Human Corneal Endothelial Cell Expansion for Corneal Endothelium Transplantation: An Overview

Abstract

The monolayer of cells forming the human corneal endothelium is critical to the maintenance of corneal transparency and is not known to regenerate in vivo. Thus, dysfunction of these cells constitutes the most often cited reasons for the 150,000 or so corneal transplants performed yearly. Although current corneal transplantation is more than 90% successful at 1 year, longer term results are not as encouraging with approximately 70% success at 5 years. Nonimmunologic graft failure and allograft endothelial rejection are the main problems. Furthermore, the global shortage of donor corneas greatly restricts several corneal transplantations performed. With advances in understanding corneal endothelial cell biology, it is now possible to cultivate human corneal endothelial cells (HCECs) in vitro, thus providing new opportunities to develop novel tissue-engineered human corneal endothelium. This review will provide an overview of (a) the characteristics of human corneal endothelium; (b) past and present HCECs isolation and culture protocols; (c) various potential carriers for the generation of tissue-engineered corneal endothelium, together with some of the functional studies reported in various animal models; and (d) the current rapid advancements in surgical techniques for keratoplasty. A successful combination of tissue-engineered human corneal endothelium coupled with innovative and groundbreaking surgical procedures will bridge basic research involving cultured HCECs, bringing it from bench to bedside.

The cornea is a highly organized, transparent tissue with five distinct layers: (a) epithelium; (b) Bowman's membrane; (c) stroma; (d) Descemet's membrane (DM); and (e) endothelium (Fig. 1). Overall, the cornea is approximately 520-μm thick with the epithelium constituting approximately 10% of the tissue, but the bulk of the cornea is the collagenous stroma that is approximately 450-μm thick (¹). Collectively, the cornea protects the delicate internal intraocular contents of the eye (^{2, 3}), and more importantly, its unique composition is the primary refracting element in the optical pathway, focusing visible light onto the retina (⁴). The stratified, squamous, nonkeratinized epithelium acts as the main protective barrier of the cornea (⁵). The Bowman's membrane forms the anterior boundary of the stroma. It is approximately 15-μm thick and is composed of randomly orientated collagen type I fibrils supported within a proteoglycan matrix (⁶). The corneal stroma is a densely interlaced, transparent connective tissue composed primarily of collagen type I fibrils and proteoglycans (^7–9). Stromal transparency has been attributed to the scattering and destructive interference of light by its unique arrangement of evenly spaced collagen fibrils (see the “destructive interference” theory of transparency) (¹⁰). The DM, a true basal lamina, is located at the posterior boundary of the stroma. It is synthesized by the corneal endothelium and the thickness of the DM has been reported to increase with age (¹¹). For the purpose of this review, greater focus will be placed on the form and function of the corneal endothelium.

FIGURE 1.: The human cornea. (A) The human cornea is a transparent, layered structure comprising five distinctive layers: (a) epithelium, (b) Bowman's membrane, (c) stroma, (d) Descemet's membrane, and (e) endothelium. (B) Freshly peeled human corneal endothelial cell monolayer still adhered to its Descemet's membrane (scale bar=100 μm).

THE CORNEAL ENDOTHELIUM

The human corneal endothelium is physiologically the most important monolayer of the cornea. These unique hexagonal cells act as a barrier for fluid moving into the stroma, which because of the glycoaminoglycan composition can adsorb large amounts of fluid creating an edematous cornea that can lead to the loss of vision (^{12, 13}). As the most metabolically active cells in the cornea, fluid pumps operate continuously to actively move fluid from the stroma back into the anterior chamber of the eye (^14–16). The dynamic balance between the “leaky” barrier and active pump regulates corneal hydration, keeping the cornea transparent (^{13, 15, 17, 18}). Corneal blindness, the situation where the retina is normal but the cornea becomes edematous, is often caused by endothelial dysfunction and is the second leading cause of visual blindness.

There is an inverse relationship between age and corneal endothelial cell (CEC) density (^19–22). The cell density of tightly packed corneal endothelium of 2-month-old infants has been reported to be as high as 5624 cells/mm², with a mean of 4252 cells/mm² within the first year from birth (²³). CEC density decreases rapidly during early childhood and is associated with the increase in corneal size with normal eye growth (¹⁹). At 5 years of age, corneal endothelium density of approximately 3591±399 cells/mm² falls to approximately 2697±246 cells/mm² by 10 years of age (²⁰). The rapid attrition rate of corneal cell density slows to a gradual decline throughout adulthood, to approximately 0.6% per year (^{19, 21, 24}), which also indicates that human corneal endothelial cells (HCECs) have a limited, if not restricted, capacity to proliferate in vivo. Cells of the human corneal endothelium have normal component of telomeres, therefore their inability to proliferate in vivo is not due to short telomeres (^{25, 26}). Joyce et al. (²⁷) showed that HCECs are arrested in the G1-phase of the cell cycle in vivo. Their subsequent studies suggested that contact-dependent inhibition plays a major role in the induction of cell-cycle arrest, and together with transforming growth factor-β2 (found within the aqueous humor), it maintains the corneal endothelial layer in a nonproliferative state (^28–30). Although the human corneal endothelium is held in a nonreplicative state within the eye, ex vivo mechanical wounding studies and treatment of HCECs using EDTA showed that the CECs retain the capacity to proliferate (^{31, 32}).

As the human corneal endothelium is not able to actively divide within the eye, it cannot undergo any form of regenerative healing to replace dead or injured CECs. Instead, existing CECs physiologically spread out to maintain the functional integrity of the corneal endothelium, sustaining cornea deturgescence and maintaining corneal transparency (^{19, 30, 33, 34}). The phenomenon of cell spreading can be associated with the variability of CEC shape (pleomorphism) and cell size (polymegathism) observed in older individuals (²²). Here, even with the age-associated decline of the corneal endothelium, its average reserve is usually enough to sufficiently maintain the critical barrier and pump function for a lifetime, without the need for clinical intervention (³³). However, in cases of accelerated or acute endothelial cell loss due to conditions such as genetically based endothelial dystrophy (^35–37), accidental or surgical trauma (³⁸), or from previous refractive or intraocular surgery (^{39, 40}), decompensation of the corneal endothelium may occur leading to its inability to efficiently pump fluid out of the stroma. The function of the endothelium becomes compromised once the CEC density falls below a critical threshold range of 500 to 1000 cells/mm² (^41–43). This results in stromal edema, corneal clouding, loss of visual acuity, and will eventually lead to corneal blindness. In such cases, the only option left to restore vision is to replace the ineffective endothelium with healthy, functional donor corneal endothelium through corneal transplantation (⁴²).

CORNEAL TRANSPLANTS IN HUMANS

The first successful full-thickness human corneal allograft was performed in 1905 by Dr. E. Zirm (reviewed in Ref. ⁴⁴). The high success rates observed in more than 90% of corneal transplant cases 1 year after surgery have been attributed to a certain degree of immune privilege of the avascular cornea, and the mechanisms behind ocular immune privilege have been thoroughly reviewed previously (^45–47). However, long-term corneal graft survival is not promising. The observed 5- and 10-year corneal graft survival estimates decrease to 74% and 64%, respectively (⁴⁸), comparable with the 5-year graft survival rates for cardiac, renal, and hepatic transplantation (⁴⁹). Recently, endothelial cell loss reported by the Cornea Donor Study from the United States demonstrated 70% attrition rate at 5 years after penetrating keratoplasty (PK) surgery (⁵⁰).

A GROWING DEMAND FOR CORNEAL TRANSPLANTATION

Population projections of developed nations indicate that the number of people aged 50 years and older has increased substantially during the past several decades (⁵¹), and the stress on healthcare systems worldwide will be unprecedented through the 21st century and beyond. This will most likely see a gradual increase in older individuals suffering from age-related cornea ailments and an increased demand for corneal grafts.

Cadaver corneal tissue for transplantation must meet stringent guidelines for acceptability that include the serological tests and medical history of the donor (^52–54). However, many potential donor corneas, often from elderly donors, are rejected for transplantation because of the lower endothelial cells count and possible age-related diseases. The availability of tissue is also affected by potential cultural, logistical, and technical difficulties (³), possibly rendering a transplant-grade donor graft material unsuitable because of high postmortem time or damage occurred during the handling of these fragile donor corneas (⁴²). These are some of the confounding problems that add to the global shortage of suitable transplant-grade cornea tissues. Even under the best conditions for transplantation, subsequent graft failure due to immunologic rejection, infection, or nonimmunologic endothelial decompensation may still occur (^{40, 55}) and will require a repeat graft. This is made worse by the fact that several studies have shown that repeat corneal transplantation have significant worse prognosis compared with primary procedures (^{56, 57}).

To cope with global shortage of donor cornea, the concept of using one donor cornea for the treatment of multiple patients has been realized with customized component corneal transplantation (⁵⁸). Here, a good-quality donor cornea can be divided into three parts using a microkeratome and a trephine to obtain (i) the peripheral corneoscleral rim with viable limbal stem cells for cadaveric limbal stem-cell transplantation; (ii) an anterior lamellar disc consisting of partial-thickness corneal stromal tissue for automated lamellar therapeutic keratoplasty; and (iii) a posterior lamellar disc consisting of corneal stroma and endothelium for posterior lamellar grafts (⁵⁸). Recent efforts have also promoted partial-thickness posterior lamellar grafts or endothelial keratoplasty (EK) to replace the more invasive PK for surgical intervention of endothelial-related corneal diseases (Fig. 2) (^{59, 60}). Furthermore, because of permanent structural changes to the stromal layer of the cornea after laser-assisted in situ keratomileusis procedure for myopia (^{61, 62}), potential donor corneas unsuitable for full-thickness corneal transplantation can theoretically still be harvested and used for these posterior lamellar grafts. To further alleviate the projected growing demand for transplant quality tissue, there is considerable clinical interest in the development of suitable alternatives for donor graft material through tissue engineering (³). Such advancement will require collaborative multidisciplinary research. This can only be realized by the expansion of primary HCECs in vitro and the development of an innovative system that enables the transplantation of functional tissue-engineered CECs by specialized surgical techniques such as EK.

FIGURE 2.: Montage of penetrating keratoplasty versus endothelial keratoplasty. (A) Sutured cornea after full-thickness penetrating keratoplasty. (B) Noninvasive endothelial keratoplasty 7 days post-Descemet stripping automated endothelial keratoplasty (DSAEK) showing attached donor disc and a clear cornea. (C) Optical coherence tomography 1 month post-DSAEK. (D) One year post-DSAEK showing a clear cornea with a cell count of 2145 endothelial cells (inset).

ISOLATION AND CELL CULTURE OF HCEC

It is generally agreed that the human corneal endothelium does not undergo any functional regeneration in vivo (^{19, 21, 24}). Various laboratories demonstrated the capacity for HCECs to proliferate in vitro. However, the procedures involved in the isolation and subsequent cultivation of these primary HCECs varied greatly between laboratories in their isolation protocols, extracellular matrices (ECM) used to aid cell attachment, and most importantly, the different culture media used in their culture and propagation.

The isolation and cultivation of primary HCECs have evolved over four decades from a gentler explant culture method, which enabled cellular migration and outgrowth of the endothelial cells (^{63, 64}) to a harsher scraping method, where the endothelial cells for culture were scrapped off the DM using a surgical blade (^{65, 66}). Currently, isolation of HCECs involves a two-step, peel-and-digest method whereby the DM-endothelial layer is peeled off from the donor cornea before being subjected to an enzymatic digestion using collagenase (⁶⁷), dispase (⁶⁸), or trypsin/EDTA (⁶⁹). However, this isolation technique may inevitably result in the co-isolation of contaminating stromal keratocytes (⁴²). As discussed earlier, the thickness of the DM can vary from one donor cornea to the another, and a thin or “sticky” DM (often found in cadaveric tissue from young donors) is difficult to peel, requiring extensive manipulation. In some cases, a thin layer of posterior stroma may be partially ripped off during the peeling process, remaining inconspicuously adherent to the DM-endothelial layer. Subsequent enzymatic treatment will release both the CECs from the DM and the undesired keratocytes trapped within the thin layer of stromal collagen. Because of the slow proliferative nature of HCECs, these contaminating stromal keratocytes will become fibroblastic and overtake the culture (Fig. 3). Engelmann et al. (⁷⁰) described a method to eliminate fibroblastic contamination using a selective l-valine-free medium, supplemented with d-valine. The substitution of l-valine by d-valine in culture suppresses fibroblast growth because fibroblasts lack the required d-amino acid oxidase needed for the conversion of d-valine to l-valine (⁷¹). However, elimination of l-valine from a culture medium can be cumbersome, especially with the use of complex supplements such as serum and bovine pituitary extract in some of the reported HCEC culture systems. Instead, refinement and improvement in the DM-endothelium peeling technique by incorporating the use of a vacuum suction cup to hold the corneal tissue in place (^{72, 73}) and improved training of the technician performing the isolation procedure will enable the isolation of a pure Descemet's layer with the attached single cell layer of endothelial cells.

FIGURE 3.: Cultivation of human corneal endothelial cells. (A) Contaminating stromal keratocytes/fibroblasts overgrowth within a culture of slow proliferative human corneal endothelial cells (scale bar=100 μm). (B) Human corneal endothelial cells derived from donor, aged 7 years; fourth passage, losing its unique hexagonal morphology, becoming elongated and fibroblastic-like (scale bar=50 μm). (C) Human corneal endothelial cells derived from donor aged 70 years; second passage, showing irregular morphology and grossly vacuolated suggestive of replicative senescence (scale bar=50 μm).

Other enhancements to the isolation and expansion of HCECs include the use of ECM-coated surfaces to encourage endothelial cell attachment. After the enzymatic dissociation of the DM-endothelial layer, isolated CECs have been plated on coated culture surfaces that include collagen (⁶⁷), ECM derived from bovine CECs (^{74, 75}), a mixture of laminin and chondroitin sulfate (⁷⁰), or a commercially available FNC Coating Mix (Athena Environmental Sciences Inc., Baltimore, MD) containing fibronectin, collagen, and albumin (⁷⁶). However, it has also been suggested that matrix coating may not be mandatory for the culture of HCECs (⁶⁹).

A wide range of complex serum-supplemented culture media developed from different basal media, together with the use of various growth factors have been shown to support HCEC growth and proliferation (Table 1), some with more success than others. Mitogenic factors such as basic fibroblast growth factor, epidermal growth factor, and nerve growth factor are added to enhance HCECs expansion (⁶⁹). However, the effect of basic fibroblast growth factor has been suggested to play a role as a differentiation factor and has not significantly improved the growth and expansion of the HCECs (⁴²). Still, many groups have found it challenging to establish consistent long-term culture of HCECs (^{30, 42, 69}). For example, established HCEC cultures tend to become heterogeneous, with cultured HCECs taking up a more fibroblastic morphology over each passage (⁴²). Taking into consideration donor-to-donor variability, established HCEC cultures from older donors proliferate not only slower but also to a lesser extent and display greater heterogeneity when compared with corneal endothelial cultures derived from younger donors (^{31, 75}). Furthermore, cellular aging leading to replicative senescence of the HCECs has been postulated, at least to a percentage of the endothelial cells that are morphologically larger, irregularly shaped, grossly vacuolated, and in some cases, multinucleated (^{30, 77}). However, in some cases, CECs cultured from older donors performed relatively well compared with CECs established from younger donors. This may be due to the factors pertaining to the preparation of the donor corneas and the relative health of the donor before death (^{76, 78}). In general, the outcome of a successful primary corneal endothelial culture will be affected by the length of time taken between death, enucleation, and preservation of donor corneas to the isolation and culture of CECs (⁷⁶).

TABLE 1: Supplemented media used in the culture of human corneal endothelial cells

A recent study has shown that the use of Y-27632, a synthetic compound that selectively inhibits the p160-Rho-associated coiled-coil kinase (ROCK) signaling pathway (⁸⁴), promoted the attachment and proliferation of CECs isolated from cynomolgus monkeys (⁸⁵). Although the underlying mechanism of the observation is unclear, it is plausible that ROCK inhibition of high-intensity ROCK activity occurring in cells under stressed conditions (including anoikis-induced cell death) may have prosurvival effects (^{86, 87}). Hence, the incorporation of such a kinase inhibitor in culture may improve the survivability of the CECs throughout the isolation procedure and passaging process, which in turn increases the initial adhering and starting several CECs for expansion.

The methodologies for the isolation and establishment of primary HCECs culture in vitro, together with the culture media that support its growth and proliferation, are constantly evolving and improving. It has been reported that HCEC aggregates isolated from the DM can be maintained in a suitable high-calcium, serum-free medium in a suspension culture as spheroids for up to 3 weeks (⁶⁷). However, long-term cultivation and expansion of HCECs require a critical component: serum. The addition of serum, although essential, introduces a complex protein mixture of unknown composition, at undefined concentration, and exhibits vast variability of biologically active compounds between batches (⁸⁸). Such inconsistency complicates and hinders characterization studies aimed at determining critical factors required for the growth and expansion of HCECs. From a therapeutic standpoint, it is also important that HCECs are propagated under xeno-free conditions (i.e., free of components derived from animal sources) so as to eliminate the potential risk of animal pathogen transmission into the human population. Recently, Ludwig et al. (⁸⁹) developed a fully defined, animal product-free culture medium for the derivation, maintenance, expansion, and differentiation of human embryonic stem cells. Perhaps a similar approach to develop a fully defined culture medium able to support the growth and expansion of HCECs, as well as to preserve its unique function, is now a feasible proposition.

PROSPECTS FOR CORNEAL ENDOTHELIAL TRANSPLANTATION USING CULTIVATED HCEC

The idea of using cultured HCECs as an alternative to full-thickness keratoplasty in the replacement of defective corneal endothelial layer was conceptualized over 3 decades ago (^{90, 91}). Potentially, the use of such tissue-engineered corneal endothelium substitutes can possibly alleviate the shortage of donor human corneal tissue faced globally. This is especially so for the treatment of bullous keratopathy; a notion now made possible with the establishment of EK techniques (see below). Hence, the ability to cultivate HCECs in vitro with relative consistency will stimulate further research into the development of a suitable delivery system in the form of a synthetic or biological carrier.

Development of full-thickness collagen-based synthetic carriers as corneal substitutes was initiated more than 10 years ago (^{92, 93}). Recent advancement involves the use of collagen including its vitrification and rehydration to form a thin transparent collagen vitrigel membrane (⁹⁴). Some studies have examined synthetic scaffolds made from fibrin– agarose (⁹⁵) and collagen–chondroitin sulfate foams (⁹⁶) as potential carriers. However, most of these reports have not been functionally validated in an in vivo model until recently. Koizumi et al. (^{97, 98}) transplanted primate CECs grown on vitrified collagen type 1 carrier into the anterior chamber of the primate's eye with its corneal endothelium mechanically scraped off and postulated that the cultivated primate CECs migrated from the collagen membrane onto the host DM, restoring corneal clarity. A further extension to this report could see the replication of this research using cultured primary HCECs. Ultimately, for synthetic carriers to be usable in any form of EK, it is crucial that its stability and efficacy can be robustly tested independently.

Thin biological carriers such as human amniotic membrane (^{68, 99}) and biodegradable gelatinous membranes (^{100, 101}) have been considered as potential replacements. However, human amniotic membrane is not fully transparent and its quality is known to be inconsistent, rending it clinically irrelevant in this context. Studies have shown that the “lifting” of confluent HCECs cultured on a thermoresponsive polymeric culture surface as an intact HCEC sheet without the use of enzymatic dissociation is possible (^{102, 103}). Although the intact gelatinous membrane- supported HCEC sheets were shown to be functional in a rabbit model (^{100, 101, 103}), such thin and fragile cell sheets are difficult to handle, and the fine manipulation required may easily result in irreversible damages to the delicate HCEC sheet. If a sturdier biodegradable alternative, such as a fibrin-based matrix, can be incorporated into these studies, preclinical xenotransplantation of HCEC sheet into a primate model will be the next step. Alternatively, instead of thin biological carriers, other researchers have taken the approach of using endothelium-denuded corneal buttons as a carrier (^104–106). The advantage of using such a carrier is that both normal corneal shape and corneal transparency are maintained. Here, cultivated HCECs can be dissociated enzymatically to form a cell suspension and seeded at a desired cell density onto the denuded DM (¹⁰⁷) directly onto the stromal layer (^{83, 108}). Tissue-engineered feline corneal endothelium using cultured feline CECs seeded onto devitalized cadaver human stromal cornea achieved a cell density of 2272±344 cells/mm² and expressed characteristic function-related markers such as Na⁺K⁺-ATPase and ZO-1 (¹⁰⁵). A subsequent report showed functional success when the tissue-engineered feline corneal endothelium, reconstructed on a devitalized human stromal carrier, was transplanted into a feline model (¹⁰⁴). Elsewhere, Honda et al. (¹⁰⁶), using cultured HCECs seeded onto fresh human corneal stromal discs, showed promising results when HCECs-populated stromal disc was transplanted into a rabbit model. Although promising, further refinement of using human corneal stroma as a carrier in achieving a clinically relevant endothelial cell density for transplantation and the mode of delivery and long-term functional assessment as to how the transplanted endothelial cell density may decrease over time is required.

CORNEAL TRANSPLANTATION—POSTERIOR LAMELLAR KERATOPLASTY

Corneal transplantation has advanced rapidly during the last decade with a shift in paradigm from full-thickness PK procedures to partial-thickness posterior lamellar graft or EK, specifically for corneal endothelium-related ailments (⁶⁰). Such partial-thickness transplantation replaces the necessary affected tissue instead of the entire cornea and has clear advantages over PK surgery in recovery of visual acuity and tectonic strength (^{59, 109–112}). Surgical procedures for posterior lamellar graft have evolved from deep lamellar endothelial keratoplasty (¹⁰⁹) to Descemet stripping endothelial keratoplasty (^{59, 112}) or Descemet stripping automated endothelial keratoplasty (¹¹⁰) to Descemet membrane endothelial keratoplasty (¹¹¹). To date, Descemet stripping endothelial keratoplasty and Descemet stripping automated endothelial keratoplasty are the most commonly applied posterior corneal lamellar procedures that essentially replace the recipient's diseased endothelium and DM with a donor endothelium/DM/thin strip of posterior stroma complex. The latter produces a donor lenticule (EK graft) of between 50 and 250 μm depending on the donor harvesting technique used. EK procedures offer better outcomes in term of faster visual rehabilitation with lower risk of intraoperative complications compared with PK surgeries (⁵⁹). However, issues such as postoperative endothelial cell attrition (^{113, 114}) and high primary graft failure rates remain (¹¹⁵). These have somewhat been reduced with the development of new insertion devices for EK grafts, which minimized intraoperative cell damage during donor graft insertion and decreased rates of postoperative endothelial cell loss (¹¹⁶). These insertion devices also offer the potential as instruments that can be used to deliver the cultivated, tissue-engineered HCECs into the anterior chamber of the eye for grafting.

A FEASIBLE FUTURE FOR TISSUE-ENGINEERED CORNEAL ENDOTHELIAL TRANSPLANTATION?

It is well established that HCECs retain their capacity to proliferate in vitro (^{30, 64, 97, 104}), and their growth dynamics are now better understood (^{29, 42, 76}). Although primary HCECs cannot be propagated indefinitely, in the context of tissue-engineered corneal endothelial grafts, the use of nontransformed cells is advantageous over the use of transfected or immortalized HCEC-lines in the uncontrollable CEC growth observed (⁴²). Continual advances and improvements in HCECs culture system may in the future enable the culture and expansion of the patient's own CECs from tissue taken from the periphery corneal endothelium, which was reported to be more proliferative compared with cells of the central corneal endothelium (^{117, 118}). Transplantation of tissue-engineered cornea endothelial derived from the patient's own endothelium may also improve the long-term outcome of corneal graft survival.

As outlined above, promising proof of concept studies have shown that primary HCECs cultivated on various carrier systems retain their functionality within various animal models (^{98, 103, 106}). However, it is necessary to identify a suitable carrier that is also clinically feasible and to establish a robust protocol to validate the selected carrier system within an in vivo primate model. Furthermore, development of a defined serum-free, xeno-free culture system for the expansion of HCECs may also be required for future clinical trials.

To conclude, innovative breakthroughs in technology and advancement in pharmaceuticals will continually improve surgical procedures and their outcomes. Prospective novel treatment strategy for EK procedure using HCECs cultured in vitro is now plausible and research thus far has been promising. Indeed, this holds great potential in advancing the field of human corneal endothelial transplantation toward a new frontier, bringing cultivated HCECs from bench to bedside.