The morphology of tadpole (Rana catesbeiana) liver under varying conditions of organ culture.

The Morphology of Tadpole (Rancr ccrfesbeiana) Liver
under Varying Conditions of Organ Culture
THOMAS PETER BENNETT AND HENRY XREIGSTEIN
Biological Science, Florida State University, Tallahassee, Florida 32306
and Stanford University Medical School, Stanford, California 94305
ABSTRACT
Primary explants from Rana catesbeiana tadpole liver have been
maintained in organ culture for six days using methods of cultivation which are
described in detail. The organization and morphology of the cells in culture have
been defined by light and electron microscopy. By adjustment of the experimental
conditions of culture, after six days, the cells are morphologically identical to
those freshly excised from tadpole liver. Soon after being put into culture, however, the chromatin of the nuclei becomes condensed, and only after about 30
hours in culture do t h e original euchromatic characteristics of the nucleus reappear. The effects of changes in the culture conditions on these morphological
characteristics are presented.
The system that has been developed is discussed with a view toward elucidating the basis of the cytological changes which occur during anuran metamorphosis.
Recent studies in this laboratory (Bennett et al., '70; Bennett and Glenn, '70)
have provided morphological criteria for
differentiation of tadpole ( R a m catesbeiana) liver cells during thyroxine-induced and natural metamorphosis. Cytological alterations include change of the
nuclei from a euchromatic to heterochromatic condition, an increase in the size
of mitochondria and changes in their
cristae from broad lamellar to a smaller
more tubular appearance, a proliferation
of the rough endoplasmic reticulum
through the cytoplasm, and changes in the
Golgi complex. In preliminary reports,
Cohen ('70) and Spiegle and Spiegle
('70) have made some of these observations on respectively R. catesbeiana and
R . pipiens.
Because of individual differences among
tadpoles in their time of response to thyroxine (Weber, '67) and tissue variability,
the development of organ or cell cultures
of their tissues is of considerable importance for elucidating the mechanisms of
thyroxine-induced changes in biochemical
and cytological patterns during metamorphosis.
ANAT. kc., 176: 461-474.
The extensive literature (Garvey, '61;
Hillis and Bang, '62; Majno, '64; Blatt et
al., '69; Simnet and Balls, '70; Balls, Simnett and Arthur, '69; Monnickendam,
Miller and Balls, '70; Shambaugh et al.,
'69; Rafferty, '69) about culturing parenchymal cells suggest that i t is necessary
that very careful morphological studies on
cells in culture must precede or be closely
correlated with biochemical studies. It is
clear that if liver cells or liver tissue are
placed in sterile culture medium, stable
enzyme activities may be measured for
days and weeks even though cells may
be dying, lysing and releasing their contents into the sterile medium (Majno, '64).
Although both biochemical and cytological studies have been published about organ culture of tadpole tail (Tata, '66;
Gross and Lapiere, '62; Weber, '69), frog
skin (Vanable, '65) and frog lens (Rothstein et al., '65), and other anuran cells
(Freed and Mezger-Freed, '69; Freed,
Mezger-Freed and Schatz, '69), only biochemical data on tadpole liver in culture
(Bennett et al., '69; Shambaugh et al., '69;
Balinsky et al., '70) along with preliminary
Received Sept. 21, '72. Accepted Mar. 6 , '73.
461
462
THOMAS PETER BENNETT AND HENRY KREIGSTEIN
statements (Bennett, '71) about the cy- dium, the tissue was sliced (0.5 mm, thicktology of the tissues have been reported. ness) with a Stadie-Riggsmicrotome which
In at least one study the cells were stated had been washed in 95% ethanol followed
to be in a degenerating state (Blatt et al., by air drying during exposure to UV. The
slices were gently rinsed from the micro'69; Cohen, '70).
The purpose of this study was to define tome with fresh culture medium into a
the techniques for preparing and condi- Petri dish. When all the liver had been
tions for maintaining R . catesbeiana tad- sliced with a microtome, the pieces which
pole liver cells in vitro over a period of had been transferred to the medium in
six days in a close approximation to their the Petri dish were then transferred to
native morphological state. This period is culture medium on bite wax. These larger
sufficient to observe in vivo effects of thy- slices of tissue were diced into pieces about
roxine (Bennett et al., '70; Frieden and 1 mm square with razor blades. Thus the
Just, '70) and should be sufficient time for final tissue fragments were approximately
in vitro study of the mechanism of thy- 1 mm X 1mm X 0.5 mm. In early studies
the size of tissue was monitored with a
roxine action.
plastic centimeter ruler or ocular micromMATERIALS AND METHODS
eter. The final tissue size was judged to
All animals used were Class 1 (Ben- be 1 mm 0.5. Subsequently the size was
nett and Glenn, '70), that is Stage IX-XI11 judged by eye with occasional standardiof Taylor and Kollros ('46) R. catesbeiana zations as described above. One dimension,
tadpoles (Connecticut Valley Biological determined by the Stadie-Riggs microtome,
Supply Co., Southampton, Mass.). These was fixed. The slices were gently removed
tadpoles have short hind limbs but no fore- with a stainless steel micro-spatula to a
limbs and are said to be in late pre- container of medium. From here two fragments were transferred with a spatula into
metamorphosis.
The culture medium used in these ex- a culture tube (12 mm X 75 mm, 2003,
periments was based on the formulation Falcon Plastics, Los Angeles, Calif.) conof Wolf and Quimby ('64) and was pur- taining 0.4 ml of culture medium. The
chased from the Grand Island Biological tube was capped and when all tubes had
Supply Company, Grant Island, N.Y. This been so prepared (about 50-75 tubes per
medium was modified to contain 100 mg 30 minutes per tadpole) they were placed
glucose per liter except as indicated in in a Dubnoff metabolic shaking incubator
(Precision Scientific Co., Chicago, Ill.).
individual experiments.
Organ cultures of liver explants (ap- All operations were conducted at 22"proximately 1 mm X 1 mm X 0.5 mm), 24°C. Incubation was at 26"C, at a slow
were prepared from tadpole liver under the shake setting (50 shakes per minute), with
following conditions. All instruments were continuous gas flow (95% air-5% CQI)
autoclaved and sterile disposable pipettes, for the time indicated in the experiments.
Petri dishes, etc., (Falcon Plastics, Los Contaminated culture tubes, as determined
Angeles, Calif.) were used. The equip- by cloudiness or pH change of the medium,
ment was further sterilized by ultra-violet were discarded.
(UV) radiation in a hood where all operaFig. 1 Phase contrast micrographs of liver
tions were performed. The tadpole was cells
during a six day culture experiment. X 400.
killed by decapitation, the abdomen was Scale shows 25 $I.
Fig. 1A Liver immediately after being put
swabbed with 95% ethanol and the liver
was removed by dissecting it free of the into culture. Note the cord-like arrangement of
cell*$(Co) and the large sinusoids (Si). Arrows
gallbladder and blood vessels. It was put point
out nuclei.
into a s m d pool (5-6 ml) of sterile culFig. 1B Appearance of cells after 48 hours
ture medium on a sterile bite wax plate in culture.
Fig. 1C After 96 hours of culture.
("Byte-Ryte," Mizzy, Incorp., Clifton Forge,
Fig. 1D After 144 hours of culture. Note that
Va.). After being momentarily placed on cord-like
arrangement of hepatocytes has been
sterile filter paper to remove excess me- preserved.
*
MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE
Figure I
463
464
THOMAS PETER BENNETT AND HENRY KREIGSTEIN
Fig. 2 Electron micrographs of cells a t varying periods in culture during a six day
culture experiment such as shown in figure 1. x 13,300. Scale shows 1p.
Fig. 2A Tadpole liver recently excised and #containing rough endoplasmic reticulum
(RER), Golgi ( G ) , bile canaliculi (bc), mitochondria ( M ) , a euchromatic nucleus ( N ) ,
glycogen (gl), and lysosomes (ly).
Fig. 2B A similar view after 48 hours in culture.
MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE
465
Fig. 2C After 96 hours in culture.
Fig. 2 D After 144 hours of culture. Note the preservation at increasing times of cytoplasmic features and characteristics.
466
THOMAS PETER BENNETT AND HENRY KREIGSTEIN
Tissue for microscopy was fixed in general morphology is not changed noticeglutaraldehyde, post-fixed in osmium te- ably. The examination of large areas of
troxide, dehydrated and embedded in randomly selected cultured tissue showed
Epon; sections were stained with lead cit- cells that were more closely packed than
rate and uranyl acetate (Glenn and Ben- those in freshly excised tissue and slightly
nett, '68; Bennett and Glenn, '70). Electron more elongate cells with typical nuclei.
microscopy was carried out with a Siemens Only seldom were necrotic areas observed.
Elmiskop or a Philips 300 at primary magElectron microscopy of cultured cells
nifications of 2000 to 30,000 X. For light
microscopy, sections 0.5 to 3 p in thickness
The representative electron micrographs
were cut and stained with Toluidine Blue shown in figure 2 summarize the features
for transmitted light optics, or mounted of cells from a six day time course exunstained for phase contrast optics.
periment similar to that described above.
Explants were cultured for a period of In figure 2A, the ultrastructural character72 hours in Wolf and Quimby medium istics of pre-metamorphic tadpole liver are
containing varying amounts of glucose : clearly displayed and correspond to those
800 mg/liter, 100 mg/liter and 10 mg/ earlier reported (Bennett and Glenn, '70).
liter. For biochemical analysis, tissues from Small, rod-shaped mitochondria occur
four culture tubes were pooled for each scattered throughout the cytoplasm and
assay and processed as described above.
they have prominent, tubulo-lamellar
Glycogen was determined by the method cristae. The occurrence of lysosomes, glyof Dubois et al. ('56) after homogeniza- cogen rosettes, and occasional stacks of
tion of weighed pieces of cultured tissue. rough endoplasmic reticulum (RER) are
features characteristic of R. catesbeiana
RESULTS
liver. A bile canaliculus is also shown,
Light microscopy of organ cultured cells
as are several multi-vesicular Golgi com(fig. 1 ) shows tadpole liver tissue as it plexes. The chromatin of the nucleus, alappears after 0, 48, 96, and 144 hours of
though not prominently shown here, is
culture. The cells of liver tissue at the bewell dispersed and the nucleus can be
ginning of the culture period (fig. 1A)
characterized as euchromatic.
occur in clusters with two or three cells
Micrographs of cells in culture for 48,
in contact with each other. The sinusoids 96, and 144 hours, respectively, are preare prominent and leucocytes and red
blood cells are clearly visible. Individual sented in figures 2B-D for comparison
with the freshly excised tissue. As can be
cells are trapezoidal or polygonal in shape
seen, the appearance of the organelles and
and have a single prominent nucleus. The their arrangements are preserved essennuclei are asymetrically located and have tially as in the fresh sample. For example,
several densely staining areas which
represent nucleolar regions and clusters of
Fig. 3 Electron micrographs of the major orheterochromatin, as revealed by electron ganelles in liver cells after three days in culture.
Fig. 3A Nucleus ( N ) with nucleolus (Nu).
microscopy (see below),
hucleoplasm is predominantly euchromatic (Eu),
After two days in culture (fig. l B ) , one with occasional areas of diffuse heterochromatin
striking change is a more close-packing of (He). AROWS emphasize fine structure of the
the cells and reduction of the well defined nuclear envelope. x 9,380. Scale shows 1 p .
Fig. 3B Field showing mitochondria (M),
sinusoidal spaces. Some cell division has surrounded
by glycogen (91) and rough endooccurred as evidenced by clusters of plasmic reticulum (RER). x 27,600. Scale show6
smaller, more tightly packed cells. De- 0.5 p.
Fig. 3.2 Rough endoplasmic reticulum in
struction of the blood cells in the sinus- close
juxtaposition to mitochondria (M), glycogen
oids has occurred to a large extent and (91 and arrow) L, lipid. Other arrows point to
there is considerable debris surrounding ribosomes. x 38,600. Scale shows 0 . 2 5 , ~ .
Fig, 3D Golgi complex with cisternae (GC)
the parenchymal cells. After longer periods and
vesicles distinctly resolved. Also shown are
in culture, 96 and 144 hours (fig. l C , D ) , glycogen
(91) granules. x 29,300. Scale shows
cells remain closely packed, but their 0.5 p.
~~
MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE
Figure 3
467
468
THOMAS PETER BENNETT AND HENRY KREIGSTEIN
mitochondria1 cristae remain distended
and lamellar; the chromatin of the nucleus remains dispersed.
Electron microscopy of organelles
Figure 3 summarizes the results of
higher magnification studies of selected
organelles; i.e., the nucleus, mitochondria,
RER, and Golgi in three day culture experiments. The nucleus, as shown in figure 3A, is predominantly euchromatic,
with some condensed chromatin associated with the nucleolar region. Figure 3B
reveals that the morphology of pre-metamorphic mitochondria has been preserved
during this culture period. Profiles of the
cristae remain broad, flat and irregular; dramatic changes in the sizes and
shapes of mitochondria do not occur as
happens during metamorphosis (Bennett
and Glenn, ’70).
A higher magnification view of RER is
shown in figure 3C. In addition, mitochondria and clusters of glycogen may be
discerned between the stacks of cisternae
of the RER. Two well developed Golgi
complexes are shown in figure 3D. They
consist of stacks of cisternae, in close opposition to glycogen, smooth endoplasmic
reticulum, etc.
Glycogen content
In vivo, liver glycogen levels are under
complex controls. In organ culture, however, this balance may be disrupted because of different levels of hormones in
serum contained in the culture medium or
because of the level of glucose in the medium. It is therefore necessary to adjust
the culture conditions of the cells in order
to obtain normal levels of glycogen coiitent. Our approach was to try to regulate
glycogen content by regulating the glucose
concentration in the medium. The following experiments established glycogen levels
in freshly excised liver tissue and in organ
cultured tissue under varying conditions
of glucose concentration in the culture
medium.
Figure 4 compares low magnification
electron micrographs of liver cells before
and after 72 hours of culture in media
with various concentrations of glucose.
Glycogen rosettes are prominent in the
cytoplasm of parenchymal cells at zero
time., After 72 hours in culture medium
with the “standard concentration of glucose, i.e., the concentration in Wolf and
Quimby medium (800 mg/l), the cells
become m e d with glycogen and sections
thus appear abnormal by comparison with
control tissue (fig. 4A,B). At the lower
concentrations of 100 mg/liter or 10 mg/
liter, the liver maintains a more normal
appearance with regard to glycogen content
and glycogen rosettes occur with a frequency comparable to that seen in tadpole
liver cells.
Quantitative results are presented in
table 1. The various glycogen contents, expressed on the basis of pg protein or milligram wet weight of tissue, confirms the
qualitative microscopic impression that
lower glucose concentrations result in a
situation more like that in vivo.
TABLE 1
Effect of glucose concentration on glycogen
content of orgaia cultured liver1
~
Glucose
concentration
pg glycogen per
pg protein
gg glycogen per
m g hssue
~
ms/l
0.059 ‘-c 0.0030
16.5 k 0.10
100
0.072 k0.018
17.622.4
800 2
0.12 i- 0.018
32.8 f6.6
10
1 Freshly excised tissue gave values of 0.084 +- 0.014
pg per pg protein.
ZConcentration present in Wolf and Quimby Medium.
Fig. 4 Electron microscore survey pictures of
liver from the same tadsole at zero time and
after three days culture in media with various
concentrations of glucose. x 5,930. Scale shows
1 P.
Fig. 4A Zero time. Freshly excised and prepared for culture.
Fig. 4B Cultured with 800 m % / l glucose, the
“standard’ content of glucose in Wolf and Quimby
medium for three days. Note the superabundance
of glycogen rosettes (91).
Fig. 4 C Cells in tissue cultured with 100
m g / l glucose.
Fig. 4D Cells as they appear after three days
with 10 m g / l glucose. The heterochromatic nucleus in ( A ) is due to pre-chilling of the tadpole,
as described by Kriegstein and Bennett (’73). N,
nucleus; gl, glycogen; L, lipid.
MORPHOLOGY OF TADPOLE LIVER I N ORGAN CULTURE
Figure 4
469
470
THOMAS PETER BENNETT AND HENRY KREIGSTEIN
Reversible chromatin condensation of liver
cell nuclei during short-term
organ culture
Since the rates of amino acid, uridine
and thymidine incorporation by tadpole
liver showed slight decreases during the
first 24-40 hours in culture, after which
incorporation rates recovered (Bennett
and Kriegstein, ’73), it was of interest to
investigate morphological correlates of
these responses. Cultures were prepared
according to METHODS and the early time
periods were monitored by microscopy.
Figure 5 summarizes the results of a
time course experiment during 36 hours
in culture with the tissue being monitored
with phase contrast optics. Figure 5A illustrates fresh tissue with its characteristic
parenchymal cell nuclei which are euchromatic. After 12 hours in culture (fig. 5 B ) ,
the nuclei are altered in appearance by
having a greater distinction between euchromatic and heterochromatic regions. At
24 hours (fig. 5C) the even, grey euchromaticity of the nuclei has been restored,
and by 36 hours (fig. 5D) the nuclei look
like those observed in the starting tissue
(fig. 5A).
The electron micrographs in figure 6
compare a cell nucleus from recently excised liver tissue with a nucleus from a
cultured tissue cell after 12 hours in culture. The former nucleus is predominantly
euchromatic, with a homogeneous distribution of chromatin, gradually becoming
more heterochromatic near the nuclear
membrane and about the nucleolus. In
contrast, the nucleus from short-term cultured tissue, has distinct condensed chromatin as well as euchromatic regions. The
areas of condensed chromatin along the
inner surface of the nuclear membrane
and that associated with the nucleolus are
both more extensive and more denseiy
staining than the corresponding chromatin
in freshly excised tissue.
DISCUSSION
A standard amphibian cell culture medium (Wolf and Quimby, ’64) which has
been modified to contain a lower concentration of glucose, has been used under
defined experimental conditions to main-
tain liver fragments (1 mm X 1 mm X
0.5 mm) from a single tadpole for an experimental period of six days. Approximately 50 to 75 experimental tubes from
a single tadpole, each containing two
pieces of liver tissue, are incubated under
an air-COz atmosphere in a Dubnoff
Shaker.
Our culture conditions, in contrast to
those of Shambaugh et al. (’69) in which
“viability of the liver cubes was not altered
significantly by incubation in oxygen, type
of medium used, or use of smaller cube
size,” had to be precisely regulated, as described in METHODS, with regard to these
parameters in order to achieve the quality
of morphological and biochemical integrity
described in the present publication. Under
many other different conditions, we observed anomolous cell morphology, large
areas of necrotic tissue and biochemical
features comparable to those reported by
other investigators for a dying liver cell
population (see review by Majno, ’64).
These other conditions that were tested included modifications of the culture medium (e.g., pH, ionic strength, serum content) of the ratio of amounts of medium
to tissue, size of tissue fragments, incubation atmosphere and the conditions for
preliminary handling of tissue. In concurrence with Shambaugh et al. (’69) we observed that “viability was adversely affected
by higher temperatures of incubation, inadequate shaking of the flasks during incubation, and prolonged preparation time
after liver removal ( 2 to 4 hours).” On
the basis of light microscopy studies, medium containing 10% fetal calf serum
proved superior to that containing frog or
iadpole serum; the latter resulted in large
Fig, 5 Light micrographs showing nuclear
changes in liver cells durins their first 36 hours
in culture, All tissues are from one animal. rbc,
red blood cell; hp, hepatocytes. x 1,000. Scale
shows l o p .
Fig. 5A Appearance of freshly excised parenchymal cells. Arrows point to nuclei, which are
euchromatic.
Fig. 5B Tissue after 12 hours in culture. The
nuclei (see arrows) are now heterochromatic.
Fig. 5C After 24 hours the cell have nuclei
that are becoming euchromatic again.
Fig. 5D Thirty-six hour cultured tissue has
cells with euchromatic nuclei as in A.
MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE
Figure 5
471
472
THOMAS PETER BENNETT AND HENRY KREIGSTEIN
Figure 6
MORPHOLOGY OF TADPOLE LIVER IN ORGAN CULTURE
accumulations of lipid (Kriegstein, ’71).
Pieces of tissue that exceeded 2 mm in
size generally showed large areas of necrosis. Two pieces of tissue per 0.4 ml of
medium could be satisfactorily maintained
for six days. One piece of tissue could be
used in this volume of medium, however,
three or four pieces generally showed
necrosis.
The results reported here demonstrate
that the normal premetamorphic fine structure features of parenchymal cells and
their organelles are well preserved for a
period of six days in culture. However, on
being put into culture medium and during
the early period (up to about 30 hours)
in culture, parenchymal cell nuclei become, according to the terminology of Fawcett (’66) and Porter and Bonneville (’70),
heterochromatic; later they return to their
so called euchromatic state.
O u r results suggest that in our case
the “trauma,” often mentioned in connection with cell culture work, that occurs
when tissue adapts to in vitro conditions
is reflected in defined fine structural
changes (i.e., chromatin condensation) in
the cultured cells. After an adjustment
period these changes are reversed completely or in part. The most obvious suggestion, following current writings, is that
chromatin condensation occurs as a result
of perhaps changes in ionic strength, of
cations, or other ingredients in the in vitro
milieu and that this condensed chromatin
is genetically inactive. Thus the biochemical, morphological and culture effects become interrelated. One is seemingly left
with a broad spectrum of possible causes
for which perhaps the best term is
“trauma” and the cell morphology and
biochemistry (Bennett and Kriegstein, ’73)
reflect recovery of the cells from the
“shock” of ‘lag” phase which occurs when
fresh tissue “adapts” to in vitro conditions.
Fig. 6 Comparison of control and incubated
(12 houre) liver cell nuclei. x 6,370. Scale
shows 1 fi.
Fig. 6A Control. Eucliromatin ( E u ) , heterochromatin (He), and the nucleolus ( N u ) are
clearly shown in this cross-section of a typical
nucleus.
Fig. 6B Incubated nucleus. Heterochromatic
( H e ) regions, together with densely stlaining spots
of heterochromatin (Ch), occur i n formerly euchromatic areas. The euchromatic regions are
now more distinct.
473
In the present work, emphasis has been
placed upon determining the viability and
also the maintenance of differentiated features of premetarnorphic tadpole liver in
culture. The assessment of these states for
tissues rests on indirect biochemical criteria and more direct morphological criteria. As reported elsewhere (Bennett and
Kriegstein, ’73) the patterns for biochemical constituents (e.g., dry weight, wet
weight, DNA, RNA and protein content)
during periods in culture as well as the
gross metabolic patterns reflected by incorporation of radioactive amino acids,
uridine, and thymidine, corroborate our
morphological observations about the
tissue.
The tadpole liver organ culture system
as here reported is convenient to set up,
uses a slightly modified medium which is
readily available, and allows as many as
50 to 75 individual tubes of liver explants to be prepared from a single tadpole
liver. This insures genetic uniformity and
a reduction in so-called “biological variability.” These features make the organ culture system suitable for further development directed toward the in vitro study of
metamorphic responses of tadpole liver
and the mode of action of thyroxine.
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