BackgroundThere has been no effective treatment or agent that is available for corneal injury in promoting corneal wound healing. Previous studies on edible bird's nest extract (EBN) had reported the presence of hormone-like substance; avian epidermal growth factor that could stimulate cell division and enhance regeneration. This study aimed to investigate the effects of EBN on corneal keratocytes proliferative capacity and phenotypical changes.MethodsCorneal keratocytes from six New Zealand White Rabbits were isolated and cultured until Passage 1. The proliferative effects of EBN on corneal keratocytes were determined by MTT assay in serum-containing medium (FDS) and serum-free medium (FD). Keratocytes phenotypical changes were morphologically assessed and gene expression of aldehyde dehydrogenase (ALDH), collagen type 1 and lumican were determined through RT-PCR.ResultsThe highest cell proliferation was observed when both media were supplemented with 0.05% and 0.1% EBN. Cell proliferation was also consistently higher in FDS compared to FD. Both phase contrast micrographs and gene expression analysis confirmed the corneal keratocytes retained their phenotypes with the addition of EBN.ConclusionsThese results suggested that low concentration of EBN could synergistically induce cell proliferation, especially in serum-containing medium. This could be a novel breakthrough as both cell proliferation and functional maintenance are important during corneal wound healing. The in vitro test is considered as a crucial first step for nutri-pharmaceutical formation of EBN-based eye drops before in vivo application.

Retinoic acid has recently been shown to control the phenotype and extracellular matrix composition of corneal stromal cells cultured in vitro as monolayers. This study set out to investigate the effects of retinoic acid on human corneal keratocytes within a 3D environment. Human corneal keratocytes were encapsulated in collagen gels, which were subsequently compressed under load, and cultured in serum-free media supplemented with 10 µM retinoic acid or DMSO vehicle for 30 days. Cell proliferation was quantified on selected days, while the expression of several important keratocytes markers was evaluated at day 30 using RT-PCR and immunoblotting. The weight and size of the collagen constructs were measured before and after hydration and contraction analyses. Retinoic acid enhanced keratocyte proliferation until day 30, whereas cells in control culture conditions showed reduced numbers after day 21. Both gene and protein expressions of keratocyte-characteristic proteoglycans (keratocan, lumican and decorin), corneal crystallins and collagen type I and V were significantly increased following retinoic acid supplementation. Retinoic acid also significantly reduced the expression of matrix metalloproteases 1, 3 and 9 while not increasing α-smooth muscle actin and fibronectin expression. Furthermore, these effects were also correlated with the ability of retinoic acid to significantly inhibit the contractility of keratocytes while allowing the build-up of corneal stromal extracellular matrix within the 3D constructs. Thus, retinoic acid supplementation represents a promising strategy to improve the phenotype of 3D-cultured keratocytes, and their usefulness as a model of corneal stroma for corneal biology and regenerative medicine applications.

To this day, the concept of continuous bioprocessing has been applied mostly to the manufacture of molecular biologics such as proteins, growth factors, and secondary metabolites with biopharmaceutical uses. The present work now sets to explore the potential application of continuous bioprocess methods to source large numbers of human adherent cells with potential therapeutic value. To this purpose, we developed a smart multifunctional surface coating capable of controlling the attachment, proliferation, and subsequent self-detachment of human corneal stromal cells. This system allowed the maintenance of cell cultures under steady-state growth conditions, where self-detaching cells were continuously replenished by the proliferation of those remaining attached. This facilitated a closed, continuous bioprocessing platform with recovery of approximately 1% of the total adherent cells per hour, a yield rate that was maintained for 1 month. Moreover, both attached and self-detached cells were shown to retain their original phenotype. Together, these results represent the proof-of-concept for a new high-throughput, high-standard, and low-cost biomanufacturing strategy with multiple potentials and important downstream applications.