layer became almost unrecognizable. Ultrastructurally, these cells were acquiring characteristics
suggestive of greater maturation than the controls
without TGF-b1, and their polarization closely resembled that of the normal cells in vivo; the tight
intercellular infoldings and cellular attachment devices, such as tight junctions, intermediate junctions,
and desmosomes, were well developed. Mitochondria
and rough endoplasmic reticulum were abundant. Microvilli were dense and pinocytic vesicles or invaginations were increased beneath the microvilli. The luminal surfaces of the multicellular cysts after the addition
80
Y. NAKANUMA ET AL.
Fig. 6. Three-dimensional culture of rabbit gallbladder epithelial
cells in collagen gel. A: One day after being cultured, epithelial cell
clump shows PAS-negative vacuoles (small arrows) and positive dot
(large arrow). B: Two days after being cultured, PAS-positive vacuoles
(arrows) increase in size and become multicellular cysts. C: Three days
after being cultured, a multicellular cyst is formed (arrow). A, B, PAS
staining after diastase digestion and hematoxylin; C, H&E staining.
Fig. 7. Ultrastructures of the rabbit gallbladder epithelial cells
cultured in collagen gel. A: One day after being cultured, there is a
small intracytoplasmic microcyst covered by microvilli (C), 34,000. B:
Four days after being cultured, the wall of the multicellular cyst is
composed of cuboidal or low columnar epithelial cells showing dense
microvilli on the luminal surface (L) and basally situated nuclei (N),
33,000.
of TGF-b1 showed the same mucin-histochemical reactions and lectin-binding patterns as the nontreated
multicellular cysts.
HGF is also known to cause branching and tree-like
growing patterns in such cultured biliary epithelial
cells (Nakamura, 1994), though it is unclear whether
this property is applicable in vivo in any species.
Effects of Cytochalasin B (CB) on the Multicellular Cysts. After the addition of CB which disrupts
intracellular microfilaments (Phillips et al., 1983) in
the three-dimensional culture of gallbladder epithelial
cells (Yoshida et al., 1995), the multicellular cysts
disintegrated and within 48 hours some epithelial cells
became more or less separated (Fig.8B). These epithe-
CULTURE OF GALLBLADDER EPITHELIAL CELLS
81
Fig. 8. Multicellular cyst of rabbit gallbladder epithelial cells
cultured in collagen gel for three days. A: After addition of transforming growth factor B1, the epithelial cells of the cyst wall become taller
and resemble more the in vivo gallbladder epithelium. H&E staining.
B: After addition of cytochalasin B, the epithelial cells consisting of the
cyst become disintegrated and PAS-positive and -negative vacuoles
appear in their cytoplasm. C: Ultrastructure of multicellular cyst after
addition of cytochalasin B show many small intracytoplasmic cysts
covered by microvilli (A) and also those covered by long villi resembling intercellular lateral infoldings (B). L, cystic lumen. 34,500. D:
Ultrastructure of multicellular cyst after addition of cytochalasin B,
showing of intracytoplasmic covered by microvilli (A). There is a thick
zone of amorphous substances around these intracytoplasmic cysts.
Arrows denote intercellular space covered by infoldings, 38,000 from
Yoshida, K., Katayanagi, K., Kawamura, Y., Saito, K., and Nakanuma,
Y. (1996) Re-establishment of rabbit gallbladder epithelial cells in
collagen gel cultures and their alterations by cytochalasin B and
transforming growth factor beta-1: A morphologic study. Pathol. Res.
Pract. 192:634–645.
lial cells also showed a vacuolated cytoplasm after 48
hours. About half of these vacuoles were mucinpositive, while the remainder were not. Ultrastructurally, the intercellular lateral spaces were wide, and only
those cellular adhesion structures facing the lumen
were visible. Some of the vacuoles were covered by
dense microvilli, resembling those of the luminal surface, and were surrounded by a thick amorphous zone
containing glycocalyceal substances, while other vacuoles were covered by a few longer and more slender
infoldings resembling the lateral cell surface as if they
were empty (Fig.8C,D). The former correspond to mucin-
82
Y. NAKANUMA ET AL.
positive vacuoles and the latter to mucin- negative
vacuoles.
Interestingly, the mucin-positive vacuoles resembled
the microvillous inclusions of enterocytes in microvillous inclusion disease (MID)(Bell et al., 1991; Carruthers et al., 1986; Rhoads et al., 1991), in which
poorly developed surface brush border microvilli and
the formation of intracytoplasmic inclusions lined by
microvilli are found. MID is also known to involve the
gallbladder (Rhoads et al., 1991). In a preliminary
experiment, similar cytoplasmic inclusions were reproduced in cultured human fetal intestinal epithelium,
using CB to disrupt the microfilaments. Carruthers et
al. (1986) speculated that MID may be attributable to a
defect in the cytoskeletal elements of the brush border.
It seems likely that microfilaments are essential for the
maintenance of the microvilli of the brush border of
cultured biliary epithelial cells, and the impairment of
these microfilaments may lead to the formation of
microvillous or related cytoplasmic inclusions.
Addition of Cytotoxic Agents. In a preliminary
experimental study we showed that several cytotoxic
agents may cause cytopathic damage in gallbladder
epithelial cell cultured in collagen gel (Kawamura et
al., 1989). This culture procedure might be valuable as
an experimental model for clinical conditions with
important epithelial damage. For example, gallbladder
epithelial cells cultured in the collagen gel for 1 week
could be exposed to mitomycin C for 48 hours, then and
the epithelial cells could be histologically examined.
Mitomycin C is an alkylating agent (Ito, 1979). At
concentrations of 100 and 1,000 µg/ml mitomycin C,
almost all epithelial cells lining the cysts became
necrotic and dropped into the cystic cavities. Almost all
cysts cultured in the collagen gel showed similar
changes.
LIVING TISSUE (ORGAN) CULTURE
OF GALLBLADDER
Tissue cultures (also called organ cultures) offer a
convenient and reliable source of living tissue for
adequate experimentation. The use of culture system is
widely accepted and has been applied to the biological,
physiological, developmental and pathological analysis
of the gastrointestinal and urogenital tracts (Menard et
al., 1993; Okumura et al., 1988). Similar systems have
been applied to human and animal gallbladders. They
allow longer time periods during which steady-state
conditions can be maintained (Gall and Bathal, 1987;
Grant and Billings, 1977; Johzaki et al., 1989; Kumar
and Jordan, 1986; Okumura et al., 1988). In studies of
the gallbladder, tissue culture is neither popular nor
satisfactory when compared to cell culture. One of the
reasons is that degenerative changes are likely to occur
in tissue-cultured explants of the gallbladder.
However, there have been several interesting reports
on tissue culture of the biliary tract, including the
gallbladder (Gall and Bathal, 1987; Grant and Billing,
1977; Kumar and Jordan, 1986; Okumura et al., 1988).
In this review, the most representative experimental
models will be examined.
For the preparation of explants or fragments of the
gallbladder for tissue culture, the gallbladders are
aseptically opened and washed gently several times
with iced 0.15 M NaCl solution or phosphate buffered
saline. Then, adequate samples of the gallbladder
showing no or minimal necroinflammatory changes are
selected and placed in iced culture medium (see below)
and then sectioned into small pieces (explants).
Okamura’s Model
For the culture media, L-15 and/or Connaught Medical Research Laboratories 1066 with added fetal calf
serum, growth factors and antibiotics are used (Okumura et al., 1988). The explants are gently plated on
Gelform gelatin sponges which are previously placed on
the culture dishes with the mucosal surface upwards.
The culture dishes are kept in a shaking incubator in a
5% CO2 atmosphere and the culture media are changed
twice a week.
In these conditions the gallbladder epithelial cells
grew and spread in all directions from the explants; the
epithelium was generally flat 7 days after being cultured, then became cuboidal or columnar. Growth also
extended into the gelatin sponge. However, regressive
changes such as cytoplasmic vacuoles were also detected 14 days after the beginning of culture. Okamura
et al. (1988) subsequently transplanted these cultured
tissues into the subcutaneous tissue of nude mice. The
viability and proliferative activities of these transplanted tissues could be maintained for 29 weeks.
Okamura et al. concluded that their method may be
applicable for studies on experimental carcinogenesis.
Elhamady’s Model
Elhamady et al. (1983) reported a tissue culture
model of guinea pig gallbladder mucosa that could be
maintained up to 2 weeks. The gallbladder of other
experimental animals may also be used. The gallbladder was placed in sterile medium, was opened and
vigorously rinsed in a series of culture dishes containing one or other of the culture media described below.
After the last rinse, the gallbladder was minced to 1
mm2 pieces with a scalpel. The pieces were placed in
tissue culture dishes covered by 2 ml complete culture
medium and with a coverslip. The dishes were placed in
plastic containers, gassed with 5% CO2, sealed with
insulating tape and placed in an incubator at 37oC.
The following five different culture media were tried:
Dulbecco’s modified Eagle’s medium, Eagle’s minimum
essential medium with Hank’s salts, NCTC 135 medium, Medium 199, and Ham’s F12. In each instance,
penicillin, streptomycin and glutamine were added to
the medium.
There was morphological evidence of satisfactory
survival of the gallbladder epithelium for up to 14 days
of culture in all of the media used without marked
morphological difference between the media. Only the
surface epithelium survived as tall columnar cells and
some thinner epithelium appeared to be migrating and
growing over the surface of the underlying connective
tissue after 2 days. The areas of outgrowth were mainly
composed of epithelial-like cells with only a small
percentage of fibroblasts.
Elhamady et al. (1983) observed outgrowth between
days 1 and 2. At the end of week 2, however, the cells
underwent dedifferentiation. The overall surface structure became generally simplified: the microvilli became
sparse with a few very long ones left between bald
areas. This aspect was clearly put in evidence by
CULTURE OF GALLBLADDER EPITHELIAL CELLS
scanning electron microscopic (SEM). The glycocalyx
was observed until day 14.
The SEM confirmed that new cells colonized the free
surface of the explant. Transmission electron microscopy showed good preservation of the original tall
epithelial cells for up to 2 weeks. The new migrating
cells were flatter than before but they kept the morphologic features of the columnar cells. Secretory granules
were absent after day 1, but increased amounts of
glycogen and lipids began to appear later.
According to our preliminary study of tissue culture
using human or mouse explants with a slight modification of Elhamady’s model, the epithelial cells grew and
spread on collagen gel (Fig.3A). These spreading cells
near the explant were taller and resembled the epithelial cells on the microexplants, while the epithelial cells
at the front of spreading epithelial cell sheet were flat.
On the contrary, the stromal cells began to grow mainly
into the collagen gel (Fig.3B).
Other Models
To date, short-term incubation of tissue fragments
from the biliary tract including the gallbladder has
been used in the experimental study of physiological
aspects of the gallbladder. Such studies require demonstration of physiologic functions similar to those found
in the native organ, but the proliferation and redifferentiation of gallbladder epithelial cells are not mandatory.
For example, Hosono (1950) cultured fragments of
guinea pig gallbladder mucosa in blood plasma or
Ringer’s solution. Blom and Helander (1977) used
pieces of rabbit gallbladder which were incubated in
vitro for 1 hour in Ringer’s solution at 37oC and studied
correlations of the ultrastructures and water or electrolytes transport and demonstrated the pericellular channels in living rabbit gallbladder epithelium.
Wright and Dimond (1968) and Geralden and Rose
(1974) showed that 95% of the potential difference in
the intact organ is lost upon removal of the mucosa. In
vitro preparations of the gallbladder have significantly
advanced our knowledge of mucosal function, particularly with respect to both monovalent ion and divalent
ion transport. In terms of secretory functions, it is
well-recognized that the gallbladder epithelium secretes mucin and other glycoproteins, and in the past
decade it has been recognized that the epithelium also
secretes H ions. The latter contributes significantly to
acidification of bile during the concentration process.
The tissue culture method has certain inherent shortcomings. Such studies necessarily involve anesthesia,
surgery, manipulation, and care of animals at a relatively high cost. In addition, these experiments require
further manipulation and diligent attention to maintain mucosal cell viability.
Advantages, Shortcomings and Applications
of Tissue Culture
Short-term quantitative observation of biliary epithelial cells is possible using this tissue culture model. In
particular, a more physiological approach is possible
with this system than with the monolayer or threedimensional cell culture. However, the long-term maintenance of this tissue culture of the gallbladder is
generally uneasy and limited to 1 week.
83
Although Elhamady et al. (1983) failed to detect
significant differences in epithelial growth by morphological observation after changing the culture medium
or adding several hormones like insulin and hydrocortisone, further studies with growth factors could be
interesting. Adhesion molecules and proinflammatory
cytokines which have been recently discovered could
also be used in this model to determine whether they
significantly affect the morphology of the gallbladder
mucosa.
OVERVIEW
Both in vivo and in vitro experiments and studies
have not only advantages but also shortcomings. The
use of monlayer and three-dimensional cell culture as
well as tissue culture systems has become an accepted
method in hepatobiliary research namely for investigating the gallbladder. For cultivation, several adequate
models of preparing and isolating epithelial cells from
human and animal gallbladders are available and are
widely used. Monolayer and three-dimensional cell
culture have their own advantages and shortcomings.
Considering the various models of isolating gallbladder
epithelial cells and the variety of culture methods, the
aim and design of the experiment should guide the
selection of the most appropriate isolation and cultivation technique. Appropriate selection will facilitate
analysis of the physiology of the gallbladder as well as
the pathophysiology and pathogenesis of gallbladder
disease including gallstone disease.
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