10 research outputs found
Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells
Purpose To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents. Methods Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect immunofluorescence using antibodies against pan-, N-, or E-cadherins or a- or ß-catenins. Alternatively, stromal equivalents were dissected, to separate the proteins from the epithelium, endothelium, and stroma with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blots of the transferred proteins exposed to these primary antibodies were detected with chemiluminescence. Native corneas were processed similarly. Results Three or four layers of epithelial cells reminiscent of the native cornea (basal cuboidal and superficial flatter cells) lay over a stromal construct containing fibroblastic cells under which an endothelium is present. Western blots and indirect immunofluorescence revealed that, similarly to the native cornea, the epithelium reacted positively to antibodies against catenins (a and ß) and E-cadherin. The endothelium of corneal constructs, whether of human or bovine origin, reacted mildly to catenins and N-cadherin. Conclusions This collagen-based corneal equivalent simulated the native cornea. Cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell–cell contacts and the existence of polarized morphology of these layers over corneal equivalents
MMP-2 expression in uveal melanoma : differential activation status dictated by the cellular environment
Purpose: MMPs are recognized to play a major role in tumor progression and metastasis of many forms of cancers. The
purpose of this study was to compare the expression and activity of MMP-2 in uveal melanoma cell lines grown either in
vitro on plastic culture plates or in vivo as tumors produced in chick embryos.
Methods: The chick chorioallantoic membrane (CAM) model was used to evaluate the tumorigenic potential of uveal
melanoma cell lines derived either from the primary uveal melanoma tumor isolated from three different patients (cell
lines SP6.5, SP8.0, and TP31) or from a metastatic lesion derived from the liver of a patient diagnosed with uveal melanoma
(cell line H79). The presence of MMP-2 in the vicinity of the tumor cells was determined by immunofluorescence
analyses. Gelatin zymography was used for the detection of latent and activated forms of MMP-2 in uveal melanoma cell
lines when grown in vitro on plastic, or in the solid tumors these cell lines produced in vivo on the CAM of the chick
embryo. The gelatinase activity was quantified by densitometric analyses and the active/(active+pro-form) ratio was
calculated as the MMP-2 activation ratio. Western blot analyses were performed to confirm the zymographic profile.
Results: Only the inactive form of MMP-2 was expressed and secreted in vitro by all uveal melanoma cell lines, higher
levels being found for the liver-derived H79 cell line whereas SP8.0 only expressed MMP-2 to a very low level. On the
other hand, all solid tumors produced in the CAM from these cell lines expressed and secreted, although to varying levels
(SP6.5 and SP8.0, TP31 and H79), primarily the active form of MMP-2. Gelatinolytic activities of active MMP-2 were
significantly higher in uveal melanoma tissues than in the non-neoplastic CAM, as revealed by the measurement of the
activation ratio. The immunolocalization of MMP-2 revealed that all cell lines were MMP-2-positive although a reduced
and more diffuse staining was observed for H79 and SP6.5 than in SP8.0 and TP31 cells.
Conclusions: These results suggest the activation of proMMP-2 as an important event in the process of uveal melanoma
progression. An elevated active to inactive MMP-2 ratio in the tumor environment of uveal melanoma suggests that a
potential MMP-2 activity could be related to the progression of this type of cancer
Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction
Purpose: Regeneration of the corneal epithelium could be severely impaired in patients suffering from limbal stem cell
deficiency. The purpose of this study was to evaluate the restoration of the corneal epithelium by grafting onto denuded
corneas autologous limbal cells cultured on fibrin gels. The rabbit model was chosen to allow the microscopic evaluation
over time after grafting.
Methods: Rabbit limbal epithelial cells (RLECs) were isolated and cultured from small limbal biopsies (3 mm2
). The
epithelium was separated from stroma after dispase digestion and put in culture on lethally irradiated fibroblasts used as a
feeder layer. At the first passage, RLECs were cultured on a fibrin gel matrix. At confluence, the cultured epithelia were
grafted in vivo on denuded autologous rabbit corneas. At different postoperative times, grafted and control (without graft
or grafted with fibrin gels only) rabbit corneas were compared in vivo with a slit lamp microscope, and in situ by histological
and immunohistological microscopy of harvested biopsies.
Results: A small limbal biopsy was sufficient to generate enough RLECs to prepare several grafts and to perform cell
analysis. Only two weeks were required to produce a cultured epithelium suitable for autologous transplantation. One
month after grafting, a normal corneal phenotype was observed on the ocular surface of grafted rabbits in contrast to the
control rabbits (ungrafted or grafted with fibrin gel only) where histological signs of conjunctivalization were found. The
absence of goblet cells and negative staining for keratin 4 confirmed that the cultured cells persisted and that the epithelium
regenerated after grafting was not from conjunctival origin.
Conclusions: Our results demonstrate that an autologous epithelium cultured on a physiologically biodegradable matrix
can be prepared from a small biopsy and grafted on denuded cornea. The autologous graft allows epithelial regeneration
from cultured cells and promotes corneal healing of unilateral total stem cell deficiency
Impact of cell source on human cornea reconstructed by tissue engineering
Purpose: To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro.
Methods: Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined.
Results: The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis.
Conclusions: The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis
Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types
Purpose: The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using
all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach.
Methods: Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and
formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each
side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology
and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane
components, Na+/K+-ATPase α1, and collagen type I was also performed.
Results: Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the
corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also
expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed
epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen
VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K
+-ATPase α1.
Conclusions: This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar
to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous
biomaterial
Optimization of culture conditions for porcine corneal endothelial cells
Purpose : To optimize the growth condition of porcine corneal endothelial cells (PCEC), we evaluated the effect of coculturing with a feeder layer (irradiated 3T3 fibroblasts) with the addition of various exogenous factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), bovine pituitary extract (BPE), ascorbic acid, and chondroitin sulfate, on cell proliferation, size, and morphology.
Methods : PCEC cultures were seeded at an initial cell density of 400 cells/cm2 in the presence or absence of 20,000 murine-irradiated 3T3 fibroblast/cm2 in the classic media Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Mean cell size and bromodeoxyuridine incorporation was assessed at various passages. Growth-promoting factors were studies by seeding PCEC at 8,000 cells/cm2 in DMEM with 20% FBS or Opti-MEM I supplemented with 4% FBS and one of the following additives: EGF (0.5, 5, 25 ng/ml), NGF (5, 20, 50 ng/ml), BPE (25, 50, 100, 200 ÎĽg/ml), ascorbic acid (10, 20, 40 ÎĽg/ml) and chondroitin sulfate (0.03, 0.08, 1.6%), alone or in combination. Cell number, size and morphology of PCEC were assessed on different cell populations. Each experiment was repeated at least twice in three sets. In some cases, cell cultures were maintained after confluence to observe post-confluence changes in cell morphology.
Results : Co-cultures of PCEC grown in DMEM 20% FBS with a 3T3 feeder layer improved the preservation of small polygonal cell shape. EGF, NGF, and chondroitin sulfate did not induce proliferation above basal level nor did these additives help maintain a small size. However, chondroitin sulfate did help preserve a good morphology. BPE and ascorbic acid had dose-dependent effects on proliferation. The combination of BPE, chondroitin sulfate, and ascorbic acid significantly increased cell numbers above those achieved with serum alone. No noticeable changes were observed when PCEC were cocultured with a 3T3 feeder layer in the final selected medium.
Conclusions : Improvements have been made for the culture of PCEC. The final selected medium consistently allowed the growth of a contact-inhibited cell monolayer of small, polygonal-shaped cells
Characterization of wound reepithelialization using a new human tissue–engineered corneal wound healing model
Purpose. The reepithelialization of the corneal surface is an important process for restoring the imaging properties of this tissue. The purpose of the present study was to characterize and validate a new human in vitro three-dimensional corneal wound healing model by studying the expression of basement membrane components and integrin subunits that play important roles during epithelial cell migration and to verify whether the presence of exogenous factors could accelerate the reepithelialization.
Methods. Tissue-engineered human cornea was wounded with a 6-mm biopsy punch, and the reepithelialization from the surrounding margins was studied. Biopsy samples of the reepithelialized surface were harvested 3 days after wounding and were processed for histologic, electron microscopic, and immunofluorescence analyses. The effects of fibrin and epithelial growth factor (EGF) on wound reepithelialization were also studied.
Results. Results demonstrated that this in vitro model allowed the migration of human corneal epithelial cells on a natural extracellular matrix. During reepithelialization, epithelial cell migration followed a consistent wavelike pattern similar to that reported for human corneal wound healing in vivo. This model showed a histologic appearance similar to that of native tissue as well as expression and modulation of basement membrane components and the integrin subunits known to be main actors during the wound healing process. It also allowed quantification of the reepithelialization rate, which was significantly accelerated in the presence of fibrin or EGF. The results indicated that αvβ6 integrin expression was increased in the migrating epithelial cells compared with the surrounding corneal tissue.
Conclusions. The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in corneal reepithelialization. Moreover, this model may also be used as a tool to screen agents that affect reepithelialization or to evaluate the effect of growth factors before animal testing
Tissue Engineering of Feline Corneal Endothelium Using a Devitalized Human Cornea as Carrier
The difficulties in obtaining good quality tissue for the replacement of corneas of patients suffering from
endothelial dysfunctions have prompted us to evaluate the feasibility of producing a tissue-engineered (TE)
corneal endothelium using devitalized human stromal carriers. Thus, corneal substitutes were produced by
seeding cultured feline corneal endothelial cells on top of previously frozen human corneal stromas. After two
weeks of culture to allow attachment and spreading of the seeded cells, the TE corneal endothelium was stained
with alizarin red for endothelial cell count and fixed for histology, immunofluorescence labeling, scanning and
transmission electron microscopy. Histology and Hoechst staining showed that there were no remaining cells in
the devitalized stroma. After seeding, histology and transmission electron microscopy showed that the TE
corneal endothelium formed a monolayer of tightly packed cells that were well adhered to Descemet’s membrane.
Scanning electron microscopy corroborated that the cells covered the entire posterior corneal surface and
had an endothelial morphology. Alizarin staining showed that mean cell counts were 2272 ± 344 cells=mm2
,
indicating that the cell density was appropriate for grafting. The TE feline corneal endothelium also expressed
the function-related proteins Na+
=HCO3
, ZO-1, and Na+
=K+
-ATPase a1, and could easily be marked with a
fluorescent tracker. This study demonstrates the feasibility of reconstructing a highly cellular and healthy corneal
endothelium on devitalized human corneal stromas
Surface topography induces 3D self-orientation of cells and extracellular matrix resulting in improved tissue function
The organization of cells and extracellular matrix (ECM) in native tissues plays a crucial role in their functionality. However, in tissue engineering, cells and ECM are randomly distributed within a scaffold. Thus, the production of engineered-tissue with complex 3D organization remains a challenge. In the present study, we used contact guidance to control the interactions between the material topography, the cells and the ECM for three different tissues, namely vascular media, corneal stroma and dermal tissue. Using a specific surface topography on an elastomeric material, we observed the orientation of a first cell layer along the patterns in the material. Orientation of the first cell layer translates into a physical cue that induces the second cell layer to follow a physiologically consistent orientation mimicking the structure of the native tissue. Furthermore, secreted ECM followed cell orientation in every layer, resulting in an oriented self-assembled tissue sheet. These self-assembled tissue sheets were then used to create 3 different structured engineered-tissue: cornea, vascular media and dermis. We showed that functionality of such structured engineered-tissue was increased when compared to the same non-structured tissue. Dermal tissues were used as a negative control in response to surface topography since native dermal fibroblasts are not preferentially oriented in vivo. Non-structured surfaces were also used to produce randomly oriented tissue sheets to evaluate the impact of tissue orientation on functional output. This novel approach for the production of more complex 3D tissues would be useful for clinical purposes and for in vitro physiological tissue model to better understand long standing questions in biology