Category: Formulation and Quality
Purpose: Human eye is multiplex organ which has unique anatomy and compartment organization. It has anatomical barriers such as epithelium, stroma, lymph flow, conjunctiva blood and tears drainage along with physiological barriers such as reflex blinking and naso-lachrymal drainage. Due to these barriers only 5% of the topical formulations are bioavailable. Conventional invitro models are limited in truly mimicking the ocular environment. Hence, better mechanical invitro screening model are needed which can accommodate for constant tear flow, tear replenishment as well as blink rate. 3D bio-printing is an emerging method of tissue fabrication in which cells are extruded within a hydrogel matrix to form a precise cell scaffold resembling the tissues. In this study we designed high-throughput 3D bioprinting of corneal equivalents and developed mechanical blinking eye model (with perfusion system simulating for tear flow) for screening of ocular formulations.
Methods: We designed our own digital 3D cornea models based on reported dimensions of adult patient cornea averages (Fig.1a). Cornea dimension were converted to 3D shapes using Autodesk fusion 360 software (Fig.1b). Exporting the data through slicr3r converted it to gcode files (Fig.1c). Gcode files were read by BioX extrusion 3D printer (Cellink) to print the cornea. In order to maintain the curvature and dimensions of cornea, a support scaffold was designed using photocurable clear resin in stereolithographic (SLA) printer (Forms lab) (Fig.2). The support scaffold we designed could facilitate the printing of 6 corneas at a time thus enabling high throughput printing of smooth uniform corneas. Appropriate bioink with desirable rheology, printability, clarity and biocompatibility was optimized. Our final bioink comprised of 3.25%w/v sodium alginate, 4% gelatin and 10mg/ml collagen. Human corneal epithelial cells and (HCE) and human corneal keratocytes (HCK) cells were incorporated in the optimized bioink and cell laden corneal stromal equivalents were printed at a pressure of 35 kPa, temperature 23°C and print speed of 10mm/s. Printed structures were cross-linked by calcium chloride 100mM, washed with HBSS and incubated at 37°C in fibroblast media. Live dead assay and Alamar assay were performed on the corneal equivalents at different time points i.e. day 0, 1, 5, 7, 14. 3D printed corneas were placed within a blinking apparatus. DC motor operated eyelid was placed over 3D printed corneal equivalents (Fig.3c) that will simulate mechanical stress occurring in vivo. The inner eyelid was coated with a hydrogel and incorporated with adjustable tear fluid mechanism to allow smooth blinking over the cornea. Blinking rate will resemble human eyelid. The model will be evaluated for screening of ocular formulations for their permeability across the corneal layers.
Results: High-throughput printing of corneas was achieved successfully by using SLA printed support scaffold. SLA printing offered resolution up to 25 microns and resulted in more precise accurate prototyping of curved structures compared to FDM printing. Triangulated tessellation resulted in smoother corneas. Support scaffold could hold 6 corneas at a time. This high-throughput support scaffold not only accelerated the printing process but also led to more accurate and uniform corneas. The corneas were able to maintain their structure, integrity and clarity. HCE and HCKs maintained high viability ( >95%) (Fig.3a) for two weeks as confirmed from live dead assay. Also, alamar assay showed cell proliferation for 9 days (Fig. 3b). Printed cornea was placed on the insert. Eyelid made of PAM-Alginate wrapped with Ecoflex elastomer for flexibility. Inner side of lid was coated with PEGDA. The blink rate was maintained at 15-20 blinks / minute. Perfusion lines were SLA printed within the eyelid. Simulated Tear fluid was perfused through these lines at rate of 1.4ul/min to simulate for lacrimal excretory ducts.
Conclusion: The current study provides proof of concept to use 3D bioprinting as a tool for high-throughput fabrication of corneal equivalents which can be further developed into a blinking eye model. 3D printed corneal equivalents have potential for multiple applications ranging from human transplants to realistic invitro screening model for the ophthalmic formulations.