Characterization of morphology and cellular metabolism during the spheroid formation by fibroblasts.

Normal adult human dermal fibroblasts were cultured at 37 degrees C on a thermoresponsive substratum composed of poly-N-isopropyl acrylamide (PNIPAAm) and type I collagen. The confluent fibroblasts were forced to detach from the substratum as a monolayer cell sheet made of polygonal cells linked together with many microvilli by decreasing an ambient temperature from 37 degrees C to a temperature below the lower critical solution temperature of PNIPAAm (about 30 degrees C). The floating cell sheet shrank and condensed into an aggregate with gap and tight junctions within several hours and finally formed a spheroid by 2 days. Spheroids were covered with squamous cells and contained cuboidal cells inside. Cycloheximide and actinomycin D reversibly inhibited the spheroid formation. Extracellular matrix fibrils deposited among inner cells when the culture period extended over 7 days. A 28-day-old spheroid with a diameter of about 600 microns contained live cells even in the central region. Cellular metabolic activities such as glucose consumption, lactic acid production, and ATP content of spheroids were significantly lower than those of monolayers. The spheroid described in the present study seems to be a useful in vitro model of connective tissues.

[Bochdalek diaphragmatic hernia in the elderly: a case report].

We successfully treated surgically an elderly patient, a 80-year-old woman, with Bochdalek diaphragmatic hernia. She had intermittent episodes of intestinal obstruction. A preoperative chest roentgenograph revealed a loop of gas-filled bowel in the left chest and elevation of the left diaphragm. CT scanning of the thorax revealed the bowels filled with gastrografin in the left thoracic cavity. She was treated surgically, through transabdominal and then transthoracic approaches. The herniated large bowel was reduced from the thoracic cavity and the hernial sac was excised. The posterolateral defect of the diaphragm, 5 x 6 cm in size was closed with interrupted mattress sutures. The patient has been well without any symptoms 6 months after the surgery. Bochdalek diaphragmatic hernia is the most common problem in infants with risky respiratory distress and high mortality, however it is preferable to carry out surgical treatment for adult patients because of good postoperative results. As a surgical route in the management of Bochdalek diaphragmatic hernia in adults, a transthoracic approach is preferable since it provides easy separation and reduction of herniated bowels from the thoracic cavity and easy closure of the diaphragmatic defect, furthermore there are no other serious gastrointestinal complications which are required specific repair.

Morphological and immuno-cytochemical characterization of a hetero-spheroid composed of fibroblasts and hepatocytes.

A novel method for the preparation of spheroids containing two types of cells (hetero-spheroid) has been successfully developed by utilizing a collagen-conjugated thermo-responsive polymer, poly-N-isopropyl acrylamide (PNIPAAm), as a cell substratum. PNIPAAm solidifies above its lower critical solution temperature (LCST, about 30 degrees C), and instantly dissolves into the culture medium below its LCST. We firstly seeded and cultured human dermal fibroblasts on the substratum up to a confluent state and then seeded rat primary hepatocytes onto the fibroblast monolayer. The heterospheroid was prepared by detaching the hepatocyte-attached fibroblast monolayer at a temperature below LCST and culturing it on the non-adhesive substratum. The surface area of the substratum and the seeding population ratio of each cell precisely and reproducibly regulated the size and the cell composition of the resulting hetero-spheroid, respectively. Histological and immuno-cytochemical observations of spheroids revealed characteristic organizations of fibroblasts and hepatocytes within a spheroid because the latter cells expressed albumin for up to at least 3 weeks. TEM study of the hetero-spheroid showed the presence of structures morphologically similar to the Disse's space and the bile canaliculus, which are features characteristic of liver. These findings suggest that the method described above is useful for making a hetero-spheroid that morphologically and functionally resembles tissues or organs in vivo, i.e. an organoid.

Reexamination of photoreceptor-bipolar connectivity patterns in carp retina: HRP-EM and Golgi-EM studies.

On- and off-center bipolar cells were identified in the carp retina by means of intracellular recording, intracellular injection of HRP, and Golgi silver-chromate impregnation. Light and electron microscopy revealed that these functionally different bipolar cells make synaptic contacts with both rods and cones, and that both on- and off-center cells can be further divided into two subtypes (I and II) according to the relationship between the position of their dendritic processes and the synaptic ribbons in the photoreceptor terminal. The type I on-center bipolar cell is characterized by a large cell body, a thick primary dendrite, and a big swelling of the axon terminal in the innermost part of the inner plexiform layer (IPL). Dendritic processes of this cell type make predominantly ribbon contacts with rods and nonribbon contacts with cones. The type II on-center cell, having a large dendritic tree in the outer plexiform layer and a large ramification of the axon terminal extending over the inner part of the IPL makes mostly nonribbon contacts with rods and cones. Many of these type II cell processes, however, terminate very close to cone synaptic ribbons. The type I off-center cell shows two varieties in the axon terminal structure; a large terminal swelling or a large flat ramification of the terminal in the outermost part of the IPL. These cells make predominantly ribbon contacts with rods and cones. Usually, but not always, the process of a type I off-center cell runs parallel to the synaptic ridge apex of cones. The type II off-center cell, showing a large ramification of the axon terminal extending over the outer half of the IPL, makes mainly nonribbon contacts with rods and cones. The results from the HRP-EM study generally agree with those from the Golgi-EM study. A few discrepancies between the results obtained with these two techniques are noted and their implication is discussed.

Electron microscopic studies on tissue distribution of lipid microspheres used as drug delivery carriers.

The tissue distribution of lipid microspheres (LMs), drug carriers for targeting therapy of anti-inflammatory drugs, was morphologically studied by electron microscope. In areas of inflammation in rats and mice, LMs were taken up by macrophages and accumulated around endothelial cells of blood vessels, and were observed to penetrate to the outer layer of blood vessels. LMs were also observed in reticuloendothelial cells such as Kupffer cells and splenic macrophages. Furthermore, the uptake of LMs by polymorphonucleocytes (PMNs) in areas of inflammation was enhanced 2-3 fold when LMs were coated with homogeneous IgG. These findings are in agreement with the tissue distribution results previously reported by the authors in studies using radioisotope-labelled LMs. The present and previous reports indicate that LMs could be used as a novel drug carrier, similarly to liposomes, in a drug delivery system specific for areas of inflammation and reticuloendothelial systems.

[Studies on minimum antibiotic concentration of cephapirin against clinically isolated strain SMK-101 of Klebsiella pneumoniae].

Using strain SMK-101 of K. pneumoniae its nephelometric absorbencies, viable cell numbers and morphological changes were studied during the time course cultured in a broth medium containing cephapirin (CEPR), and following results were obtained. After 1 to 3 hours culture in the presence of varying concentration of the antibiotic, the absorbency increased in spite of without change in the viable cell number. Morphologically, elongation and swelling of central portion of the cells were observed though differences of the degree of these findings varied depending upon the concentration of the antibiotic. At the concentration higher than 1/4 MIC, indistinct structure was shown in cytoplasm. After 6 hours culture, 3 directions of absorbence curves, ascending, descending and no change, and 2 directions of viable cell numbers, decreasing and increasing were shown. As the morphological changes of the cells, filamentation, leaking of intracellular components were shown in rather upper concentration of the antibiotic. Fission was demonstrated around the end of cells cultured in rather lower concentration of the antibiotic. After 9 hours culture, absorbency and viable cell number were parallel. In this period, structural findings of cytoplasm became clear and fission was also demonstrated by light microscope except for the cells cultured in more than 1 MIC of the antibiotic. After 24 hours culture, both absorbency and viable cell number increased again and fission was observed in the cell which showed filamentation in 1 MIC of the antibiotic.

Connections between photoreceptors and horseradish peroxidase-injected bipolar cells in the carp retina.

We describe two physiologically and morphologically distinct types of on-center bipolar cell in the carp retina. These cells can be distinguished from one another in the following respects: (1) "type I" cells respond to moderate light intensities with a transient depolarization followed by a plateau, while the response of "type II" cells is approximately rectangular in shape; (2) latency of type II cell responses is shorter than that of type I cell responses; (3) mean diameter of type II cell dendritic fields (114 micron) is larger than that of type I cell dendritic fields (65 micron) in the outer plexiform layer; (4) mean diameter of type II cell bodies (7 micron) is smaller than that of type I cell bodies (9 micron); (5) axons of type II cells show extensive arborizations (51 micron), while those of type I cells show a single swelling with a few short collaterals (31 micron) in the proximal half of inner plexiform layer. HRP-injected on- and off-center bipolars were examined by electron microscopy. The dendrites of these bipolar cells invaginate into rod spherules and often occupy the central position of the triad. The dendrites of such bipolar cells also invaginate into cone pedicles, but in quite distinct patterns. Type I on-center bipolars do not send processes to the synaptic ribbons. In contrast, off-center bipolars and type II on-center bipolars send processes to the synaptic ribbons. The dendrites of off-center bipolars occupy the central position of the triad. Although type II on-center bipolar dendrites approach the ribbon very closely, there is usually a process from an off-center bipolar interposed between them.