(M1430-05-30) High-Throughput 3D Bioprinting of Corneal Stromal Equivalents

Purpose: In this study, we propose for the first time a novel high-throughput approach to develop corneal stromal models by optimizing bio-ink printability with gelatin, generating G-code that enables the rapid production of corneas, and printing within a corneal support fabricated by SLA printer. We wish to develop a novel method of 3D printing human corneal stoma models to create better in vitro models for drug development or as a possible solution to limited corneal transplants.

Methods: In this study, we designed a novel approach to 3D bioprinting corneal stroma in tandem with micro-transfer molding, optimized bio-inks with quality by design (QbD) techniques and utilized G-code to replicate tissue models in a high-throughput fashion. Our tissue model was designed taking average dimensions of the adult corneal stroma, converting them to a 3D shape that could be further sliced into G-code, and then printing with a BIO X 3D printer. A support scaffold was manufactured by SLA printer to help maintain the curvature of the cornea during printing and to mold high-resolution features that would be difficult to achieve by extrusion alone. This support scaffold could facilitate high-throughput printing of 6–12 corneas and enabled using bio-inks of optimized mechanical properties to minimize printing time.

Results: Viscosity was plotted against increasing shear stress to determine the elastic and storage modulus of the formulations (Figure 2a). Printing at temperatures higher than 37°C resulted in the melting of gelatin and a loss in storage modulus of the bio-ink. Temperatures lower than 20°C resulted in using a higher pressure needed to extrude the filament and thus exerting more force on the cells which is not desirable. Oscillation temperature ramps of each bio-ink allowed us to find their respective optimal printing temperature due to the ratio between storage and loss moduli. The flow sweep tests showed that our gels displayed shear thinning behavior (Figure 2b). This was beneficial in the extrusion process because the gels could flow more easily when pressure was applied. The reduction in viscosity is also beneficial to reduce shear stresses experienced by the cells, which has also shown to be positive for cell viability. Live dead assay showed the percent cell viability was 96 ± 5% and 86 ± 2% on Days 1 and 14, respectively (Figure 3a, b). Alamar assay also demonstrated that cells maintained their viability and growth in printed corneal scaffold for 2 weeks. Cell growth rate was calculated as percentage of growth on Day 1. Growth rate was observed as 101 ± 9% on Day 5, 98.5 ± 13% and 93.8 ± 7% on Day 10 and Day 14, respectively (Figure 3c).

Conclusion: Taken together, we designed a model for high-throughput 3D printing and established proof of concept for the feasibility of rapid printing of corneal stromal scaffolds. Due to the optimum ratio of collagen, gelatin, and sodium alginate in the bio-ink, the printed corneas were transparent, smooth, possessed desired curvature, and also supported the growth of HCKs for 2 weeks. This study offers a novel avenue for optimization and design of highly organized and biomimicking structures in integration with composition and component design to obtain 3D printed tissues on large scale.

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Acknowledgements:We would like to thank Dr. Eric Lochner for helping us with AFM studies and Florida State University, Department of Physics, for allowing us to use their facilities. The research was supported by the National Institute on Minority Health and Health Disparities of National Institutes of Health under Award number U54 MD007582 (U54 RCMI grant) and NSF-CREST Center for Complex Materials Design for Multidimensional Additive Processing (CoManD) award # 1735968.
Funding information
NSF-CREST Center for Complex Materials Design for Multidimensional Additive Processing (CoManD), Grant/ Award Number: 1735968; the National Institute on Minority Health and Health Disparities of National Institutes of Health, Grant/Award Number: U54 MD007582

The development of human corneal stroma and base support scaffolds apparatus. (a) Average corneal stroma dimensions modeled in Autodesk Fusion 360. (b) Bright-field image of single well of support scaffold showing its radius. (c) 3D model of 6-well corneal scaffold. (d) Support and raft preparation with Preform software for SLA printing of 12-well scaffold. (e) Printing path of 12-well corneal scaffold. (f) High-throughput printing of 12 corneas with a BIOX printer. (g) Printed cornea after crosslinking and placing within media. (h) Picture of cornea showing its transparency through the letter “C.” (i) Bright-field image of 3D printed cornea using inverted microscope on Day 1

Characterization of bioinks (a) Flow sweep test using rheometer to determine the viscosity at varying shear stress. Ink 2 exhibited higher viscosity as compared to others. (b) Shear stress vs shear rate behavior of bioinks showing shear thinning. (c) Light transmittance of bio-ink 2, 3D printed cornea at varying wavelengths compared with control (water).

Evaluation of cell viability and immunofluorescence staining of 3D bioprinted corneas. (a) Live dead assay in 3D printed corneas bearing human corneal keratocyte (HCK) cells on Day 1 (merged image). (b) Live dead assay in 3D printed corneas bearing HCK cells on Day 14 (merged image). (c) Alamar assay on 3D printed corneas on Days 1, 5, 10, and 14. Immunofluorescence staining of 3D printed corneas using a fluorescent microscope for (d) fibronectin (red), (e) F-actin (green), (f) DAPI (blue), and (g) merge image