Pupal ovaries of the wild oak silkworm Antheraea yamamai Guerin-Meneville were cultured in MGM-448 (Modified Grace Medium-448) medium containing 10% fetal bovine serum. After the primary culture was set up in 1988, a continuous cell line was obtained in 1991, designated as NISES-Anya-0611 (Anya-0611). The population doubling time was 54 hrs. and 19 min. at 96 passages and 88 hrs. and 29 min. at 387 passages. Spindle-shaped and spherical cells coexisted in the cell group. The cell line karyotype line was typical of lepidopteran cell lines, consisting of numerous small chromosomes. The cell line was distinguished from other lepidopteran cell lines by comparing malic enzyme, phosphoglucose isomerase, phosphoglucose mutase, and isocitric dehydrogenase isozyme patterns. The cell line was highly infected to the Antheraea yamamai nuclear polyhedrosis virus (Anya NPV). The luciferase gene of recombinant Bm NPV (BmNPVP6ETL) was able to express in the cell line, too, so that luciferase recombinant products were able to be detected in the cell body and in supernatant. The Anya NPV clone group was isolated on the cell seat using plaque purification.

The use of animal serum in cell culture is vital for providing the nutrient factors required to promote proliferation and function. Fetal calf serum has become the preferred choice because of its abundance, reasonable cost, and ability to sustain human cells in vitro. Although a wide variety of serum sources have been tested and used, little is known about the ability of serum obtained from the American black bear (Ursus americanus) to support human cell growth in culture. The American black bear, an animal comparable in size to humans, is unique in that it hibernates for mo at a time but does not experience extensive bone loss normally associated with extended immobility. The aim of this study was to analyze the effect of bear serum on human osteoblast cultures. We discovered that three of the eight bear serum samples induced significantly higher proliferation rates in osteoblasts than did fetal calf serum over a 24-h period. Osteoblasts incubated in bear serum displayed higher messenger ribonucleic acid levels for phenotype markers osteocalcin and type I collagen than did those incubated in fetal calf serum. The mitogenic activity of the bear serum was reduced when heated at 56° C for 30 min before use in culture. The molecular weight of the mitogenic factors was found to be primarily greater than 50 kDa. The present work demonstrates the capability of serum from American black bears to support human osteoblast proliferation in vitro.

Treatment with fat body extract (FBX) from pupae of the tobacco hornworm, Manduca sexta, caused mortality in larvae of two pest lepidopterans, the gypsy moth, Lymantria dispar, and the cotton leafworm, Spodoptera littoralis. In FBX-treated larvae, the feeding rate was depressed, causing reduced weight gain and then larval death. Their midgut showed formation of multicellular layers of midgut epidermis, indicating stem-cell hyperplasia. Hence, the integument of FBX-treated larvae had a double cuticle, indicating induction of premature molting. But radioimmunoassay measurements confirmed that the amount of ecdysteroids in FBX was too low to be responsible for the molt-inducing effects observed after treatment with FBX. With midgut stem cell cultures in vitro, addition of FBX to the culture medium stimulated cell proliferation and differentiation in a concentration-dependent manner. This effect was compared with those of insect molting hormones, ecdysone and 20-hydroxyecdysone; an ecdysteroid agonist, RH-2485; and a purified protein from FBX (multiplication factor). This article describes the mode of action of FBX and possible interplay between fat body factor(s) and insect hormones in the development and metamorphosis of the insect midgut.

Rat hepatocytes were cultured initially as spheroids on culture plates and then transferred into a rotating wall vessel (high–aspect ratio vessel [HARV]) for further culturing. Morphological evaluation based on electron microscopy showed that hepatocyte spheroids cultured for 30 d in the HARV had a compact structure with tight cell–cell junctions, numerous smooth and rough endoplasmic reticulum, intact mitochondria, and bile canaliculi lined with microvilli. The viability and differentiated properties of the hepatocytes cultured in the HARV were further substantiated by the presence of both phase I oxidation and phase II conjugation drug-metabolizing enzyme activities, as well as albumin synthesis. Homogenates prepared from freshly isolated hepatocytes and hepatocytes cultured in the HARV showed similar cytochrome P450 2B activities measured as pentoxyresorufin-O-dealkylase and testosterone 16β-hydroxylase. Further, intact hepatocytes cultured in the HARV were found to metabolize chlorzoxazone to 6-hydroxychlorzoxazone; dextromethorphan to dextrorphan, 3-methoxymorphinan, and 3-hydroxymorphinan; midazolam to 1-hydroxymidazolam and 4-hydroxymidazolam; and 7-hydroxycoumarin to its glucuronide and sulfate conjugates. In conclusion, we found that hepatocyte spheroids could be cultured in a HARV to retain cellular and physiological properties of the intact liver, including drug-metabolizing enzyme activities, plasma protein production, and long-term (1 mo) maintenance of viability and cellular function.

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of dilute cell culture media as an environmental or physiological simulant. Gill epithelia were cultured on cell culture inserts under symmetrical conditions (L15 apical–L15 basolateral) for 6–7 d. The following experiments were then conducted. (1) To mimic a gradual lowering of environmental salinity, apical L15 medium was progressively diluted with FW (first to 2/3 L15 for 8 h and then to 1/3 L15 for 6 h) before the introduction of apical FW (FW apical–L15 basolateral, analogous to a fish in a natural FW environment). Dilute apical media had no significant effect on the electrophysiological properties of preparations compared with symmetrical culture conditions, and no evidence for active Na or Cl⁻ transport was observed. Preparations subsequently exposed to apical FW exhibited a negative transepithelial potential and evidence of active Cl⁻ uptake and slight Na extrusion. (2) To mimic the extracellular fluid dilution that occurs in euryhaline fish after abrupt transfer from saline to FW, the osmolality or ionic strength (or both) of basolateral media was reduced by 20–40% (using either FW or FW mannitol) while simultaneously replacing apical media with FW. Under these conditions, Na and Cl⁻ influx rates were low compared with efflux rates, while the Ussing flux ratio analysis generally indicated active Cl⁻ uptake and Na extrusion. The Na –K adenosine triphosphatase activity was not affected by alterations in basolateral osmolality. Our studies indicate that cultured trout gill epithelia are tolerant of media dilution from both the apical and the basolateral direction; however, neither treatment alone appeared to increase ion influx rates or stimulate active Na uptake in cultured trout gill epithelia.

The genus Morone is an important one for U.S. aquaculture, but there has been no available cell line from this genus. We report here a cell line (the WBE line) derived from white bass embryos that has been grown for more than 80 passages over 21 mo in Dulbecco modified Eagle medium supplemented with fetal bovine serum. The WBE line showed epithelial features with positive immunohistochemical staining for cytokeratin and intercellular junctions when observed by electron microscopy. The doubling time, transformation characteristics, response to cryopreservation, and karyotype were determined. The WBE line was also tested by polymerase chain reaction to verify the species of origin and to screen for mycoplasma infection. This epithelial cell line may prove useful for the study of host–pathogen interactions that occur at the epithelial surface in this commercially important fish species.

Pharmacological treatments for psoriasis are generally based on antiproliferative, anti-inflammatory, or differentiation-modifying activity, or a combination of two or more of these actions. Potentially new drugs for treatment of psoriasis, which act on proliferation, can be identified by screening large compound libraries in a cell proliferation model that allows for characterization of drug effects on in vitro growth of normal human keratinocytes. High-throughput programs based on biological testing of diverse collections of compounds can rapidly identify leads for potential drug candidates in the treatment of psoriasis. In this study, we describe nonradioactive measurement of keratinocyte proliferation in the exponential growth phase in a 96-well format, using a sensitive deoxyribonucleic acid–binding dye to analyze drugs that are pharmacologically active in growth inhibition. Release of lactate dehydrogenase was used to exclude cytotoxic effects. We examined a number of compounds in a test range of 10⁻⁷ to 10⁻⁵ M, including known antipsoriatic drugs, and experimental drugs that are potentially useful in the treatment of psoriasis. We found strong concentration-dependent growth inhibition by dithranol, an antipsoriatic compound that is presumed to target the epidermal compartment. Methotrexate, cyclosporin A, and all-trans retinoic acid did not significantly affect proliferation at therapeutically relevant concentrations. The p38 mitogen-activated protein kinase inhibitor, SB220025, and curcumin, a natural phytochemical, inhibited keratinocyte proliferation at 10⁻⁵ M. We conclude that this assay, in combination with the previously developed assays for psoriatic differentiation, provides a useful tool for identification of antipsoriatic drugs.

Given the utility of murine models and the physiological and pathological significance of the aortic endothelium, we developed a simplified, nonenzymatic method for isolation and culture of murine aortic endothelial cells (MAECs). Aortic explants were initially cultured on fibronectin-coated plastic. Murine aortic endothelial cells migrated from the explants and proliferated. This expansion allowed for cultures to be established from the aortas of one or three mice. Murine aortic endothelial cells were then purified from expanded cultures by fluorescence-activated cell sorting for the uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate–labeled acetylated low-density lipoprotein. The majority of the cells in expanded cultures were as positive as human umbilical vein endothelial cells labeled in the same way. The most positive half of the labeled MAEC population was placed back in culture, and the cells formed “cobblestone” monolayers at confluence. Smooth muscle α-actin, which was present in aortic tissue and to a lesser extent in explant cultures before sorting, was not detected in selected MAECs. Western blotting and immunostaining also demonstrated the presence of the endothelial markers, platelet endothelial cell adhesion molecule-1, factor VIII–related antigen, and Bandeiraea simplicifolia lectin 1 binding. Murine aortic endothelial cells retained expected inflammatory functions: vascular cell adhesion molecule-1 protein was induced by bacterial endotoxin, and NO production was synergistically induced by the combination of endotoxin and interferon-γ. Our simple, efficient method will facilitate investigations of aortic endothelial cell function in vitro using murine models.

Optical measurements from epithelial cells grown on clear solid surfaces (e.g., coverslips, petri dishes) are often compared with other measurements (e.g., short-circuit current; I_sc) obtained from cells grown on opaque porous surfaces (inserts). However, the relative levels of differentiation of cells grown under the two conditions are usually unknown. To address this issue, we grew primary cultures of human tracheal epithelium on solid surfaces or on porous inserts and compared their total levels of protein and deoxyribonucleic acid, electrical properties in Ussing chambers, and ultrastructure. To measure ion transport across cells grown on solid supports, cells were grown on inserts placed on parafilm. Later, separation of insert from parafilm allowed the cells' I_sc to be measured in Ussing chambers. Four different media were used. Cells grown in one medium showed very low levels of differentiation on all growth supports. In the other media, growth on inserts markedly enhanced differentiation as compared with solid supports. Baseline I_sc of cells grown on either clear or opaque inserts was at least 30 times greater than that of cells grown on solid supports, though I_sc with clear inserts averaged ∼30% lower than that with opaque inserts. We conclude that though differentiation of cells may vary slightly depending on the insert used, cells on any type of insert are much better differentiated than cells grown on solid surfaces. Thus, it is both possible and desirable to make all functional measurements on cells grown on clear porous supports.

The purpose of the this study was to find media that supported high levels of differentiation in primary cultures of human tracheal epithelium. We tested six previously described, partially defined media and three nondefined media. Cells were grown with an air interface on porous-bottomed inserts, and differentiation was assessed from electrophysiological properties, levels of total protein and deoxyribonucleic acid, and histology. In all media, cells polarized and developed tight junctions, as assessed from transepithelial electrical resistance and were better differentiated at 14 d after plating than at 7 d. The partially defined media described previously by Gray et al. (Am. J. Respir. Cell. Mol. Biol. 14:104–112; 1996) and Matsui et al. (J. Clin. Invest. 102:1125–1131; 1998) and an undefined medium containing Ultroser G serum substitute produced the most highly differentiated epithelial cells, as revealed by a high short-circuit current (I_sc) and a ciliated, pseudostratified appearance. In other media, cells tended to be either squamous or stratified squamous, with I_sc levels <25% of those obtained with the three optimal media. Though no key factor in the composition of the partially defined media could be identified, two of the four media with high concentrations of retinoic acid produced good differentiation. In contrast, the two media with the lowest [Ca] (0.11 mM) produced poorly differentiated cells, as did the two partially defined media with low or no retinoic acid concentration.

Isolation and growth of malignant cells from solid tumors have often met with disappointing results. Consequently, we have developed a cell culture methodology based on ex vivo explantation of tumor tissue, with subsequent monolayer cell outgrowth. In an attempt to assess methods for detection of malignant cells in these cultures, we analyzed and compared the results of cytopathology, growth in soft agar, and detection of telomerase activity with those of standard immunohistochemistry (IHC) techniques for the detection of cytokeratins, tumor marker p53, and proliferation marker Ki-67. The sensitivity of detection of malignant cells was 85% (22/26) for cytopathological examination, 30% (3/10) for soft agar growth, and 100% (12/12) for detection of telomerase activity. From these data, we concluded that both cytopathological examination and assessment of telomerase activity contribute to the detection of malignant cells in primary cultures of human solid tumors, whereas growth in soft agar was not a good indicator of malignant cells. Although not specific for malignant cells per se, IHC detection for epithelial cell cytokeratins showed a high degree of sensitivity (100%, 23/23), whereas the sensitivity for detection of tumor marker p53 and proliferation marker Ki-67 was 30% (7/23) and 70% (16/23), respectively. These data also provide proof that malignant tumor cells, derived from a diverse number of human solid tumors, can be isolated and grown in primary cell culture.

Xenopus laevis A6 cells, which are cloned epithelial cells from the Xenopus kidney, differentiate into a dome structure when the cells reach confluence. We investigated the gravitational responses of A6 cellular motility during normal differentiation and differentiation under hypergravity conditions using centrifugation (1–100 × g). Progression to dome formation was analyzed by time-lapse micrography. Dome formation and increased expression of Na /K –adenosine triphosphatase were used as markers of differentiation. Interestingly, a high rate of cellular proliferation was observed at a low level of hypergravity (5 × g). Despite this, there was no difference in the time to dome formation between the control cells at primary cell density and those that differentiated under hyper- or hypogravity conditions. In conclusion, this experiment on amphibian cells revealed that the proliferation of A6 cells was strongly affected by gravity conditions, but the differentiation step appears to be controlled by an intra- or intercellular clock.

Recently, a sweet taste receptor family, the T1R family, that recognizes some carbohydrates including sucrose was identified. Although the T1R3 molecule is known to participate in heterodimers that are used as sweet- and umami-tasting receptors, there is no evidence that T1R3 alone recognizes similar ligands. We demonstrate for the first time that the candidate sweet taste receptor T1R3 is essential for the recognition and response to the disaccharide trehalose. Our system is a valuable tool not only for understanding the relationship between sweeteners and their receptors but also for exploring the diversities of their receptors, resulting in the design of new high-potency sweeteners.

A three-dimensional (3D) clinostat is a device for multidirectional G force generation. By controlled rotation of two axes, a 3D clinostat cancels the cumulative gravity vector at the center of the device and produces an environment with an average of 10⁻³ G over time. We cultured a human osteoblast cell line in a 3D clinostat and examined the growth properties and differentiation of the cells, including morphology, histological detection of calcification, and mitogen-activated protein kinase (MAPK) cascades. In a normal 1 G condition, alkaline phosphatase (AlPase) activity was detected on day 7 of culture, bone nodules were formed on day 12, and calcium deposits were seen on day 20. In the 3D clinostat, the cells looked larger and bulged. AlPase activity was detected on day 10 of culture. However, neither bone nodules nor calcification was found in the 3D clinostat up to day 21. The expression levels of core-binding factor A1 (a transcription factor for bone formation) and osteocalcin (a bone matrix protein) increased in the control culture but decreased in culture in 3D clinostat. Phosphorylation of p38^MAPK (p38) was repressed in culture in 3D clinostat, whereas total p38 as well as total and phosphorylated forms of extracellular signal–regulated kinases and stress-activated protein kinase/jun N-terminal kinase were not changed in the 3D clinostat. When a p38 inhibitor, SB 203580, was added to the culture medium in a normal 1 G environment, AlPase activity and formation of bone nodules and calcium deposits were strongly inhibited. On the other hand, they were inhibited only partially by a MAPK kinase inhibitor, U-0126. On the basis of these results, it is concluded that (1) osteoblast differentiation is inhibited in culture in a 3D clinostat and (2) this inhibition is mainly due to the suppression of p38 phosphorylation.

Cleft palate is the most common craniofacial anomaly. Affected individuals require extensive medical and psychosocial support. Although cleft palate has a complex and poorly understood etiology, low maternal folate is known to be a risk factor for craniofacial anomalies. Folate deficiency results in elevated homocysteine levels, which may disturb palatogenesis by several mechanisms, including oxidative stress and perturbation of matrix metabolism. We examined the effect of homocysteine-induced oxidative stress on human embryonic palatal mesenchyme (HEPM) cells and demonstrated that biologically relevant levels of homocysteine (20–100 μM) with copper (10 μM) resulted in dose-dependant apoptosis, which was prevented by addition of catalase but not superoxide dismutase. Incubation of murine palates in organ culture with homocysteine (100 μM) and CuSO₄ (10 μM) resulted in a decrease in palate fusion, which was not significant. Gelatin gel zymograms of HEPM cell–conditioned media and extracts of cultured murine palates, however, showed no change in the expression or activation of pro–matrix metalloproteinase-2 with homocysteine (20 μM–1 mM) with or without CuSO₄ (10 μM). We have demonstrated that biologically relevant levels of homocysteine in combination with copper can result in apoptosis as a result of oxidative stress; therefore, homocysteine has the potential to disrupt normal palate development.