Corneal Endothelial Cell Sheets Research Articles

Detachment of bovine corneal endothelial cell sheets by cooling-induced surface hydration of poly[(R)-3-hydroxybutyrate]-based thermoresponsive copolymer coating.

Cell sheet technology (CST) is a fascinating scaffoldless tissue engineering technique to generate a physiologically representative tissue replacement from autologous sources. As compared to conventional enzymatic cell harvesting methods, CST enables the preservation of important cell-to-cell junctions and extracellular matrix (ECM) components. However, covalent grafting methods are often employed for CST. In this study, a series of triblock copolymers with a hydrophobic and biocompatible poly[(R)-3-hydroxybutyrate] (PHB) central block flanked by varying lengths of terminal poly(N-isopropylacrylamide) (PNIPAAm) blocks (PNIPAAm-PHB-PNIPAAm) was synthesized via atom transfer radical polymerization of NIPAAm. The thermoresponsive triblock copolymers were explored as a non-covalent surface coating for culturing and detaching bovine corneal endothelial cell (BCEC) sheets. Aqueous solutions of the triblock copolymers produced thermosensitive micelles which can be drop-casted on glass substrates, resulting in a temperature-responsive surface. Importantly, incorporating a central hydrophobic PHB block enabled the anchoring of the coating to the bare substrate and enhanced the proliferation rate of the BCECs studied. Effective detachment of an intact cell sheet was also demonstrated via a cooling treatment at 4 °C for 20 min, and the viability of the detached cell sheet was found to be unaffected by the cooling. This work may potentially inspire more studies involving the non-covalent thermoresponsive polymer coatings for corneal tissue engineering applications.

Corneal endothelial cell sheet bioengineering from neural crest cell-derived adipose stem cells on novel thermo-responsive elastin-mimetic dendrimers decorated with RGD

Due to the limitations in in vivo division of Human Corneal Endothelial Cells (HCECs), and a shortage of cornea donations, there is increasing focus on bioengineering Corneal endothelium (CE) using alternative cells. Cell sheet engineering allows for the maintenance of the cellular junctions necessary for CE function. The aim of this study was to generate CE-like cell sheets on innovative thermo-responsive biopolymer, using hADSC-derived CE-like cells.Thermo-responsive biopolymers were fabricated by conjugating Elastin-like polypeptide (ELP) functionalized with RGD to a G4 dendrimer as a nanoplatform, named elastin-mimetic dendrimer (EMD). The hADSCs were first reprogrammed into neurospheres on Poly-L-lysine, induced by small molecules. Then, neural crest cells (NCCs) were differentiated into CE-like cells on EMD, and cultured until the CE-like cell sheet formed.EMD exhibited excellent biocompatibility improving cell spreading, adhesion, proliferation and NCCs differentiation into CE-like cells, and Lower critical solution temperature (LCST) near physiological temperature ideal for CE-like cell sheet harvesting. NCCs and CE-like cells markers were confirmed by qRT-PCR, immunocytochemistry, and western blot. After 18 days, cell sheets with cellular junctions were harvested by decreasing the temperature. This study thus represents the successful generation of CE-like cell sheets from hADSC-derived CE-like cells on EMD.

New developments in corneal endothelial cell replacement.

Corneal transplantation is currently the most effective treatment to restore corneal clarity in patients with endothelial disorders. Endothelial transplantation, either by Descemet membrane endothelial keratoplasty (DMEK) or by Descemet stripping (automated) endothelial keratoplasty (DS(A)EK), is a surgical approach that replaces diseased Descemet membrane and endothelium with tissue from a healthy donor eye. Its application, however, is limited by the availability of healthy donor tissue. To increase the pool of endothelial grafts, research has focused on developing new treatment options as alternatives to conventional corneal transplantation. These treatment options can be considered as either 'surgery-based', that is tissue-efficient modifications of the current techniques (e.g. Descemet stripping only (DSO)/Descemetorhexis without endothelial keratoplasty (DWEK) and Quarter-DMEK), or 'cell-based' approaches, which rely on invitro expansion of human corneal endothelial cells (hCEC) (i.e. cultured corneal endothelial cell sheet transplantation and cell injection). In this review, we will focus on the most recent developments in the field of the 'cell-based' approaches. Starting with the description of aspects involved in the isolation of hCEC from donor tissue, we then describe the different natural and bioengineered carriers currently used in endothelial cell sheet transplantation, and finally, we discuss the current 'state of the art' in novel therapeutic approaches such as endothelial cell injection.

In vitro expanded corneal endothelial cell sheets: towards a new therapeutic approach

AbstractCorneal transplantation is currently the only effective treatment solution to restore corneal clarity in patients with endothelial disorders. Descemet membrane endothelial keratoplasty (DMEK) is the most recent corneal transplantation technique that selectively replaces only Descemet membrane (DM) and endothelium with the same layers of a healthy donor. However, its application is limited by shortage of high‐quality healthy donor tissue. In order to increase the pool of endothelial grafts, new strategies have been developed, which include surgical‐based and tissue‐engineering based approaches.Tissue‐engineered approaches such as endothelial cell sheet transplantation and cell injection rely on the in‐vitro expansion of cultured corneal endothelial cells (CEC). For endothelial cell sheet transplantation suitable cell carriers are required, which could either be natural carriers (e.g. human anterior lens capsules (HALC)) or bioengineered matrices (e.g. 20 µm thick collagen‐based carriers (LK20)).Within the ARREST BLINDNESS project, CEC were cultured on different carriers and the suitability of the cell‐carrier sheets as tissue‐engineered grafts for DMEK‐like procedures was evaluated by performing in‐vitro surgeries. Upon in‐vitro DMEK surgery, CEC‐HALC sheets behaved similarly to a standard DMEK‐graft on all scored aspects such as graft implantation, unfolding and adherence to bare stroma, whereas graft adhesion and surgical handling, respectively, are issues that still need to be addressed for CEC‐LK20 and CEC‐‘denuded DM’ sheets. First results of in‐vivo tests in an animal model will be presented as well as measures to approach legislative issues.

Optimization of Cultured Human Corneal Endothelial Cell Sheet Transplantation and Post-Operative Sheet Evaluation in a Rabbit Model

ABSTRACTPurpose: To optimize cultured human corneal endothelial cell (cHCEC) sheet transplantation technique for maintenance of cHCEC viability.Materials and Methods: cHCEC sheets cultured on a collagen scaffold were covered with or without Viscoat® and exposed to humidified air in the incubator. cHCEC sheets with or without Viscoat® were transplanted into cadaveric porcine eyes by the DSAEK technique with full air tamponade and incubated for various time periods. Then cell viability was determined by using the live/dead assay kit. cHCEC sheets with Viscoat® were transplanted into rabbit eyes and the sheets were histologically evaluated before and 14 days after transplantation.Results: A collagen scaffold and Viscoat® were effective for protecting cHCEC from damage due to air exposure in vitro. All cells died after 18 hours of air exposure in porcine eyes in Viscoat® untreated control. In contrast, Viscoat® treatment sustained full cell viability following 2 hours and could maintain approximately 80% viability after 18 hours. In a rabbit model, transplanted cHCEC sheet with Viscoat® maintained cell density at 2803 ± 229 mm2 (18% cell loss) and expression of N-cadherin, zonula occludens-1, and actin-filament localized to cell boundary as similar as donor HCEC.Conclusion: Viscoat® can contribute to cHCEC protection from damage caused by exposure to air.

Hyaluronic acid concentration-mediated changes in structure and function of porous carriers for corneal endothelial cell sheet delivery

In this study, the effects of hyaluronic acid (HA) concentrations (0.05–1.25wt.%) on the properties of porous carriers for corneal endothelial tissue engineering were investigated. The pore size and porosity gradually increased with decreasing solid content. However, at relatively low HA concentration (i.e., 0.05wt.%), the material samples contained small interior pores and a dense surface skin layer, probably due to no gas bubble effect on the stirring processing of porous microstructures of freeze-dried polysaccharide hydrogels. The carriers prepared from 0.25wt.% HA solution had the highest freezable water content and oxygen and glucose permeability among the samples evaluated. Results of cell viability assays and quantitative real-time reverse transcription polymerase chain reaction analyses showed that the HA concentration-related alteration of porous microstructure dictates the compatibility of biopolymer carriers with corneal endothelial cell (CEC) cultures. In vivo studies demonstrated that the CEC sheet/HA carrier construct implants are therapeutically efficacious in the reconstruction of endothelial scrape-wounded corneas. It is concluded that the polysaccharide concentration is the major factor for affecting the processing of carriers and their structure and function. Porous hydrogels prepared from 0.25wt.% HA solution are capable of delivering bioengineered CEC sheets to the posterior surface of cornea.

Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering.

Hyaluronic acid (HA) is a linear polysaccharide naturally found in the eye and therefore is one of the most promising biomaterials for corneal endothelial regenerative medicine. This study reports, for the first time, the development of overrun-processed porous HA hydrogels for corneal endothelial cell (CEC) sheet transplantation and tissue engineering applications. The hydrogel carriers were characterized to examine their structures and functions. Evaluations of carbodiimide cross-linked air-dried and freeze-dried HA samples were conducted simultaneously for comparison. The results indicated that during the fabrication of freeze-dried HA discs, a technique of introducing gas bubbles in the aqueous biopolymer solutions can be used to enlarge pore structure and prevent dense surface skin formation. Among all the groups studied, the overrun-processed porous HA carriers show the greatest biological stability, the highest freezable water content and glucose permeability, and the minimized adverse effects on ionic pump function of rabbit CECs. After transfer and attachment of bioengineered CEC sheets to the overrun-processed HA hydrogel carriers, the therapeutic efficacy of cell/biopolymer constructs was tested using a rabbit model with corneal endothelial dysfunction. Clinical observations including slit-lamp biomicroscopy, specular microscopy, and corneal thickness measurements showed that the construct implants can regenerate corneal endothelium and restore corneal transparency at 4 weeks postoperatively. Our findings suggest that cell sheet transplantation using overrun-processed porous HA hydrogels offers a new way to reconstruct the posterior corneal surface and improve endothelial tissue function.

Development of a Bioengineered Corneal Endothelial Cell Sheet to Fit the Corneal Curvature

To evaluate a novel bioengineered corneal endothelial cell sheet that fits the curvature of the posterior corneal surface. A spherically curved gelatin hydrogel sheet (SCGS) was prepared by the dehydrothermal cross-linking method, and its permeability to water and protein was tested. Monkey corneal endothelial cells (MCECs) were seeded onto these hydrogel sheets, and the cells were examined by immunohistochemistry. Then MCEC-SCGS constructs were transplanted in monkeys with bullous keratopathy to assess the efficacy of the hydrogel sheets as a scaffold. The hydrogel sheets showed similar permeability to water and protein as that of atelocollagen and vitrigel sheets. After transplantation, the SCGS did not show wrinkling and adhered tightly to the posterior corneal surface, whereas the flat sheets developed wrinkles that inhibited tight adhesion. Monkey corneal endothelial cells grown on hydrogel sheets expressed anti-zonula occludens-1 (ZO-1), N-cadherin, and sodium, potassium, and adenosine triphosphatase (Na,K-ATPase) along the plasma membrane. In a monkey model of bullous keratopathy, transplanted MCEC-SCGS constructs showed good adhesion to the posterior corneal surface, with subsequent improvement of corneal edema and transparency. A novel MCEC-SCGS construct was effective in a monkey model of bullous keratopathy. The SCGS achieves close adhesion to the posterior corneal surface without wrinkling and may contribute to clinical transplantation of corneal endothelial cell sheets.

N-isopropylacrylamide-co-glycidylmethacrylate as a thermoresponsive substrate for corneal endothelial cell sheet engineering.

Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate) polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.

Tissue engineering of the corneal endothelium: a review of carrier materials.

Functional impairment of the human corneal endothelium can lead to corneal blindness. In order to meet the high demand for transplants with an appropriate human corneal endothelial cell density as a prerequisite for corneal function, several tissue engineering techniques have been developed to generate transplantable endothelial cell sheets. These approaches range from the use of natural membranes, biological polymers and biosynthetic material compositions, to completely synthetic materials as matrices for corneal endothelial cell sheet generation. This review gives an overview about currently used materials for the generation of transplantable corneal endothelial cell sheets with a special focus on thermo-responsive polymer coatings.

Regenerative medicine of cornea by cell sheet engineering using temperature-responsive culture surfaces

Recently, regenerative medicine has been focused on as next-generation definitive therapies for several diseases or injuries for which there are currently no effective treatments. These therapies have been rapidly developed through the effective fusion between different fields such as stem cell biology and biomaterials. So far, we have proposed “cell sheet engineering” through our core technology that simply applies alterations of the temperature which allows regulation of the attachment or detachment of living cells on the culture surfaces grafted with the temperature-responsive polymer “poly(N-isoproplyacrylamide)”. This technology enables us to construct bioengineered sheet-like tissues, termed “cell sheets”, without the need of using biodegradable scaffolds and protease treatments that are traditionally used. Therefore, our carrier-free cell sheets not only are independent of the issues observed in conventional methods, but also showed further advantages in the reconstruction of the corneal epithelium or endothelium (e.g. improvement of optical transparency and cell-cell interactions to host stroma in reconstructed tissues). Moreover, our approach does not have issues such as immune rejection or limited number of donors, due to the use of cell sheets derived from autologous limbal (in patients with unilateral disease) or oral mucosal epithelial cells (in patients with bilateral disorders). Indeed, we have successfully performed the clinical application for the reconstruction of ocular surfaces through the transplantation of our carrier-free corneal epithelial cell sheets, as evidenced by improved visual acuity as well as long-term maintenance of postoperative health conditions on ocular surfaces in all patients. We have also proposed a novel approach for the reconstruction of the corneal endothelium using corneal endothelial cell sheets by demonstrating its successful application. Thus, our cell sheet engineering technique provides a breakthrough in the field of regenerative medicine applied to the cornea.

Human corneal endothelial cell sheets for transplantation: Thermo-responsive cell culture carriers to meet cell-specific requirements

Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options.

EVALUATION OF CROSS-LINKING TIME FOR POROUS GELATIN HYDROGELS ON CELL SHEET DELIVERY PERFORMANCE

To overcome the drawbacks posed by surgical manipulation of bioengineered corneal endothelial cell (CEC) sheets, a simple stirring process combined with freeze-drying method was recently developed for the production of cross-linked porous gelatin hydrogels that can provide the support structure and improve the aqueous humor circulation. In this study, we further evaluated the influence of cross-linking time (0–48 h) on the delivery performance of carbodiimide modified gelatin carriers. It was found that smaller pore size, lower porosity, and larger superficial area were associated with increasing extent of cross-linking of the carrier discs. Although the hydrogels treated for short reaction time (i.e., below 6 h) had low resistance to initial nutrient permeation, these materials exhibited rapid swelling, implying a potential anterior segment tissue squeezing effect for use as intraocular implants. In addition, the delivery carriers with limited extent of cross-linking degraded too fast to be effective for retention of cell sheet grafts at the site of injury. By contrast, the gelatin samples with cross-linking degrees greater than 50% showed slower degradation rates and smaller porous structure, thereby possibly causing a significant inhibition of CEC proliferation. Cell sheet transfer studies demonstrated that the carrier discs with a high cross-linking degree (59.4 ± 1.3%) were more difficult to achieve stable cell attachment than their counterparts with a low cross-linking degree (48.3 ± 1.5%). Our findings suggest that among the cross-linked porous samples studied, 12 h is the best cross-linking reaction time for preparation of cell sheet carriers with suitable delivery performance.

Development of new therapeutic modalities for corneal endothelial disease focused on the proliferation of corneal endothelial cells using animal models

This review describes our recent attempts to develop new therapeutic modalities for corneal endothelial disease using animal models including non-human primate model in which the proliferative ability of corneal endothelial cells is severely limited, as is the case in humans. First, we describe our attempt to develop new surgical treatments using cultivated corneal endothelial cells for advanced corneal endothelial dysfunction. It includes two different approaches; a “corneal endothelial cell sheet transplantation” with cells grown on a type-I collagen carrier, and a “cell-injection therapy” combined with the application of Rho-kinase (ROCK) inhibitor. Recently, it was reported that the selective ROCK inhibitor, Y-27632, promotes cell adhesion and proliferation and inhibits the apoptosis of primate corneal endothelial cells in culture. When cultivated corneal endothelial cells were injected into the anterior chamber of animal eyes in the presence of ROCK inhibitor, endothelial cell adhesion was promoted and the cells achieved a high cell density and a morphology similar to corneal endothelial cells in vivo. We are also trying to develop a novel medical treatment for the early phase of corneal endothelial disease by the use of ROCK inhibitor eye drops. In rabbit and monkey experiments using partial endothelial dysfunction models, corneal endothelial wound healing was accelerated by the topical application of ROCK inhibitor to the ocular surface, and resulted in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. We are now trying to advance the clinical application of these new therapies for patients with corneal endothelial dysfunction.

Carbodiimide cross-linked hyaluronic acid hydrogels as cell sheet delivery vehicles: characterization and interaction with corneal endothelial cells

It was reported that cell-adhesive gelatin discs have been successfully used as delivery vehicles for intraocular grafting of bioengineered corneal endothelial cell sheets. Development of alternative biomaterials to bovine-based gelatin vehicles can potentially eliminate the risk of bovine spongiform encephalopathy. In the present work, to investigate whether it was appropriate for use as cell sheet delivery vehicles, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) cross-linked hyaluronic acid (HA) hydrogels were studied by determinations of morphological characteristic, mechanical and thermal property, water content, in vitro degradability and cytocompatibility. Glutaraldehyde (GTA) cross-linked HA samples were used for comparison. It was found that HA discs after cross-linking significantly increased its tensile stress but reduced its tensile strain, water uptake and enzymatic degradability. The results of differential scanning calorimetry demonstrated that cross-linking could lead to the alteration of polymer structure. In addition, the EDC-cross-linked HA discs had a smoother surface structure, a faster degradation rate and a relatively lower cytotoxicity as compared to the GTA cross-linked counterparts. It is concluded that EDC can be successfully applied for HA cross-linking to fabricate structurally stable, mechanically reinforced, readily deformable, transparent and cytocompatible HA hydrogel discs with the potential to be applied as delivery vehicles for corneal endothelial cell therapy.

Tissue-Engineered Human Corneal Endothelial Cell Sheet Transplantation in a Rabbit Model Using Functional Biomaterials

This study was performed to investigate whether transplantation of bioengineered human corneal endothelial cell (HCEC) sheet grafts into corneas denuded of endothelium could restore corneal function and clarity in a rabbit model. After being labeled with PKH26 fluorescent dye, the adult HCECs derived from eye bank corneas were cultivated on the thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surfaces for 3 weeks at 37 degrees C, and were harvested as transplantable cell sheets after incubation for 45 min at 20 degrees C. Attached by gelatin hydrogel discs, the bioengineered cell monolayers were transplanted to rabbit corneas denuded of endothelium (HCEC sheet group). Traumatized rabbit corneas were served as controls. Postsurgical corneas underwent clinical observations and histological examinations for 6 months. By transmission electron microscopy and Western blot analysis of zonula occludens-1 and Na+,K+ -adenosine triphosphatase proteins, the structure and function of HCEC sheets resembled those of native corneal endothelium. After endothelial cells were removed, corneas of each group turned severe edematous and opaque. In the HCEC sheet groups, corneal clarity was gradually restored and corneal thickness was significantly less than that in the control groups (P<0.05). The attached PKH26-positive HCECs spread on rabbit Descemet's membrane after receiving cell sheet grafts. Intraocular delivery of HCEC sheets by means of a minimally invasive technique (i.e., small-incision surgery using biodegradable hydrogels) demonstrated long-term graft integration with damaged corneas. These results indicate that using cultured HCECs and functional biomaterials, PNIPAAm and gelatin, an effective cell sheet-based therapy can be developed for the treatment of corneal endothelium deficiency.

Functional biomedical polymers for corneal regenerative medicine

Recent progress in biomedical polymer science has greatly contributed to rapid development of corneal regenerative medicine. In the past decades, scientists have achieved several major breakthroughs in corneal tissue reconstruction. Studies regarding the findings of core-and-skirt keratoprostheses for visual rehabilitation, biosynthetic tissue replacements for corneal transplantation, and thermo-responsive cell-detachable substrates for corneal cell sheet engineering have been reported by several groups of investigators. This brief overview focuses on the contributions of functional polymers in the applications of corneal regenerative medicine. The keratoprosthetic devices developed by our group using heterobifunctional silicone rubber membranes grafted with different bioactive functional groups showed promising results in animal studies. In addition, the fabrication and transplantation of bioengineered human corneal endothelial cell sheets by utilizing the functional biomedical polymers such as poly( N-isopropylacrylamide) and gelatin are discussed, especially for the significance of gelatin in the development of a potential intraocular delivery system for cell sheet grafts.

Thermo‐responsive poly(NiPAAm‐co‐DEGMA) substrates for gentle harvest of human corneal endothelial cell sheets

Gentle harvesting of corneal endothelial cell sheets grown in culture is of interest for the development of cornea replacement strategies. Thin films of a fast responding copolymer of N-isopropylacrylamide (NiPAAm) and diethyleneglycol methacrylate (DEGMA) with a phase transition temperature of 32 degrees C were prepared and evaluated for that purpose. The polymer layers were immobilized onto fluorocarbon substrates using low pressure argon plasma treatment. Cell culture and detachment experiments were performed with L929 mouse fibroblasts and human corneal endothelial cells (HCEC) at standard conditions. The hydrogel-coated supports were found to permit adhesion, spreading, and proliferation of both cell types. Harvesting of cell sheets was achieved upon lowering the temperature to about 30 degrees C. The formation of a closed monolayer as a crucial prerequisite for maintaining ionic pump function in HCEC was proven by ZO-1 immunostainung. Labeling of fibronectin indicated that the vast majority of the extracellular matrix is detached from the hydrogel coatings together with the cell layer. Inspired by this result, the reuse of the hydrogel-coated culture carriers was investigated confirming the suitability of the substrates for repeated cell harvesting. Altogether, the introduced thermoresponsive coating was found advantageous for the efficient generation of HCEC sheets and will be further utilized in transplantation strategies.

Effect of Charge and Molecular Weight on the Functionality of Gelatin Carriers for Corneal Endothelial Cell Therapy

Cell transplantation strategies usually involve the use of supporting carrier materials because of the soft and fragile nature of these grafts. In this work, a cell-adhesive gelatin hydrogel carrier was fabricated to deliver cultivated human corneal endothelial cell (HCEC) sheets, which were harvested from thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted culture surfaces. The carrier disks, consisting of gelatins with a different isoelectric point (IEP = 5.0 and 9.0) and a molecular weight (MW) ranging from 3 to 100 kDa, were subjected to 16.6 kGy gamma irradiation for sterilization. The effect of IEP and MW of the raw gelatins (i.e., before irradiation) on the functionality of sterilized disks was studied by determinations of mechanical property, water content, dissolution degree, and cytocompatibility. Irrespective of the IEP of raw gelatin, hydrogel disks prepared with high MW (100 kDa) exhibited a greater tensile strength, lower water content, and slower dissolution rate than those made of low MW gelatin (8 and 3 kDa). From the investigation of cellular responses to the disks, the negatively charged gelatin (IEP = 5.0) groups were more cytocompatible when compared with their positively charged counterparts (IEP = 9.0) at the same MW (100 kDa). Additionally, in the negatively charged gelatin groups, only a slight increase in pro-inflammatory cytokine expression was observed with increasing MW of gelatin from 3 to 100 kDa. It is concluded that the gamma-sterilized hydrogel disks made from raw gelatins (IEP = 5.0, MW = 100 kDa) with appropriate dissolution degree and acceptable cytocompatibility are capable of providing stable mechanical support, making these carriers promising candidates for intraocular delivery of cultivated HCEC sheets.

Functional human corneal endothelial cell sheets harvested from temperature‐responsive culture surfaces

This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen-coated dishes and, after several passages, expanded cells were then seeded onto novel temperature-responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+-ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H-ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+-ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.