Table 2 Recent innovative protocols for ex vivo expansion of corneal endothelial cells (from cadaveric corneas)

R-G corneas:

n = 269;

mean donor age: 53 ± 16 y;

DTI: 7 ± 3 d

• Source tissue: CE + DM from: whole cornea (Ø 11.5 mm), Cen (Ø 8 mm) or Per (3.5 mm); 1 cornea/1 culture

• Culture models: explants (n = 13) or cell suspension (P&D method); enzymes tested: 0.1%/0.2% Col II, 0.05% Trypsin/EDTA, 0.1% dispase

• Surface: 6-well culture plates—uncoated, or coated (collagen IV, fibronectin or FNC)

• Two types of culture media tested:

Medium 1: EGM-2, 2%-10% FBS, EGF, VEGF, FGF, IGF, HC, Gent, Amp-B;

Medium 2: DMEM, 10% FBS, FGF

• P0: 14 days

• P1: successful culture, when cuboidal CECs formed confluent layer at P1

• P2-P3 also performed (enzymes: 0.05% Trypsin/EDTA)

• Successful cultures: 31.97% (86 of 269);

• Effect of donor age: successful (47 ± 18 y) vs. unsuccessful (58 ± 14 y) cultures (p < 0.001)

• Source tissue (successful cultures): whole cornea (47%, 8/17), Per (28%, 64/230), Cen (64%, 14/22), variable sample sizes

• General selection criteria for successful CEC culture: young donors (≤ 30 y), cell suspension model (0.2% Col II digestion), mitogen-rich medium (e.g., EGM-2), coating substrate (FNC)

• CECs from both Cen and Per regions reached confluence and could be passaged (up to P3)

[17]

R-G corneas:

n = 33 pairs (mean donor age: 20 y; DTI: 5–14 d) + n = 4 single corneas (age: 60–66 y; DTI: 4–8 d);

ECD ≥ 2000;

• Source tissue: paired corneoscleral tissues; 1 cornea/1 culture condition;

• Culture model: P&D method—peeling of CE + DM from whole cornea + enzymatic digestion: 2 mg/ml Col A and 1X TrypLE

• Surface: FNC

• Overnight pre-stabilization of isolated CECs in SM (Endo-SFM + 5% FBS)

• Dual-media: PM (M199 + F12, 5% FBS, 20 mg/ml Asc, 1X ITS, 10 ng/ml bFGF); effect of Y-27632 (3–1000 µM in SM) also analyzed

• P0: PM (∼14 d) + SM (7 d)

• P2 or P3 used for experiments

• subculture (enzyme: TrypLE)

• Dual-media culture led to confluent monolayers with polygonal/hexagonal CECs that expressed Na⁺/K⁺-ATPase, ZO-1, GPC-4, CD-200, detected by IHC

• Y-27632 in culture medium improved adherence, cell morphology, and overall cell yield compared to donor-matched control cultures; optimal concentration of Y-27632 was 10 µM

• Y-27632 enhanced cell proliferation in cultures derived from young, but not from old (≥ 60 y) donors

[128]

R-G corneas:

n = 24 pairs;

mean donor age: 64 ± 14 y;

DPT: 17 ± 6 d;

DTI: 32 ± 7 d;

ECD < 2200

• Source tissue: paired corneas/1 culture

• Culture model: P&D method—peeling of CE + DM from whole cornea + enzymatic digestion: 2 mg/ml Col I and 1X TrypLE

• Surface: Lab-Tek II, coated with FNC

• Plating: 100µL of the cell suspension per well

• Dual-media: PM (M199 + F12, 5% FBS, 1% Asc, 0.5% ITS, 25 µg/mL rh-bFGF, 10 µM Y-27632, antibiotics); SM (Endo-SFM, 5% FBS, antibiotics)

• P0:∼15 d; no passages;

• 4-plating conditions: 2 conditions: cells covered with sodium hyaluronate after plating (forced attachment) + 2 conditions without forced attachment

• Effect of forced attachment: CECs grew faster in PM in the first week of culture (average ECD 2500 ± 94 cells/mm² at day 15), compared to CECs grown without forced attachment; CECs cultured with forced attachment were vital (low level of apoptosis), highly metabolically active, and had a higher number of focal adhesions (vinculin) compared to untreated controls

• Conclusion: CECs derived from older donor corneas can be effectively expanded ex vivo, and the outcome of such culture can be improved by forced attachment, using non-toxic viscoelastic solution

[45]

R-G corneas:

n = 35 pairs;

DTI: 10 d (median);

ECD ≤ 2200

• Source tissue: paired corneas/1 culture;

• Culture model: P&D method—peeling of CE + DM from whole cornea + enzymatic digestion: Col I, or Liberase, or TrypLE

• Surface: coatings tested: FNC, collagen IV, rLaminin-511/521;

• Animal model for Tx: rabbits with induced bullous keratopathy

• Pre-stabilization (≤ 48 h) of isolated CECs in SM (Endo-SFM + 5% FBS)

• Dual-media: PM (M199 + F12, 5% FBS/EquaFetal/hu-serum, 20 μg/ml Asc, 1 × ITS, 10 ng/ml bFGF); SM (Endo-SFM, 5% serum)

• P0: SM (2d) + PM (∼14 d) + SM (2 d)

• P2-P3 used for experiments (IHC etc.)

• P1-P2 used for preparation of T-E graft = CECs seeded onto decellularized DM/stroma lenticule at ECD 3,000 and kept in SM for 5–7 d prior Tx)

• Effect of enzymes: Liberase performance was comparable to Col I

• Surface coatings: attachment profiles of collagen IV and rLaminin-511/521 were similar to FNC, but laminins (mainly rLaminin-511) were better substrates than collagen IV and FNC

• Effect of serum: FBS and EquaFetal—similar performance; hu-serum led to inconsistent cell growth, worsened morphology and fibroblastic changes of CECs during culture

• IHC: Human CECs (up to 3rd passage) formed CE monolayer, and expressed Na⁺/K⁺-ATPase, ZO-1, CD166, PRDX-6 + had normal karyotype

• Tx results: functional mosaic of CE and stromal hydration re-established at day 28 post-Tx into the rabbits' eyes

[30]

C-G corneas:

n = 36,

donor age: 10–70 y (74% donors ≤ 40 y old);

DPT: ≤ 24 h;

DTI: ≤ 14 d;

ECD ≥ 2500 (89% corneas)

• Source tissue: 1 cornea/1 culture

• Culture model: P&D method—peeling of CE + DM from whole cornea + overnight pre-incubation of lamella in PM + enzymatic digestion: 0.02% EDTA

• Surface 12-well culture plate (1–3 wells) coated with FNC

• Dual-media: PM (Opti-MEM I, 8% FBS, 5 ng/mL rh-EGF, 20 ng/mL rh-NGF, 100 µg/mL BPE, 0.5 mM L-Asc 2-P, 200 mg/L CaCl₂, 0.08% CHS, 50 µg/mL Gent, 1X AA); other tested supplements: L-Asc, Y-27632, SB-154352, rh-R-spondin-1

• SM: SM1 (Endo-SFM, 4% FBS, 50 µg/mL Gent, 1X AA); SM2 (Opti-MEM I, 4% FBS, 50 µg/mL Gent, 1X AA)

• P0: PM (∼2–4 weeks) + SM (7 d)

• P2–P4 used for experiments

• Effect of ascorbate: higher cell counts in medium with L-Asc 2-P than in the media with L-Asc;

• Y-27632: no effect on CECs proliferation; higher doses of the inhibitor led to EndMT

• SB-154352: no effect on proliferation or survival of CECs

• R-spondin-1: no significant effect on proliferation of CECs

• SM: SM1 better than SM2; SM1 supported canonical cell morphology (expression of CD56 marker) and barrier function of CE; CECs maintained in PM for a prolonged period: low CD56 expression and initiation of EndMT

[40]

C-G corneas;

donor age: 7–29 y;

DTI: ≤ 14 d;

ECD ≥ 2500;

Note: first in-human clinical trial

• Source tissue: 1 cornea/1 culture

• Culture model: P&D method—peeling of CE + DM from whole cornea + enzymatic digestion: 1 mg/mL Col A

• Surface: 6-well culture plate (2 wells coated with collagen type I)

• Patients (Tx): 11 patients (age: 20–90 y) with bullous keratopathy

• Proliferation medium only: PM (Opti-MEM I, 8% FBS, 200 mg/L CaCl₂, 0.08% CHS, 50 mg/mL Gent, 5 ng/mL EGF, 20 µg/mL Asc, 10 µM Y-27632; 10 µM SB-203580, 1 µM SB-431542)

• P0: PM (∼4 weeks);

• P2-P3 used for Tx

• Cell lots for clinical application were examined to verify that they met criteria for surgical use and 1 × 10⁶ (Patients 2–11) or 5 × 10⁵ (Patient 1) CECs resuspended in 300 μl of modified Opti-MEM I medium with Y-27632 were injected into the anterior chamber of bullous keratopathy patients; patients were kept in a prone position for 3 h to enhance the adhesion of the injected CECs

• 24 weeks after cell injection, ECD increased in all (11/11) patients and visual acuity improved in most patients (9/11)

• Prospective observational study confirmed that at 5 years after surgery, the CE function was restored in 10 of the 11 eyes, whose mean central corneal ECD was 1257 ± 467 cells/mm²

[24]

R-G corneas:

n = 18 pairs (mean donor age: 19 y) + n = 6 single corneas (donor age: 19–69 y);

DTI: 9 d (median);

ECD ≤ 2200

• Source tissue: whole cornea;

• Culture model: P&D method—peeling of CE + DM from whole cornea + enzymatic digestion: Col I, and TrypLE

• Surface: collagen IV, seeding density: 1 × 10⁴ cells/cm²

• Animal model (Tx): rabbits (n = 20) with bullous keratopathy

• Tissue-engineered (T-E) group: Group A (n = 3, received T-E graft), Group B (n = 3, no DM + no T-E graft), Group C (n = 3, T-E graft = DM/stroma lenticule with no cultured CECs)

• Cell-injection (C-I) group: Group 1 (n = 5, no CE, intact DM, C-I graft), Group 2 (n = 3, no CE, no DM, C-I graft), Group 3 (n = 3, no CE, intact DM, treatment: only Y-27632)

• Overnight pre-stabilization of isolated CECs in SM (Endo-SFM + 5% FBS)

• Dual media: PM (M199 + F12, 5% serum, 20 μg/ml Asc, 1 × ITS, 10 ng/ml rh-FGF); SM (Endo-SFM, 5% serum)

• P0: SM (1d) + PM (∼2–4 weeks) + SM (≤ 48 h)

• P2-P3 used for experiments;

• T-E graft: P1-P2 CECs seeded onto decellularized DM/stroma lenticule at ECD 3,000 (∼8.5 × 10⁴ cells) and kept in SM for 5–7 d prior Tx

• C-I graft: approx. 6.0 × 10⁵ CECs (P2) re-suspended in 150 μl of SM + Y-27632

• IHC, flow-cytometry: CECs (P2-P3) expressed Na⁺/K⁺-ATPase, ZO-1, CD166, PRDX-6

• Corneal transparency (3 weeks post-Tx): T-E group—Group A: Cen region of cornea, corresponding to the graft remained clear, but Per region (= no DM, no cells), remained hazy throughout the study period; Group B–C: both remained hazy; C-I group: signs of intraocular inflammation after Tx, which resolved one week after Tx; unlike Group 2 and 3 corneas, Group 1 corneas remained clear throughout the follow-up period (21 d)

• Central corneal thickness (at week 3): T-E group had thinner corneas than C-I group (NS difference)

• Mean ECD post-Tx (at week 3): T-E group: ECD 1248 ± 64; C-I group: 1409 ± 128

• Characterization of excised corneas: only Group A (T-E group) and Group 1 (C-I group) CE were reactive to anti-human specific nuclei antibody attributing corneal recovery to the functional human CECs

• Both T-E and C-I grafting can be used for cell-based therapy of diseased CE, but further evaluation of long-term safety and efficacy of the two methods is necessary

[21]

R-G corneas:

n = 19;

mean age: 72 ± 5 y;

DTP: 10 ± 5 h;

ECD < 2200

• Source tissue: 1 donor/1 culture

• Culture model: P&D method—peeling of CE + DM from Cen (Ø 8.25 mm) and Per (2.75 mm) regions of CE + enzymatic digestion: 2 mg/mL Col I, 1X TrypLE

• Surface: Lab-Tek II (FNC); 100 ul cells/1 well

• Proliferation medium only: PM (M199 + F12, 5% FBS, 1% Asc, 0.5% ITS, 10 ng/mL rh-bFGF, 10 μM Y-27632, 1% PenStrep)

• P0: 9 days; no passages

• Cen vs. Per: CECs from both zones reached confluence (18/19 cultures); higher ECD (p < 0.05) in the Per; positive IHC signal for PRDX-6, ZO-1, Ki-67 in both regions; NS difference in proliferation rate, cell area, and hexagonality between regions

• One R-G cornea allowed preparation of two full grafts (from Cen and Per) usable for Tx

[39]

R-G corneas:

n = 22 (n = 16 pairs + n = 6 single corneas);

mean donor age: 53 y;

DTI: median 12 d;

• Method 1: peeling of CE + DM, pre-incubation in SM or PM (≥ 48 h), TrypLE digestion, non-propagated CECs collected for Tx (cell-injection)

• Method 2: peeling of CE + DM, digestion with Col; ex vivo propagation of CECs prior cell injection; pooled cells from 1 paired donor corneas/1 culture

• Animal model for Tx: rabbits (n = 20) with bullous keratopathy

• Dual-media (Method 2): PM (M199 + F12, 5% FBS, 20 μg/ml Asc, 1 × ITS,10 ng/ml bFGF); SM (End-SFM, 5% FBS, antibiotics)

• Tx: 6 × 10⁵ cells CECs (P2) collected and resuspended in 150 µL of SM with Y-27637 for injection into the rabbit eyes

• 48-h pre-incubation of peeled CE + DM lamellas in SM vs PM: unlike PM, the SM improved cell morphology and cellular yields after cell isolation

• Tx of CECs prepared by Method 1 and Method 2: both methods were comparable–both treated groups of rabbits formed CE monolayer and maintained corneal clarity after Tx throughout the study period; corneas that did not receive any cells remained significantly (p < 0.05) thicker compared to corneas treated with cells (Method 1 and 2)

• Authors showed that a direct collection of a good quality CECs from donor R-G corneas is possible and allows utilization of R-G corneas for Tx purposes

[20]

R-G cadaveric corneas:

n = 28;

mean donor age: 18 y;

DPT: ˂ 24 h;

DTI: ˂ 15 d;

ECD ≥ 2300

• Culture model: P&D method—peeling of CE + DM, followed by two variants of isolation: Method 1 (trypsin + laminin coated surface), or Method 2 (Col A + Collagen IV coated surface)

• Two types (conditions) of cell culture: Condition 1 (Single medium): PM (M199 + F12 medium, FBS, rh-insulin, Asc, PenStrep, rh-bFGF) or Condition 2 (Dual media): SM (Endo-SFM, FBS, PenStrep, Amp-B) and PM (M199 + F12, FBS, L-Asc-2-P, PenStrep, ITS, Amp-B, rh-bFGF)

• P0 until confluence

• P1-4 also used for experiments

• Condition 1 vs. Condition 2: CECs (P0) maintained in SM (Condition 2) had a robust expression of the CEC-specific genes in P0, i.e., 85.6% (87/97) of the selected CEC-specific genes were expressed, while CECs (P0) cultured in Condition 1 expressed only 78.4% (76/97) genes

• Continuous passaging induced replicative cell senescence and loss of CECs identity, e.g., by P4, only 75.3% (73/97) CEC-specific genes were expressed in Condition 2-cultured CECs

• SLC4A11 and CD44 may represent the optimal markers of high-quality cultured (by Method 2 + Condition 2) CECs

[18]