[CANCER RESEARCH 36, 1003-1010, March 1976]
Long-Term Organ Culture of Human Bronchial Epithelium'
Lucy A. Barrett, Elizabeth
M. McDowell, Arthur L. Frank, Curtis C. Harris, and Benjamin F. Trump2
Cellular Pathobiology Laboratory, Department of Pathology, University of Maryland, Baltimore, Maryland 21201 (L. A. B., E. M. McD., B. F. TI, and Lung
Cancer Branch (A. L. F.J and Human Tissue Studies Section (C. C. H.), National Cancer Institute, Bethesda, Maryland 20014
SUMMARY
MATERIALS AND METHODS
Human bronchial epithelium has been maintained in or
gan culture in serum-supplemented medium for 4 months.
After 4 to 6 weeks in culture, various changes in morphol
ogy were apparent. There was an increase in autophagic
vacuoles in mucous, ciliated, and basal cells, a reduction in
the height of the columnar cells, a decrease in the number
of goblet mucous cells, and an increase in cells with small
mucous granules. After 3 months in culture, the basal lam
ma was frequently covered by 2 or 3 layers of epithelial cells
consisting of nonkeratinizing squamous cells with short
microvilli and small mucous granules. Less frequently, kera
tinizing squamous cells were seen.
Differentiated epithelium incorporated precu rsors into
macromolecules in serum-free medium, supplemented with
vitamin A, at 1 week of culture. These explants exhibited
changed epithelium by 2 weeks, similar to that described for
epithelium in serum-supplemented medium after 4 to 6
weeks.
Organ Culture. The bronchus was dissected from the
lung by aseptic technique and placed in cold Leibowitz
Medium L-15 (11) for transportation to the organ culture
laboratory. The bronchus was further trimmed of lung tissue
and cut into flat 2-sq cm pieces. In all cases, bronchus that
was not grossly involved with tumor was used. Pieces of
bronchus were placed, with the epithelium uppermost, into
60-mm plastic Petri dishes. The medium used was CMRL
1066 (Grand Island Biological Co., Grand Island, N. V.) con
taming 0.1 @g
hydrocortisone hemisuccinate per ml (Schwarz/
Mann, Rockville, Md.), 1 j.@gbovine recrystallized insulin per
ml (Schwarz/Mann), 100 units penicillin per ml, 100 @g
streptomycin per ml (Grand Island Biological), 1 @g
ampho
tericin B per ml (Grand Island Biological), 2 mM L-glutamine
(Grand Island Biological) and 5% heat-inactivated (30 mm at
56°)fetal calf serum (Grand Island Biological). In selected
short-term culture experiments, 0.1 @g
j3-retinyl acetate per
ml (Hoffman-LaRoche, Nutley, N. J.) was added, and the
inactivated fetal calf serum and amphotericin B were
omitted. The culture dishes were placed in a controlled
atmosphere chamber (BelIco Glass Co., Vineland, N. J.)
INTRODUCTION
This paper represents part of a larger study designed to
study the effects of carcinogens on human bronchial epi
thelium in vivo and in vitro (6, 7, 23). This and the following
paper (6) demonstrate the potential usefulness of an in vitro
method in elucidating the biological and metabolic effects
of chemicals on human bronchial epithelium in carcinogen
esis studies. In this paper we describe conditions for the
organ culture of human bronchial epithelium obtained from
either surgically resected lungs or from “immediate
autop
sies―
(24). The results indicate that human bronchial epithe
hum can be cultured for at least 4 months and that the
epithelial cells can be studied with respect to carcinogen
metabolism.
Tracheas from hamsters (15), rats (5, 8, 17), and other
animals (25) have been used extensively in organ culture.
Lasnitzki (9) is credited with being the first to maintain
human respiratory epithelium in organ culture. Since then,
others (3, 4, 13, 16, 20) have maintained human bronchus in
organ culture for up to 21 days in vitro. Human bronchial
epithelial cells have been cultured for 4 months (21).
â€T̃his work was supported
in part by Contract
NO 1 CP 43237 from the
National Cancer Institute. This is Contribution 306 from the Cellular Patho
biology Laboratory.
2Towhom
requests
forreprints
should
besent.
Received July 14, 1975; accepted November 24, 1975.
MARCH1976
(Fig.
1) and
gassed
with
45% 02, 50%
N2, and
5% CO2 for
approximately 5 mm. The chamber was then placed on a
rocker platform which rocked at 10 cycles/mm causing the
media to flow intermittently over the epithelial surface. The
explants were incubated at 36.5°.The media and atmos
phere were replaced 3 times each week.
Electron Microscopy. Explants were fixed in 4% glutaral
dehyde buffered with 0.1 M sodium cacodylate (pH 7.4) at
room temperature for 4 to 6 hr just prior to and after inter
vals of culture in medium with serum. In most cases, the
samples were sliced perpendicular to the epithelium, prior
to posthxation with 0504 using a Smith-Farquhar tissue
chopper so as to give sections that were 100 @.tm
thick.
Subsequent to glutaraldehyde fixation, the tissue sections
were washed in 0.1 M sodium cacodylate buffer, postfixed in
1% 0504 buffered with 0.1 Ms-collidine, and stained en bloc
with uranyl acetate. The tissues were dehydrated in a
graded ethanol series and embedded in Epon 812. Areas of
epithelium were chosen for thin sectioning by examining
0.5-jim sections stained with toluidine blue. Thin sections
were double-stained on the grids with uranyl magnesium
acetate and lead citrate and examined in a JEOL 100B
electron microscope.
Radioautography. After 1 week in culture in a serum-free
medium, bronchial pieces were incubated for 6 hr with
either: (a) [3H]uridine (15 pCi/mI; specific activity, 27.8 Cu
mmole; New England Nuclear, Boston, Mass.); (b)
1003
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L. A. Barrett et a!.
[3H]leucine (15 MCi/mI; specific activity, 58.2 Cu/mmole;
New England Nuclear); or (c) [3Hjthymidine (50 @tCi/ml;
specific activity, 20 Cu/mmole; New England Nuclear). Fol
lowing fixation in 1.33% 0504 buffered by 0.1 M s-collidine,
pH 7.4, and embedding
in Epon,
sections
1 @tmthick
were
coated with NTB-2 photographic emulsion, exposed in the
dark for 2 weeks, and autoradiograms were prepared as
previously described (2).
Histochemistry. Histochemical demonstrations of cell
surface, Golgi apparatus, and lysosomes were made on
bronchus cultured from 3 patients, with and without lung
neoplasms, at varying time intervals of culture, ranging from
4 days to 16 weeks.
All procedures were performed on tissues fixed in either
2% formaldehyde-1% glutaraldehyde or in 4% formalde
hyde-1% glutaraldehyde prepared with 200 mOsmole phos
phate buffer (12). Following fixation for 3 hr at 4°,the bron
chial epithelium was removed from the underlying connec
tive tissue and washed overnight in 0.2 M cacodylate buffer
containing 7.5% sucrose. Small pieces of epithelium were
incubated in the appropriate histochemical reaction mix
tures. Acidic groups of the glycocalyx were demonstrated,
using the Rinehart and AbuI-Haj dialyzed iron method (19)
as advocated by Wetzel et a!. (26). Following incubation, the
tissues were washed in 0.2 M cacodylate-sucrose buffer and
postfixed in 1% 0504 buffered with 0.1 M s-collidine. For
demonstration of acid phosphatase activity, tissues, fixed as
above, were washed in 0.2 M cacodylate-sucrose buffer
overnight at 4°and incubated for 1 hr at 37°in a modified
Gomori lead medium (14). Following incubation, the tissues
were washed 3 times in 0.05 M acetate buffer, briefly rinsed
in 0.5% (NH4)@S,
washed in cacodylate buffer, and postfixed
in 1% 0504 as described above. The epithelium was stained
en bloc in uranyl acetate for 1 hr. Tissues stained for acidic
groups and acid phosphatase activity were dehydrated and
embedded in Epon. Thin sections mounted on carbon-sta
bilized, collodion-coated copper grids were double-stained
with uranyl acetate and lead citrate before examination in
the electron microscope. Reactive sites presumed to be
glycoproteins were demonstrated using PTA3 (18). Tissues
were fixed as above, washed in 0.2 M cacodylate-sucrose
buffer, and embedded in glycolmethyacrylate (10). Thin sec
tions, mounted on carbon-coated copper grids were stained
by flotation for 15 mm on 3% PTA at pH 1.5 (0.2 N HCI). They
were then washed in distilled water, dried, and examined in
the electron microscope.
RESULTS
Morphology of Bronchial Epithelium Fixed at Zero Time.
For the most part, the epithelium consisted of a pseudo
stratified columnar epithelium with goblet cells, ciliated
cells, basal cells, small granule or neurosecretory cells, and
cells with small mucous granules that were tall and colum
nar with a dark cytoplasm and short microvilli; they pos
sessed small mucous granules (Fig. 2). These will be termed
SMGC.
As described by Auerbach et a!. (1), areas of abnormal
3 The abbreviations
mucous granule cells.
1004
used
are: PTA, phosphotungstic
acid;
SMGC,
small
epithelium were sometimes encountered in the specimens
fixed prior to culture, referred to as the zero time samples.
These areas showed focal abnormalities, including squa
mous metaplasia and dysplasia in scattered areas. The most
common types of focal abnormality consisted of additional
layers of basal or intermediate cells, decrease in goblet
cells, shortening of columnar cells, both ciliated and mu
cous types, and at times loss of cilia and squamous meta
plasia, both keratinizing and nonkeratinizing.
The age, sex, and smoking habits of the patients and the
type of tumor seen in the cases used in this study are shown
in Table 1.
Organ Culture. The normal appearance of the epithelium
was maintained for only 1 week when cultured in serum-free
medium. After 2 weeks, there was a decrease in number and
height of ciliated cells and a decrease in goblet cells, but an
increase in number of dark SMGC, which were cuboidal or
squamous and lined the lumen. Because of this, culture was
not made for longer than 2 weeks in serum-free medium.
After 1 week, incorporation of precursors into macromole
cules could be demonstrated. This included incorporation
of labeled uridine (Fig. 3), leucine (Fig. 4), and thymidine
(Figs. 5 and 6). Labeling was seen in various cells of the
epithelium. Labeled thymidine incorporation occurred in
basal cells and in SMGC (Fig. 5) but was not observed in
ciliated cells. Sometimes thymidine incorporation was seen
in many of the cells at the edge of the culture which ap
peared to be growing around the edge of the explants
(Fig. 6).
Explants cultured in medium containing serum maintained
differentiation longer and the epithelium consisted of cil
iated columnar cells, with a reduced number of goblet cells,
up to 1 month. This was observed most consistently in
explants from patients exhibiting minimally changed areas
at the start of culture (Fig. 7). As seen in zero-time samples,
microvilli and luminal surface of the apical membrane of the
goblet cells were always coated more thickly with colloidal
iron than either the cilia or the microvilli of the ciliated cells
(Fig. 7, inset). Early during culture there was an increase in
the number and size of autophagic vacuoles in some mu
cous, ciliated, and basal cells (Fig. 8). Acid phosphatase
activity was demonstrated in these vacuoles (Fig. 9). During
the 1st 2 months of culture, there tended to be a reduction
in the height of columnar cells (Fig. 8, inset). The Golgi
apparatus was very prominent in these cuboidal cells and
the lamellae of the maturing face were stained with PTA; cell
surface, mucous granules, and lysosomes were also stained
(Fig. 10). Cuboidal SMGC with short microvilli and a promi
nent glycocalyx (Fig. 11, inset) were frequently encountered
after 2 months in culture, and mucus was present only as
small granules at the luminal surface (Fig. 11). Ciliated and
goblet cells were rare, although in 2 cases they were rela
tively numerous after 3 months. Cuboidal cells containing
both cilia and mucus were seen more frequently than in
zero-time samples (Fig. 12).
After 3 months in culture, the basal lamina was often
covered by 2 or 3 layers of cells. The surface cells in such
areas consisted mainly of flattened nonkeratinizing squa
mous cells with short microvilli and small mucous droplets.
A few of these cells showed early keratinization. The cyto
plasm contained mitochondria, profiles of endoplasmic re
CANCERRESEARCHVOL.36
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Organ Culture of Human Bronchial Epithelium
Table 1
Age, sex, diagnoses,and smoking histories of patients used in this study
Case Age (yr)
Sex
20 cigars/day for 15 yr
30 cigarettes/day for 30 yr
2
48
M
SCC
3
58
M
SCC
4
76
M
AC
5
6
52
52
F
M
14
15
17
21
57
48
59
64
M
M
M
M
AC
Chronic
inflammation
and fibrosis
AC
Abscesses and fibrosis
LCC
AC
23
64
M
5CC
24
25
63
63
M
M
LCC
AC
27
51
M
AC
29
17
M
HI
30
53
M
AC
31
32
37
61
39
54
M
M
5CC
Headtrauma
M
5CC
44
60
M
AC
46
56
M
LCC
a 5CC,
squamous
cell
Smoking habitsb
Diagnosisa
40 cigarettes/day
5 cigarettes/day
for 50 yr
30 cigarettes/day
20 cigarettes/day for 30 yr
10 cigarettes/day for manyyr
40 cigarettes/day for 40 yr
10 cigarettes/day
for 40 yr
60 cigarettes/day for 25 years;
nonsmokerfor last 5 years
20 cigarettes/day for 50 yr
40 cigarettes/day for manyyr
30 cigarettes/day for 12 yr;
nonsmokerfor last 12 years
No information
Smoked cigarettes for many
years
10 cigarettes/day for 50years
No information
60-80 cigarettes/day for many
years, nonsmoker for last 2
years
20 cigarettes/day for 46 yr
No information
carcinoma;
AC,
adenocarcinoma;
obtained
from
a retrospective
LCC,
large
cell
carcinoma,
HI,
head injury.
ii Smoking
habits
record in hospital
were
review
of the
patient's
medical
charts.
ticulum, and a very extensive Golgi apparatus (Fig. 14).
Secondary lysosomes and autophagic vacuoles were also
seen in these cells. Similar cells were seen at earlier time
intervals in some cultures. The small stubby microvilli at the
luminal surface had a prominent glycocalyx that was thickly
stained with colloidal iron (Fig. 13). The staining was similar
in degree to that seen on microvilli of differentiated goblet
cells (compare Fig. 7, inset, with Fig. 13).
Patchy areas of nonkeratinizing squamous cells at the
luminal surface were also seen very early in some cultures,
probably from loss of the ciliated and mucous cells due
either to mechanical removal or acute cell death. In these
cases, the few remaining basal cells probably divided and
formed a thin epithelial layer over the denuded basal lam
ma.
DISCUSSION
The present observations show that human bronchial epi
thelium can be maintained for prolonged periods of time in
organ bulture, with preservation of viability as evidenced by
ultrastructural characteristics and incorporation of labeled
precursors into macromolecules. Using the conditions de
scribed here, epithelium consistent with that seen at zero
time could be maintained in many cultures for between 1
and 2 months. After 2 months, however, conversion of the
epithelium into an abnormal type occurred with general loss
of goblet cells loss of ciliated cells, and appearance of
shorter surface cells, nonkeratinizing squamous or cuboidal
SMGC that had short microvilli and a prominent glycocalyx.
An important factor in the interpretation of the changes
that occur under these conditions is the nature of the epi
thelium at the beginning of the culture period. As is well
known from the work of Auerbach (1), the bronchial epithe
hum of human patients with smoking histories, especially
those with bronchogenic squamous cell carcinomas, show
variable numbers of abnormal areas including metaplasia,
dysplasia, and other changes. Although such changes were,
indeed, present in the starting material used in this study,
careful evaluation of all 50 cases so far selected indicated
that focal areas of abnormality were noted in about 10 to
20% of the area of the surface epithelium. The morphology
of the epithelium was variable throughout the 1st 2 months
of culture. This may have been due at least in part to the
variable state of the epithelium at the start of culture. After 2
months of culture, increasing occurrence of nonkeratiniz
ing (SMGC) and keratinizing squamous cells suggests that
the culture conditions, although maintaining viability after
that time, could not maintain normal differentiation. Al
though the mechanism of this morphological change is not
presently known, it is conceivable that it involves the vi
tamin A content (27) which, in the present study, only con
sisted of that present in the fetal calf serum added. Further
more, since the fetal calf serum was heat inactivated, this
would tend to further decrease the vitamin A content.
It is important to note, however, that the maintenance of
viable human epithelium in organ culture for 17 weeks and
of differentiated epithelium for 2 months is considerably
longer than has been previously reported, although others
MARCH 1976
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1005
L. A. Barrett et a!.
(21) have maintained explanted bronchial epithelial cells,
some with cilia, for 4 months without additional vitamin A.
Modifications of the culture medium such as adding vitamin
A could perhaps result in even better maintenance. The
principal changes occurring during the early time intervals
consisted of decreased numbers of goblet cells and in
creased numbers of autophagic vacuoles. The former is
thought to represent effective discharge of the mucus and
the latter is a common accompaniment of organ culture
(22).
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4. Crocker, T. T., O'Donnell, T., and Nunes, L. L Toxicity of Benzopyrene
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Fig. 1. Controlled atmosphere chamber used to culture bronchial samples in 45% 0,, 50% N,, arid 5% CO,.
Fig. 2. Zero-time human bronchial epithelium, Case 23, showing pseudostratified columnar epithelium with goblet cells, ciliated cells, basal cells, and
SMGC (at arrow). Toluidine blue, x 290.
Fig. 3. One-week culture, Case 2. Incorporation of [3H]uridmneinto the nuclei of various cell types. Toluidine blue, x 880.
Fig. 4. One-week culture, Case 2. Incorporation of [3Hjleucine into cytoplasmic macromolecules. Toluidine blue, x 880.
Fig. 5. One-week culture, Case 30. Incorporation of @H]thymidmne
into the nuclei of basal cells and SMGC. Toluidine blue, x 880.
Fig. 6. One-week culture, Case 30. The edge of a piece of bronchus showing the incorporation of @H]thymidmne
into the nuclei of cells growing around the
edge of the explant. Toluidine blue, x 320.
Fig. 7. Six-week culture, Case 17. Ciliated columnar cells showing excellent morphology after 6 weeks in organ culture. x 14,000. Inset, 4-week culture,
Case 44. Colloidal iron deposits stain the glycocalyx of a ciliated and goblet cell. The microvilli and luminal surface of the luminal membrane of the goblet cell
(large arrow) are coated more thickly than either the cilia or microvilli of the ciliated cell (small arrow). x 8,600.
Fig. 8. Ten-day culture, Case 17. Autophagic vacuoles containing myelin whorls are very numerous and the Golgi apparatus (G) is prominent in these
cuboldal cells. x 8,600. Inset, 6-week culture, Case 2. Columnar cells show a reduction in cell height. Note SMGC at arrow. Toluidine blue, x 600.
Fig. 9. Four-day culture, Case 29. Acid phosphatase activity is demonstrated in the numerous lysosomes and large autophagic vacuoles(arrows). x 5,000.
Fig. 10. Ten-day culture, Case 17. The Golgi apparatus (G) is prominent in a cuboidal cell, and the Iamellae of the maturing face are stained with
phosphotungstic acid. Cell surface (arrows), lysosomes (L), and small apical mucous granules (MG) are also stained. x 12,000.
Fig. 11. Eleven-week culture, Case 2. A portion of SMGC. Note the small mucous granulss(MG) at the luminal border of the cell. x 12,800. Inset, 11-week
culture, Case 2. Microvilli showing a prominent glycocalyx. x 29,000.
Fig. 12. Six-week culture, Case 17. A cuboidal ciliated cell with mucous granules. Ciliary basal bodies are present (arrows). x 30,000.
Fig. 13. Sixteen-week culture, Case 44. The glycocalyx of the stubby microvilli of the luminal surface of the squamous cell is thickly stained with colloidal
aron(arrow). Other cell surfaces are less intensely stained (arrowhead). x 12,000.
Fig. 14. Sixteen-week culture, Case 2. Nonkeratinizing squamous cell. Note the large Golgi apparatus (G) and mucous granules (MG). x 9,000.
1006
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Organ Culture of Human Bronchial Epithe!ium
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Long-Term Organ Culture of Human Bronchial Epithelium
Lucy A. Barrett, Elizabeth M. McDowell, Arthur L. Frank, et al.
Cancer Res 1976;36:1003-1010.
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