Culturing cells

With several patients regenerating anatomically perfect corneas and recovering 20/20 vision in an early trial, injecting cultured corneal endothelial cells (CECs) shows great promise for treating Fuchs’ and other corneal endothelial dysfunction. However, many challenges must yet be overcome for the technology to move from the lab to the clinic, Naoki Okumura MD, PhD told the 6th EuCornea Congress in Barcelona, Spain.

Developing CEC injection therapy from concept to the first clinical trial took nearly a decade, said Dr Okumura, of Doshisha University, Kyoto, Japan. Several more years will be required to conduct clinical trials comparing CEC injection to endothelial keratoplasty, and to develop processes for reliably culturing high-quality cells and efficiently distributing them for clinical use.

ROCK inhibitor breakthrough

Early injections of cultured rabbit CECs into rabbit eyes with induced endothelial dysfunction failed to regenerate transparent corneas, Dr Okumura said. Analysis revealed that removing CECs from the culture plate activated a Rho kinase (ROCK) pathway that interfered with their ability to attach to a substrate and enabled anoikis, or programmed cell death induced by separation from the extracellular matrix.

Dr Okumura and colleagues hypothesised that countering this activation with a ROCK inhibitor might allow injected CECs to adhere to Descemet’s membrane. And sure enough, damaged corneas of rabbit eyes injected with both CECs and the ROCK inhibitor Y-27632 cleared and thinned significantly after 48 hours, with CECs densely adhering, while those injected with CECs only remained as clouded and thick as untreated controls, with very few CECs adhering.

Subsequently, six of six monkeys with induced corneal dysfunction regenerated completely transparent and anatomically normal corneas after injection with cultured monkey CECs and Y-27632. “This was proof of concept,” Dr Okumura said.

Beginning in 2013, Dr Okumura and colleagues injected human CECs and Y-27632 into the anterior chambers of patients with Fuchs’ endothelial dystrophy in an as-yet unpublished clinical trial. Several of these patients regenerated anatomically normal corneas with vision returning to 20/20, Dr Okumura reported. One year after treatment, one patient saw better with the CEC injection-treated eye than his fellow eye, which had received Descemet’s stripping automated endothelial keratoplasty (DSAEK) five years earlier – showing that tissue engineering has the potential not only to relieve donor cornea shortages, but also improve visual outcomes for patients with endothelial diseases.

However, treating patients with CEC injection requires very large quantities of cultured cells. In theory, five passes of seeding and expansion can produce enough cells from a single donor cornea to treat more than 200 patients. In practice this has proven quite difficult, Dr Okumura said.

Early attempts found limited proliferation of human CECs in vitro, and the cells that did develop often transformed into non-functioning fibroblastic phenotypes. As cells underwent multiple expansion passes, their quality also fell, with many cells in later passes becoming senescent. This limited their density, ability to create tight intercellular junctions, and their ion pumping capacity, all of which are needed to create an effective epithelial barrier with pump action that prevents corneal oedema.

ROCK inhibitors helped by promoting in vitro cell proliferation and adhesion.

Conditioning support medium with human bone marrow mesenchymal stem cells also promoted cell proliferation. Coating the culture substrate with Laminin 511-E8 significantly enhanced cell proliferation and promoted rapid growth with high cell density.

Analysis also found that CEC fibroblastic transformation could be interrupted by inhibiting TGF-β receptors. This resulted in culturing hexagonal phenotype cells that expressed normal pumping and intracellular adhesion proteins, Dr Okumura said.

Centrifuging cultured cells has also proven an effective way to remove senescent cells, which have a lower density. Higher cell density has proven to be an important predictor of better clinical outcomes in animal models, he added.

Incorporating these features has produced a CEC culturing protocol sufficient for research. “We are now routinely culturing human CECs for clinical trial use,” Dr Okumura said.

But many steps remain before cultured human CECs become clinically available.

“We do understand the long and winding road waiting for us. But we have to move forward to commercialisation and industrialisation, because we believe that to provide the cultured cells by eye banks or companies is the only way to make this therapy a real therapy that helps patients,” added Dr Okumura.

References available on request

Naoki Okumura:
nokumura@koto.kpu-m.ac.jp