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The Literature | April 2021

Alternatives to Human Corneal Donor Tissue

Using discarded lenticules as scaffolding for engineered corneal tissue.

Decellularization and recellularization of cornea: Progress towards a donor alternative

Fernández-Pérez J, Ahearne M1
Industry support: No


This review article explores corneal bioengineering as a method of generating transplantable tissue. Fernández-Pérez and Ahearne examine various techniques of decellularization, analytical techniques to confirm decellularization efficiency, different cell sources for recellularization, and recellularization methods. These alternative sources of tissue are being developed in response to a global shortage of human corneal donor tissue. The issue of immune rejection is also being addressed through the use of other transplantable tissues.2

Study in Brief

A worldwide scarcity of corneal tissue is spurring research on alternative tissue options. This review article examines multiple sources of tissue, including animals, human hydrogel, and decellularized and recellularized extracellular matrix tissue.


Patients who are blind from bilateral corneal disease can benefit from corneal transplantation. More viable options for corneal tissue would assist a greater number of these patients.


Collagen hydrogel and extracellular matrix (ECM) are the main components of transplantable tissue. Biologic tissue alternatives use collagen-based materials, scaffolding with hydrogel matrices, and, most recently, 3D printing.3,4

ECM can be created in vitro, but the process is expensive and takes longer than desired.5 ECM derived from decellularized corneas is an attractive alternative. In decellularization, mechanical agitation and detergents, chemicals, biologic agents, or organic acids are used to break down the cells.6,7 Decellularization is validated in three main ways: (1) the absence of cell nuclei is demonstrated, (2) anti–double-stranded DNA is quantified, and (3) the length of DNA remnants below a certain length is maintained.8 The structure, transparency, and composition of the corneal tissue must be maintained for it to be usable. Thus far, the results with different techniques have not been reproducible.

Tissue from other species has been used to study decellularization, but it can be highly immunogenic to humans if transplanted.9 For research purposes, human tissue has recently become available in the form of corneal stromal tissue from refractive surgery procedures that would normally be discarded.10

Recellularizing decellularized corneas with human cells is a process that can be used to create more viable tissue for possible transplantation.7 Because keratocytes are derived from neural crest mesenchyme, there are several sources for corneal recellularization. Autologous cells from the contralateral eye, for example, are a safe source of cells. The process of recellularization is difficult, however, and requires seeding the cells directly into the stroma.11

Must corneal tissue be recellularized before transplantation? This is currently unknown. Acellular transplants have been successful in animal models.12 The few human studies conducted involved patients who needed anterior lamellar corneal transplants; researchers reported no benefit from using recellularized tissue versus acellular matrix tissue.13

Additional studies are required to determine proper protocols for and to standardize methods of decellularizing and recellularizing corneal tissue to be used for human transplantation.

Engineering a Corneal Stromal Equivalent Using a Novel Multilayered Fabrication Assembly Technique

Fernández-Pérez J, Madden PW, Ahearne M14
Industry support: No


In an attempt to fabricate corneal substitutes for transplantation simply and quickly, Fernández-Pérez et al sought to construct mature tissue from decellularized porcine corneal sheets layered with collagen hydrogel. They evaluated three key parameters to determine utility: (1) transparency, (2) cellular viability, and (3) phenotype.

Study in Brief

This study evaluated decellularized porcine corneal sheets layered with human hydrogel tissue as a possible alternative to corneal tissue. Investigators measured key parameters of corneal viability, including transparency, anti–double-stranded DNA content, histology, suturability, and tissue strength.


New sources of human cells for recellularization of the corneal extracellular matrix may increase the availability of viable corneal transplant tissue.

The investigators reported success with decellularization and recellularization of the lenticules used—high cell viability, keratocyte-like phenotype, and integration into the host cornea with acceptable optical properties. Tissue could be surgically manipulated and sutured without tearing.


Globally, one human donor cornea is available for every 70 needed.15 Fernández-Pérez et al interlaced layers of decellularized porcine cornea with hydrogels that were laden with human corneal cells to produce a new corneal substitute. Tissue was created by decellularizing porcine corneal tissue. DNA content and degradation were tested, and human cell cultures were isolated. Four gels and five sheets of matrix were combined and allowed to grow for 3 weeks.

The cells were prepared and examined with laser scanning confocal microscopy, transparency and transmittance were tested, and a polymerase chain reaction was performed to measure the amount of cell RNA remaining. Histologic and immunohistochemical analyses were done on the tissue as well. Additionally, tissue was sutured to assess its strength and clinical viability.

Results showed highly transparent cells with no cell nuclei remaining and demonstrated a mild effect on corneal thickness from swelling during decellularization. Based on the parameters measured, this corneal stromal equivalent appears to be a viable alternative to corneal tissue.

1. Fernández-Pérez J, Ahearne M. Decellularization and recellularization of cornea: progress towards a donor alternative. Methods. 2020;171:86-96.

2. Matthyssen S, Van den Bogerd B, Dhubhghaill SN, Koppen C, Zakaria N. Corneal regeneration: a review of stromal replacements. Acta Biomater. 2018;69:31-41.

3. Liu W, Merrett K, Griffith M, et al. Recombinant human collagen for tissue engineered corneal substitutes. Biomaterials. 2008;29(9):1147-1158.

4. Fagerholm P, Lagali NS, Merrett K, et al. A biosynthetic alternative to human donor tissue for inducing corneal regeneration: 24-month follow-up of a phase 1 clinical study. Sci Transl Med. 2010;2(46):46ra61.

5. Guo X, Hutcheon AEK, Melotti SA, Zieske JD, Trinkaus-Randall V, Ruberti JW. Morphologic characterization of organized extracellular matrix decomposition by ascorbic acid–stimulated human corneal fibroblasts. Invest Ophthalmol Vis Sci. 2007;48(9):4050-4060.

6. Oh JY, Kim MK, Lee HJ, Ko JH, Wee WR, Lee JH. Processing porcine cornea for biomedical applications. Tissue Eng Part C Methods. 2009;15(4):635-645.

7. Diao JM, Pang X, Qiu Y, Miao Y, Yu MM, Fan TJ. Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata. Exp Eye Res. 2015;132:216-224.

8. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials. 2011;32(12):3233-3243.

9. Wang RG, Ruan M, Zhang RJ, et al. Antigenicity of tissues and organs from GGTA1/CMAH/Β4GalNT2 triple gene knockout pigs. J Biomed Res. 2018;33(4):235-243.

10. Yin H, Qiu P, Wu F, et al. Construction of a corneal stromal equivalent with SMILE-derived lenticules and fibrin glue. Sci Rep. 2016;6:33848.

11. Gonzalez-Andrades M, de la Cruz Cardona J, Ionescu AM, Campos A, Del Mar Perez M, Alaminos M. Generation of bioengineered corneas with decellularized xenografts and human keratocytes. Invest Ophthalmol Vis Sci. 2011;52(1):215-222.

12. Xu YG, Xu YS, Huang C, Feng Y, Li Y, Wang W. Development of a rabbit corneal equivalent using an acellular corneal matrix of a porcine substrate. Mol Vis. 2008;14:2180-2189.

13. Alió Del Barrio JL, El Zarif M, Azaar A, et al. Corneal stroma enhancement with decellularized stromal laminas with or without stem cell recellularization for advanced keratoconus. Am J Ophthalmol. 2018;186:47-58.

14. Fernández-Pérez J, Madden PW, Ahearne M. Engineering a corneal stromal equivalent using a novel multilayered fabrication assembly technique. Tissue Eng Part A. 2020;26(19-20):1030-1041.

15. Gain P, Jullienne R, He Z, Aldossary M, Acquart S, Cognasse F, Thuret G. Global survey of corneal transplantation and eye banking. JAMA Ophthalmol. 2016;134(2):167-173.

Section Editor Edward Manche, MD
  • Director of Cornea and Refractive Surgery, Stanford Laser Eye Center, California
  • Professor of Ophthalmology, Stanford University School of Medicine, California
  • edward.manche@stanford.edu
  • Financial disclosure: None
Lisa Martén, MD, MPH
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