1. Himi Tripathi 1, Deepika Gupta 2, Sujata Mohanty 3, Seema Sen 4, Seema Kashyap 4, Radhika Tandon 1 1 Department of Ophthalmology, Dr Rajendra Prasad Centre for Ophthalmic Sciences; 2 SMITA Research Labs, Department of Textile Technology, Indian Institute of Technology, New Delhi ; 3 Stem Cell Facility, All India Institute of Medical Sciences; 4 Department of Ocular Pathology, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences Submission ID: 14408 The authors have no financial interests to disclose Tissue Engineered Decellularized Human Corneal Stroma for Reconstructing the Corneal Endothelium in-Vitro

2. Introduction  The lack of an intact endothelium of sufficient cell density and functionality results in vision loss  Pseudophakic bullous keratopathy, aphakic bullous keratopathy, corneal endotheliopathy and Fuchs’ dystrophy are the diseases in which endothelium loss occurs and require only corneal endothelium replacement 1.  The approach to corneal transplantation has changed as the concept of customized corneal component replacement and therapy is advocated corneal tissue engineering emerges as a new frontier  Biological scaffolds are difficult to handle during the transplantation surgery and synthetic scaffolds often integrate poorly with host tissues 2  Besides synthetic polymers and biomaterials, naturally occurring biomaterials like cornea after decellularization, itself can be more promising as it has a unique extracellular matrix(ECM) organization 3 Use of different methods using mechanical, chemical and enzymatic protocols for decellularization of corneal stroma has been reported 4,5  Aim of our study was to evaluate decellularized corneal stromal scaffolds for cultivating human corneal endothelial cells (HCECs)

3. Materials and Methods Human donor corneas not suitable for surgery were used in the study After removing epithelium and endothelium mechanically, stromas were decellularized using six different protocols (mechanical, chemical and enzymatic using different concentrations of detergents, alcohol and trypsin EDTA) Those decellularized corneas were subjected to histological analysis to visualize cellular remnants and the general histoarchitecture of tissues; DNA extraction to estimate DNA concentration in untreated and decellularized tissues; scanning electron microscopy (SEM) for visualization of cellular morphology; alcian blue staining to preserve ECM immuhistochemistry for major structural proteins collagen I, II, IV, fibronectin; tensile strength to estimate the mechanical strength using uniaxial load testing equipment & optical transparency via UV- visible recording spectrophotometer to check the transparency of decellularized tissues 10,000 HCECs were seeded on the decellularized corneal tissue and placed in culture medium for 14 days and then analyzed for recellularization by histology SEM, live-dead staining and immunocytochemistry for endothelial specific markers ZO-1, Na + /K + -ATPase, VE-Cadherin & Connexin-43

4. Table 1: Protocols Used for Decellularization S.No.ProtocolsReagents usedTime @ temperature 1.Protocol I A (PIA)0.1%SDS&75% ethanol27 hrs; 20+ 2°C 2.Protocol I B (PIB)0.5%SDS&75% ethanol27 hrs; 20+ 2°C 3.Protocol II (PII)75% ethanol& trypsin-EDTA (0.05%)109 hrs;20+ 2°C 4.Protocol III (PIII)2% Triton X-100& 0.1% NH 4 OH72 hrs; 4°C 5.Protocol IV A (PIV A)0.5% trypsin 0.05%EDTA, 0.02mg/ml DNAse, 100 U/ml penicillin & 100μg/ml streptomycin 24 hrs; 37 °C 6.Protocol IV B (PIV B)0.25% trypsin 0.05%EDTA, 0.02mg/ml DNAse, 100 U/ml penicillin & 100μg/ml streptomycin 24 hrs; 37 °C

5. Results Histological analysis of corneas by H&E staining in untreated and corneas treated with different decellularization treatments: Keratocyte nuclei were observed in (A) untreated corneas while nuclei or cellular debris were absent from the corneas processed according to (B) protocol PIA, (C) PIB, (D)PII, (E)PIII, (F)PIVA, (G)PIVB while fibrillar structures were preserved DNA estimation of decellularized and untreated corneas 1. Verification of cell removal 1.1) Histological evaluation of decellularized corneas 1.2) DNA estimation

6. 2. Characterizations of Decellularized for Extra Cellular Matrix (ECM) Architecture The alcian blue staining confirmed an intact and well organized extracellular matrix in (A) untreated cornea and approximately unchanged stromal matrix in the decellularized corneas processed according to (B) protocol PIA, (C) PIB, (E)PIII, (F)PIVA, (G) PIVB except in PII (D) 2.1 Alcian blue staining 2.2 Collagen I staining

7. 2.3 Collagen II Staining2.4 Collagen IV Staining Immunohistological characterization of decellularized human corneal stromas for various ECM proteins : (A)Untreated and decellularized corneal stromas treated according to protocol (B) PIA, (C) PIB, (D) PII, (E) PIII, (F) PIVA and (G) PIVB were immunostained with collagen I, collagen II, collagen IV & fibronectin; the decellularized corneal stromas showing similar staining as untreated cornea for various ECM 2.5Fibronectin Staining

8. Scanning electron micrographs showing images of untreated and decellularized corneal stromas. The collagen fibrils appear to be interconnected and they formed collagen bundles regular and parallel to corneal surface. The results were similar in both the groups;(A)untreated & decellularized corneal stromas treated according to protocol (B) protocol PIA, (C) PIB, (D) PII, (E) PIII, (F) PIVA and (G) PIVB except in PII 3. Scanning Electron Micrographs Showing Morphology of Collagen Fibrils in Untreated & Decellularized Corneal Stromas 5. Mechanical Strength of Decellularized Corneal Stromas 4. Transmittance Assay for Optical Transparency Stress-Strain curve of decellularized corneal stroma Representative plot showing light transmittance percentage of all the decellularized &untreated corneas

9. 6. Repopulation of Decellularized Human Corneas HCECs cultured on decellularized corneal stroma: H&E images of decellularized tissue (A) unseeded & (B) HCECs repopulated on decelluarized corneal human stromas showed monolayer of cultured HCECs on decellularized corneal stroma; Cell viability staining of cultured human corneal endothelial cells over decellularized corneal stroma PIVB for two weeks: Viable monolayers was determined by staining with Live/Dead Viability Kit in which the live cells fluoresce green 6.3 SEM of Repopulated Decellularized Corneas Scanning electron microscopy images of HCECs cultured over decellularized stroma PIVB to evaluate the attachment ability at different time intervals for 2 weeks: (A) after 1 day, (B) after 3 days, (C) after 7 days and (D) after 14 days; (E) image of decellularized stroma (F) HCECs adhered to decellularized stroma and showing smooth cell sheet and close association with neighbourhood cells (G) Cultured cells showing numerous microvili at higher magnification. 6.1 Hematoxylin &Eosin Staining6.2 Live/Dead Cell Viability Staining

10. 6.4 Immunocytochemistry to Check the Phenotypic Expression of Repopulated Cells for HCECs Specific Markers Human corneal endothelial cells (HCECs) were cultured on decellularized corneal stroma PIVB for 2 weeks and immunocytochemistry results showed that they maintained their phenotypic properties by expressing Connexin-43 (A-C), VE-Cadherin(D-F), Zona occludens-1 (G-H) and Na + K + ATPase (J-L).

11. H&E staining confirmed efficient elimination of cellular components whereas alcian blue confirmed good preservation of ECM and major structural proteins collagen type I, II, IV& fibronectin. SEM results indicate that all the decellularization treatments except corneas treated with alcohol(PII) preserved native structure of the stromal collagen fibres and they were tight and had parallel arrangement similarly to untreated corneas. PII showed disorganized, irregular and loosen collagen fibers The estimated DNA concentration decreased in the decellularized corneas treated with different protocols (p<0.001). Decellularization did not compromise optical transmittance of the tissues treated with trypsin EDTA method. No significant difference was observed between untreated and decellularized corneas treated with PIII (p= 0.1), PIVA (p= 0.4)and PIVB (p= 0.1) tensile strength H& E staining of decellularized cornea with endothelial cells revealed that cells were able to form uniform monolayer of endothelial cells on the scaffold this result was supported by SEM. Cell viability staining revealed that decellularized corneas were biocompatible and support expansion of HCECs Moreover, they also expressed the endothelial specific markers Na + /K + -ATPase, ZO-1, VE- Cadherin& Connexin-43

12. Conclusions, References &Correspondence Decellularized stromas supported endothelial cell expansion and they retained phenotypic properties mainly intact barrier and ionic pump function References 1.Chen K.H. et al. Cornea 2001;20(7):731-737 2.Koizumi N et al. I.O.V.S 2007;48(10):4519-4526 3.Crapo P.M. et al. Biomaterials.2011;32:3233-3243 4.Choi J. et al Biomaterials.2010;31:6738-6745 5.Shao Y. et al Int. J. Ophthalmol. 2012; 5:415-418. Correspondence Dr. Radhika Tandon, Professor of Ophthalmology Dr Rajendra Prasad Centre for Ophthalmic Sciences All India Institute of Medical Sciences, New Delhi-110029, India email id : radhika_tan@yahoo.com 1.P.M. Crapo et al. Biomaterials 32 (2011) 3233e3243