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Development of connective tissue fibers in epithelium-containing cultures.

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DEVELOPMENT O F CONNECTIVE TISSUE FIBERS
IN EPITHELIUM-CONTAINING CULTURES
WILLIAM BLOOM AND RUTH H. SANDSTROM
Department or Anatomy, The University of Chicago
In 1928 and 1929 Maximow reported that he had obtained
the development of reticular and collagenous connective tissue
fibers in cultures of adult rabbit thymus. He believed these
were true connective tissue fibers f o r they resisted digestion
by pancreatin and were stained blue with the Mallory-Azan
method and black or brown with Foot’s modification of the
Bielschowsky silver impregnation technic. He also noted that
the early fibers remained unchanged on the addition of dilute
formic acid to the living cultures.
Maximow held that his cultures brought evidence in favor of
the view that connective tissue fibers develop extracellularly.
He based this conclusion on the facts that the developing network of new fibers only occasionally followed the outline of
the cells at the edge of the migration zone (see figs. 3 and 4,
and plates 7 and 8 of his 1929 report) and that peculiar short
fibrils were present in the fibrin f a r beyond the points reached
by the outwandering cells (see figs. 1 and 2 of the same
paper) ; these short fibrils he believed to be the first stages in
the formation of the fibrous network. He found no evidence
f o r the transformation of fibrin into fibers and no indication of
the existence of an amorphous ground substance or an
exoplasma.
These experiments of Maximow were the first time that connective tissue fibers satisfying all the tests for these structures
were obtained in tissue culture, and gave impetus to the study
This investigation was aided by a grant from the Rockefeller Foundation to
the Biological Sciences Division of The University of Chicago.
75
76
WILLIAM BLOOM AND RUTH H. SANDSTROM
of this question by many workers. Their work has recently
been reveiwed by Gt. Levi ( '34).
All of the subsequent workers on this question have confirmed Maximow's observation that connective tissue fibers
develop in great profusion in cultures of most tissues, and
most of them agree with him that the fibers develop extracellularly in the cultures.
McKinney ('29), working in this laboratory, noted the extensive development of reticular fibers in a few days in vitro
of rabbit lymphatic tissue. But he did not observe any of the
peculiar, clearly extracellular, short, wavy, spirochete-like
figures that Maximow found and figured. McKinney found
that all of the newly-formed fibers were in contact with preexisting fibers. A similar inability to find such figures has
since been commented on by the other workers on this theme
in tissue culture, and especially by Momigliano-Levi ; although
in his latest report ('32) this author holds that the peculiar
structures that Maximow found may represent an atypical
type of collagen.
I n the extensive study of the question of the origin of connective tissue fibers in a wide variety of tissues of several
species which we have made, we soon found that the observation made by McKinney and others was correct, and that in
most tissues the new fibers always were connected with the
original fibers of the explant, and that the peculiar isolated
fibrils that Maximow found did not exist, As we still have
Maximow's original preparations and have seen in them exactly the structures he saw and described, and as one of US
drew some of these figures for the detailed posthumous report
of this work (Maximow, '29), we thought it necessary to repeat Maximow's technic exactly on the same tissue that he
had used, because all of those who were unable to repeat his
observations had studied tissues other than the thymus which
he used.
I n our study of these thymus cultures we believe we have
found the cause of the disparity in the results obtained by
FIBERS IN TISSUE CULTURE
77
Maximow and the subsequent workers on the question of the
mode of origin of fibers in tissue cultures. These are the
findings which we shall report here.
MATERIAL AND METHOD
This report is based on cultures of adult rabbit thymus,
embryonic guinea pig gall bladder, and several strains of
subcutaneous connective tissue.
The thymic tissue was taken from young adult rabbits and
explant ed in heparinized rabbit plasma and rabbit embryonic
extract diluted 1to 8 with Tyrode’s solution, One hundred to
150 cultures were made in each series of thymus cultures and
five to eight cultures were fixed each day after explantation.
The cultures were made with Maximow’s double coverslip
method so that after 3 or 4 days the culture could be washed
in Tyrode’s solution and given fresh embryonic extract, and
plasma when liquefication had occurred. By this method the
explanted tissue is not disturbed and the original explant remains for the life of the culture, Maximow’s youngest cultures were fixed at 12 days, at a time when he could see
new-formed fibers in the living cultures. We fixed cultures
in each series at daily intervals to determine the time and
appearance of the peculiar, isolated fibrils.
The embryonic gall bladders were cultured from 8 em.
guinea pig embryos. The technic used here was the same as
for the rabbit thymus except that guinea pig plasma and extract were used.
Cultures from adult and embryonic rabbit and chick connective tissue were prepared by Carrell’s method which involved cutting the cultures every 2 or 3 days; in aU cases
homogenous plasma and extract was used. After the cultures
had obtained a uniform appearance, consisting only of spindleshaped, fibroblast-like cells, growing a t a fairly uniform rate,
they were carried on by Maximow’s double cover-slip method,
and fixed at various time intervals.
All of the cultures were fixed with Zenker-formol. Some of
each series were impregnated in toto; others after being
78
WILLIAM BLOOM AND RUTH H. SANDSTROM
serially sectioned were impregnated by Foot’s modification of
the Bielschowsky method f o r reticular fibers as adapted to
tissue culture in toto preparations by McKinney (’29).
OBSERVATIONS
The development of adult rabbit thymus in tissue cultures
has been described by Popoff ( ’27) and Tschassownikow (’26,
’29). Briefly, these consist in migration of lymphocytes (small
thymocytes) into the surrounding medium during the first 24
hours after explantation. This migration of lymphocytes is
so extensive as to suggest that the culture is from a lymph
node. After this time ameboid macrophages appear in the
migration zone. According to both Popoff and Tschassownikow, most of these are of fixed macrophage (histiocyte) origin
while some of them arise from hypertrophic lymphocytes.
Our observations agree with these conclusions.
During the second day in vitro, clear, pale areas begin to
appear within most of the explants. These are ‘islands’ of
epithelium. Their development from some of the epithelial
reticular cells of the thymus has been traced by both Popoff
and Tschassownikow. In cultures vitally stained with lithium
carmin, these epithelial cells remain free from dye granules in
contrast to the macrophages which store large amounts of the
dye.
I n most thymus cultures, the fibrin of the medium begins
to liquefy during the third or fourth day in vitro. These gaps
in the supporting fibrin network were always repaired by the
addition of fresh plasma, but fresh liquefication soon began
again in most of the cultures. The destruction of the fibrin
may be so extensive as to cause great gaps in the medium and
the cultures contract down to small, rounded bits of tissue.
I n serial sections, impregnated for fibers, of thymus cultures
during the first few days after explantation, there is an increasing denity of the fibers within the explant and the beginning of networks of fibers in the migration zone. To a
small degree the increasing denseness in the explant is due
to the migration of cells, but for the most part it is due to the
FIBERS IN TISSUE CULTURE
79
development of new fibrils. The process here is much the
same as described by Maximow in thymus and blood leukocyte cultures, and by McKinney for lymphatic tissue cultures,
and by Momigliano-Levi and others for connective tissue cultures. The original wide meshes of the fibrous reticulum become smaller and smaller due to the development of great
numbers of thin, new fibrils. These form progressively larger
bundles so that soon the original meshwork is completely obscured by the dense masses of new fibers.
The newly developed fibers in the migration zone are in
contact with the fibers of the mother piece. Until 8 days have
elapsed, there is nothing suggestive of the isolated fibrils and
spirochete-like figures that Maximow described in his 12-day
thymus cultures. Aftcr 8 days they are very abundant and
correspond in all details with those described by Maximow.
When thymus cultures of 8 to 12 days, or more, are impregnated in toto, these black-staining ‘fibrils’ appear as irreguIar,
thin wavy lines or clumps of lines radiating from a focus.
They are clearly not connected with the dense mass of fibers
in the explant and migration zone. Between the portion of
the culture medium not invaded by cells and the migration
zone, these isolated fibrils begin to fuse into larger connected
masses which in turn become continuous with those in the
center of the explant.
The fibrils in the migration zone lie in several layers which
are completely separated from one another. It was first
thought that these lamellae of fibers were due to successive
coatings of plasma when the cultures were washed, but the
evidence for this is not conclusive. The direction of the
fibrils in the different layers varies from one layer to the next
so that the fibrils do not always assume a primarily radial
arrangement,
Our failure to find the atypical fibrils with dark-field illumination of the living cultures is probably due to the fact that
the fibrin of the cultures makes observation with this technic
almost impossible. We have no evidence of the occurrence of
these fibrils in fibrin-free cultures.
80
WILLIAM BLOOM AND RUTH H. SANDSTROM
I n cultures of embryonic gall bladder, new reticular fibers
form very rapidly and in some instances in which liquefaction
has occurred in such cultures, figures comparable to those
described by Maximow for thymus cultures can be found in
varying numbers.
When bits of loose connective tissue are cultured from adult
or embryonic animals, and repeatedly subcultured by Carrell’s
cutting method, many generations may elapse without liquefaction occurring. But in some cultures liquefaction of the
fibrin suddenly develops. The reason for this is not apparent;
but, once liquefaction has started, we have found it almost
impossible to prevent it recurring in successive generations
developing from this particular culture. In several of these
connective tissue cultures which had developed liquefaction,
peculiar short wavy fibrils may be found beyond the zone
reached by migrating cells, much like those Maximow found in
thymus cultures. This is in sharp contrast to the development
of the fibrils in similar cultures in which liquefaction has not
occurred, for in the latter the fibrils always radiate more or
less by direct continuity from the center of the explant toward the periphery of the migration zone, reaching almost
to the outermost cells.
DISCUSSION
When the connective tissues are explanted in vitro and allowed to live by Maximow’s method of culturing-that
is
by simple washing and addition of fresh nutritive material to
the medium, and not by cutting-connective tissue fibers appear in great numbers. The fibrils in the center of the explant become more numerous and arranged in progressively
denser bundles which stain blue with Mallory’s aniline blue
method, and, with progressive aging of the culture, gradually
lose their ability to be impregnated with silver. In addition,
in the migration zone new fibrils appear, sometimes close to
the cell bodies but frequently without apparent connection
with them. These fibrils form a network whose connections
with the central explanted mass of fibrils can be traced without
FIBERS IN TISSUE CULTURE
81
difficulty in in toto preparations. When a tissue containing epithelium which liquefies the fibrin is explanted, or sometimes
when liquefaction occurs spontaneously in connective tissue
cultures there appear the interrupted, atypical fibrils which
Maximow considered to represent one of the first stages in
the development of connective tissue fibers in vitro.
The failure of other investigators to find these peculiar
fibers is undoubtedly due to the fact that all of them have
cultured various types of connective or reticular tissue. In
such cultures, for instance, Momigliano-Levi has described the
development of the fibers as occurring extracellularly, giving
figures quite similar to those which Maximow gave as occurring in some of his cultures. We shall discuss elsewhere
whether these are the first stages in the development of connective tissue fibers. The only point which we want to make
here is that the peculiar, spirochete-like figures which
Maximow found in his thymus cultures are to be found after
8 days with great regularity in cultures of this organ. They
may also be found at times in cultures of other tissues containing epithelium and occasionally in some of those connective tissue cultures in which liquefaction of the fibrin occurs
spontaneously. And we have not seen them in any cultures
unless liquefaction was present. The explanation of the mode
of origin of these peculiar figures is not clear. Some of them
may be small portions of the reticular framework which have
broken free, due to the disruption of the culture resulting from
the liquefaction of the fibrin, and have lodged in the culture
medium. We do not believe that this is the sole explanation of
the cause of the atypical fibers that Maximow described, for,
as we shall show elsewhere, the first fibers to develop in a
fiber-free culture often have the same appearance as the
peculiar figures which Maximow found in his thymus cultures.
The suggestion may be offered that these peculiar figures remlt from the solution of the collagenous material in those
cultures in which liquefaction occurs and that after a time this
dissolved collagenous material precipitates in the form of
star-shaped or wavy, spirochete-like fibrils. A somewhat
82
WILLIAM BLOOM AND RUTH H. SANDSTROM
similar process has been described by Nageotte ('31) for the
collagen which he has extracted from tendons in the tail of
the rat. At present we cannot give a definite answer to this
question.
It seems unlikely that the conditions that produce the first
fibers in fiber-free cultures are the same as those which cause
the development of the peculiar branching extracellular fibers
in cultures with liquefaction. The point to be emphasized is
that the peculiar figures described by Maximow can be duplicated at will in rabbit thymus cultures. The significance of
these structures in the question of fiber formation is still undetermined.
There is one more point to be noted: Although liquefaction
of the fibrin may appear as early as the third day in cultures
of the thymus, the atypical fibrils do not appear before the
eighth or ninth day.
CONCLUSIONS
The peculiar star- and crystal-like fibrils which Maximow
found beyond the migration zone in cultures of rabbit thymus
are found also in embryonic guinea pig gall bladder cultures
and in some subcutaeous connective tissue cultures in which
liquefaction of the fibrin has occurred.
These peculiar fibrils do not appear in cultures of reticular
o r collagenous tissues. As they fail to appear in such cultures
and as large numbers of typical reticular and collagenous
fibers do appear in them, these atypical fibrils cannot be looked
upon as evidence of an extracellular origin of connective tissue
fibers in general.
On the other hand, the first fibers which do appear in
originally fiber-free cultures often have the same appearance
as the atypical fibrils which Masirnow found in cultures of
thymus.
FIBERS IN TISSUE CULTURE
83
LITERATURE CITED
L m , G. 1934 Explantation, besenders die Struktur und die biologisehen
Eigenschaften der in vitro gezlichteten Zellen und Gewebe. Ergeb. d.
Anat. u. Entwicklungs., vol. 62, p. 105.
MAXIMOW,A. 1928 Development of argyophile and collagenous fibers in tissue
cultures. Proc. SOC.Exp. €601. and Med., vol. 25, p. 439.
1928 Ueber die Entatehung von argyrophilen und kolIagenen Fasern
in vitro. Central. f. allg. Path., vol. 43, p. 145.
1929 Uber die Entwicklung argyrophiler und Kollagener Faaern in
Kulturen von erwachsenen Siugetiergewebe. (Nach des Verf assers
Tode geschrieben und verSffentlicht von W. Bloom.) Zeit. f. mikrosk.anat. Forsch., vol. 17, p. 625.
MCKINNEY,R. L. 1929 Studies on fibers in tissue culture. 111. The development of reticulum into collagenous fibers in cultures of adult rabbit
lymph nodes. Arch. f. exper. Zellforschung, Val. 9, pp. 14-35.
MOMIQLIANO-LBVI,
G. 1932 Formazione e maturazione delle fibre collagene nelle
colture in vitro. Zeit f. Zellforsch., vol. 16, p. 389.
N A G E J.
~ , 1931 Essais de reproduction in vitro de la trame collagene et
bypothasea relatives a la construction de cette trame in vivo. Annals
d'Anatomie Pathol!gique
et d 'Anatomie Normale Medico-Chmrg.,
vol. 8, no. 1, Janvier.
Popom, N. W. 1927 The histogenesis of the thymus as shown by tissue cultures.
Arch. f. exp. Zellforsch., vol. 4, p. 395.
TSCHASSOWNIKOW,
N. 1926 ijber die in vitro-Kulturen des Thymus. Arch. f.
exper. Zellforsch., vol. 3, pp. 250-276.
1929 Ueber in vitro-Ziichtung von riintgenisiertem Thymus.
Ebenda., vol. 8, p. 189.
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