共查询到20条相似文献,搜索用时 15 毫秒
1.
Builles N Bechetoille N Justin V André V Barbaro V Di Iorio E Auxenfans C Hulmes DJ Damour O 《Cell biology and toxicology》2007,23(4):279-292
We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for
use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent,
keratocytes from human corneas were cultured in collagen–glycosaminoglycan–chitosan foams. Limbal stem cell-derived epithelial
cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological
counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63ΔNα,
and the expression of keratin 3 and 14-3-3σ in the upper cell layers. In addition, the cuboidal basal epithelial cells were
anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes
colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular
matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to
have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas.
Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions
by including the inducing effects of mesenchyme and cell–matrix interactions on epithelial cell morphogenesis. 相似文献
2.
Silvia Díaz-Prado Mª Esther Rendal-Vázquez Emma Muiños-López Tamara Hermida-Gómez Margarita Rodríguez-Cabarcos Isaac Fuentes-Boquete Francisco J. de Toro Francisco J. Blanco 《Cell and tissue banking》2010,11(2):183-195
The human amniotic membrane (HAM) is an abundant and readily obtained tissue that may be an important source of scaffold for
transplanted chondrocytes in cartilage regeneration in vivo. To evaluate the potential use of cryopreserved HAMs as a support
system for human chondrocytes in human articular cartilage repair. Chondrocytes were isolated from human articular cartilage,
cultured and grown on the chorionic basement membrane side of HAMs. HAMs with chondrocytes were then used in 44 in vitro human
osteoarthritis cartilage repair trials. Repair was evaluated at 4, 8 and 16 weeks by histological analysis. Chondrocytes cultured
on the HAM revealed that cells grew on the chorionic basement membrane layer, but not on the epithelial side. Chondrocytes
grown on the chorionic side of the HAM express type II collagen but not type I, indicating that after being in culture for
3–4 weeks they had not de-differentiated into fibroblasts. In vitro repair experiments showed formation on OA cartilage of
new tissue expressing type II collagen. Integration of the new tissue with OA cartilage was excellent. The results indicate
that cryopreserved HAMs can be used to support chondrocyte proliferation for transplantation therapy to repair OA cartilage. 相似文献
3.
Abdullah B Shibghatullah AH Hamid SS Omar NS Samsuddin AR 《Cell and tissue banking》2009,10(3):205-213
This study was performed to determine the microscopic biological response of human nasal septum chondrocytes and human knee
articular chondrocytes placed on a demineralized bovine bone scaffold. Both chondrocytes were cultured and seeded onto the
bovine bone scaffold with seeding density of 1 × 105 cells per 100 μl/scaffold and incubated for 1, 2, 5 and 7 days. Proliferation
and viability of the cells were measured by mitochondrial dehydrogenase activity (MTT assay), adhesion study was analyzed
by scanning electron microscopy and differentiation study was analyzed by immunofluorescence staining and confocal laser scanning
electron microscopy. The results showed good proliferation and viability of both chondrocytes on the scaffolds from day 1
to day 7. Both chondrocytes increased in number with time and readily grew on the surface and into the open pores of the scaffold.
Immunofluorescence staining demonstrated collagen type II on the scaffolds for both chondrocytes. The results showed good
cells proliferation, attachment and maturity of the chondrocytes on the demineralized bovine bone scaffold. The bovine bone
being easily resourced, relatively inexpensive and non toxic has good potential for use as a three dimensional construct in
cartilage tissue engineering. 相似文献
4.
Lucie Gregoire Adnan Munkarah Raja Rabah Robert T. Morris Wayne D. Lancaster 《In vitro cellular & developmental biology. Animal》1998,34(8):636-639
Summary The objective of this work was to establish an in vitro multidimensional culture system for human ovarian surface epithelial (HOSE) cells as a model for ovarian carcinogenesis.
The epithelial origin of cell outgrowth from cells obtained from the ovarian surface was confirmed by keratin staining. Two
cultures from two different patients were established, HOSE-A and HOSE-B. Cultures were infected with a retrovirus expressing
human papillomavirus genes E6 and E7 to extend their life span. HOSE cells were seeded onto collagen gels containing NIH3T3-J2
fibroblasts as feeder cells and grown to confluence submerged in growth medium. The collagen bed was then raised to the air-medium
interface for 7 d (organotypic culture). Microscopically, fixed cultures revealed a single layer of flat cells growing on
the collagen surface, reminiscent of HOSE cells in vivo. Infected HOSE-A and HOSE-B cells exhibited aberrant growth because they stratified. In addition, established ovarian cancer
lines grown in this fashion stratified and showed malignant phenotypes. Thus, cells grown in organotypic culture resemble
their in vivo counterparts, providing a basis for establishing a system to study growth, proliferation, differential gene expression, and
perhaps malignant transformation of HOSE cells. 相似文献
5.
This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air-liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 mum) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes. 相似文献
6.
Maria Iris Hermanns Sabine Fuchs Michaela Bock Katja Wenzel Eckhard Mayer Kai Kehe Fernando Bittinger C. James Kirkpatrick 《Cell and tissue research》2009,336(1):91-105
In order to delineate individual pathomechanisms in acute lung injury and pulmonary toxicology, we developed a primary coculture
system to simulate the human alveolo-capillary barrier. Human pulmonary microvascular endothelial cells (HPMEC) were cocultivated
with primary isolated human type II alveolar epithelial cells (HATII) on opposite sides of a permeable filter support, thereby
constituting a bilayer. Within 7–11 days of coculture, the HATII cells partly transdifferentiated to type-I-like (HATI-like)
cells, as demonstrated by morphological changes from a cuboidal to a flattened morphology, the loss of HATII-cell-specific
organelles and the increase of HATI-cell-related markers (caveolin-1, aquaporin-5, receptor for advanced glycation end-products).
Immunofluorescent analysis detected type-II-like and type-I-like alveolar epithelial cells mimicking the heterocellular composition
of alveolar epithelium in vivo. The heterocellular epithelial monolayer showed a circumferential staining of tight-junctional
(ZO-1, occludin) and adherens-junctional (E-cadherin, β-catenin) proteins. HPMEC on the opposite side also developed tight
and adherens junctions (VE-cadherin, β-catenin). Under integral barrier properties, exposure to the proinflammatory cytokine
tumour necrosis factor-α from either the endothelial (basolateral) or the epithelial (apical) side caused a largely compartmentalized
release of the chemokines interleukin-8 and monocyte chemoattractant protein-1. Thus, the established coculture provides a
suitable in vitro model to examine barrier function at the distal lung, including the interaction of microvascular endothelial
cells with ATII-like and ATI-like epithelial cells. The compartmentalization of the barrier-forming bilayer also allows mechanisms
of lung injury to be studied in both the epithelial (intra-alveolar) and the endothelial (intravascular) compartments.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
This study was supported by the BMVg Grant E/B41G/1G302/1A402 and the 6th Framework program of the European Union, NanoBioPharmaceutics. 相似文献
7.
Summary A novel culture system included a self-designed bi-layer 3-D collagen scaffold with different pore size on both sides and
specific culture media for different culture stages. This skin equivalent culture model provides a new investigating system
to study the role of extracellular matrix and growth factors including epidermal growth factor (EGF), keratinocyte growth
factor (KGF), transforming growth factor beta 1 (TGF-β1), in the cell–cell and cell–matrix interactions. Keratinocytes were
seeded onto the dermal equivalent and incubated under submerged condition for 5 days then proceeding to air–liquid interface
cultured either with or without EGF addition. In this study, EGF has a positive effect on the keratinocyte migration and proliferation
in the submerged stage. However, when 10 ng per ml of EGF was continual added in the air-lifted stage, a less organized and
thin differentiated keratinocyte layers were found. Continual 10 ng per ml of EGF addition in the air-lifted stage resulted
in uneven cell–matrix interface, and disorganization of the suprabasal layers. On the contrary, in the air-lifted stage without
excess EGF, the epithelium cells will stratify, differentiate, and form an epidermis completed with basal, spinous, granular,
and cornified layers. The results showed that time scale modulation of EGF on keratinocyte cell behavior depend on the expression
of paracrine or autocrine growth factors (e.g. KGF and TGF-β1). 相似文献
8.
用低温酶消化法分离兔气管上皮细胞,具有细胞损伤小,活力及纯度高的优点,成纤维细胞污染极低。人胎盘胶原提高了气管上皮细胞贴壁性。无血清培养基能促进细胞增殖,分化和成熟。气液界面培养方式更好地模拟了气管上皮细胞的天然生长环境,在膜上呈复层生长,有利于细胞的分化成熟及功能表达。光镜下细胞形态及免疫组化细胞角蛋白染色阳性证实培养细胞为气管上皮细胞。本文所建立的兔气管上皮细胞体外气液界面无血清培养方法为研究气 管上皮细胞的生理和病理提供了一个十分有用的模型。 相似文献
9.
Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. We have investigated the ability of implanted chondrocytes to attach at the site of injury and to be incorporated into the decellularized host matrix adjacent to a defect in an in vitro human explant model. Human osteochondral dowels received a standardized injury, were seeded with passage 3 chondrocytes labelled with PKH 26 and compared with two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Seeded chondrocytes adhered to the standardized defect and areas of lamina splendens disruption but did not migrate into the adjacent acellular region. A difference was noted in viable-cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present. The results indicate that cultured human chondrocytes do indeed adhere to regions of AC matrix injury but do not migrate into the host tissue, despite the presence of viable cells. This human explant model is thus an effective tool for studying the interaction of implanted cells and host tissue. 相似文献
10.
Coraux C Zahm JM Puchelle E Gaillard D 《American journal of physiology. Lung cellular and molecular physiology》2000,279(2):L224-L234
Development of human fetal airways requires interaction of the respiratory epithelium and the extracellular matrix through integrins. Nevertheless, the specific roles of beta(1)-integrins during development and tubular morphogenesis are still unknown. To analyze beta(1)-integrin localization and influence during migration, we developed a model of human fetal tracheal explants growing on collagen and overlaid with a second layer of collagen to form a sandwich. In this configuration, cord and tubule formation proceeded normally but were inhibited by incubation with anti-beta(1)-integrin subunit antibodies. On a collagen matrix, beta(1)-integrins were immunolocalized on the entire plasma membrane of migrating epithelial cells and almost exclusively on the basal plasma membrane of nonmigratory epithelial cells. In a sandwich configuration, beta(1)-integrins became detectable in the cytoplasm of epithelial cells. Coating cultures with collagen transiently altered the morphology of migrating cells and their speed and direction of migration, whereas incubation with anti-beta(1)-integrin subunit antibodies irreversibly altered these parameters. These observations suggest that the matrix environment, by modulating beta(1)-integrin expression patterns, plays a key role during tubular morphogenesis of human fetal tracheal epithelium, principally by modulating epithelial cell migration. 相似文献
11.
Elevated expression of hormone-regulated rat hepatocyte functions in a new serum-free hepatocyte-stromal cell coculture model 总被引:6,自引:0,他引:6
Ries K Krause P Solsbacher M Schwartz P Unthan-Fechner K Christ B Markus PM Probst I 《In vitro cellular & developmental biology. Animal》2000,36(8):502-512
Summary The specific performance of the adult hepatic parenchymal cell is maintained and controlled by factors deriving from the stromal
bed; the chemical nature of these factors is unknown. This study aimed to develop a serum-free hierarchical hepatocyte-nonparenchymal
(stromal) cell coculture system. Hepatic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal
growth factor, basic fibroblast growth factor, and hepatocyte-conditioned medium; cell type composition changed during the
2-wk culture period. During the first wk, the culture consisted of proliferating sinusoidal endothelial cells with well-preserved
sieve plates, proliferating hepatic stellate cells, and partially activated Kupffer cells. The number of endothelial cells
declined thereafter; stellate cells and Kupffer cells became the prominent cell types after 8 d. Hepatocytes were seeded onto
stromal cells precultured for 4–14 d; they adhered to stellate and Kupffer cells, but spared the islands of endothelial cells.
Stellate cells spread out on top of the hepatocytes; Kupffer cell extensions established multiple contacts to hepatocytes
and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte
differentiation became evident after 48 h; a strong improvement of cell responsiveness toward hormones could be observed in
cocultured hepatocytes. Hierarchial hepatocyte coculture enhanced the glucagon-dependent increases in phosphoenolpyruvate
carboxykinase activity and messenger ribonucleic acid (mRNA) content three- and twofold, respectively; glucagon-activated
urea production was elevated twofold. Coculturing also stimulated glycogen deposition; basal synthesis was increased by 30%
and the responsiveness toward insulin and glucose was elevated by 100 and 55%, respectively. The insulin-dependent rise in
the glucokinase mRNA content was increased twofold in cocultured hepatocytes. It can be concluded that long-term signals from
stromal cells maintain hepatocyte differentiation. This coculture model should, therefore, provide the technical basis for
the investigation of stroma-derived differentiation factors. 相似文献
12.
Vitrification is a promising alternative to tissue preservation, in which the tissue is permeated with cryoprotective agents (CPAs) in order to circumvent the hazardous effects associated with ice formation. In this study, we evaluate the effect of vitreous cryopreservation of rabbit trachea, by comparing vitrification procedure with conventional computer-programmed slow freezing approaches. Harvested rabbit trachea were tailored and divided into groups and cryopreserved by vitrification and programmed freezing, respectively. The morphology and ultrastructure of the thawed tracheal fragments including HE dyes, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) staining, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were studied to assess the integrity of the tracheal fragments. Morphological studies demonstrated that both cryopreservation procedure retained the integrity of trachea, both epithelial cells, cilia and cartilage cells were in good shape. Compared with slow freezing methods, vitrification was less detrimental to cartilage cells and had a higher survival rate of chondrocytes and coverage of epithelium and cilia. Therefore, vitrification procedure can be a more satisfactory method to preserve trachea and the survival of chondrocytes in situ in cartilage tissue is adequate and respiratory epithelium is soundly present. 相似文献
13.
We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on
tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm × 4 mm) were seeded with bovine articular
chondrocytes and cultured under normoxic (21% O2) or hypoxic (5% O2) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10–20%; 0.5 Hz) was
applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (±25°; 0.5 Hz). Culture
under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression
of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in
normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic
than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan
mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen
tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the
chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis.
Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype
and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating
cartilage-like tissue in vitro.
This work was supported by the Swiss National Science Foundation (grant no. 3200B0-104083). 相似文献
14.
Feng Xie WenJie Zhang FanFan Chen GuangDong Zhou Lei Cui Wei Liu YiLin Cao 《中国科学C辑(英文版)》2008,51(9):774-778
In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic environment provided by mature
chondrocytes was investigated. Flk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine
articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold
complexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type II collagen antibody staining.
Positive staining of mouse Major Histocompatibility Complex class I molecules confirmed that part of the chondrocytes were
derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation. 相似文献
15.
Atsushi Ochiai Makito Emura Margrit Riebe-Imre Ulrich Mohr Jörn Hilfrich Eiichi Tahara Donald L. Dungworth 《Virchows Archiv. B, Cell pathology including molecular pathology》1992,61(1):217-226
A human fetal bronchial epithelial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions.
Despite a somewhat fibro-blastic shape the cells maintained immunoreactivity to cytokeratin, carcinoembryonic antigen and
epithelial membrane antigen. When grown on a collagen gel in a growth-hormone-supplemented medium, their spindle shape became
more conspicuous. With an additional supplement of vitamin A (6 μg/ml), most of the cells underwent differentiation by producing
many bright inclusion bodies which proved to be strongly positive with periodic acid-Schiff and weakly positive with alcian
blue staining. Electron microscopy revealed a well-developed rough endoplasmic reticulum, an enlarged Golgi apparatus and
many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE
cells cultured on collagen gel with vitamin A secreted hyaluronic acid and neutral glycoproteins containing mainly N-linked
glycoproteins whose glycans were of a complex type. A monoclonal antibody (SEC-41) generated against the neutral glyco-proteins
detected a glycoprotein of approximately 52 kDa in the spent culture medium of differentiated HFBE cells. This antibody also
reacted with the intracytoplasmic secretory granules in these cells. When tested on frozen sections of lung tissue, the immunohistochemical
reactivity of the SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung, and respiratory
epithelial cells in the fetal lung. More-over, this antibody could detect secretory glycoprotein in broncho-alveolar lavages
from two patients. This paper clearly demonstrates that cells derived from human fetal bronchial epithelium can be cultivated
in an undifferentiated precursor state and, under appropriate culture conditions, can be stimulated to undergo differentiation
into a Clara cell type. 相似文献
16.
Gao Nan Wang ZhiChong Huang Bing Ge Jian Lu Rong Zhang KeFei Fan ZhiGang Lu Li Peng Zhan Cui GuangHui 《中国科学C辑(英文版)》2007,50(1):101-110
Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like
cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal
of this study was to investigate the potential of epidermal stem cells for trans-differentiation into corneal epithelium-like
cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis,
immunohistology and RT-PCR were conducted to identify the expression of specific markers (β1m α6 integrin, K15, K1/K10, K3/K12
and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal
epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression
difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10.
Putative epidermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant
in rapid attaching cells by type IV collagen attachment isolation. Before being cocultured, the rhesus putative epidermal
stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12
(a marker of corneal epithelial cells), K15 and β1 integrin, but no K1/K10. Cells being not coculture converted into terminally
differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate
into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for
the construction of bioengineered corneas. 相似文献
17.
A double staining method combining Ulex europaeus agglutinin I lectin (UEA-I) and collagen type IV staining was used to determine the capillary density and the number of capillaries
relative to different fibre types in human skeletal muscles. The result of this combined staining was compared with that of
other staining methods including amylase-periodic acid Schiff (PAS), UEA-I, anti-collagen type IV and anti-von Willebrand
factor. Muscle biopsy specimens, 12 from M. vastus lateralis and 6 from M. soleus, were obtained from 18 healthy young men. Compared with amylase-PAS staining, double staining showed a larger number of capillaries
surrounding type I (+9.6%), type IIA (+8.6%) and type IIB (+11.6%) fibres in the M. vastus lateralis specimens (P<0.001 for all differences). The capillary to fibre ratio (cap·fibre–1) and the capillary density (cap·mm–2) were 8.3% (P<0.002) and 7.9% (P<0.001) larger, respectively. In the M. soleus specimens, cap·fibre–1 and cap·mm–2 were 7.4 and 9.9% larger, respectively, by double staining compared with PAS staining. Further comparisons showed that the
cap·fibre–1 and cap·mm–2 obtained with double staining were similar to the values determined by the UEA-I staining, but greater than that measured
by the collagen type IV method. The double staining gave a more marked stain of capillaries and revealed muscle fibre borders
clearly, which is an advantage in studies that require comparisons between serial sections using computerised image analyses.
It is concluded that the double staining method is superior to either the UEA-I, collagen type IV or the traditional amylase-PAS
staining methods in analysing capillary density of normal human skeletal muscle.
Accepted: 17 October 1996 相似文献
18.
Paquette JS Tremblay P Bernier V Auger FA Laviolette M Germain L Boutet M Boulet LP Goulet F 《In vitro cellular & developmental biology. Animal》2003,39(5-6):213-220
We have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serum-free medium supplemented with retinoic acid (5 x 10(-8) M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models will be useful for studying human bronchial properties in vitro. 相似文献
19.
Thorsten Kirsch Bernd Swoboda Klaus von der Mark 《Differentiation; research in biological diversity》1992,52(1):89-100
In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis. 相似文献
20.
XIE Feng ZHANG WenJie CHEN FanFan ZHOU GuangDong CUI Lei LIU Wei & CAO YiLin 《中国科学:生命科学英文版》2008,51(9):774-778
In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic envi-ronment provided by mature chondrocytes was investigated. Flk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold com-plexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type II collagen antibody staining. Positive staining of mouse Major Histocompatibility Complex class I molecules confirmed that part of the chondrocytes were derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation. 相似文献