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Continuous-Perifusion tissue culture of fetal and adult pancreas of the lizard Anolis carolinensis.

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THE ANATOMICAL RECORD 203:165-173 (1982)
Continuous-PerifusionTissue Culture of Fetal and Adult
Pancreas of the Lizard Anolis carolinensis
WILLIAM R. RHOTEN
Department of Anatomy, CMDNJ-New Jersey Medical School, Newark, NJ
07103
ABSTRACT
The differentiation of the fetal saurian pancreas in continuousperifusion tissue culture (CPTC)was examined. Splenic pancreases from 24-day
postoviposition fetuses of the green anole,Anolis carolinensis, were grown for 8 to
31 days by CPTC following successful preliminary studies with adult pancreas.
Adult anolian endocrine pancreas was maintained for up to 7 days by CPTC. The
pancreatic explants were examined morphologically by light and electron microscopy. The functional integrity of the endocrine cells was evaluated by measuring
hormone levels of the explants and in the basal medium and by determining the
kinetics of hormone release. The pancreatic endocrine cells from fetal and adult
anoles were functionally and morphologically intact after CPTC. The exocrine
pancreas was not maintained during culture. This study demonstrates for the first
time the growth of the reptilian endocrine pancreas in culture.
The splenic pancreas of the adult green
anole, Anolis carolinensis, is rich in endocrine
cells and contains about 300 ng of insulin
(Rhoten, 1973a) and more than 3,000 ng of
glucagon (Rhoten, 1976). The glucagon-containing alpha cells are the predominant cell
type, followed in number by somewhat fewer
insulin-containing beta cells, still fewer
somatostatin-containing delta cells (10%15%), and a small percentage of pancreatic
polypeptide-containing F cells (Rhoten, 1973a;
Rhoten and Smith, 1978; Rhoten and Hall,
1981). The functional attributes associated
with these anolian pancreatic endocrine cells
include attenuated insulin secretion, in comparison to mammals, in response to a glucose
challenge, a relative insensitivity of the alpha
cells to changes in glucose levels, and hyperglycemia (Rhoten, 1973a, 1973b, 1974a, 1978;
Marschall and Gist, 1973). Thus, the fetal
anolian pancreas may be a useful model with
which to explore the cellular mechanisms
underlying the development of reduced islet
cell sensitivity to glucose and hyperglycemia.
An attenuated insulin secretory response to
glucose is also found in diabetes mellitus and
in fetal beta cells of a number of mammalian
species including man (see Rhoten, 1980).Furthermore, hyperglycemia is the sine qua non
for the diagnosis of diabetes. Therefore, the
morphological and functional differentiation of
0003-276X/82/2031-0165$03.000 1982 Alan R. Liss, Inc.
fetal anolian pancreas grown in continuousperifusion tissue culture was examined. Prior
to using fetal pancreas, fragments of adult
splenic pancreas were grown in continuousperifusion tissue culture (CPTC).Results with
the adult pancreas were encouraging and perifusion cultures of 24-day postoviposition
splenic pancreases were initiated. The fetal
pancreases were grown for up to 31 days by the
CPTC method. After 31 days in CPTC, the endocrine cells were intact and alpha, beta, and
delta cells could be identified structurally with
ease. The presumptive acinar component was
nongranulated and appeared to be in various
stages of degeneration. The results demonstrate for the first time the growth of the reptilian endocrine pancreas in culture.
MATERIALS AND METHODS
Adult males and gravid females of Anolis
carolinensis were obtained from a biological
supply house (The Snake Farm, LaPlace, La).
The animals were maintained in an environmental chamber (Calumet Scientific) at a day
temperature of 32 f 1"C. The day length (light
cycle) was 14 hours and the night length (dark
cycle)was 10 hours. The relative humidity was
maintained at about 60%. The above condi_
_
_
.
~
Received October 1, 1981; accepted December 23, 1981
166
W.B. RHOTEN
tions are consistent with observations on the
preferred body temperature and effects of environment on Anolis carolinensis (Licht, 1968,
1971; Crews e t al., 1974). The animals were offered food, instars of Galleria mellonella (the
wax moth) or Tenebrio molitor (mealworms),
daily. The cages of the gravid females were examined a t least four times daily for eggs. The
eggs were incubated on gauze wicks a t 28 rn
2°C wet bulb a s described previously (Rhoten
and Hall, 1982).
Aseptic technique was used during excision
of the pancreas from adult and fetal anoles.
The splenic pancreases were rinsed in sterile,
Hanks’ balanced salt solution containing penicillin (200 IUiml),streptomycin (200 pggiml) and
an antimycotic (5 pgiml). The tissue was cut
into small pieces, about 0.5 mm on a side, as required. The tissue was transferred to culture
media and injected via a valve into the perifusion chamber of a sterile, filled and operating
continuous-perifusion tissue culture (CPTC)
system (see below). Fragments of adult splenic
pancreas were utilized with two different culture media prior to using fetal splenic pancreas. The medium was either CMRL-1066 or
Ham’s F-12 (Gibco),both containing fetal calf
serum (lo%), 2 mgiml glucose, and penicillinstreptomycin (100 IUiml and 100 pgiml, respectively). The concentration of glucose
selected (2 mgiml) evokes no significant increase in insulin release from perifused splenic
islets of adult anoles (Rhoten, 1974a) and is
only slightly less than the normal plasma glucose levels of adult anoles (Rhoten, 1973a).
Media were changed daily or every other day.
Since it is well known that temperature plays
an important role in the metabolism of poikilothermic vertebrates like the reptiles, the
anolian cultures were kept in an environmental
chamber under the conditions described above.
In particular, i t should be noted that the temperature regimen included 14 hours a t 32°C
and 10 hours a t 24°C.
The perifusion system used in this study
(CPTC) was developed in the laboratories of
Dr. Paul E. Lacy (Lacy et al., 1976). Two basic
perifusion systems have been developed: one
system requires a carbon dioxide incubator,
the other does not. In the latter system, as
modified for use in this study, a closed, external “lung” (permitting gas exchange) is required. The lung consists of a 500-ml Erlenmeyer flask with a #7 silicone rubber stopper
with multiple inlet and outlet ports for the tissue culture media within Silastic tubing and
the gas phase of 7% carbon dioxide-93% air,
saturated with water. The tissue fragments
were grown on Nitex, a monofilament wire
cloth (Tetko, Inc.), in standard perifusion
chambers (Swinnex) that are connected by a
series of valves and couplers to the culture
media. Circulation of media is maintained by
an infusion pump (Extracorporeal Medical
Specialties, Inc.). The entire assembly was
placed in the environmental chamber. The dynamics of insulin and glucagon release from
some cultures were determined during shortterm perifusion with Krebs-Ringer-bicarbonate solution of intact explants. The details
concerning the perifusion procedures are available elsewhere (Rhoten, 197313; Clements and
Rhoten, 1976). Hormone levels were measured
by radioimmunoassay (Wright et al., 1968;
Heding, 1971).
Explants taken for electron microscopy were
fixed either in a dilute glutaraldehyde-formaldehyde solution (Karnovsky, 1967)consisting
of 3% glutaraldehyde, 2% formaldehyde, 0.09
M sodium cacodylate, and 4.5 mM calcium
chloride, or in 2.5% glutaraldehyde in 75 mM
phosphate buffer, both a t pH 7.2. The explants were postfixed in 170osmium tetroxide,
dehydrated, and embedded in araldite ACM
(Fliika, A.G., Switzerland). Thin sections were
stained with 2% aqueous uranyl acetate and
lead citrate (Reynolds, 1963). Tissue for light
microscopy was fixed in 3% glutaraldehydeBouin’s solution and processed routinely for
embedding in glycol methacrylate (JB-4, Polysciences, Inc., Warrington, Pa). One part promoter was added to 20-25 parts catalyzed
plastic. Thick (1.5-pm) sections were stained
with aldehyde fuchsin-trichrome (Epple, 1967).
RESULTS
Adult pancreas
The pancreatic endocrine cells of adult
anoles were maintained in the continuous-perifusion tissue culture system in good condition
for 7 days when the cultures were terminated
(Fig. 1).At least two pancreatic endocrine cell
types were seen a t low magnification in aldehyde fuchsin-trichrome (AFT)preparations of
the explants after 7 days of CPTC (Fig. 1).The
A hhrwia tions
~
~
~~
a , acinar cells
A. alpha cell
H. beta cell
CPTC. continuous-perifusion tissue culture
D. delta cell
e. endocrine cells
s. ”small granule” cell
PERIFIJSION CULTURE O F ANOLIAN PANCREAS
Fig. 1. Adult splenic pancreas grown by CPTC for 7 days
in nutrient mixture F-12. The endocrine pancreas is mostly
intact, whereas the acinar pancreas is degenerating or has
disappeared. Aldehyde fuchsin-trichrome staining of glycol
methacrylate-embedded explant. x 112.
Fig. 2. Adult splenic pancreas grown by CPTC for 7 days
in medium CMRL-1066. Aldehyde fuchsin-trichrome-stained section of glycol methacrylateembedded explant. Endocrine cells make up most of the field with the aldehyde
fuchsin-reactive beta cells appearing black. The alpha cells
light microscopic appearance of the endocrine
cells was similar for cells grown in medium
CMRL-1066 (Fig. 2) or in nutrient mixture
F-12 (Fig. 1).At higher magnification, the cytoplasm of the endocrine cells was colored in one
of three ways with the AFT procedure: blueblack (aldehyde fuchsin stained), reddishorange, or grayish-pink. These tinctorid properties of the CPTC pancreas were typical of
those seen in the normal adult anolian pancreas (Fig. 3). Another striking feature of the
pancreas maintained in CPTC was the nearly
complete absence of readily identifiable exocrine pancreas (Fig. 1 and Fig. 2). The blood
vascular spaces, which were characteristic of
the endocrine areas in the normal pancreas
(Fig. 3), were not preserved in CPTC (Fig. 2).
Insulin and glucagon content were similar
with the two different culture media: insulin,
about 1 2 pgiexplant of splenic pancreas for
both media (range of 10.0 to 13.7, n = 4), and
glucagon 62 and 120 nglexplant of splenic pan-
167
and the delta cells are gray. The acinar cells are degenerating or have disappeared (compare with Fig. 3). X 280.
Fig. 3. Normal adult splenic pancreas. The endocrine
cells consist of aldehyde fuchsin-reactive beta cells (black
cytoplasm). alpha cells, and delta cells (both with a gray
cytoplasm). The acinar cells have relatively large zymogen
granules (black)in the apical region. Compare with Figure 2.
Aldehyde fuchsin-trichrome-stainedsection of glycol methacrylateembedded tissue. X 280.
creas for CMRL-1066 and F-12, respectively (n
= 1 each). Insulin was released into the media
during CPTC and the levels were similar for
the two different media (Fig. 4). At day 7 of
CPTC, the insulin release rate was 6.1 ngiday
in CMRL-1066 and 10.2 ngiday in F-12. The insulin release rates appeared t o stabilize at 3-4
days of CPTC and showed only a modest decline thereafter (Fig. 4).Since these results indicated that adult beta cells had adapted to the
culture conditions by 5 days of CPTC, another
set of cultures was initiated to examine the
kinetics of hormone release a t day 5 of CPTC.
Explants grown in CPTC for 5 days showed
enhanced insulin and glucagon secretion when
challenged with appropriate secretagogues
during perifusion with Krebs-Ringer-bicarbonate solution in vitro (Fig. 5). In response to a
low concentration of glucose (0.5 mgiml) and
arginine (10 mM), the explants released about
50% more glucagon than in the basal medium
(Fig. 5). Insulin secretion was increased when
168
-
W.B. RHOTEN
100 T
m
n
M R L-I088
+I
; 80
aQ.
Y
n
4
80
w
-I
40
E
-1
a
a
20
z
1
2
9
4
5
6
7
1
2
3
4
5
8
Days
Fig. 4. Insulin release from the splenic pancreas of
Anolis carolinensis during CPTC. The splenic pancreases
were maintained in either medium CMRL1066 (left side)or
nutrient mixture F-12 (right side). Day 1 of CPTC was used
a s 10070and the amount of insulin in the medium on subsequent days was related to day 1.
the glucose concentration was raised (Fig. 5).
Insulin levels in the perifusate were essentially
undetectable before exposure of the explants
to a high concentration of glucose (Fig. 5).
Glucagon secretion became attenuated during
perifusion with the medium containing a high
concentration of glucose (Fig. 5). Ultrastructurally, the explants showed variable degrees
of cytoplasmic granulation after perifusion
(Fig. 6). In summary, the endocrine cells of the
adult anolian splenic pancreas were functionally and structurally intact after being grown for
up to one week in CPTC. Therefore, the growth
of fetal anolian pancreas under similar conditions was attempted.
Fetal pancreas
Twenty-four-day postoviposition splenic
pancreases were grown for 8 to 31 days by
CPTC in medium CMRL-1066. After 8 days in
CPTC, the endocrine cells were moderately to
well granulated (Fig.7). The presence of cells of
the acinar pancreas was not apparent. The
three major endocrine cell types (alpha, beta,
and delta cells) could readily be identified in
the electron microscope after 8 days in CPTC
(Fig. 7). These endocrine cells resembled closely those present in neonates (postoviposition
time to birth was 30.1 + 0.2 days at 28°C wet
bulb) and adults, with the exception of a more
heterogeneous appearance of the secretory
granules in some cells. An additional endocrine-like cell type was occasionally observed.
The secretory granules were relatively small,
2 5 30
40
55
70
80
T I M E (mi")
Fig. 5. Hormone secretion from adult splenic pancreas
grown by CPTC for 5 days. After 5 days of CPTC in medium
CMRI,-1066. the chamber containing the explants was disconnected from the culture apparatus and connected t o the
short-term perifusion system. The explants were perifused
with 1 mg of glucoseiml from 0 t o 28 minutes, and with 10
mM arginine from 28 t o 90 minutes in the presence of either
a low concentration of glucose, 0.5 mgiml (28-58 minutes).
or a high concentration of glucose, 6 mgiml(58-90 minutes).
Insulin release ( 0)was stimulated markedly in the presence
of high glucose. Insulin levels in the perifusate were not consistently detectable before exposure t o high glucose. Glucagon secretion (H)
was elevated in a low concentration of glucose and reduced in a high concentration of glucose. The
dead space of the system was equivalent t o 2 minutes of
perifusion. An explant from this perifusion can be seen in
Figure 6.
electron dense and elliptical in profile in these
endocrine-like cells (Fig. 7). The presumptive
acinar component lacked zymogen granules.
Explant insulin content was 14 ng a t 10 days
and had increased to 42 n g at 20 days (mean of
two separate perifusion cultures). After 31
days in CPTC, the endocrine cells were intact
and alpha, beta, and delta cells could be identified (Fig. 8). Many alpha cells exhibited an unusual degree of electron density differences in
their granules (Fig. 8). Some endocrine-like
cells could not be identified on the basis of
their secretory granule morphologies (Fig. 8).
A few cells contained small, elliptic and electron-dense granules (Fig. 8). Insulin secretion,
as reflected in insulin levels of basal media,
tended to stabilize at low levels after an initial
period of decline lasting about 4 days (Fig. 9).
Insulin release was about 1nglday per explant
at days 8-10 of CPTC and was 0.5 nglday per
explant at day 31 in one culture (Fig. 9). Thus,
the endocrine cells of fetal anolian pancreas
can be maintained structurally and functionally for up to 31 days by CPTC.
PERIFUSION CULTURE O F ANOLIAN PANCREAS
Fig. 6. Adult splenic pancreas grown by CFTC for 5 days
and perifused in vitro. This explant was from the cultures
used to obtain the hormone release data in Figure 5. The de-
169
g e e of granulation of the endocrine cells varies considerably. Alpha cells and beta cells are present. X 6,000.
were present. The insulin content of cultured
fetal anolian pancreas increased more than
The anolian endocrine pancreas has been 10-fold from the time CPTC was initiated at
grown and maintained long-term in continu- day 24 postoviposition (3.1 i 0.75 nglsplenic
ous-perifusion tissue culture (CPTC). Fetal pancreas, n = 3) until 20 days of CPTC had
pancreas was grown for up to 31 days by CPTC elapsed (42 ngiexplant). This amount of insulin
and morphologically intact endocrine cells is, however, considerably less than the levels of
DISCUSSION
170
W.B. RHOTEN
Fig. 7. Day 24 postoviposition pancreas grown by CPTC
for 8 days. Alpha, beta, and delta cells are identified easily
by their morphognomonic secretory granules. A portion of
an endocrine-like cell with electron-dense and relatively
small secretory granules is present (arrow). Most of t h e endocrine cells are moderately to well granulated. X 3.750.
insulin found in the adult splenic pancreas
(about 300 ng; Rhoten, 1973a). The glucagon
content a t the time of initiation of CPTC, 10.8
+ 1.3 nglsplenic pancreas, was 3- to 4-fold
greater than that found for insulin, hut explant
levels of glucagon were not measured during
CPTC. Levels of insulin released into the basal
media were quite low during CPTC (about 1
ngiday per explant) hut tended to stabilize a t
about day 4 of culture. Cells with alpha
granules, which contain glucagon (Rhoten and
Hall, 198l), were present in explants after 31
days of CPTC; however, many alpha cells had
an unusual degree of electron density differ-
PERIFUSION CULTURE OF ANOLIAN PANCREAS
171
Fig. 8. Day 24 postoviposition pancreas grown for 31
days by CPTC. Some of the endocrine-like cells can be identified as alpha or beta cells on the basis of their secretory
granule morphologies. An endocrine-likecell with small elec-
tron-dense granules can also be seen. The cell type of the
other cells present (e)cannot be determined by morphology
alone. X 4.500.
ences in their granules (see Fig. 8). F cells
which contain pancreatic polypeptide (PP)
may have been present in the cultures, but
they would not have been distinguished with
the techniques employed in this study. As reported recently (Rhoten and Hall, 19811, the
secretory granules of the PP-containing F cells
of the adult anole cannot be identified with certainty, especially with regard to the secretory
granules of the D cells, unless immunocytochemical techniques are used. The small, elliptic and electron-dense granules that were occasionally observed in the cultured explants
appear to be similar to those found in the endocrine-like cells during the early postoviposition
period (Rhoten and Hall, 1982). Cells with
these small, electron-dense granule profiles are
found rarely in the adult and in the cultured
adult pancreas. Granules of similar electron
microscopic appearance are found in the early
postoviposition pancreas and their contents
can be identified by immunocytochemistry
(Rhoten and Hall, 19821, but the content of
these diminutive granules in the cultured pancreas and in the adult pancreas is not known.
The apparent remnants of the acinar component lacked zymogen granules after 31 days of
CPTC, suggesting the failure of the acinar cells
to continue development during culture since
these cells are well granulated in the neonate
and in the adult. The nearly complete absence
of readily identifiable acinar cells and the numerous vacant areas in the cultured explants of
adult splenic pancreas indicate that the adult
172
W.B. RHOTEN
0 DAYS O F CULTURE
15
20
25
2 4 6 8 10
,’
/I-\,;
;
\
o
I 2 3 4 5 6 7 8 9 1 0
0 DAYS
30
\
12
14
16
OF CULTURE
Fig. 9. Insulin release into basal medium from fetal
splenic pancreas grown by CPTC for up to 31 days. The
growth medium used was CMRL-1066 supplemented with
fetal calf serum and glucose (see Materials and Methods).
Media were changed daily or every other day and insulin
levels measured by radioimmunoassay. The number of cultures ( < > I included in the data (n)decreased during CPTC as
the cultures were terminated (days 1-10. n = 4; day 11. n =
3; days 12-16, n = 1). Insulin release was determined for
only one culture grown for 31 days ( 0 ). Vertical lines indicate the standard error of the mean.
exocrine pancreas degenerates and disappears
during CPTC (compare Figs. 1 and 2 with Fig.
3). Thus, neither the presumptive acinar component of the fetal pancreas nor the exocrine
pancreas of the adult appears to be maintained
in CPTC under the present conditions. Disappearance or dedifferentiation of the exocrine
component during culture of mammalian pancreas is not uncommon (see Lazarow et al.,
1973; Lacy and Gingerich, 1977; Hellerstrom
et al., 1979). In contrast, the endocrine component of both the fetal and adult pancreas
was preserved in CPTC.
The morphological integrity of the endocrine
pancreatic cells during CPTC was evident at
both the light microscopic and the electron microscopic levels. The functional integrity of the
endocrine cells grown in CPTC was demonstrated by the maintenance of, or the increase
in, hormone levels during culture, the release of
insulin into the basal medium, and the characteristic secretory response of alpha cells and
beta cells to conditions that normally are
either stimulatory or inhibitory. The finding of
stabilized insulin release at 3-4 days of CPTC
with adult explants (see Fig. 4) indicates that
the beta cells have adapted to the culture environment. I t seems likely that a substantial
portion of the insulin release, which occurs at
days 1 and 2 of CPTC, reflects the nonphysiologic release of insulin as the cells adapt to culture conditions. If so, then a more meaningful
comparison in terms of insulin release is between day 7 of CPTC and day 4.Insulin secretion into basal medium by adult explants at
day 7 was about 75% of that at day 4 with
either medium CMRL-1066 or nutrient mixture F-12 (Fig. 4). Insulin secretion was enhanced by an elevated concentration of glucose
in the presence of arginine as has been found
with short-term incubation of anolian splenic
pancreas (Rhoten, 1973b, 1973c, 1974a.
197413).Glucagon secretion, on the other hand,
was reduced in the high glucose containing medium, and enhanced in a medium with a low
concentration of glucose (see Fig. 5). These results are in accord with our prior findings on
glucagon secretion by perifused splenic
pancreas of the anole (Rhoten, 1978).
The results indicate that the anole has additional utility as a model with which to study
the regulation of pancreatic endocrine cells in a
naturally occurring state of attenuated insulin
release and a reduced population of pancreatic
beta cells. Such investigations with the anolian
pancreas appear to be desirable in efforts to
understand similar physiologic and anatomic
states related to the etiology and pathogenesis
of diabetes mellitus. Furthermore, the growth
and differentiation of pancreatic endocrine
cells is potentially of great importance in considering the transplantation of islets or individual islet cell types because for transplantation it would be desirable to harvest cells with
known secretory and synthetic characteristics
and known mitotic potential. Such parameters
can be evaluated readily in continuous-perifusion tissue culture.
In summary, the splenic pancreas from adult
and fetal green anoles has been grown in continuous-perifusion tissue culture. The pancreatic endocrine cells were functionally and morphologically intact after long-term culture (up
to 31 days for the fetal explants). The exocrine
pancreas virtually disappears during culture.
ACKNOWLEDGMENTS
Supported in part by NSF grant No. PCM7617997, Public Health Service grant AM19538,
and a grant from the New Jersey Affiliate of
the American Diabetes Association. The generous gifts of antisera by Drs. Tager and
Wright, and the gift of insulin and glucagon by
PERIFUSION CULTURE OF ANOLIAN PANCREAS
Dr. Root are gratefully acknowledged. The
author thanks Marge Pascavage for typing the
manuscript.
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