Paired Human Corneas Research Articles

Advances in corneal preservation using an active storage machine

Since the 1980s, corneal storage has been carried out by eye banks throughout the world, mainly by two methods: short‐term storage at 4°C (America, India), and medium‐to‐long‐term organoculture at 31–34°C (Europe, Australia). In these two techniques, known as passive, the cornea is simply immersed in a storage medium. However, the control of corneal hydration in vivo depends both on the proper functioning of the corneal endothelial cells (responsible for the imbibition pressure IP) and on the presence of a normal intraocular pressure (IOP), both of which are opposed to the stromal swelling pressure (SP): IP = SP‐IOP. By restoring IOP ex vivo in an active storage machine (ASM) that separates an endothelial chamber from an epithelial chamber, corneal hydration is better controlled, thus preventing the formation of deep endothelial folds responsible for endothelial over mortality. The active control of corneal hydration eliminates the need to add macromolecules to deswell the cornea (continuously for cold storage or during the last 48–72 h for organoculture). The toxicity of this forced deswelling has been demonstrated for organ culture.We performed two successive preclinical studies comparing ASM and organoculture with paired human corneas having more than 2000 cells/mm2 at the time of the first endothelial count. The organoculture medium (CorneaMax, Eurobio, les Ulis, France) was identical in both groups. The ASM was an experimental version from our laboratory, including a PEEK (polyetheretherketone) corneal support, a peristaltic pump, an Edwards pressure sensor, a microsolenoid valve and a microcontroller managing the flow rate (2.6 μL/min) and the IOP (21 mmHg). In the first study, for 52 pairs of corneas stored for 4 weeks, the ASM corneas had, at the end of storage, +23% viable endothelial cells compared to passive organoculture. Furthermore, ASM also significantly increased Na+/K+/ATPase protein expression by 3.8 ± 1.4. In the second study, for 12 pairs of corneas stored for 3 months, the ASM corneas had +53% viable endothelial cells at the end of storage compared to passive organoculture. One third of corneas still had 2000 cells/mm2 or more in ASM versus none in organoculture. The ASM significantly increased Na+/K+/ATPase protein expression by 2.9 ± 0.8.The ASM has 2 transparent windows which allow all quality controls to be performed without deconditioning the cornea, and in particular to perform endothelial counting by specular microscopy on a cornea which remains thin. The ASM thus combines the advantages of both methods of storage: long‐term storage without massive oedema and without toxic dextran and a closed system allowing non‐invasive controls. As with the perfusion machines for vascularized organs, the restoration of near‐physiological conditions improves grafts' quality.We have transferred this innovation to THEA laboratories (Clermont‐Ferrand, world specialist in ophthalmology) which is industrializing the ASM via its spin‐off SINCLER. The industrial version will take the form of a single‐use cassette incorporating a flow/pressure regulator, a media reservoir and a waste bin. The cassette will be plugged into a computer‐controlled multi‐place incubator for IOP and media flow management. The ASM could revolutionize eye banking worldwide.

Epithelial alternate exposure to air and liquid further improves human corneas stored in the active storage machine

AbstractPurposeThe standard version of our corneal active storage machine (ASM) maintains corneas viability much better than standard organ culture over 1 and 3 months (Am J Transplant 2019, Transplantation 2020). While it aims at recreating a physiologic environment (IOP and medium renewal), corneas were nevertheless immersed from both sides, probably contributing to slightly higher thickness than in vivo.AimTo assess whether epithelial alternate exposure to air and liquid (AirLifting, AL) could further improve quality of corneas stored into the ASM.MethodsTwenty paired human corneas (initial ECD > 2000 cells/mm2 and <10% difference between right and left) were randomized to be stored for 1 month in the ASM either fully immersed or with AL. All other parameters were similar: IOP (21 mmHg), endothelial medium flow (2.6 μL/min) and nature (CMax, Eurobio). AL was done with CMax (6 cycles air‐liquid/min). Specular microscopy ECD, OCT thickness, transparency were monitored at Day (D)2, D26, D28. Pancorneal viable ECD (Hoechst/Ethidium/Calcein‐AM), histology, immunostaining, western blot for epithelial cells and endothelial cells (ECs) structure/functions proteins were done at D28.ResultsAt D2, ECD was 2412 ± 222 versus 2385 ± 251 cells/mm2 (p = 0.349) respectively with and without AL and 2109 ± 165 versus 2119 ± 199 cells/mm2 (p = 0.621) at D26. The viable ECD at D28 was 2170 ± 321 versus 2185 ± 275cells/mm2 (p = 0.794). All corneas remained thin transparent during storage and at D28 only, corneas were thinner with AL: 667 ± 60μm versus 695 ± 55μm without (p = 0.048). Epithelial quality was similar, but the epithelium was thicker with AL (49 ± 20) than without (23 ± 7μm) (p = 0.001) and had more cell layers. Endothelial cells structure/functions were similar.ConclusionsBy mimicking blinking, AL enhances epithelium structure during storage into the ASM. It may also slightly help controlling corneal thickness. ASM with AL may be useful for penetrating and anterior keratoplasty as well as for research works.

Similar Biomechanical Cross-linking Effect After SMILE and PRK in Human Corneas in an Ex Vivo Model for Postoperative Ectasia.

To evaluate the biomechanical effect of corneal cross-linking (CXL) in paired human corneas following small incision lenticule extraction (SMILE) or photorefractive keratectomy (PRK) in an ex vivo model for postoperative ectasia. Twenty-six paired human corneas preserved in tissue culture medium were equally divided into two groups: right and left corneas were treated with PRK and SMILE, respectively. Corneal thickness was measured in all eyes before surgery. Corneas were stretched using an extensometer with two cycles of up to 9 N (570 kPA stress), followed by accelerated CXL with irradiance of 9 mW/cm2 for 10 minutes (fluence 5.4 J/cm2) in both groups. The elastic modulus was evaluated using two-dimensional stress-strain extensometry. Following accelerated CXL, the ectatic cornea model showed a mean effective elastic modulus of 17.2 ± 5.3 MPa after PRK and 14.1 ± 5.0 MPa after SMILE. Although the elastic modulus in corneas previously subjected to PRK was higher, there was no significant biomechanical difference between the two groups (P = .093). Under similar conditions, both experimental groups (PRK followed by CXL and SMILE followed by CXL) were characterized by similar biomechanical stability as measured experimentally on ex vivo human fellow corneas. The data suggest that, in the event of postoperative ectasia, the biomechanical improvement achieved by CXL may be similar after PRK and SMILE. [J Refract Surg. 2020;36(1):49-54].

Preclinical validation of an innovative corneal bioreactor versus organ culture for long term storage: a randomized controled study

PurposeThe organ culture (OC) is a passive corneal storage, gold standard in Europe for decades. The bioreactor (BR) is a sterile transparent device that allows active storage: transcorneal pressure gradient and fluid renewal. Aim: To compare quality of human corneas stored for 1 month in our BR versus (vs) OC.MethodsRandomized controled preclinical study, authorized by the French Biomedicine Agency and an IRB, including only fresh scientific paired human corneas >2000cells/mm2 and <10% difference between both corneas at day (D)2. Immediately at retrieval, one cornea was randomized and stored in OC and the other in BR, both with the same commercial medium (CorneaMax). At D26 only OC corneas were transferred to a deswelling medium (CorneaJet). We calculated that 49 pairs were needed to show a difference of 500 viable cells/mm2 between groups. Assessments were performed at D2, 26, 28. Main criterion: viable ECD (vECD) determined by triple staining Hoechst/Ethidium/Calcein at D28, blind of storage method. Secondary criteria: average transparency (T), central corneal thickness (CCT) and microbiology.ResultsWe retrieved 60 pairs, included 52 pairs at D2, analyzed 50 pairs (1 contamination per group). At D2 ECD in BR was 2607±340 vs 2576±357cells/mm2 in OC (p=0.243). At D26 ECD was >2000cells/mm2 for 92% of corneas in BR vs 58% in OC (p<0.001). At D28 vECD was higher in BR 2188±379 vs 1687±389cells/mm2 in OC, with 76% of corneas >2000 viable cells/mm2 in BR vs 22% in OC (p<0.001); T didn't differ and CCT was higher in BR with 684±52 vs 608±53μm in OC (p<0.001).ConclusionsRestoring IOP and renewing medium significantly improves endothelial cell survival while avoiding deleterious deswelling necessary in OC: after 1 month‐storage, the BR provides much more corneas suitable for transplantation and higher quality grafts that could survive longer in recipients.

VIP and VIP Gene Silencing Modulation of Differentiation Marker N-Cadherin and Cell Shape of Corneal Endothelium in Human Corneas Ex Vivo

Vasoactive intestinal peptide (VIP) is expressed by corneal endothelial (CE) cells and is present in the aqueous humor, which bathes CE cells in vivo. This study demonstrated the role of CE cell VIP in maintaining the expression level of a CE differentiation marker, N-cadherin, and the hexagonal cell shape. To determine the most effective VIP concentration, bovine corneoscleral explants were treated with 0 (control) and 10(-12) to 10(-6) M VIP. Paired human corneas (nine donors) from an eye bank were used as control; the other corneas were treated with VIP. To silence endogenous VIP, paired fresh human donor corneas (from seven cadavers) were transduced with VIP shRNA or the control lentiviral particles and then bisected/quartered for quantitative analysis by semiquantitative RT-PCR (for mRNA) and Western blot analysis/immunocytochemistry (for protein), whereas alizarin red S staining revealed CE cell shape. VIP concentration dependently increased bovine CE cell N-cadherin mRNA levels, with the maximal effect observed between 10(-10) (1.47 +/- 0.06-fold; P = 0.002) and 10(-8) M VIP (1.48 +/- 0.18-fold; P = 0.012). VIP (10(-8) M) treatment increased N-cadherin protein levels in bovine and human CE cells to 1.98 +/- 0.28-fold (P = 0.005) and 1.17 +/- 0.10 (range, 0.91-1.87)-fold (P = 0.050) of their respective controls. VIP antagonist (SN)VIPhyb diminished the VIP effect. VIP silencing resulted in deterioration of the hexagonal cell shape and decreased levels of VIP protein and mRNA, N-cadherin (but not connexin-43) mRNA and protein, and the antiapoptotic Bcl-2 protein. Through its autocrine VIP, CE cells play an active role in maintaining the differentiated state and suppressing apoptosis in the corneal endothelium in situ.

Use of Poloxamers for Deswelling of Organ-Cultured Corneas

Dextran T500, routinely used as a deswelling supplement in organ culture (OC), has been suspected of being toxic to corneal endothelial cells (ECs). This study was conducted to evaluate the innovative use of poloxamers compared with dextran for deswelling OC corneas. Five poloxamers (P124, P188, P237, P338, and P407) were dissolved respectively in a standard OC medium to reach 350 mOsmol/kg. In vitro cytotoxicity of these media was tested by MTT assay on human corneal epithelial and endothelial cell lines and on primary human corneal fibroblasts. Paired human corneas stored in OC for at least 21 days were assigned for 48 hours to a poloxamer medium or to a standard deswelling medium containing 5% dextran T500. Corneal EC density, morphometry, visualization, mortality, stromal thickness, transparency, and folding were evaluated before and after deswelling. Corneas were finally cut into three parts for histologic and ultrastructural observation. Besides similar corneal transparency improvement and thickness deswelling, poloxamers (except P124) reduced EC loss and facilitated endothelial visualization, but improved stromal folding less than dextran. The similar ultrastructures observed in the two groups were epithelial shedding, normal collagen fiber diameter and organization, uptake of deswelling agents by ECs, vacuolization but normal organelles in ECs and keratocytes, and endothelial surface modifications. P188, P237, P338, and P407 performed similarly in preserving ECs, improving EC visualization, deswelling corneal stroma and inducing moderate injuries to corneal ultrastructure. They appear superior to dextran for corneal deswelling in OC.

005 Analyses histologiques et ultra structurales des cornées humaines conservées en Corneamax et déturgées durant 48 heures dans les poloxamers

Poloxamers (P) are various synthetic block copolymers widely used in pharmaceutic industry. We previously described the efficiency of P188, 237, 338 and 407 to deswell human corneas at the end of organ culture (OC) and their ability to reduce the endothelial cell loss compared to 5% T500 Dextran in CorneaMax (with 2% foetal calf serum). We aimed to further assess the cellular effects of 48 hours incubation in P using histology and electron microscopy (EM). The P were dissolved in CMax (Eurobio, Les Ulis, France) at 350 mOsmol/L. Five paired human corneas with more than 2000 endothelial cells/mm2 used for each poloxamer. After 3 weeks of OC in CMax, one cornea was incubated for 48 hours in CMax + poloxamer, and the mate cornea in CorneaJet (CMax+ 5% T500 Dextran). Controls: fresh corneal button from keratoconus and lattice dystrophy (for normal unstored endothelium) and organ cultured corneas without deswelling. Corneas were divided into 3 parts for cross sectional histology (epithelial thickness measurement and counting of keratocytes), transmission EM of the posterior stroma and endothelium (cell morphology and interfibrillar spaces measurement) and scanning EM of the endothelium (morphology). Cross sectional histology showed differences in epithelial thickness. For both types of macromolecules (poloxamers and dextran) TEM revealed cytoplasmic vacuoles in endothelial cells and keratocytes and SEM confirmed the integrity of endothelial monolayer. Histological and ultrastructural findings, along with capacity of thinning, transparency improvement and endothelial cell preservation will allow selecting poloxamers to be added to CorneaMax for a future clinical application. This complementary validation using light and electron microscopy reasonably confirms the promising use of Poloxamer to replace T500 Dextran to deswell corneas during organ culture.

Animal Compound–Free Medium and Poloxamer for Human Corneal Organ Culture and Deswelling

Eliminating fetal calf serum (FCS) from corneal organ culture (OC) media has long been a challenge. This study was an assessment of a new animal compound-free (ACF) medium for corneal storage and of its combination with poloxamer for end-of-storage corneal deswelling. A randomized controlled study with masked assessment compared the ACF medium to standard commercialized media containing 2% FCS and their combination with dextran for deswelling. Paired human corneas were randomly allocated at procurement, one to the ACF medium and the other to the FCS media, and then assessed at day (D)2 and D30 of OC storage and after 48 hours of deswelling. Comparison criteria were endothelial cell density (ECD) and morphometry by a corneal analyser, quality of endothelial visualization (using saline), EC mortality (trypan blue), corneal thickness, corneal transparency, and folding. Fifty-six corneas (28 pairs) with ECD of 2000 cells/mm(2) or more were enrolled. Data were compared using paired tests with P < 0.01 deemed significant. Parameters were similar at baseline (D2) between groups. Daily EC loss during the 30 days of storage was reduced with the ACF compared with standard (-0.31% +/- 0.30% vs. -0.88% +/- 0.38%, P < 0.001). With poloxamer 188 (Lutrol F68; BASF, Ludwigshafen, Germany), EC loss was substantially reduced (-1.43% +/- 3.60 vs. -15.41% +/- 10.13%, P < 0.001) and morphometry better preserved, despite thickness reduction, transparency improvement and folding reduction comparable to dextran. After 30 days of storage in ACF medium and deswelling in poloxamer 188, ECD was 30% higher (2466 +/- 447 cells/mm(2) vs. 1729 +/- 281 cells/mm(2), P < 0.001). ACF medium alone and combined with poloxamer 188 considerably facilitated EC visualization at D30 and after deswelling. The ACF medium combined with poloxamer 188 for deswelling showed superiority over standard FCS medium in its ability to preserve EC viability and facilitate endothelial visualization. This innovative use of poloxamer for deswelling appears far less toxic than does dextran.

Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium.

To investigate the effect of FGF-2 on corneal endothelial cell survival in porcine and human corneas during corneal storage in a serum-free medium. Porcine and paired human corneas were stored at 32 degrees C for 9 and 22 days, respectively. One cornea of each pair was stored in a serum-free culture medium, and the mate was preserved in the same medium supplemented with 10 ng/mL FGF-2. Quantitative analysis of corneal damage after storage was determined by the Janus green photometry technique. 5-Bromo-2-deoxyuridine (BrdU) labeling of the endothelium determined the effect of FGF-2 on endothelial proliferation during storage. Additional cell culture studies were performed to elucidate the role of FGF-2 on the incidence of endothelial apoptosis after serum deprivation. When FGF-2 was added to the serum-free medium, the damage rates of porcine endothelia were reduced from 15.1% +/- 8.7% (control) to 6.4% +/- 2.0% after 9 days and from 25.3% +/- 10.2% to 13.6% +/- 4.2% after 22 days of storage. In the human corneas stored during 22 days in FGF-2-supplemented medium, the amount of endothelial damage was 11.8% +/- 3.2%, which was significantly less damage than in the control fellow corneas stored in unsupplemented serum-free medium (19.3% +/- 6.3%; P < 0.01). DNA synthesis was not enhanced in corneas stored in serum-free medium, serum-free medium+FGF-2, or medium containing 10% FCS. Only a few (3.8%) TUNEL-positive endothelial cells were detected in cultures maintained in FGF-2-supplemented serum-free medium compared with a high number (48%) of apoptotic cells in control cultures. FGF-2 efficiently reduces human corneal endothelial damage that occurs during organ culture storage in a serum-free medium. This effect is truly protective, because no proliferative activity and a decreased rate of apoptosis were determined. FGF-2 emerges as an important component of a future serum-free corneal organ-culture medium established to replace fetal calf serum (FCS) as a potential source of recipient infection.

Ex vivo phosphorus magnetic resonance spectroscopy on eye bank corneas and corneal metabolic health.

Since a potential exists for untoward effects on the cornea from the high magnetic fields and radio-frequency energies, and the further manipulation required for phosphorus-31 magnetic resonance spectroscopy (31P-MRS), we determined the effects of this technology on tissues using paired human corneas (n = 4) meeting criteria acceptable for transplantation. Slit-lamp biomicroscopy, pachometry, specular microscopy, and redux fluorophotometry were performed on all corneas. One cornea of each pair was examined (< 30 min) by 31P-MRS. Following 31P-MRS, slit-lamp biomicroscopy, pachometry, and redox fluorophotometry were again performed. Data tabulated included the 31P energy modulus (1.37 +/- 0.28), the ATP/Pi (2.92 +/- 0.59) and SP/Pi (0.76 +/- 0.04) ratios, and the intracorneal pH (7.24 +/- 0.09). Since there were no significant differences in slit-lamp biomicroscopy, endothelial density and morphometry, cell counts, and pachometric and redox fluorophotometric measurements between corneas of each pair before and after 31P-MRS analysis, it was concluded that there was no detectable metabolic damage secondary to such analysis. This study suggests that MRS analysis of human eye-bank tissues does not damage the cornea metabolically and may provide a practical evaluation of the health of the cornea at the biochemical level.

Clinical Comparison of Grafts Stored in McCarey-Kaufman Medium at 4°C and in Corneal Organ Culture at 31°C

Twenty-eight paired human corneas were preserved in minimal essential medium at 31 degrees C and in McCarey-Kaufman medium at 4 degrees C. These grafts were then transplanted in pairs of patients with keratoconus who were age matched as closely as possible. These pairs received donor corneas from the same donor, so for each pair the donor age and time from death to preservation were the same. Visual acuity, central corneal thickness, and endothelial cell counts were compared. During the 1- to 2-year study period, no statistically significant difference in visual acuity, corneal thickness, or endothelial cell density was found between grafts stored in minimal essential medium and those stored in McCarey-Kaufman medium.

Antimicrobial Efficacy and Corneal Endothelial Toxicity of DexSol Corneal Storage Medium Supplemented with Vancomycin

AbstractDespite the presence of gentamicin in corneal storage medium, postoperative endophthalmitis may result from the transmission of bacteria via contaminated corneal tissue. The authors evaluated the antimicrobial activity and endothelial toxicity of vancomycin (10 µ/ml) in combination with gentamicin (100 µ/ml) in DexSol corneal storage medium. When tested against a panel of common ocular pathogens, vancomycin combined with gentamicin proved more effective than gentamicin alone for inhibiting the growth of Streptococcus pneumoniae at 4° C after 1- and 5-day incubation periods. Ultrastructural and morphometric analyses of paired human corneas stored for 5 days in either DexSol supplemented with vancomycin combined with gentamicin or DexSol with gentamicin alone did not show a significant difference in endothelial toxicity between the two groups. These results suggest that vancomycin combined with gentamicin has superior antimicrobial effect and similar corneal toxicity when compared with media supplemented with gentamicin alone.

Air/liquid corneal organ culture: a light microscopic study

Air/liquid organ culture of tissues with stratified epithelial layers has been shown to encourage tight packing of cells and promote cellular differentiation. In this study human corneas cultured in a air/liquid environment were compared to paired, conventionally-cultured corneas to determine if the long-term morphology could be improved. Fourteen paired human corneas were cultured at 37 degrees C in covered culture dishes for 1 to 3 weeks. Air/liquid cultured corneas were placed epithelial-side up in a fixed position and culture medium was added to a level so that during rocking the corneal epithelia were intermittently exposed to air/liquid environments. Mate corneas were cultured using the conventional method. In this method corneas are fully submerged, epithelial-side down, in culture medium. After 3 weeks of culture significantly less epithelial intercellular edema was noted for the air/liquid cultures (p = 0.033), compared to conventional cultures. Significant improvements in cellular structure of the endothelial layers, after 1 and 3 weeks incubation (p = 0.029 and 0.000) and stromal layers, after 3 weeks in culture (p = 0.024), were also noted. We have shown that slight modifications of the organ culture environment lead to improvements in corneal morphology. Air/liquid corneal organ culture has promise for use in corneal wound healing studies and long-term culture.

Tolerance of human corneal endothelium to glycerol

As an initial step in the development of a method for corneal cryopreservation by vitrification, we attempted to establish the maximum concentration of glycerol to which human corneal endothelium could be exposed at 4 °C for 15 min without damage. Damage was defined as an increase in mean endothelial cell size or the inability to maintain corneal thickness for 1 week after exposure to glycerol. Using a system for long-term corneal perfusion, we perfused 24 paired human corneas with glycerol at 4 °C. The concentration of glycerol increased at a rate of 20% ( w v ) (2.2 M) per hour until the desired maximum concentration was reached for that cornea, stabilized for 15 min, and then decreased at the same rate. The corneas were then perfused at 37 °C with Dulbecco's medium at a rate of 5 μl/min under 18 mm Hg intracameral pressure for 7 days with daily measurements of corneal thickness. Endothelial morphology was examined by specular microscopy and by scanning electron microscopy. After 7 days of perfusion at 37 °C, there was a statistically significant direct relationship between the maximum concentration of glycerol to which the experimental eyes had been exposed and the increase in mean endothelial cell size. The mean endothelial cell size increased in corneas exposed to glycerol concentrations of 40, 50, and 60% ( w v ), but did not differ significantly from baseline measurements in the corneas exposed to 30% glycerol or less. Thus, there was no detectable damage to human corneas exposed to 30% ( w v ) (3.3 M) glycerol in this system. Tolerance of higher concentrations may be achieved by changes in the rates of addition and removal of glycerol or in the composition of the perfusate.

Human Corneal Endothelial Layer Repair During Organ Culture

Circular freeze-thaw endothelial wounds were created on paired human corneas. Ultrastructural and physiological studies were performed after organ culture (OC) incubation at 37 C from 1 to 21 days as well as on fresh noncultured controls. As early as 24 hours after injury, OC corneas demonstrated ultrastructurally intact endothelial cells at the margin of the wound, elongating and sliding toward its center. All OC corneas were completely covered by ultrastructurally intact and physiologically functioning endothelial cells by seven days of OC. These cells were approximately twice normal size. Enlarged endothelial cells that maintained deturgescence function were seen in the wounded area after 14 and 21 days of OC. None of the fresh controls demonstrated deturgescence function and in none could ultrastructurally intact endothelial cells be found in the area of the wound. This confirms our hypothesis that during 37 C OC incubation, human corneal endothelium repairs defects in its layer by cells that are physiologically and ultrastructurally intact.