Chantal Perrache

Delivery of macromolecules into the endothelium of whole ex vivo human cornea by femtosecond laser-activated carbon nanoparticles

Background The targeted delivery of drugs or genes into corneal endothelial cells (ECs) during eye banking could help improve graft quality and quantity. Physical methods raising less safety concerns than viral ones, we previously adapted, for in vitro ECs, a recent innovative technique of drug delivery based on the activation of carbon nanoparticles (CNPs) by a femtosecond laser (fsL). The aim of the present pilot study was to adapt this method to enable molecule delivery into the intact endothelium of ex vivo human corneas. Methods ECs from 40 organ-cultured corneas were perforated by photoacoustic reaction induced by irradiation of CNPs by a fsL. This enabled intracellular delivery of Alexa Fluor 488 dextran, a 4000 Da fluorescent macromolecule. The influence of increasing laser fluences (15, 20, 30 and 40 mJ/cm2) and of protective additives (ROCK inhibitor and poloxamer 407) on delivery and mortality rates was quantified using ImageJ. Results No dextran was delivered with a fluence lower than 20 mJ/cm2. Dextran was delivered into 3% (range 0%–7%) of cells at 20 mJ/cm2, 7% (range 2%–12%) at 30 mJ/cm2 and reaching a median 13% (range 3%–24%) for 40 mJ/cm2, showing that dextran uptake by ECs increased significantly with fluence. Induced mortality varied from 0% to 53% irrespective of fluence, but likely to be related with the endothelial status (EC density and morphometry, donor age, storage duration and presence of Descemets folds). ROCK inhibitor slightly increased uptake efficiency, unlike poloxamer. However, none of them decreased the mortality induced by laser. Conclusions This study shows that a macromolecule can be delivered specifically into ECs of a whole organ-cultured human cornea, using fsL-activated CNPs. The delivery rate was relatively high for a non-viral method. Further optimisation is required to understand and reduce variability in cell mortality.

Storage of Porcine Cornea in an Innovative Bioreactor

Purpose To quantify cell survival and tissue structure preservation of porcine cornea stored in a bioreactor (BR) that recreates a transcorneal pressure gradient equivalent to intraocular pressure (IOP) and renews the medium. Methods A BR comprising endothelial and epithelial chambers was machined in a biocompatible material. The porcine cornea, securely held, separated the chambers. Medium flow and pressure inside the endothelial chamber were maintained by a peristaltic pump. In the epithelial chamber, the corneal surface was alternatively exposed to air and a specific medium. Two transparent windows allowed thickness measurement by optical coherence tomography without opening the BR. Porcine corneas stored in the BR-on (pressure 20 mm Hg, flow 5 μL/min, temperature 31°C) were compared with (1) BR-off (no pressure or flow); (2) organ culture; and (3) Petri dish with agar on the endothelial side. Epithelial and limbal structure and differentiation, corneal transparency and thickness, and endothelial viability were compared after 7 days of storage and with fresh corneas. Results Corneas stored in the BR-on were thinner and more transparent than those stored with the other methods. The BR-on preserved a stratified and differentiated (K3/K12+) corneal epithelium and undifferentiated basal limbal cells with stemness markers (K3/K12-; ABCB5, K14, p75+), as well as endothelial integrity. Conclusions By recreating equivalent IOP and medium renewal, the BR obtained unprecedented storage quality of porcine corneas and preserved their main epithelial, limbal, and endothelial characteristics.

Cutting and Decellularization of Multiple Corneal Stromal Lamellae for the Bioengineering of Endothelial Grafts

Purpose Engineered corneal endothelial grafts able to provide numerous functional endothelial cells for the restoration of corneal transparency would be a worthwhile way of replacing donor tissue, which is extremely scarce. The grafts are simply constructed: a biocompatible thin and transparent carrier colonized by a monolayer of cultured endothelial cells (ECs). Here we describe a process able to obtain appropriate carriers by recycling human corneas unsuitable for graft in their original state, but liable to provide multiple thin lamellae when cut with a femtosecond laser as used in refractive surgery. Methods We selected a robust method of stromal decellularization. To demonstrate that neither this process nor long-term storage hindered cell adherence, lamellae were endothelialized with an EC line. Results The constructs achieved up to very high EC density (the main quality criterion for regular donor corneas) while remaining transparent and thin. We verified that they could be inserted in the anterior chamber of a human eye, like a conventional endothelial graft. Human decellularized cornea will likely be directly compatible with the recipient cornea and comply with the requirements of health regulatory authorities. Conclusions This study demonstrates that thin human corneal lamellae could have high potential as carriers in next-generation therapy for endothelial dysfunctions.

Immunosuppression by a subconjunctival implant releasing dexamethasone in a rabbit model of penetrating keratoplasty

Aims To evaluate the efficacy of a subconjunctival dexamethasone-releasing implant in preventing rejection of penetrating keratoplasty (PK) in an animal model. Methods Twenty-two rabbits underwent allogenic PK. After randomisation, they received either a 700 µg dexamethasone implant under the conjunctiva at the end of surgery (n=10), one dexamethasone 1 mg/mL eye-drop thrice daily (n=6) or a placebo thrice daily (n=6). The suture was left in place. Animals were observed weekly by slit-lamp and optical coherence tomography with quantification of transparency, neovascularisation and central corneal thickness (CCT). At 5–6 weeks, they were euthanised for histology. The residual dexamethasone concentration in ocular tissues was measured with an ultra-performance liquid chromatography-tandem mass spectrometer. Results Placebo group: early neovascularisation was systematic, penetrating the graft by 270–360° at 5–6 weeks. Rejection occurred in 50% of cases. Eye-drop and implant groups: similar course without rejection at 6 weeks and normal CCT. Neovascularisation was observed in 5/6 rabbits in the eye-drop group and in 6/8 in the implant group, with two cases of new vessels penetrating the graft from week 3. Neovascularisation scores did not differ significantly between the two treatments and were significantly lower than for the placebo. Histology was in agreement in all cases. Implants disappeared after 3–5 weeks. No local side effect was observed. Tissue concentrations were all higher at day 8 (n=2) in the implant group than in the eye drop group and lower at 6 weeks (n=8). Conclusions In this PK model characterised by a high rejection rate, a subconjunctival dexamethasone implant was for 6 weeks as effective as the topical form in preventing allograft rejection.