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.