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The most commonly used tissue substitute for ocular surface reconstruction is human amniotic membrane (AM). Because of its low biomechanical strength and intransparency there is a need to search for alternatives of consistent quality. This study, further explored the biocompatibility of Keratin Film (KF) and its ability to sustain corneal epithelial wound healing. In three equal groups of 5 New Zeeland white rabbits a 4 mm superficial keratectomy was created in the right eye. Five eyes received a KF, five a human AM graft and the remaining five no implant. All eyes were treated with ofloxacin and dexamethasone eye drops and followed up for 10 days. Corneal fluorescein staining, vascularization, and transparency were assessed using slit lamp biomicroscopy according to a standardized grading score during and at the end of follow-up. The corneal-scleral-button was excised and processed for histology. After 10 days all eyes which had received a KF showed complete epithelial healing and no signs of neovascularization. In the AM group 1 eye showed a persistent epithelial defect at day 10 and 2 eyes showed neovascularization at day 7 resolving at day 10. Transparency improved progressively both in the KF group as well as in the AM group towards the end of the follow. Histology showed a multilayer epithelium firmly adherent to the KF with no evidence of keratocyte migration or inflammatory reaction in the corneal stroma. In this study on rabbit eyes KF better supported corneal epithelial wound healing than amniotic membrane., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Conventional safety and efficacy test models, such as animal experiments or static in vitro cell culture models, can often not reliably predict the most promising drug candidates. Therefore, a novel microfluidic cell culture platform, called Dynamic Micro Tissue Engineering System (DynaMiTES), was designed to allow online analysis of drugs permeating through barrier forming tissues under dynamic conditions combined with monitoring of the transepithelial electrical resistance (TEER) by electrodes optimized for homogeneous current distribution. A variety of pre-cultivated cell culture inserts can be integrated and exposed to well controlled dynamic micro flow conditions, resulting in a tightly regulated exposure of the cells to tested drugs, drug formulations and shear forces. With these qualities, the new system can provide more relevant information compared to static measurements. As a first in vitro model, a three-dimensional hemicornea construct consisting of human keratocytes (HCK-Ca) and epithelial cells (HCE-T) was successfully tested in the DynaMiTES. Thereby, we were able to demonstrate the functionality and cell compatibility of this new organ on chip test platform. The modular design of the DynaMiTES allows fast adaptation suitable for the investigation of drug permeation through other important cellular barriers., (Copyright © 2017. Published by Elsevier B.V.)

The present overview deals with current approaches for the improvement of in vitro models for preclinical drug and formulation screening which were elaborated in a joint project at the Center of Pharmaceutical Engineering of the TU Braunschweig. Within this project a special focus was laid on the enhancement of skin and cornea models. For this reason, first, a computation-based approach for in silico modeling of dermal cell proliferation and differentiation was developed. The simulation should for example enhance the understanding of the performed 2D in vitro tests on the antiproliferative effect of hyperforin. A second approach aimed at establishing in vivo-like dynamic conditions in in vitro drug absorption studies in contrast to the commonly used static conditions. The reported Dynamic Micro Tissue Engineering System (DynaMiTES) combines the advantages of in vitro cell culture models and microfluidic systems for the emulation of dynamic drug absorption at different physiological barriers and, later, for the investigation of dynamic culture conditions. Finally, cryopreserved shipping was investigated for a human hemicornea construct. As the implementation of a tissue-engineering laboratory is time-consuming and cost-intensive, commercial availability of advanced 3D human tissue is preferred from a variety of companies. However, for shipping purposes cryopreservation is a challenge to maintain the same quality and performance of the tissue in the laboratory of both, the provider and the customer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present study, a formerly designed Dynamic Micro Tissue Engineering System (DynaMiTES) was applied with our prevalidated human hemicornea (HC) construct to obtain a test platform for improved absorption studies of the anterior eye (Ocular DynaMiTES). First, the cultivation procedure of the classic HC was slightly adapted to the novel DynaMiTES design. The obtained inverted HC was then compared to classic HC regarding cell morphology using light and scanning electron microscopy, cell viability using MTT dye reaction and epithelial barrier properties observing transepithelial electrical resistance and apparent permeation coefficient of sodium fluorescein. These tested cell criteria were similar. In addition, the effects of four different flow rates on the same cell characteristics were investigated using the DynaMiTES. Because no harmful potential of flow was found, dynamic absorption studies of sodium fluorescein with and without 0.005%, 0.01% and 0.02% benzalkonium chloride were performed compared to the common static test procedure. In this proof-of-concept study, the dynamic test conditions showed different results than the static test conditions with a better prediction of in vivo data. Thus, we propose that our DynaMiTES platform provides great opportunities for the improvement of common in vitro drug testing procedures., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, a shipping protocol for our 3D human hemicornea (HC) construct should be developed to provide quality-maintaining shipping conditions and to allow its ready-to-use application in drug absorption studies. First, the effects of single and multiple parameters, such as the type of shipping container, storage temperature and CO 2 supply, were investigated under controlled laboratory conditions by assessing cell viability via MTT dye reaction and epithelial barrier properties via transepithelial electrical resistance (TEER) measurements. These investigations showed that TEER is more susceptible to shipping parameters than cell viability. Furthermore, the results were used to determine the optimal shipping conditions and critical values for subsequent overnight, real-time shipping experiments. Epithelial barrier properties were then investigated via TEER and the permeation of sodium fluorescein for shipped and not shipped HC. The results underscore that acceleration forces and changes in position may have a great impact on the epithelial barrier of 3D models. Low acceleration values and short changes in position caused only minor impairments. However, combined or intensive separate effects resulted in considerably low yields after shipping. Consequently, barrier-maintaining shipping of 3D in vitro models seems to be challenging, as mechanical forces have to be reduced to a minimum., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The pharmacokinetic behaviors of novel ophthalmic drugs are often preliminarily investigated in preclinical studies using ex vivo animal cornea or corneal cell culture models. During transcorneal passage, topically applied drugs may be affected by drug metabolizing enzymes. The knowledge regarding the functional expression of metabolic enzymes in corneal tissue is marginal; thus, the aim of this study was to investigate cytochrome P450 activity in an organotypic three-dimensional human cornea construct and to compare it with porcine and rabbit corneas, which are commonly used ex vivo cornea models. The total cytochrome P450 activity was determined by measuring the transformation of 7-ethoxycoumarin. Furthermore, the expression of the cytochrome P450 enzyme 2D6 (CYP2D6) was investigated at the protein level using immunohistochemistry and western blotting. CYP2D6 activity measurements were performed using a d-luciferin-based assay. In summary, similar levels of the total cytochrome P450 activity were identified in all 3 cornea models. The protein expression of CYP2D6 was confirmed in the human cornea construct and porcine cornea, whereas the signals in the rabbit cornea were weak. The analysis of the CYP2D6 activity indicated similar values for the human cornea construct and porcine cornea; however, a distinctly lower activity was observed in the rabbit cornea., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: The purpose of this study was to analyse P-glycoprotein (P-gp) expression in different human in-vitro cornea models (HCE-T epithelial model and Hemicornea construct) after stimulation with P-gp substrates (rhodamine 123, levofloxacin and acebutolol)., Methods: The influence of P-gp substrates on mRNA expression was analysed using reverse transcriptase polymerase chain reaction (PCR) and real-time PCR. The effect of stimulation on the transporter functionality was estimated with a digoxin efflux assay. The Caco-2 cell line was used as positive control., Key Findings: The reverse transcriptase PCR results showed an increase in band intensity compared with the control medium for all substrates. The real-time PCR for the Caco-2 and HCE-T epithelial model yielded a similar outcome, in which all tested substrates upregulated P-gp. In contrast, the Hemicornea construct showed no significant increase in the mRNA expression after stimulation. Both in-vitro models possessed similar drug transport profiles after stimulation. A significantly increased efflux of digoxin was measured after 24 and 72 h of stimulation with levofloxacin and acebutolol., Conclusions: The expression and functionality of the P-gp in corneal tissue can be influenced through time exposure with specific substrates. However, the exact mechanism still requires further elucidation., (© 2015 Royal Pharmaceutical Society.)

Unlabelled: In vitro studies of ocular bioavailability of active pharmaceutical ingredients (API) from colloidal drug delivery systems do not consider physiological shear stress generated by eyelid wiping and tear flow. The present study introduces a live cell imaging approach which enables the investigation of model drug uptake from various formulations under shear stress by using custom-made microchannels for the cultivation of human corneal epithelial cells (HCE-T). Coumarin-6 (C-6) was used as a model API incorporated into solid lipid nanoparticles and liposomes, and as an aqueous crystalline suspension. Confocal laser scanning microscopy visualized C-6 uptake into HCE-T cells in a time-resolved manner with an applied shear stress of 0.1 Pa. Static conditions were also studied for comparative purposes. Additionally, solid lipid nanoparticles (SLN) were labeled with a fluorescent phospholipid to check whether C-6 uptake was associated with SLN incorporation into the cells., Results: Intact SLN were not incorporated into the cells, i.e., C-6 was passively redistributed from SLN to lipophilic cellular compartments. C-6 was enriched up to a given limit in HCE-T cells within 5 min of contact with the dispersions both under static and under flow conditions. The C-6 delivery rate from liposomes was superior to that from SLN whereby the suspension exhibited the lowest rate. C-6 release rates were comparable for static and flow conditions. Alternate flushing with formulations and buffer revealed that cells accumulated C-6. The results suggest that combining microfluidics with live cell imaging provides a valuable option for in vitro studies of ocular drug delivery.

Preclinical studies addressing the transcorneal absorption of ophthalmic drugs are mainly performed using ex vivo animal corneas and in vitro corneal cell culture models, leaving open the question of transferability to humans in an in vivo situation. While passive drug absorption through corneal tissue is well understood, little is known about the expression of transporter proteins and active drug transport in human and animal corneas as well as corneal cell culture models. Therefore, the aim of this study was to conduct an expression analysis of four multidrug resistance-associated proteins (MRP1, 2, 4 and 5) in various in vitro and ex vivo corneal models, leading to a better understanding of the comparability of different corneal models regarding drug absorption and transferability to humans. Two well-established in vitro human corneal models, the HCE-T epithelial model and the more organotypic Hemicornea construct, both of which are based on the SV40 immortalized human corneal epithelial cell line HCE-T, were analyzed, as were excised rabbit and porcine cornea. Specimens of abraded epithelia from human donor corneas were also tested. MRP mRNA expression was determined via reverse transcriptase polymerase chain reaction. Protein expression was examined using Western blot experiments and immunohistochemistry. The functional activity of the MRP efflux transporter was detected in transport assays using specific marker and inhibitor substances. The functional expression of all of the tested MRP transporters was detected in the HCE-T epithelial model. Hemicornea constructs displayed a similar expression pattern for MRP1, 4 and 5, whereas no MRP2 protein expression or activity was detected. However, excised animal corneas exhibited different expression profiles. In porcine cornea, no functional expression of MRP1, 2, or 5 was observed, and we failed to detect MRP4 expression in rabbit cornea. The results suggest that MRP1, 2, 4, and 5 are expressed in the human corneal epithelium and confirm that the transfer of data obtained from animal experiments to an in vivo situation in humans should be performed with caution.

Ocular drug absorption studies are required for the development of new drugs or drug delivery systems for eye treatment. Such preclinical investigations on transcorneal drug absorption are performed ex vivo with the excised corneas of experimental animals or in vitro using corneal cell culture models. The data currently available on the expression of ABC transporter proteins in corneal tissue is limited or contradictory. This study describes, for the first time, the comparison of the expression of ABC transporters, in particular, MDR1, BCRP and MRP3, between human cornea cell culture models and the most commonly used ex vivo models, namely, rabbit and porcine corneas, conducted in the same laboratory. The expression levels and functionality were determined by means of PCR, western blot, immunohistochemistry and bidirectional permeation studies using specific substrates and inhibitors. The results clearly indicate species-dependent expression of the studied efflux transporters. In the rabbit cornea, the expression and activity of MDR1 transporter was confirmed, whereas human cell culture models and porcine corneas did not show MDR1 expression. However, human cornea models possessed MRP3 and BCRP expression, whereas no functional expression was found in rabbit and porcine corneas. Therefore, the translation of transcorneal permeation data from animal experiments to humans should be performed with caution., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: Drugs from ophthalmic formulations are mainly absorbed into the eye via the corneal route. However, little is known about drug metabolism during the transcorneal passage. The objective of this study was to determine the mRNA expression of phase I and II isoenzymes in human corneal epithelial tissue, corneal cell lines, and a tissue-engineered cornea equivalent (a hemicornea construct) as in vitro model for drug absorption studies., Methods: The reverse transcription polymerase chain reaction was used to profile the mRNA expression of 10 cytochrome P450 enzymes (CYP) and seven phase II enzymes in the three human corneal cell lines and the hemicornea construct. The human corneal epithelial cell line (HCE-T), human corneal keratocyte cell line (HCK-Ca) and human corneal endothelial cell line (HENC) were used. Human liver tissue, human corneal epithelium from donor corneas, and the human colon adenocarcinoma cell line Caco-2 were also investigated., Results: All the phase I and II mRNAs were expressed in the human liver tissue. The Caco-2 cell line showed an expression pattern similar to the liver tissue, although the signals for CYP1A2 and CYP3A4 were absent. In the case of the donor human corneal epithelium, all the detected phase I mRNAs had lower levels than did the liver tissue. By contrast, the phase II mRNA expression pattern was heterogeneous to the liver tissue. The expression patterns in the three human corneal cell lines were comparable, although the signals for a few phase I enzymes and N-acetyltransferase (NAT2) mRNAs were only detectable in the HCE-T. In the hemicornea construct, all the investigated phase I and II mRNA (except for CYP1A2, CYP2B6, CYP2C19, and NAT2) were expressed., Conclusions: Overall, the mRNA expressions of the tested phase I and phase II enzymes in the hemicornea construct and the three corneal cell lines correlated well with the expression patterns of the ex vivo human corneal epithelium.

Introduction: Many diseases of the anterior eye segment are treated using topically applied ophthalmic drugs. For these drugs, the cornea is the main barrier to reaching the interior of the eye. In vitro studies regarding transcorneal drug absorption are commonly performed using excised corneas from experimental animals. Due to several disadvantages and limitations of these animal experiments, establishing corneal cell culture models has been attempted as an alternative., Areas Covered: This review summarizes the development of in vitro models based on corneal cell cultures for permeation studies during the last 20 years, starting with simple epithelial models and moving toward complex organotypical 3D corneal equivalents., Expert Opinion: Current human 3D corneal cell culture models have the potential to replace excised animal corneas in drug absorption studies. However, for widespread use, the contemporary validation of existent systems is required.

Purpose: The aim of the study was to formulate and evaluate surface-modified solid lipid nanoparticles sustained delivery system of timolol hydrogen maleate, a prototype ocular drug using a human cornea construct., Materials and Methods: Surface-modified solid lipid nanoparticles containing timolol with and without phospholipid were formulated by melt emulsification with high-pressure homogenization and characterized by particle size, wide-angle X-ray diffraction, encapsulation efficiency, and in vitro drug release. Drug transport studies through cornea bioengineered from human donor cornea cells were carried out using a modified Franz diffusion cell and drug concentration analyzed by high-performance liquid chromatography., Results: Results show that surface-modified solid lipid nanoparticles possessed very small particles (42.9 +/- 0.3 nm, 47.2 +/- 0.3 nm, 42.7 +/- 0.7 nm, and 37.7 +/- 0.3 nm, respectively for SM-SLN 1, SM-SLN 2, SM-SLN 3, and SM-SLN 4) with low polydispersity indices, increased encapsulation efficiency (> 44%), and sustained in vitro release compared with unmodified lipid nanoparticles whose particles were greater than 160 nm. Permeation of timolol hydrogen maleate from the surface-modified lipid nanoparticles across the cornea construct was sustained compared with timolol hydrogen maleate solution in distilled water., Conclusions: Surface-modified solid lipid nanoparticles could provide an efficient way of improving ocular bioavailability of timolol hydrogen maleate.

Purpose: To provide a model to be used for in vitro studies on drug effects in dogs, this study was conducted to establish a protocol for the construction of a three-dimensional corneal construct. Primary canine corneal cells and a rabbit corneal epithelial (RCE) cell line were used in comparison., Methods: The corneal construct was assembled step by step in membrane inserts of a six-well plate over a total of 5 weeks, including culture at the air-liquid interface to allow a differentiation of the epithelial cells. The constructs were studied histologically. Single cell cultures of canine corneal cells as well as RCE cells were stimulated with lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS). Treatment with different concentrations of dexamethasone was used to test its effects on the cellular prostaglandin E(2) (PGE(2)) production. The same experiments were repeated with the corneal constructs and the reactions compared. Expression of the glucocorticoid receptor (GR) in the constructs was studied using immunohistochemistry., Results: A protocol for the construction of a vital corneal construct was established and the morphological similarity to the canine cornea in vivo shown. The GR protein was detected in all three cell types of the constructs. Stimulation with LPS and SDS led only in the corneal constructs to a significantly increased PGE(2) production, which could be reduced by dexamethasone., Conclusions: The corneal construct is an interesting system to test drug effects on corneal cells. It allows studies on a cornea-like system including all three major cell types.

Solid lipid nanoparticles (SLNs) were prepared with a combination of homolipid from goat (goat fat) and phospholipid, and evaluated for diclofenac sodium (DNa) delivery to the eye using bio-engineered human cornea, produced from immortalized human corneal endothelial cells (HENC), stromal fibroblasts and epithelial cells CEPI 17 CL 4. Encapsulation efficiency was high and sustained release of DNa and high permeation through the bio-engineered cornea were achieved. Results obtained in this work showed that permeation of DNa through the cornea construct was improved by formulation as SLN modified with phospholipid.

Cell culture models of the cornea are continually developed to replace the isolated animal cornea for transcorneal drug absorption studies. The aim of this study was to determine and compare epithelial tightness and permeability of currently available corneal cell culture models to avoid interlaboratory variability and to assess their usefulness for in-vitro permeation studies. Pure epithelial cell culture models (CEPI, SIRC and HCE-T cell lines), primary cultures of human corneal epithelium (HCEpiC) and the two commercially available models (RHC and Epiocular), as well as organotypic human cornea constructs (HCC, HCC-HCE-T), were investigated and data were compared with those obtained from the excised bovine cornea. Barrier properties were assessed by measurements of transepithelial electrical resistance (TEER) and permeability of three passively absorbed substances (mannitol, testosterone and timolol maleate) with different physico-chemical properties. TEER experiments revealed weak barrier functions for all of the investigated epithelial models (

Organotypic cornea equivalents are used as in vitro models for permeation studies. Many ophthalmic drugs are applied as ester prodrugs to achieve a higher bioavailability. The esterase activity of three corneal human cell lines (epithelial, stromal, endothelial cells) as well as of excised porcine cornea, human donor cornea and human cornea construct (HCC) was investigated and compared. Esterase activity was determined using p-nitrophenyl acetate and hydrocortisone acetate (HCA) as esterase substrates. Hydrocortisone acetate permeation across porcine cornea, human donor cornea and HCC was studied in vitro using Franz-diffusion cells. Corneal epithelial cells showed the highest esterase activity and only small differences to keratocytes and endothelial cells were detectable. The permeation barrier properties of the different corneal tissues were very similar in the case of HCA permeation whereas HCA metabolism rates were in the ranking order of porcine cornea > HCC > human donor cornea. Permeation and metabolism studies indicate that the in vitro permeation model HCC is able to adequately convert hydrocortisone acetate to hydrocortisone.

Transcorneal in vitro permeation studies of ophthalmic drugs are normally performed with either excised animal corneas or latterly corneal cell culture models. A good correlation between these models and excised animal corneas regarding permeation behaviour of drugs has already been shown. However, comparisons between corneal in vitro models containing human cells and excised human corneas do not exist yet. Therefore in the present study the transcorneal permeation of six different model drugs (pilocarpine hydrochloride, befunolol hydrochloride, hydrocortisone, diclofenac sodium, clindamycin hydrochloride and timolol maleate) across our previously described three-dimensional organotypic human cornea construct (HCC) was tested using Franz diffusion cells and compared with permeation data obtained from human donor corneas. The HCC showed a similar permeation behaviour compared with human donor cornea for all substances. The permeabilities (permeation coefficients P) of the human cornea equivalent versus the human donor cornea were the same in the case of diclofenac, clindamycin, timolol, but marginally decreased for hydrocortisone and slightly increased for pilocarpine and befunolol. These small differences of permeation coefficients were expressed as factors and only varied from 0.8 to 1.4. The results indicate that the HCC may be an alternative for in vitro permeation studies and appropriate for predicting drug absorption into the human eye.

The aim of this study was to develop an in vitro model of the cornea of bovine cells, to characterise the model by histochemical methods and to investigate permeation of ophthalmic drugs through the model. As in the in vivo situation, an in vitro model of the cornea should consist of all three different types of cells. In the current study, the construction of the in vitro cornea was performed using cells prepared from primary cultures. To investigate the state of the cells in the cultures, growth curves were established. Immunocytochemical determination of keratin and vimentin was performed for all three isolated and sub-cultivated cell types of the bovine cornea. To further simulate the in vivo conditions, corneal epithelial cells were seeded onto the collagen-gel base containing the stromal cells with an underlying sheet of endothelium. Permeation experiments were performed with pilocarpine hydrochloride and timolol hydrogen maleate as model drugs and excised bovine cornea and the in vitro cornea as permeation barriers. The immunohistochemical investigations show that excised bovine cornea and the in vitro model of the cornea are comparable with respect to the expression of keratin K3, indicating that the primarily isolated cells correspond to the different cell types of the cornea. Culturing of the epithelial cells on the complex basis has led to the formation of a corneal epithelium with several layers, closely resembling the morphology of the in vivo epithelium. Although the permeation rates of the drug through the in vitro cornea were always higher, the sequence in which the drugs permeate through the two types of barriers was the same. The drug permeation through the in vitro cornea may therefore be a useful predictive tool to estimate the permeability coefficients of drugs through excised cornea.

Aims: For the study of transcorneal in vitro permeation of ophthalmic drugs, excised animal cornea or corneal epithelial cell culture are frequently used as a replacement for the human cornea. The main purposes of this study were to reconstruct a complete human organotypic cornea equivalent, consisting of all three different cell types (epithelial, stromal, and endothelial); to test the barrier function of this bio-engineered human cornea using three different model drugs (pilocarpine hydrochloride (PHCl), befunolol hydrochloride (BHCl), and hydrocortisone (HC)); and to determine its usefulness as an in vitro model for prediction of ocular drug absorption into the human eye., Methods: A multilayer tissue construct was created step by step in Transwell cell culture insert using SV-40 immortalised human endothelial and epithelial cells and native stromal cells (fibroblasts). Morphology was characterised by light microscopy using routine H&E staining. Scanning electron microscopy was used to evaluate ultrastructural features. Ocular permeation of drugs across the human cornea construct was tested using modified Franz cells and compared with data obtained from excised porcine cornea and previously described porcine cornea constructs., Results: and conclusion: The cornea construct exhibited typical corneal structures such as a monolayer of hexagonally shaped endothelial cells and a multilayered epithelium consisting of seven to nine cell layers with flat superficial cells. The formation of microplicae and microvilli was also confirmed. The human cornea construct showed similar permeation behaviour for all substances compared with excised porcine cornea. However, permeability (permeation coefficients K(p)) of the human cornea equivalent (PHCl 13.4*10(-6) (SD 3.01*10(-6)); BHCl 9.88*10(-6) (SD 1.79*10(-6)); HC 5.41*10(-6) (SD 0.40*10(-6)) cm/s) was about 1.6-1.8 fold higher than excised porcine cornea. Compared with data from the porcine cornea construct the cultivated human equivalent showed a decreased permeability. The reconstructed human cornea could be appropriate to predict drug absorption into the human eye.

The purpose of this study was to develop an organotypic cornea equivalent consisting of three different cell types (epithelial, stromal and endothelial cells) and to investigate its usefulness as in vitro model for permeation studies. The different cell types of a porcine cornea were selectively isolated and a multilayer tissue construct was created step-by-step in Transwell cell culture insert. Histology, basement membrane components (laminin, fibronectin) and surfaces of cornea construct were investigated to evaluate the degree of comparability to porcine cornea from slaughtered animals. The cornea construct exhibited similarities to the original cornea. Ocular permeation of befunolol hydrochloride from different formulations across the cornea construct was tested using modified Franz cells and compared with data obtained from excised cornea. The cornea construct showed a similar permeation behavior for befunolol hydrochloride from different formulations compared with excised porcine cornea. However, permeation coefficients K(p) obtained with the construct were about three to fourfold higher for aqueous formulations and same for the w/o-emulsion. The reconstructed cornea could be an alternative to excised animal tissue for drug permeation studies in vitro.

Background: In vitro investigations of transcorneal permeation behaviour with new drugs in ophthalmology are mainly carried out using excised corneas taken from slaughtered or experimental animals. Analogous to previously used dermis models, an in vitro model was constructed from porcine corneal cell cultures and the permeation barrier properties were tested and compared with permeation data from excised corneas., Methods: Epithelial, stroma and endothelial cells were successfully isolated by treatment with various enzymes and a corneal equivalent was created step-by-step which morphologically resembled the original tissue. Five different drug formulations were investigated and pilocarpine hydrochloride was chosen as the model drug. The permeation studies were made with a modified Franz cell and analysis was performed by high performance liquid chromatography. Permeation data from excised corneas and from the cornea construct were compared whereby data obtained with lipophilic preparations did not differ (factor of 1) and those obtained with aqueous formulations were relatively similar (factor of 3-4)., Results and Conclusions: The results obtained show that the cornea construct can be used as an alternative to excised corneas for in vitro investigations of ophthalmic drug preparations.