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

From: Ex vivo expansion and characterization of human corneal endothelium for transplantation: a review

Cell source Cell isolation and plating Culture medium and culture time Main outcomes Ref
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 (31000 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 57 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 (24 weeks) + SM (7 d)
• P2P4 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: 2090 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 211) 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 57 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 BC: 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 comparableboth 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]