Introduction: The purpose of this work was to evaluate the in vitro growth capacity and functionality of human corneal endothelial cells (hCEC) expanded from corneas of elderly (>60 years) donors that were preserved using an organotypic culture method (>15 days, 31°C) and did not meet the clinical criteria for keratoplasty. Methods: Cell cultures were obtained from prior descemetorhexis (≥10 mm) and a controlled incubation with collagenase type I followed by recombinant trypsin. Cells were seeded on coated plates (fibronectin-albumin-collagen I) and cultures were expanded using the dual supplemented medium approach (maintenance medium and growth medium), in the presence of a 10 μ<sc>m</sc> Rho-associated protein kinase inhibitor (Y-27632). Cell passages were obtained at culture confluency (∼2 weeks). A quantitative colorimetric WST-1 cell growth assay was performed at different time points of the culture. Morphometric analysis (area assessment and circularity), immunocytochemistry (ZO-1, Na+/K+-ATPase α, Ki67), and transendothelial electrical resistance (TEER) were performed on confluent monolayers. Results: There was no difference between the cell growth profiles of hCEC cultures obtained from corneas older than 60 years, whether preserved cold or cultivated organotypic corneas. Primary cultures were able to maintain a certain cell circularity index (around 0.8) and morphology (hexagonal) similar to corneal endothelial mosaic. The ZO-1 and Na+/K+-ATPase pump markers were highly positive in confluent cell monolayers at 21 days after isolation (passage 0; P0), but significantly decreased in confluent monolayers after the first passage (P1). A weak expression of Ki67 was observed in both P0 and P1 monolayers. The P0 monolayers showed a progressive increase in TEER values between days 6 and 11 and remained stable until day 18 of culture, indicating a state of controlled permeability in monolayers. The P1 monolayers also showed some functional ability but with decreased TEER values compared to monolayers at P0. Conclusions: Our results indicate that it is possible to obtain functional hCEC cultures in eye banks, using simplified and standardized protocols, from older donor corneas (>60 years of age), previously preserved under organotypic culture conditions. This tissue is more readily available in our setting, due to the profile of the donor population or due to the low endothelial count (<2,000 cells/mm2) of the donated cornea.

Diseases affecting the corneal endothelium are responsible for up to 64% of corneal transplantation (keratoplasty) indications [1, 2]. Endothelial failure is generally due to mechanisms that compromise the density and physiology of human corneal endothelial cells (hCEC) [3, 4]. Clinical situations related to corneal endothelial dysfunction (CED), congenital (Fuchs endothelial dystrophy), or acquired (bullous keratopathy), lead to significant visual impairment due to progressive corneal loss of transparency secondary to corneal edema, and ultimately blindness [3, 4].

The corneal endothelium is characterized by its metabolic refinement. It forms a highly differentiated cell monolayer responsible for corneal tissue homeostasis through an active Na+/K+-ATPase pump that maintains both stromal deturgescence and transparency [5, 6]. Although the endothelium is essential to ensure proper corneal physiology, several factors limit the proliferative capacity of hCEC and its poor ability to regenerate in vivo [5‒7]. Previous research indicates that the monolayer of the corneal endothelium originates from poorly differentiated mesenchymal cells, derived from the neural crest and located at the periphery of the cornea, through a process of proliferation, differentiation, and centripetal migration [8]. Furthermore, the proliferative capacity of corneal endothelium terminates after the formation of intercellular bridges that incite a contact inhibition mechanism which is maintained throughout adult life [5, 8]. Moreover, there is a high concentration of TGF-β in the aqueous humor [5, 7], contributing to an increase in the expression of p27Kip1 (a cyclin-dependent kinase inhibitor) that prevents the transition to the S phase and maintains the cell population arrested in the G1 phase of the cell cycle [5‒7]. Meanwhile, hCEC in ex vivo corneas or under in vitro cell culture conditions might recover a certain proliferative capacity and enter the cell cycle again after the release of intercellular contacts in the presence of mitogens [5, 9‒11].

Although the ability to divide hCEC can be recovered to some extent, this relative proliferative ability is greatly affected by the donor’s age. Semiquantitative analysis revealed a significant decrease in the rate of cell cycle entry and the relative number of dividing hCEC in corneas from older donors (>50 years) [5, 6]. Furthermore, the mean in vitro doubling time for hCEC of these corneas was approximately twice that of cultured cells from younger ones (<30 years) [5, 12, 13]. Considering the intrinsic nature of hCEC, as discussed above, the dynamics of corneal donation pose a major obstacle to the purposeful implementation of cell therapy for CED since most of the corneas available for transplant come from elderly donors.

The current mainstay treatment for corneal opacities due to endothelial dysfunction is selective endothelial keratoplasty (EK) [3, 4, 14‒16]. Although evolution and standardization in keratoplasty techniques have improved postoperative management and results [17‒19], corneal supply remains the most limiting factor in the fight against corneal blindness [1, 2]. In a worldwide context, demand dramatically surpasses the availability of donated corneas for clinical applications: a single cornea is available for every 70 patients in need of keratoplasty [1]. Regenerative medicine approaches based on advanced cell therapy and tissue bioengineering emerge as rational and sustainable candidates to combat blindness secondary to CED [20‒25]. Furthermore, despite their poor proliferative properties in vivo and in vitro, growing evidence shows that the expansion of hCEC can be improved for translational purposes [22‒25]. The purpose of this study was to evaluate the growth capacity and in vitro functionality of hCEC expanded from corneas of elderly donors (>60 years) that were preserved in an organotypic culture method (>15 days, 31°C) and did not meet the criteria for keratoplasty indication.

Donor Human Corneoscleral Tissues

The local Ethics Committee approved the present study (HCB/2016/0910, Hospital Clinic, Barcelona, Barcelona, Spain), which followed the principles and ethical principles of the Declaration of Helsinki (2013; Fortaleza, Brazil). A total of 57 corneas (26 cold preserved and 31 organotypic cultivated) not suitable for clinical application were obtained from the Barcelona Tissue Bank (BTB – Banc de Sang i Teixits, Barcelona, Spain). All the information for the donation and their respective informed consents were obtained with the authorization and acceptance of the use of the samples for the corresponding purposes, according to local and EEC regulations. In addition, an informed written consent was obtained as per the BTB standard operational procedure for eye donation destined for research. The corneas were preserved in Eusol-C medium (Alchimia; Ponte San Nicolo, Italy) at 4°C, for a maximum period of 7 days (cold preserved corneas), and then in CorneaMax medium (Eurobio Scientific, Les Ulis, France) at 31°C for a maximum period of 28 days (organotypic cultivated corneas).

Isolation and Culture of hCECs

The isolation and culture of hCEC were carried out according to previously published protocols [22, 26], with some minor modifications. First, the corneoscleral rims were placed endothelium side up on a 10 mm disposable cornea punch (Janach, E. Janach SRL, Como, Italy) and mildly stabilized by vacuum suction. Next, a brief treatment (<1 min) with 0.25% Trypan Blue solution delineated the delimitation line. After the peripheral choroidal and endothelium remnants were removed, the Descemet’s membrane (DM) endothelial cell (EC) layer was carefully stripped off (descemetorhexis) under a dissecting microscope (Leica, Leica Biosystem, Wetzlar, Germany). In sequence, the DM-EC monolayer was washed three times with phosphate buffer saline (PBS) with 1% antibiotic/antimycotic solution and maintained for 24 h in a maintenance medium (as described below) at 37°C (humidified incubator, 95% O2, 5% CO2). Subsequently, the tissue was incubated with 1 mg/mL collagenase type I (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) for 1–3 h at 37°C in a humidified incubator (95% O2, 5% CO2). At this point, corneal ECs were entirely displaced from the DM in densely packed clusters. Subsequently, PBS washing was performed and hCECs were incubated for 5 min with TrypLE Select (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C and centrifuged at 400 g for 5 min. The supernatant was then removed, and the cell pellet was resuspended in the maintenance medium (S medium; M5), which consisted of human endothelial SFM (Gibco, USA), supplemented with 5% fetal bovine serum (FBS; Biowest, Nuaillé, France), 10 μm Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Sigma Aldrich, Germany) and 1% antibiotic/antimycotic solution (Sigma Aldrich, Germany). The hCEC resulting from the isolation of two paired corneas was then plated in one well (3.5 cm2) of a 12 multi-well plate, previously coated with FNC coating mix (AthenaES, Baltimore, MD, USA).

In vitro Expansion of hCECs

In vitro expansion of hCEC was carried out in two steps, using a dual media approach with a maintenance cell culture medium (S medium; M5) in combination with a growth cell culture medium (P medium; M4) [22]. The latter consisted of Ham’s F12/M199 medium (Gibco, USA), supplemented with 5% FBS, 1 mg/mL insulin, 0.55 mg/mL transferrin, and 0.5 µg/mL sodium selenite (ITS) (Sigma Aldrich, Germany), 0.2 mm ascorbic-2-phosphate (Dako; Agilent Technologies, Santa Clara, CA, USA), 10 ng/mL human basic fibroblast growth factor (Sigma Aldrich, Germany), 10 μm ROCK inhibitor Y-27632, and 1% antibiotic/antimycotic solution. Cells were first left to adhere overnight to the surface coated with FNC Coating Mix (AthenaES) in M5 medium, which was replaced by M4 medium to induce cell proliferation. Upon culture confluency (∼2 weeks), M4 medium was replaced by fresh M5 medium (∼1 week). Cell culture mediums were changed every 2 days. Passages were carried out with a plating cell density of 1 × 104 cells/cm2, as previously recommended [27], after dissociation of TrypLE Select (Gibco, USA).

Cell Growth Assay

The hCEC growth was measured using the quantitative colorimetric WST-1 assay (Abcam, Cambridge, UK). Briefly, hCEC from preserved and cultivated corneas were seeded in 12 multi-well plates. At different times during the culture period (days 1, 7, 14, and 21), the assay was carried out by adding 10% (vol:vol) WST-1 cell proliferation reagent to cell culture medium, following 3-h incubation at 37°C, and the absorbance was measured at 450 nm. The reading results of 3 replicates were expressed as mean with standard deviation (MD ± SD), as optical density (OD) of the blank well (without cells) subtracted from the OD of the wells containing hCEC. This experiment compared organotypic corneas with cold-preserved corneas which also did not meet the criteria for clinical use.

Morphometric Analysis, Area Assessment, and Circularity

Cell morphology during in vitro expansion was evaluated at different time points, with an inverted microscope (phase contrast or light field) (Leica DM IL LED; Leica, Germany) and a digital camera (Leica DFC 450C; Leica, Germany). Cell area and perimeter data were obtained after random selection of cells from phase contrast images, at the end of each cell passage in confluence, between 21 and 28 days of culture. The cell boundaries were defined using ImageJ software (https://imagej.nih.gov/ij/; National Institutes of Health, Bethesda, MD), and the circularity was determined using the following formula: circularity = 4π (area/perimeter2) (index range between 0 and 1.0) [27]. Therefore, hexagonal hCEC will have a profile closer to 1.0, while elongated fibroblast-like hCEC will have a circularity value closer to zero [27]. The circularity reference value for this study was based on previous studies of specular microscopy in normal hCEC, which is approximately 0.82 ± 0.03 [22]. Fields (n = 5) containing at least 100 cells were analyzed in 3 independent culture isolations.

Immunocytochemistry

The confluent cell monolayers of the primary culture (P0) and passage 1 (P1) cultured in 48-well plates were fixed with 4% paraformaldehyde solution or with cold absolute methanol for 10 min and rinsed three times with PBS. Subsequently, cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 min and blocking was performed with 10% goat serum for 30 min at room temperature. The following primary antibodies were used: mouse anti-ZO-1 antibody (5 µg/mL; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-Na+/K+-ATPase α antibody (5 µg/mL; Merck, Darmstadt, Germany) and rabbit anti-Ki67 (1 µg/mL; Abcam, Cambridge, UK). Primary antibodies were diluted with a blocking buffer solution and incubated at room temperature for 1 h. After three washes in PBS, cells were incubated with secondary antibodies, goat anti-mouse Alexa Fluor 488 (1 µg/mL; Invitrogen, USA), and goat anti-rabbit Alexa Fluor 568 (1 µg/mL; Invitrogen, USA) for 1 h at room temperature and protected from the light. Finally, the cells were rinsed three times with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, Germany) for 5 min. The images were taken under a fluorescence microscope (Leica DM IL LED) using a digital camera (Leica DFC 450C).

Transendothelial Electrical Resistance

Cultured cells were seeded into 12 mm (1.1 cm2) transwells (0.4 μm pore; Costar, Corning Inc., Merck KGaA, Darmstadt, Germany), previously coated with FNC Coating Mix (AthenaES) at 5 × 103 cells/cm2 density. At different time points during the culture period (21–26 days), transendothelial electrical resistance (TEER) was measured using the Millicell-ERS (Electrical Resistance System; MERS00002, Millipore, Merck KGaA, Darmstadt, Germany). The TEER values were normalized with the control wells, consisting of transwells without cells filled with M5 (maintenance medium). Three replicates were used to obtain the final values for each point. The results were presented as MD ± SD in Ω/cm2.

Statistical Analysis

Numeric data were expressed as MD ± SD or median and interquartile range. Comparisons between groups were evaluated using independent paired Student’s t tests. Results with a p value <0.05 were considered significant.

Isolation and Growth of Cultures

Thirty-one (n = 31) corneas preserved in organotypic culture (cultivated; mean of 17 ± 3 days at 31°C) were processed. The average donor age was 67 ± 3.9 years of age (43–85) and the samples were divided into three age groups: (i) 40 to 50 yo (n = 4; 13%); (ii) 50 to 60 yo (n = 4; 13%); and (iii) >60 yo (n = 23; 74%). The protocol, with some methodological modifications adapted by our group, is shown in Figure 1a and b. Furthermore, 26 corneas (n = 26) preserved at low temperature (preserved; mean of 4 ± 1.5 days at 4°C) with a mean age of 69 ± 6.1 years of age (47–85) were processed and divided into two age groups: (i) 40 to 50 yo (n = 4; 15%) and (ii) >60 yo (n = 22; 85%). Cold-preserved corneas were only used for comparative viability and culture cell growth assay. Cultivated and preserved corneas did not meet the criteria for clinical use. The main causes of exclusion were corneal stromal opacities and low EC count, ranging from 1,000 to 2,000 cells/mm2.

Fig. 1.

Isolation and in vitro attachment and growth of hCEC. a Schematic diagram showing the different phases of the in vitro culture of hCEC after enzymatic isolation from the Descemet’s Membrane (DM)-endothelial layer (EC) layer (DM-EC). b Phase contrast images showing the hCEC still attached to the DM (top) after descemetorhexis, released clusters from the DM (middle) after controlled collagenase digestion, and into monolayer in vitro cultures within the first 48 h (bottom left), after trypsin separation, and after 3–4 weeks of culture confluence (bottom right). c Viability and cell growth of in vitro hCEC cultures assessed by WST-1 assay for cultures obtained from preserved (4°C) and cultivated (31°C) corneas older than 60 years (upper) and cultivated corneas from donors younger than 60 years of age (lower). Data are expressed as MD ± SD from 3 independent experiments (***p < 0.001 compared to day 1). Scale bar, 100 µm. ON, overnight; S medium, maintenance cell culture medium (M5); P medium, growth cell culture medium (M4).

Fig. 1.

Isolation and in vitro attachment and growth of hCEC. a Schematic diagram showing the different phases of the in vitro culture of hCEC after enzymatic isolation from the Descemet’s Membrane (DM)-endothelial layer (EC) layer (DM-EC). b Phase contrast images showing the hCEC still attached to the DM (top) after descemetorhexis, released clusters from the DM (middle) after controlled collagenase digestion, and into monolayer in vitro cultures within the first 48 h (bottom left), after trypsin separation, and after 3–4 weeks of culture confluence (bottom right). c Viability and cell growth of in vitro hCEC cultures assessed by WST-1 assay for cultures obtained from preserved (4°C) and cultivated (31°C) corneas older than 60 years (upper) and cultivated corneas from donors younger than 60 years of age (lower). Data are expressed as MD ± SD from 3 independent experiments (***p < 0.001 compared to day 1). Scale bar, 100 µm. ON, overnight; S medium, maintenance cell culture medium (M5); P medium, growth cell culture medium (M4).

Close modal

Cellular viability and growth studies demonstrated the growth pattern of the cultures for 21 days (Fig. 1c). There was no difference between the growth profile of cell cultures obtained for preserved (low temperature 4°C) or cultivated (31°C) corneas with donors older than 60 years (Fig. 1c, upper). As expected, cell growth in the cultures was observed until day 14 (growth medium; P medium, M4), when the cultures reached confluency. Culture stability was observed between days 15 and 21 (maintenance medium; S medium, M5). Interestingly, we observed similar viability and cell growth profiles for cultures obtained from corneas that were preserved from donors under 60 years of age (Fig. 1c, bottom). After the proliferation phase of cultures, the change to the maintenance medium M5 allowed the hCEC of all groups to reestablish the hexagonal phenotype of the mosaic cell monolayer (Fig. 1b).

Cellular Morphometry and Circularity

To analyze the cell polymorphism in the cultures obtained, we studied the cell area (Fig. 2; Table 1) according to each group. Since hCECs exhibit a regular mosaic appearance in vivo, cell morphology was assessed by their regularity [28]. In cultures obtained from the younger group (40–50 years of age), in isolation (passage 0; P0), the average value of the number of cells per area remained below the reference value (2,000 cells/mm2), but a trend toward a more homogeneous distribution was observed (Fig. 2a, b top), indicating a certain regularity between cell areas. However, 21 days after cultures (confluence) in the first (P1) and second (P2) passages, the cell monolayers did not present this regular distribution, indicating area heterogeneity (Fig. 2a, b top). In cultures obtained from the intermediate group (between 50 and 60 years of age), regular distribution was only maintained at P0 (Fig. 2a). On the contrary, the distribution of regularity was highly variable in isolation (P0) in the cells obtained from organotypic cultures (Fig. 2a, b below) and also in those cold-preserved (not shown) from the group of older donors (>60 years). Cell areas increased in each passage (Fig. 2a).

Fig. 2.

Cellular morphometric and circularity analysis of cultured hCEC along the passages. a Scatter plots showing cellular area according to the donor age (40 to 50, >50 to 60, and >60 years of age). The variability of the cell area is shown throughout the isolation (P0), first (P1), second (P2), and third (P3) passages (each point of the graph corresponds to the area of a cell). Five fields of each cell monolayer at confluency (day 21 in culture) obtained from 3 donors per age group were analyzed to obtain the median. Data are expressed as median and interquartile ranges. b Phase contrast images of cultures over the passages from a 43-year-old donor (upper) and a 65-year-old donor (lower). c Cellular circularity analysis of expanded hCEC at P0 and P1 according to the donors age (40 to 50, 50 to 60, and >60 years of age). Data are expressed as MD ± SD. **p < 0.01; ***p < 0.001. Scale bar, 100 µm.

Fig. 2.

Cellular morphometric and circularity analysis of cultured hCEC along the passages. a Scatter plots showing cellular area according to the donor age (40 to 50, >50 to 60, and >60 years of age). The variability of the cell area is shown throughout the isolation (P0), first (P1), second (P2), and third (P3) passages (each point of the graph corresponds to the area of a cell). Five fields of each cell monolayer at confluency (day 21 in culture) obtained from 3 donors per age group were analyzed to obtain the median. Data are expressed as median and interquartile ranges. b Phase contrast images of cultures over the passages from a 43-year-old donor (upper) and a 65-year-old donor (lower). c Cellular circularity analysis of expanded hCEC at P0 and P1 according to the donors age (40 to 50, 50 to 60, and >60 years of age). Data are expressed as MD ± SD. **p < 0.01; ***p < 0.001. Scale bar, 100 µm.

Close modal
Table 1.

Area and circularity of hCECs obtained from cultured corneas and expanded in culture

PassageCell area±SD, µm2Cell circularity±SD
 40–50 years of age  
P0 977±711 0.81±0.01 
P1 2,920±2,534 0.82±0.02 
P2 3,780±3,212 0.78±0.04 
 >50–60 years of age 
P0 1,728±906 0,83±0.02 
P1 6,681±3,995 0.77±0.02 
 >60 years of age 
P0 2,443±1,767 0.80±0.04 
P1 3,600±3,180 0.53±0.14 
PassageCell area±SD, µm2Cell circularity±SD
 40–50 years of age  
P0 977±711 0.81±0.01 
P1 2,920±2,534 0.82±0.02 
P2 3,780±3,212 0.78±0.04 
 >50–60 years of age 
P0 1,728±906 0,83±0.02 
P1 6,681±3,995 0.77±0.02 
 >60 years of age 
P0 2,443±1,767 0.80±0.04 
P1 3,600±3,180 0.53±0.14 

P0, cell isolation; P1, first passage; P2, second passage.

Mean±standard deviation (SD).

Cell circularity indices remained close to 1.0 for all cell populations in cultures after isolation (P0), and this was maintained in all age groups (Fig. 2c; Table 1). This finding indicates that the hCEC from primary cultures was able to maintain a certain characteristic cell morphology resembling the mosaic of the in vivo corneal endothelium (hexagonal/polygonal), regardless of donor age. This morphological regularity was also observed in the cell populations of the cultures in P1, obtained from donor corneas over 60 years of age (Fig. 2c). However, the cell populations in P1 cultures from these donors presented a low circularity index, around 0.5 (Table 1). This fact is most likely due to the poor capacity for cell growth in vitro, the lack of a confluence of cell populations in culture, and the consequent acquisition of a fibroblastic phenotype (Fig. 2b top, passage 3 and bottom, EDGE, passage 1).

Characterization and Functionality

To verify the characteristics of the cultures obtained, we evaluated the expression of conventional hCEC surface markers for zonula occludens (ZO-1) and the Na+/K+-ATPase pump using immunocytochemical techniques (Fig. 3). In P0 cultures at 21 days, a highly positive cell monolayer was observed for both markers. The positive ZO-1 signal demonstrated the formation of intercellular adhesions, which confirmed the ability of cultured hCEC to maintain the morphology of the canonical polygonal mosaic. Similarly, the presence of ATPase suggested the maintenance of certain functional activity (Fig. 3a). These characteristics are drastically lost in cell monolayers in P1 cultures (Fig. 3a) and are even undetectable in several more peripheral areas of the cultures (not shown). On the other hand, the proliferative state of hCEC cultures was identified by the proliferation nuclear marker Ki67 (Fig. 3b). Weak expression was observed in monolayer P0 and P1, confirming the low proliferative capacity of expanded hCEC in vitro.

Fig. 3.

Characterization and functionality of hCEC monolayers. a Immunostaining for corneal endothelial markers such as zonula occludens (ZO-1) and sodium-potassium ATPase (Na+/K+-ATPase). b Immunostaining of cultured hCECs for nuclear proliferation marker Ki67. c Transendothelial electrical resistance (TEER) measurements in hCEC polarized confluent monolayers at isolation (P0) and the first passage (P1). Cultures obtained from corneas older than 60-year-old donors. *p < 0.05; **p < 0.01; ***p < 0.001 compared to day 1. Scale bar, 100 µm.

Fig. 3.

Characterization and functionality of hCEC monolayers. a Immunostaining for corneal endothelial markers such as zonula occludens (ZO-1) and sodium-potassium ATPase (Na+/K+-ATPase). b Immunostaining of cultured hCECs for nuclear proliferation marker Ki67. c Transendothelial electrical resistance (TEER) measurements in hCEC polarized confluent monolayers at isolation (P0) and the first passage (P1). Cultures obtained from corneas older than 60-year-old donors. *p < 0.05; **p < 0.01; ***p < 0.001 compared to day 1. Scale bar, 100 µm.

Close modal

Finally, the in vitro functionality of hCEC monolayers was evaluated by TEER measurements of confluent cultures that were seeded onto permeable filter systems (transwell). Monolayers at P0 showed a progressive increase in TEER values until approximately day 11 of culture. Interestingly, a more noticeable increase in TEER values was observed between days 6 and 11 of culture (Fig. 3c). Subsequently, the TEER values were stable up to day 18 of culture, indicating a state of controlled permeability in the monolayer. TEER values decreased gradually from day 18 to day 21 of culture (Fig. 3c) until the growth medium (P medium, M4) was exchanged for the maintenance medium (S medium, M5). From this point on, TEER values recovered progressively to near-peak values, although at a slower pace. Cell monolayers at P1 also showed some functional capacity but with decreased TEER values compared to the monolayers at P0 (Fig. 3c). However, once the TEER values peaked, contrary to what we observed at P0, the passaged hCEC could not maintain the barrier function, with a progressive reduction of TEER values between days 11 and 18 of culture. These results indicate that the hCECs of older donors partially lose their functional capacity once the first passage (P1) is established.

CED accounts for most transplant indications in the developed world, between 25% and 64%, with EK still being the technique of choice for the treatment [29, 30]. Although EK is quite effective in reversing corneal blindness [19, 31‒33], the availability of donor corneas addresses only 1.5% of the population in need of surgery [1]. In this context, it is only natural to pursue alternatives to EK that can alleviate the pressure on the current system of cornea donation.

Recently, expanded hCEC in vitro has been successfully used in a relatively small number of patients (n = 11) to treat different causes of ED with satisfactory mid-term survival and low rate of complications [34]. This approach has several potential advantages, as follows: (i) its general application for a variety of CED causes; (ii) certified control of culture conditions and cell functionality due to strict compliance and monitoring of local regulatory GMP rules; and (iii) most importantly, cell therapy with cultured hCEC will possibly enable treatment of multiple patients with cells recovered from a single donated cornea. However, methodological challenges, logistical hurdles, financial cost and infrastructure optimization, and local regulatory restrictions must all be considered.

In the present study, we successfully established hCEC cultures from donor corneas from elderly patients, which had been maintained under organotypic culture conditions (31°C) and did not meet the quality criteria for clinical use. We succeeded in expanding cultures from donors over 60 years of age, whose EC density (ECD) was less than 2,000 cells/mm2. Between 30% and 60% of these low ECD corneas were considered unsuitable for clinical use [35], especially for EK, and were eventually discarded [36]. Our results have also allowed us to demonstrate that the viability and cell growth profiles, as well as the evidence of functionality and maintenance of morphological profiles, are similar between the cultures obtained from corneas preserved for a short period of time at low temperature and those preserved under organotypic conditions. Likewise, the viability and growth profiles of the cultures were similar between the donor groups, according to the age groups considered.

Other studies have also been able to establish viable hCEC cultures from elderly donors, both in cornea preserved at low temperature and in organotypic culture conditions [10, 37]. The latter can maintain corneal tissue in optimal conditions for up to 3 or 4 weeks, offering considerable logistical advantages with respect to cold preservation methods. Additionally, organotypic preservation media are substantially enriched with nutrients whose composition contributes to better maintenance of endothelial metabolic conditions. The reduction of metabolic stress on the corneal endothelium and the maintenance of better cell viability would be the main advantages of obtaining cultures from corneal tissue previously preserved under organotypic conditions [10]. Although comparative analysis is beyond the scope of this manuscript, our results corroborate the previous ones. It was supported by the conservation of the morphometric evaluation of the cultured cells, as well as the verification of the sustained increases in TEER experiments where we have been able to induce a certain cell polarity and barrier function of the monolayers. However, these features have only been verified in initial passages: in isolation (passage 0; P0), first (P1), and exceptionally in the second passage (P2).

A crucial difference in our strategy was that all procedures for obtaining hCEC cultures were carried out entirely in our eye bank facilities. Both temperature changes in corneal storage between transport and surgery and a longer death-to-preservation time have significantly reduced the viability of hCEC after EK [30‒33]. To our knowledge, this is the first time that hCEC culture was performed, circumventing the need for transportation to specialized laboratories, which may contribute to optimized conditions for expanding hCEC from elderly corneas.

Like the “peel-and-digest” technique, the first step of our hCEC expansion protocol consisted of harvesting cells with a controlled enzymatic dissociation. However, a substantial modification presented here was the removal of a single DM-EC layer (diameter ≥10 mm) instead of the several small explants commonly used [10, 22, 27]. This minor difference may be the key to avoiding additional damage to hCEC due to excessive manipulation. We also optimized the procedure by introducing an initial incubation step under culture conditions in which the DM-EC layer was kept intact after descemetorhexis in a maintenance culture medium, prior to enzymatic digestion in two steps (collagenase/recombinant trypsin). The strong adhesion between the DM-EC monolayer and the extracellular matrix of the deep lamellae of the corneal stroma, which is more evident in the corneal tissue of young donors, offers less resistance to peeling in the corneas of older donors [38]. Furthermore, we observed that the peeling action to obtain a large DM-EC monolayer seemed to be facilitated by the slight stromal edema observed in corneas preserved with the organotypic culture method. After isolation, we conducted in vitro expansion of hCEC through a two-phase culture system according to the protocol described above [22]. At P0, it was possible to establish a monolayer of cultured hCEC that retained the canonical phenotypical traits of a healthy endothelium in all age groups. Furthermore, the circularity of the cells remained close to 0.80, reflecting the retention of the typical hexagonal shape, regardless of the age of the donor. On the other hand, cells from elderly donors (>60 years of age) showed a significant enlargement compared to both younger groups, probably secondary to their weaker proliferative capacity. An increase in cell area and an impairment of circularity are known to be related to a decrease in the cell density of the corneal endothelial monolayer and consequent alterations in its homeostasis [5‒7].

In general, primary cell cultures established from younger donor corneas have been shown to have higher proliferative capacity and a better ex vivo expansion profile [39, 40]. The limited regenerative capacity of the corneal endothelium has been observed not only in vivo but also in vitro [5, 6, 40]. This is especially true for hCEC derived from elderly donor corneas. Similarly, cultures from elderly donors exhibit a senescent cell phenotype which not only affects the in vitro cell morphology but also impairs their proliferative and functional capacity [5‒7, 41]. Further, the “cell contact inhibition” event observed in the monolayers in vivo and in vitro, may contribute to maintaining cells arrested in G1 phase of the cell cycle, inhibiting or decreasing their proliferative capacity [5‒7]. A practical explanation is the very low expression of Ki67 marker in our cultures when the cell monolayer is established [5]. On the other hand, corneas from young donors are much more difficult to procure and, if obtained, generally meet all the criteria of clinical applicability for transplantation. In this regard, other strategies for obtaining and optimizing primary cultures of hCECs have been described. All of this could generally explain the difficulties to stablish primary cultures, from elderly donors with endothelial counts lower than recommended for clinical application, with morphologically viable cells, and mainly with appropriate functionality.

Several relevant technical aspects and methodological challenges exist for maintaining stable and functional ex vivo long-term primary hCEC cultures, including those related to proliferative capacity and cell dedifferentiation [7, 39, 41]. It is known that adult hCEC do not spontaneously regenerate in vivo after endothelial injury. Likewise, the decrease in ECD due to senescence or disease is accompanied by a progressive change in endothelial mosaic – polymorphism and polymegathism – as a compensatory response to endothelial physiology [5, 6]. However, we expanded hCEC from elderly donors, confirming that even these fragile cells retained a certain degree of proliferative capability. It is noteworthy that, after passaging, our samples also showed a predisposition to undergo a phenomenon of cell dedifferentiation through the corneal endothelial-mesenchymal transition (EnMT), which induces the loss of its morphological characteristics. EnMT is characterized by fibroblastic transformation and disappearance of tight junctions and a consequent loss of cell monolayer barrier function [5, 6]. It poses a significant risk of premature culture failure, as these cells tend to rapidly overtake the entire culture [42].

Robust evidence indicates that the ROCK signaling pathway plays crucial physiological roles for several cell types, including hCEC. Regarding hCEC, ROCK is involved in cell proliferation and adhesion events, modulation of cytoskeleton and actin stress fibers, and corneal wound healing mechanisms [43, 44]. For example, the addition of Y-27632 to culture systems reversibly changes cytoskeleton and stress fibers, focal adhesions to the extracellular matrix, and tight cell junctions, promoting migration and facilitating cell proliferation [11, 45, 46]. Furthermore, experimentally induced endothelial lesions revealed that inflammatory mechanisms increase the production of fibroblast growth factor 2 in the aqueous humor by inactivating Rho through the PI-3 kinase pathway. This facilitates cell proliferation but also triggers fibroblast transformation [43, 47]. Interestingly, Rho-kinase inhibitors can also inhibit EnMT in hCEC cultures, reducing pleomorphism and polymegathism, while increasing the expression of intercellular adhesion molecules (ZO1) improving the Na+/K+-ATPase ion pump [45]. Therefore, when considering the propagation of hCEC from elderly donors, research on new modulating molecules may contribute to the optimization of primary hCEC cultures. Their use as a supplement in culture medium has been widely recommended to optimize proliferative and functional capacities.

To fight corneal blindness, it is essential to increase and facilitate access to corneal tissue for transplants, but this process can take time and involves not only technical but also in some cases cultural and religious obstacles. At the same time, future decisive and effective therapies can be sought through sustained research and training focused on cell-based therapies as a cost-benefit, rational, and less invasive approach. Bearing this in mind, ocular tissue banks require the support of the academic-scientific sector with the aim of facilitating the integration, innovation, and development of sustainable services. Tissue banks and research centers must collaborate to find answers and provide therapeutic alternatives such as advanced therapies, working together on technological advances in this field. In the same way, the local government regulatory framework should accelerate, accompanying the pace of translational approaches. Our results indicate that it is possible to obtain hCEC with simplified, standardized protocols and from elderly donor corneas. In our setting, this tissue is more readily available, either due to the profile of the donor population or due to a low endothelial count (<2,000 cells/mm2) of the donated cornea. At the same time, preservation of corneas with organotypic cultures has its advantages, favoring the availability of tissue for a new therapeutic modality in regenerative medicine through the expansion of hCEC in vitro in lieu of traditional keratoplasty techniques.

The author (C.A.-R.) received an individual grant from IMO Foundation (Barcelona, Spain).

The local Ethics Committee approved the present study (HCB/2016/0910, Hospital Clinic in Barcelona, Barcelona, Spain), which followed the principles and ethical principles of the Declaration of Helsinki (2013; Fortaleza, Brazil). Human tissue was obtained and processed according to the EEC guidelines for clinical use (EEC regulations 2004/23/EC and 2006/17/EC). It was also obtained, processed, analyzed, and preserved in accordance with current local regulations and in accordance with the protocol and with the legal requirements established by Law 14/2007 on biomedical research and RD 1716/2011 (Spain), which regulates the use of biological samples in research. All information for donation and their respective informed consents were obtained with the authorization and acceptance of the use of the samples for the corresponding purposes. In addition, informed written consent was obtained according to the BTB (Banc de Sang i Teixits, Barcelona, Spain) standard operational procedure for eye donation destined for research.

The authors have no conflicts of interest to declare.

This work was funded in part by a grant from Fondos de Investigaciones Sanitarias del Instituto Carlos III (FIS18-PI00355). The research project was co-financed by the European Regional Development Fund (FEDER) of the European Union.

C.A.-R. and R.P.C.-M. (a, b, c, and d); F.B.S. (a, b, and c); N.O. and A.R.-M. (a and c); and N.N.-N., J.L.G., and J.A.P.G. (b and c). (a) Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; (b) drafting the work or critically revising it for important intellectual content; (c) final approval of the version to be published; and (d) agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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