共查询到20条相似文献,搜索用时 15 毫秒
1.
Petra M. de Jong Marianne A. J. A. van Sterkenburg Johanna A. Kempenaar Joop H. Dijkman Maria Ponec 《In vitro cellular & developmental biology. Animal》1993,29(5):379-387
Summary In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies
obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies)
using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation
and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells
were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged,
low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell
cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under
submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an
appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six
layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged
and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed
in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the
culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was
not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia
appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial
cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function. 相似文献
2.
The Rose tissue culture chamber has been divided into 2 compartments by using half-thickness gaskets with an interposed Nuclepore (General Electric Co.) filter. This permits study of the interaction of 2 different cell populations on each side of the membrane. Radioiodinated serum albumin concentration studies demonstrate rapid distribution of labelled protein across the membrane while its pore size of 0.5 μ or 0.8 μ prevents migration of the cells themselves. Equilibrium between plain medium and radioactive medium was established in 7-9 hr. 相似文献
3.
Biosynthesis of respiratory-tract mucins. A comparison of canine epithelial goblet-cell and submucosal-gland secretions 总被引:3,自引:2,他引:1
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1. A modified canine tracheal organ culture system was used to investigate differences between mucous secretions of epithelial goblet cells and the submucosal glands. 2. Denuded explants were prepared by removing goblet, ciliated and basal cells from the surface epithelium leaving an intact basement membrane and viable submucosa. 3. Denuded explants actively incorporated radioactive precursors into secreted macromolecules when cultured in medium 199 containing label. 4. Chromatography on Bio-Gel A-150m and electrophoresis on 1% agarose gels indicated that epithelial goblet cell secretions were relatively more sulphated than submucosal glandular secretions. 5. The glandular structures were shown to respond to a parasympathomimetic agent. 相似文献
4.
Sweat JM Johnson CM Gibbs EP 《In vitro cellular & developmental biology. Animal》2003,39(5-6):249-256
In the present study, culture conditions that promote the growth and differentiation of manatee respiratory tract epithelial cells toward a mucociliary phenotype were determined. Characterization of a manatee-specific cell line enables investigators to conduct in vitro testing where live-animal experimentation is not possible. Cell cultures were established from both explants and enzymatically dissociated cells that were isolated from manatee bronchial tissue. To modulate their differentiation, bronchial epithelial cells were grown on Transwell collagen membranes either submerged or at an air-liquid interface. Growth on a collagen membrane at an air-liquid interface and medium supplemented with retinoic acid was required to promote a mucociliary phenotype. When cells were grown in submerged cultures without retinoic acid, they appeared more squamous and were not ciliated. Intracellular keratin proteins were detected in both submerged and interface cultures. Cultured manatee bronchial epithelial cells will facilitate future studies to investigate their potential role in pulmonary disease associated with brevetoxicosis after exposure to the red-tide organism, Karenia brevis. 相似文献
5.
Michael G. Gabridge 《Applied microbiology》1974,28(5):774-777
A modified parabiotic chamber for organ cultures is described. It consists of a modified organ culture dish, glass cover slip, a cylinder of semipermeable membrane, and a stainless-steel mesh filter support. Silicone and agar seals were used. Diffusion occurred rapidly and with consistent rates at 37 C when a ratio of 5:2 reservoir to inner chamber volume was used. The device is simple to construct and assemble and allows for continuous observation while maintaining a large surface area available for diffusion. The volumes of the reservoir, inner chamber, and agar base can be varied, and the chamber may be used with a wide variety of cell and organ culture systems. 相似文献
6.
Cyclamen persicum (Mill.) cv. Sierra Rose protoplasts were isolated from adventitious shoots regenerated in vitro from petiole
and leaf explants with yields of 1.3 × 106 protoplasts/g f.wt. of tissue. Protoplasts were embedded within agarose lenses
bathed in modified KM8p medium supplemented with 1.0 mg l-1 NAA, 0.1 mg l-1 2,4-D and 0.5 mg l-1 BA. Cell division was observed
after 4–5 days. After 6 weeks calli had grown out of the lenses which were transferred to modified MS medium for further callus
growth. The most rapid callus growth was on medium containing 0.1 mg l-1 NAA and 10 mg l-1 TDZ.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
7.
James H. Resau Ph.D. Kosaku Sakamoto John R. Cottrell Carnell Newkirk 《Cell biology and toxicology》1986,2(3):401-415
Adult Syrian Golden hamster alimentary tract maintained as explants in organ culture was studied using the model system for hamster pancreas described by Resau et al. (1983a). Explants of esophagus, stomach, duodenum and colon were maintained in organ culture on Gelfoam® sponge rafts in a high-oxygen atmosphere with serum-supplemented CMRL-1066 medium. All of the tissues were observed to show evidence of sublethal acute cell injury during the first several days of culture. Subsequently, the epithelial tissues recovered from this injury, repopulated the denuded areas of the explants and replicated within the sponge matrix. Explants were maintained in a differentiated state for 30+ days and sampled for morphology to examine the process of cell injury, repair, differentiation and replication which occurs in mucosal epithelia. The percentage of basement membrane covered by epithelia in the explants from various tissues was compared to the level of LDH in the media to reveal the relationship between viability determined by biochemical and by morphological methods. Restitution of the mucosal surface occurred in all of the explants. We conclude that adequate populations of replicating cells are maintained within the epithelium of the hamster alimentary tract tissues in vitro so that restitution can occur through migration and subsequent differentiation of the epithelial cells within the mucosa of the explants.Abbreviations 4F-1G
4% formaldehyde 1% glutaraldehyde fixative
- LDH
lactate dehydrogenase
- OsO4
osmium tetroxide
- PAS/PLH
periodic acid, periodic acid Shiff lead hematoxylin stain 相似文献
8.
Charles R. Capers 《The Journal of cell biology》1960,7(3):559-565
Healthy, mature, spontaneously contracting muscle was cultivated from explants of 13-day chick embryos for periods up to 4 months in the multipurpose chamber (Rose, 1954) using cellophane-strip technique (Rose et al., 1958) with silicone gaskets, Eagle's medium including 10 per cent horse serum reinforced with 300 mg-per cent of glucose, and the teased type of explant. This method provided optically ideal conditions for the study of muscle fibers with oil immersion, phase contrast time-lapse cinematography at 1 frame per minute without apparent damage for periods as long as 10 days. In no case was mitosis, amitosis, or nuclear "budding" observed in the course of muscle development. Multinuclear muscle fibers have been shown with cine technique to result from both myoblast fusion and polar extension of preformed (explanted) muscle tissue. Myoblast fusion was the only demonstrable way of giving rise to multinucleation. Nuclear membrane "wrinkling" was shown to be merely a temporary distortion that occurred during nuclear migration and rotation. It is suggested that this phenomenon may be responsible for numerous reports of amitosis in the genesis of muscle fibers. The histological development of new straps resulted from an orderly sequence of events. Included in these were polar extension, nuclear migration, rotation, and fixation. Following these events there was increased mitochondrial activity, myofibril formation, and cross-banding. Spontaneous contractions were seen throughout the entire course of differentiation in vitro but became more regular and stronger in the later stages. 相似文献
9.
Theodore Fainstat 《In vitro cellular & developmental biology. Plant》1972,7(5):300-303
Summary Explants of whole ovaries and oviducts from postnatal rats were completely submerged during cultivation as organ cultures
in chemically defined medium. The oxygen concentration in the culture chamber was raised to over 90%, and the stainless steel
platform, used for cultures at the surface of the medium, was abandoned and excluded from further use. Thus, all of the periphery
of an explant had equal access to nutrients and oxygen. Throughout the ovarian explants the tissues appeared uniformly viable,
and mitotic figures were distributed evenly. These observations are in contrast to those on organs cultivated at the surface
of the medium where a lack of structural uniformity had been detected. The method has general application to a variety of
organs. 相似文献
10.
Jae-Il Ahn In-Keun Jang Young-Kwon Seo Hee-Hoon Yoon Yeon-Ho Shin Jae-Chan Kim Kye-Yong Song Eun-Kyung Yang Jung-Keug Park 《Biotechnology and Bioprocess Engineering》2008,13(3):279-287
Herein, we reconstructed a rabbit corneal epithelium on a lyophilized amniotic membrane (LAM) using a modified version of
two Teflon rings (the Ahn’s supporter). We compared the corneal epithelial cells we had differentiated in vitro using air-liquid interface (6 days, 12 days) and submerged (6 days, 12 days) cultures and followed a six-day tilting dynamic
air-liquid interface culture with a six-day tilting submerged culture. We characterized the reconstructed corneal epithelium
using digital photography, histological imaging, and transmission electron microscopy. The reconstructed corneal epithelium
created under air-liquid interface culture exhibited a healthier basal corneal epithelial layer than that created under submerged
culture. The reconstructed corneal epithelium on the LAM that was produced using the tilting dymanic culture exhibited a healthy
basal layer. We therefore proposed that tilting submerged culture not only supplied nutrients from the medium to the corneal
epithelial cells on the LAM, but it also removed the horny layer in the upper part of the reconstructed corneal epithelium,
presumably by mimicking the effects of blinking. This study demonstrated that corneal epithelium reconstruction on a LAM using
a tilting submerged culture after a tilting air-liquid interface culture may be useful not only for allogeneic or autologous
transplantation, but also for in vitro toxicological test kits. 相似文献
11.
Theodore Fainstat 《In vitro cellular & developmental biology. Plant》1972,7(6):300-303
Summary Explants of whole ovaries and oviducts from postnatal rats were completely submerged during cultivation as organ cultures
in chemically defined medium. The oxygen concentration in the culture chamber was raised to over 90%, and the stainless steel
platform, used for cultures at the surface of the medium, was abandoned and excluded from further use. Thus, all of the periphery
of an explant had equal access to nutrients and oxygen. Throughout the ovarian explants the tissues appeared uniformly viable,
and mitotic figures were distributed evenly. These observations are in contrast to those on organs cultivated at the surface
of the medium where a lack of structural uniformity had been detected. The method has general application to a variety of
organs. 相似文献
12.
H. Märkl C. Zenneck A. Ch. Dubach James C. Ogbonna 《Applied microbiology and biotechnology》1993,39(1):48-52
High cell density cultivation of Escherichia coli on a glycerol-based mineral medium was studied. The cultivation was done in a dialysis reactor composed of two chambers. The inner chamber is formed and separated from an outer chamber by a membrane. Fresh medium was continuously exchanged with medium in the outer chamber so that both glycerol and other components of the medium were supplied to the inner chamber through the membrane. Inhibitory substances diffused from the inner to the outer chamber and were subsequently removed with effluent from the outer chamber. Initially, mathematical models were used to describe the process. The optimal cultivation parameters, such as the initial glycerol concentrations in the two chambers, the desired transport rate across the membrane, glycerol concentration in the feed/dialysing medium, and the time to start the medium exchange, were determined from preliminary experiments and calculations. The actual cultivation results agreed very well with the model predictions. A very high cell concentration of 174 g dry weight/1 was obtained. This cell concentration is within the range of the maximum theoretical concentration of E. coli in culture broth (160–200 g/l).Dedicated to the 60th birthday of Prof. Dr. D. Vortmeyer
Correspondence to: H. Märkl 相似文献
13.
Soluble factors and the immune response: in vitro studies of the immunosuppression induced by the graft-versus-host reaction 总被引:3,自引:0,他引:3
In vitro experiments were carried out to investigate the cause(s) of the immunosuppression induced by the graft-versus-host (GVH) reaction in F1 hybrid mice injected with parental strain lymphoid cells. A modified Marbrook culture chamber, made up of two cell compartments separated by a cell impermeable membrane, was used in these studies. Spleen cells from either normal animals (NSC) or from animals experiencing a GVH reaction (GVH-SC) were cultured with sheep red blood cells (SRBC) and the direct plaque-forming cell (PFC) response to SRBC was measured. It was found that normal thymus, lymph node and spleen cells, separated from the GVH-SC by a cell impermeable membrane, restored partially or totally the immune response of the suppressed cells, while bone marrow cells did not. It was also found that GVH-SC inhibited the PFC response to SRBC of NSC when mixed in culture at a ratio of 1:5. Conversely the inhibitory effect of GVH-SC on the immune response of NSC was abrogated when the two cell populations were separated by a cell impermeable membrane. These observations demonstrate that GVH-induced immunosuppression is caused, at least in part, by the deficiency of a T-cell derived factor which is a necessary component of the normal immune response. It is suggested that the suppressive effect of GVH-SC on the immune response of NSC is mediated by a non-T cell which regulates the release and/or production of the T-cell derived factor. 相似文献
14.
B. N. S. Murthy N. N. Vettakkorumakankav S. KrishnaRaj J. Odumeru P. K. Saxena 《Plant cell reports》1999,18(7-8):607-613
Several cultivars of hybrid seed geranium (Pelargonium×hortorum Bailey), previously shown to be recalcitrant in culture, produced somatic embryos at high frequency when explants were co-cultivated
with a morphogenesis promoting bacterium. This bacterium was isolated as an in vitro contaminant from cultures of geranium
seedling explants and identified as belonging to the genus Bacillus and species circulans. Co-cultivation of hypocotyl explants with the bacterium promoted somatic embryo formation and improved
both the frequency and quality of somatic embryos. In the cultivar Ringo Rose, the least responsive among the cultivars screened,
the embryogenic response was more than four times that of axenic cultures. Nearly 70% of these embryos converted into plantlets,
while the somatic embryos induced under axenic conditions developed poorly and plantlet formation was inconsistent. Among
the different treatments of bacterial culture tested (autoclaved culture, culture filtrate, sonicated bacterial culture, sonication
of bacterial culture followed by filtration, HPLC fractionation of crude bacterial lysate), only two HPLC fractions promoted
embryogenesis to a marginal degree. Co-cultivation of the explants with bacterium during the first week of induction was crucial
for obtaining high-frequency embryogenesis, indicating the role of bacterial stimuli during the induction process.
Received: 23 June 1998 / Revision received: 20 August 1998 / Accepted: 27 October 1998 相似文献
15.
Maras Z Yardley G Deane E Moore GP 《In vitro cellular & developmental biology. Animal》1999,35(10):606-611
Summary The secretory coil of the ovine apocrine gland is composed predominantly of two cell types, secretory cells lining the lumen
and myoepithelial cells adjacent to the basement membrane. The glands synthesize a number of hormones and growth factors,
but analysis of the functions of these molecules may be hampered by the mixing of apocrine and sebaceous secretions in the
pilary canal. The purpose of this study was to isolate the glands and devise simple culture procedures to facilitate investigations
of secretory cell function. The most successful approach involved microdissection of the secretory coils individually from
skin biopsies and culture in Dulbecco’s modified Eagle’s medium. After 1–2 wk in medium, cell outgrowths were seen from explants.
These consisted predominantly of populations of epithelial cells, many containing granules. Smaller granules were usually
concentrated around the cell nuclei and accumulated lipophilic dyes. Large granules were unreactive. Western analysis showed
that cells in culture synthesized nerve growth factor-like peptides, a feature consistent with one of the functions of the
gland in vivo. When isolated secretory coils were explanted to culture dishes coated with matrigel, highly compact, multilayered
masses of cells grew out. Subsequently, tubular structures formed. The observations suggest that some differentiated functions
of gland cells were retained in vitro and that the procedures described provide a system for the study of apocrine secretions
in isolation from those of other skin glands. 相似文献
16.
Plants were regenerated via adventitious shoot initiation from petal explants of carnation (Dianthus caryophyllus L.) cultivars Crowley Sim, Ember Rose, Orchid Beauty, Red Sim, White Sim and from stem segments of Crowley Sim, Red Sim, White Sim. Differences in cultivar response were observed, with White Sim being the most responsive for both explant types. Plants were also regenerated from receptacles of this cultivar. The effect of different cytokinins on regeneration from petal and stem explants of cultivar White Sim was compared. Thidiazuron was more effective than 6-benzylaminopurine or kinetin. In stem explants, morphogenic capacity was determined by the developmental stage of the explant. Highest percentage of shoot formation was observed in the youngest stem segments, on all the cytokinins tested. Stem-derived plants grew faster than petal or receptacle-derived plants and produced normal, flowering plants eight to ten months after culture. 相似文献
17.
The proteins, the DNA content and the brush border membrane enzyme activities of adult mouse intestinal explants have been measured during a 24-hr organ culture. These activities were not modified in comparison with the controls at the beginning of the culture. The activity of the enzymes secreted in the medium during the 24-hr culture was equal to the activity present in the explants at the start of the culture. These results show that several metabolic functions of the intestine are fully preserved in organ culture. 相似文献
18.
Micropropagation of three berry fruit species using nodal segments from field-grown plants 总被引:2,自引:0,他引:2
Plant micropropagation of blueberry (Vaccinium corymbosum L. cv. Berkeley), blackberry (Rubus sp. cv. Smoothstem) and raspberry (Rubus idaeus L. cv. Gradina) was carried out from nodal segments of adult field‐grown plants. Hardwood and softwood cuttings were studied as explant sources. The cultures successfully established were softwood from all three species, and hardwood only from blueberry. Shoot‐bud establishment from blueberry was achieved by culturing explants in WPM salts with MS vitamins for 15 days, and then 30 days in the same medium with 18 mM Zeatin. The best results of multiplication were obtained in the same medium with 25 mM 2iP. For blackberry, shoot‐bud establishment was achieved by culturing explants in MS medium for 15 days, and then in the same medium with 4 mM BA and 0.25 mM IBA. This medium was also the best for blackberry multiplication. Raspberry explants (cvs Gradina and Willamette) were cultured in MS medium for 15 days and then transferred to MS medium supplemented with 4 μm BA and 0.25 mM IBA. After 30 days of culture, only ‘Gradina’ explants survived, from which shoot‐bud establishment was obtained in a modified MS medium (Anderson's macronutrients except calcium, with Sequestrene as the iron source) with the same growth regulators. Multiplication was achieved by subculturing explants in the same medium either with 4 mM BA plus 0.25 mM IBA or with 8 mM BA plus 0.25 mM IBA. Shoots of at least 1 cm in length from all species were rooted ex vitro in a mixture of peat and Perlite (1:1, v/v) in a mist chamber, and 100% of rooting plants were acclimated. Bacterial, fungal and viral diseases were detected in stock plants, while tests carried out in both shoots and regenerated plants revealed the absence of any kind of disease. 相似文献
19.
Cylinders of carrot taproot secondary phloem were cultured on one of four media: 1) 2% sucrose + 1% agar (SA); 2) Heller's basal medium (NA); 3) NA + 10-5 g/liter 2,4-D (H4); and 4) NA + H4 + 15% coconut milk (HW). Samples were taken from the cultured explants at 3-day intervals. A morphological study of the cultured explants revealed no differences between callus-initiating explants (cultured on HW medium) and noncallus-initiating explants (cultured on SA, NA, and H4 media) within the first 3 days of culture. All explants exhibited a typical wound response. Cell division ceased in the NA and SA explants after the sixth day in culture. Extensive cell division occurred in the subsurface layer of dividing cells in the HW explants and resulted in the formation of callus by the ninth day in culture. Histochemical staining revealed that the activity of NAD diaphorase, succinic dehydrogenase, and cytochrome oxidase were closely correlated with the wound response and with callus initiation in the cultured explants. The activity of these enzymes was high in the layer of dividing cells of all explants after 3 days of culture, but with longer periods of culture the activity of these enzymes was closely correlated with the extent of cell division. Acid phosphatase activity was associated with the dividing cell layers of all explants, but comparatively little acid phosphatase activity was observed in the NA, SA, and H4 explants as compared to the HW explants, and acid phosphatase was strongly correlated with callus initiation by the HW explants. Using the nitroso reaction, “catechol tannins” were found in the surface layers of the NA, SA, and H4 explants, while no nitroso-reaction-positive substances were detected in the HW explants during the period of callus initiation. 相似文献
20.
Summary A completely liquid-filled growth chamber for axenic cultures ofTetrahymena pyriformis is described; gas exchange is ensured across a synthetic membrane. The chamber may be incorporated into a continuous flow system with inoculation and removal of cell samples under sterile conditions. Initially, the generation time of the cells was slightly prolonged, about 10%, but after some cell doublings decreased to 5%. The capacity of the cells to form food vacuoles (endocytosis) was unaltered during growth in the chamber. The synthetic membrane was highly permeable to O2 and CO2; however, cells grown in the chamber contained small refractive granules. The culture chamber permits the culture volume to be varied and it may be used for other protozoa, bacteria, and even tissue culture cells. 相似文献