A primary culture model of rabbit conjunctival epithelial cells exhibiting tight barrier properties

Saha, Pratik; Kim, Kwang‐Jin; Lee, Vincent H.L.

doi:10.3109/02713689608995151

Cited by 60 publications

(31 citation statements)

References 9 publications

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“…In fact, in terms of TEER values, an important indication of the tightness of the culture [27,30], the AIC cultures exhibit higher values ( fig. 6) than reported in the literature [1]. In contrast, the I eq is lower than the data reported from ocular cell cultures of other species.…”

Section: Discussioncontrasting

confidence: 54%

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Multilayer Primary Epithelial Cell Culture from Bovine Conjunctiva as a Model for in vitro Toxicity Tests

Civiale¹,

Paladino²,

Marino³

et al. 2003

Ophthalmic Res

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Objective: The purpose of this study was to obtain a primary cell culture of bovine origin similar to the conjunctiva in terms of morphology and cell types, which could be of use for in vitro toxicity studies. Methods: After separation from the stroma by enzymatic treatment, conjunctival epithelial cells were dissociated and plated onto collagen-coated Transwell^® filters (1.13 cm² area). One group of plates was maintained in immersion and another was cultured under air-lifted conditions. Anti-epithelial keratin antibodies (AE1/AE3, K4) and antidesmoplakin 1 and 2 were used to characterize the cells by indirect immunofluorescence. The cell layer was examined after histological processing of the Transwell filter. Ultrastructural analysis was carried out by scanning electron microscopy (SEM). The bioelectric parameters transepithelial electrical resistance (TEER), potential difference (PD), short circuit current and paracellular permeability profile of carboxyfluorescein were monitored as indices of the functional characteristics of these cultures. Cytotoxicity was evaluated on morphological and functional (TEER) grounds after treating the cultures with several test substances. Results: Morphological studies showed pure and homogeneous cell cultures. In the SEM analysis, we observed contiguous polygonal cells with numerous short microvilli, a characteristic proportion of light, medium and dark cells and a sparse population of rounded PAS-positive cells, i.e. resembling goblet cells. Air-lifted cultures also showed a tissue-like cellular organization (8–9 layers). Immersion cultures reached a maximum TEER value of around 2.95 kΩ·cm² 7 days after plating while in air-lifted cultures TEER peaked up to 5.59 kΩ·cm² 11 days after plating. With regard to the use of bovine conjunctival epithelial cells (BCECs) for cytotoxicity screening, the system responded finely to the insults and yielded morphological and functional results in accordance with data obtained in vivo. Conclusions: BCECs reproduce cell morphology and differentiation of the original tissue and should prove a useful tool for initial studies of drug toxicity.

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Section: Discussioncontrasting

confidence: 54%

“…The conjunctiva is permeable to a large variety of drugs [1][2][3]. Of particular interest in drug transport studies is the bulbar conjunctiva that constitutes the first noncorneal route for a drug to reach the posterior segment of the eye.…”

Section: Introductionmentioning

confidence: 99%

Multilayer Primary Epithelial Cell Culture from Bovine Conjunctiva as a Model for in vitro Toxicity Tests

Civiale¹,

Paladino²,

Marino³

et al. 2003

Ophthalmic Res

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“…For examCompared to the cornea, the conjunctiva is less permeable for lipophilic agents. Furthermore, tight junctions of conjunctival epithelium also reduce its permeability to hydrophilic molecules [9]. Nonetheless, dense vascularisation with blood and lymphatic vessels leads to absorption of drugs into circulation and reduces their bioavailability at the destination site.…”

Section: Drug and Formulation-related Factorsmentioning

confidence: 99%

Drug bioavailability from topically applied ocular drops. Does drop size matter?

Jünemann

Chorągiewicz²,

Ozimek³

et al. 2016

Ophthalmology J

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The application of drops containing ocular medicines to the conjunctival sac is the most common method of drug delivery to the anterior segment of the eye. Although this route of application seemingly displays numerous advantages, obtaining effective drug concentration at its site of action is challenging. The bioavailability of a topically applied drug depends on various factors related to the eye, to the drug and formulation, to the drop, and to the patient. The present article discusses their relative significance. From a drop applied to an eye, at most 5% of a drug dose enters the ocular structures. Of utmost importance for effective ocular drug delivery are patient compliance and the physicochemical properties of the drug. For a given concentration of an active substance, drop size may determine drug adverse effects but does not influence its efficacy.

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“…Since there were no reports of rat conjunctival epithelial primary cultures or immortalized cell lines, we developed SV40-transformed rat conjunctival cell lines suitable for in vitro investigation of transepithelial antigen delivery. The cells formed functional, tight, epithelial barriers when grown on permeable supports using the approach employed by Saha et al(1996). This in vitro model is expected to provide a more complete understanding the precise cellular and molecular mechanisms for antigen uptake from the ocular surface and has application to the rational design of effective ocular immunization regimes.…”

Section: Introductionmentioning

confidence: 99%

Development of immortalized rat conjunctival epithelial cell lines: An in vitro model to examine transepithelial antigen delivery

O’Sullivan¹,

Baylor²,

Montgomery³

2007

Experimental Eye Research

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The objective of these studies was to develop conjunctival epithelial cell lines for investigation of antigen translocation across a mucosal barrier. Conjunctival epithelial cells from Fischer 344 rats were immortalized with pSV3(neo) resulting in two cell lines -CJ4.1A and CJ4.3C. Each formed confluent cell layers with epithelial morphology when grown on permeable membrane filters. They expressed the SV40 T antigen, the conjunctiva-specific cytokeratin 4, the goblet cell-specific cytokeratin 7 and were negative for the corneal epithelial cell-specific cytokeratin 12. The cell lines have been in culture for over 60 passages, and the population doubling times were 22 ± 7 hours for CJ4.1A and 23 ± 9 hours for CJ4.3C. When grown on Transwell™ membranes, each cell line achieved a transepithelial electrical resistance of 600-800 Ωcm 2 by 3 to 4 days and maintained a high resistance for several days. Both cell lines expressed zona occludins-1 at confluence. At 24 h following addition of 250 μg of FITC-labeled ovalbumin to the apical chambers, 15 ± 6 μg could be detected in the basal chamber of CJ4.1A and 6 ± 1 μg in the basal medium of CJ4.3C. In contrast, 82 ± 6 μg was detected in the lower chambers of cell-free Transwells. Similarly, Transwells containing confluent CJ4.1A or CJ4.3C cells impeded passage of 0.1 μm diameter polystyrene microspheres (5 ± 1 and 4 ± 1 %, respectively, of the apical input), compared to 26 % ± 6 % of the input microspheres recovered from the basal chambers of cell-free Transwells™. Pretreatment with 4 mM EGTA for 10 min caused an increase in OVA-FITC translocation across CJ4.3C cells. Incubation in the presence of 4 mM EGTA significantly increased OVA-FITC translocation across both cell lines, relative to untreated cell layers. Morphological and functional characterization indicate that these cells provide a useful experimental tool to assess strategies for enhancing transepithelial antigen uptake.

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A primary culture model of rabbit conjunctival epithelial cells exhibiting tight barrier properties

Abstract: A functional, tight, epithelial barrier of the pigmented rabbit conjunctiva on a permeable support has been developed, which may be useful for mechanistic studies of ion and drug transport at the cellular level.

Cited by 60 publications

References 9 publications

Multilayer Primary Epithelial Cell Culture from Bovine Conjunctiva as a Model for in vitro Toxicity Tests

Multilayer Primary Epithelial Cell Culture from Bovine Conjunctiva as a Model for in vitro Toxicity Tests

Drug bioavailability from topically applied ocular drops. Does drop size matter?

Development of immortalized rat conjunctival epithelial cell lines: An in vitro model to examine transepithelial antigen delivery

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