Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions.

This study demonstrates that thin human corneal lamellae could have high potential as carriers in next-generation therapy for endothelial dysfunctions.

Abstract:A novel ultrasound (US) high-channels platform is a pre-requisite to open new frontiers in diagnostic and/or therapy by experimental implementation of innovative advanced US techniques. To date, a few systems with more than 1000 transducers permit full and simultaneous control in both transmission and receiving of all single elements of arrays. A powerful US platform for implementing 4-D (real-time 3-D) advanced US strategies, offering full research access, is presented in this paper. It includes a 1024-elements array prototype designed for 4-D cardiac dual-mode US imaging/therapy and 4 synchronized Vantage systems. The physical addressing of each element was properly chosen for allowing various array downsampled combinations while minimizing the number of driving systems. Numerical simulations of US imaging were performed, and corresponding experimental data were acquired to compare full and downsampled array strategies, testing 4-D imaging sequences and reconstruction processes. The results indicate the degree of degradation of image quality when using full array or downsampled combinations, and the contrast ratio and the contrast to noise ratio vary from 7.71 dB to 2.02 dB and from 2.99 dB to −7.31 dB, respectively. Moreover, the feasibility of the 4-D US platform implementation was tested on a blood vessel mimicking phantom for preliminary Doppler applications. The acquired data with fast volumetric imaging with up to 2000 fps allowed assessing the validity of common 3-D power Doppler, opening in this way a large field of applications.

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Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3–57.3) of CECs with low mortality (0.5%, range (0.55–1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for future cell or tissue therapy.

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Duplex ultrasonography is a mode of medical ultrasonography that allows one to visualize, at the same time, the inner structure of the body (B-mode) and the blood flow at a particular point in the body (Doppler mode). This mode requires a strategy for alternating B-mode and flow emissions. Traditional strategies either halve the maximum measurable velocity or introduce gaps in the flow data. The objective of this article is to propose a completely original method based on compressive sensing for reconstructing the Doppler signal segment by segment. Our approach is based on randomly alternating B-mode and flow emissions. The influence of the different parameters on the reconstruction quality is studied in detail. The technique is evaluated and its feasability is validated in simulation and from experimental in vivo data. It is also compared to the only method from the literature, proposed by Jensen, that reconstructs blood velocity estimates from sparse data sets.