(K-434) Bioreactor for Corneal Tissue Engineering: A Novel Approach to Corneal Cell Cultivation

Introduction 

More than 2.2 billion individuals globally have potentially preventable vision impairment, underscoring the need for innovative advancements in the field of ophthalmological care and research.[1-2] Tissue engineering combined with bioreactor culture has the potential to create new corneal tissues, which presents a significant breakthrough not only as a solution to address the burden of vision impairment but also as a platform for developing novel processes.[3]

Our work harnesses the transformative potential of bioreactor technology in advancing corneal research and biomechanical development, providing an alternative to conventional transplants. 

The cornea's complex biomechanics play an essential role in maintaining ocular structure and function. Within the eye, corneas undergo linear strain due to intraocular pressure and experience shear stress attributable to the tear film.[4] Many other groups have developed bioreactor systems to impart strain to growing tissue.[5]

In this study, our objective was to create a suitable microenvironment for culturing corneal fibroblast cells that mimics the biomechanical environment of the native cornea. We have designed, tested, and evolved a corneal bioreactor system that includes a pressure control system, a flexible membrane, and a media flow chamber with six wells to replicate corneal size (see Figure 1C). Specifically, we aimed to validate that our system could hold pressure over seven days of cell culture and that cells were viable in the system.

Materials and Methods 

Bioreactor-System: Using prior lab studies, we machined a bioreactor system utilizing polyether ether ketone (PEEK) as the primary material.[5] PEEK provides the structure necessary for the bioreactor with minimal impact on culturing cells. We used transparent plexiglass chamber lids, steel screws, washers, and O-rings for their mechanical hermetic sealing capabilities. The bioreactor was equipped with steel tubes serving as inlets and outlets. A peristaltic pump circulates the media in a radial flow system, ensuring the cells' constant supply of nutrients. 

A pressure sensor-equipped Arduino system regulated the bioreactor's pressure. Solenoid motor-equipped syringes introduced pressure into the system, allowing for adjustable pressure to mimic increasing intraocular pressures.

Cell-Culture: Bioreactor materials were pre-sterilized via autoclave. The system was assembled under a laminar flow hood to maintain a sterile environment and established within an incubator providing optimal temperature and CO₂ conditions for cell growth (Fig. 1A). Rabbit corneal fibroblasts (RCFs) were seeded onto the bioreactor membranes and in 24-well plates (approximately the same area as the bioreactor wells) at 5000 cells/cm2 and grown for 4-7 days in cell culture media (DMEM (Sigma, St. Louis, MO) supplemented with 10% Fetal Bovine Serum (VWR, Radnor, PA) and 5% penicillin/streptomycin (ThermoFisher Scientific, Waltham, MA).

Cell-Growth and Viability: We dismantled the bioreactor after seven days and dissected the membrane for imaging. A Nikon Eclipse Ti-U microscope imaged the bioreactor and control plate cells. Following the MTT protocol, cell viability assay (R&D Systems, Minneapolis, MN) quantified bioreactor and control samples by applying a standard curve.

Results and Discussions 

Design-Changes: Initial efforts establishing a viable bioreactor encountered significant challenges, the most prominent being media leakage and contamination (Fig. 1B). However, informing subsequent refinements and innovations in our system. 

Media predominantly leaked from the chambers' edges (Fig. 1C). This led to significant modifications: introducing steel washers to both chambers and thickening the plexiglass, yielding a distributed load effect. The second trial reduced leakage to ≈20ml of media per hour from ≈100ml per hour. Meticulously, monitoring allowed us to locate the remaining leakage points, predominantly associated with the inlets and outlets. We leveraged these insights to replace the seals with suitable washer-shaped O-rings at the inlets and outlets. Modifications effectively eliminated leakage, preparing the bioreactor for experimentation.

Cell-Viability: Cells were cultured for seven days without media leakage or contamination (Fig. 1D). Imaging data depicted living cells at similar densities to control samples, indicating successful viability within the bioreactor environment (Fig. 1E). An MTT assay confirmed cell viability in the bioreactor and control plates and calculated similar cell numbers to quantify these results.

Our bioreactor has been validated to maintain desired pressure and a sterile environment and supported cell growth comparable to controls.

Conclusion 

This research underscores the significance of bioreactor technology in ophthalmology, specifically for cultivating a desirable microenvironment for corneal fibroblasts. The resolution of initial challenges, particularly media leakage, contamination, and subsequent cultivation of corneal cells, presents a significant milestone in our corneal tissue engineering lab. While we continue to optimize the system further, these initial results confirm our bioreactor's potential as a promising tool for corneal tissue engineering.

In future studies, we plan to expand the range of tested pressures to explore the impact of elevated intraocular pressure on cell behavior. Our lab has published several studies looking at the potential of various signals impacting cell protein expression and, ultimately, transparency of the resulting tissue.[6-10] This bioreactor can potentially simultaneously evaluate the impact of mechanical, electromagnetic, chemical, and surface topology signals on corneal fibroblast behavior. Resulting in implications for vision restoration efforts worldwide, where tissue-engineered corneas grown in bioreactors could mitigate dependence on limited donor-derived transplants.

[1] World Health Organization. (n.d.). Vision Impairment and blindness. World Health Organization. https://www.who.int/news-room/fact-sheets/detail/blindness-and-visual-impairment
[2] Bourne, R. R. A., Flaxman, S. R., Braithwaite, T., Cicinelli, M. V., Das, A., Jonas, J. B., Keeffe, J., Kempen, J. H., Leasher, J., Limburg, H., Naidoo, K., Pesudovs, K., Resnikoff, S., Silvester, A., Stevens, G. A., Tahhan, N., Wong, T. Y., Taylor, H. R., & Vision Loss Expert Group (2017). Magnitude, temporal trends, and projections of the global prevalence of blindness and distance and near vision impairment: a systematic review and meta-analysis. The Lancet. Global health, 5(9), e888–e897. https://doi.org/10.1016/S2214-109X(17)30293-0
[3] Oie, Y., & Nishida, K. (2016). Corneal regenerative medicine. Regenerative therapy, 5, 40–45. https://doi.org/10.1016/j.reth.2016.06.002
[4]Liu, J., & Roberts, C. J. (2005). Influence of corneal biomechanical properties on intraocular pressure measurement: Quantitative analysis. Journal of Cataract & Refractive Surgery, 31(1), 146–155. https://doi.org/10.1016/j.jcrs.2004.09.031.
[5]Meyer, U., Büchter, A., Nazer, N., & Wiesmann, H. P. (2006). Design and performance of a bioreactor system for mechanically promoted three-dimensional tissue engineering. The British journal of oral & maxillofacial surgery, 44(2), 134–140. https://doi.org/10.1016/j.bjoms.2005.05.001
[6] Orwin, E., Shah, A., Voorhees, A., & Ravi, V. (2007). Bioreactor design for cornea tissue engineering: Material-cell interactions. Acta biomaterialia, 3(6), 1041–1049. https://doi.org/10.1016/j.actbio.2007.04.008
[7] Thompson, R. E., Boraas, L. C., Sowder, M., Bechtel, M. K., & Orwin, E. J. (2013). Three-dimensional cell culture environment promotes partial recovery of the native corneal keratocyte phenotype from a subcultured population. Tissue engineering. Part A, 19(13-14), 1564–1572. https://doi.org/10.1089/ten.TEA.2012.0084
[8] Thompson, R. E., Boraas, L. C., Sowder, M., Bechtel, M. K., & Orwin, E. J. (2013). Three-dimensional cell culture environment promotes partial recovery of the native corneal keratocyte phenotype from a subcultured population. Tissue engineering. Part A, 19(13-14), 1564–1572. https://doi.org/10.1089/ten.TEA.2012.0084
[9] Wray, L. S., & Orwin, E. J. (2009). Recreating the microenvironment of the native cornea for tissue engineering applications. Tissue engineering. Part A, 15(7), 1463–1472. https://doi.org/10.1089/ten.tea.2008.0239
[10] Phu, D., Wray, L. S., Warren, R. V., Haskell, R. C., & Orwin, E. J. (2011). Effect of substrate composition and alignment on corneal cell phenotype. Tissue engineering. Part A, 17(5-6), 799–807. https://doi.org/10.1089/ten.TEA.2009.0724
[11] Leonard, E. K., Pai, V. H., Amberg, P., Gardner, J., & Orwin, E. J. (2012). Design and validation of a corneal bioreactor. Biotechnology and bioengineering, 109(12), 3189–3198. https://doi.org/10.1002/bit.24587