EXPERIMENTAL STUDY OF HUMAN GALL-BLADDER EPITHELIUM BY ORGAN CULTURE IN VITRO AND XENOTRANSPLANTATION TO NUDE MICE

Abstract

This paper presents results of the long-term maintenance of human gall-bladder epithelia as xenografts following explant organ culture. The sequential changes in morphological appearance during the period of in vitro organ culture as well as in vivo xenografts were extensively studied. Surgically resected gall-bladder tissues (8 cases with cholecystolithiasis and one with cholecysto-choledocholithiasis) provided the materials used in this study. They were cut into small fragments and maintained as explant organ cultures according to the method of Resau et. al. After several weeks of organ culture, about half of the explants were xenotransplanted into nude mice. The xenografts recovered at particular intervals were studied by microscopy and histochemistry. The results were as follows.In explant tissues, sequential changes in morphology, which included degeneration, regeneration and proliferation of the epithelia, were observed during the course of in vitro culture. These findings were substantiated by the viable epithelia composed of flat, cuboidal and columnar cells. The morphological viability was well preserved during the initial three weeks. However, it began to deteriorate thereafter, and no viable epithelia were obtained after 7 weeks. In contrast to the morphological viability, lactate dehydrogenase activity in the explant media showed bi-phasic elevation. The first peak, which occurred within 24 hours, was remarkably high ; however, the second peak, at around 10 days, was relatively low. These findings in morphology and enzymology are compatible with the process generally observed after cell injury and subsequent regeneration.In the xenografted tissues, the formation of multiple cysts of various sizes was noted. These cysts and the surrounding tissue contained various kinds of morphological architecture including projection of proliferated epithelial cells in the lumen and sinus-like structures. The viability of the epithelial cells composing the inner surface of the cyst walls was excellent. These cells showed various stages of morphological differentiation (flat, cuboidal and tall columnar cells) depending on how long the xenograft was maintained in vivo. In addition, acid muco-substance or sulfomucin, which was evidenced by positive staining with periodic acid-Schiff-alcian-blue and high-iron-diamine-alcian-blue, was observed in the tall columnar epithelial cells. These findings indicate functional differentiation in these cells.These results may extend the knowledge of human gall-bladder organ culture, and indicate that the method employed in the present study is potentially applicable to the study of human gall-bladder carcinogenesis in an experimental model.