Advertisement

Fabrication of Corneal Extracellular Matrix-Derived Hydrogels

  • Mark AhearneEmail author
  • Julia Fernández-Pérez
Protocol
  • 49 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2145)

Abstract

Hydrogels derived from corneal extracellular matrix (ECM) represent a promising biomaterial for corneal repair and regeneration. To fabricate these hydrogels, first corneas need to be decellularized using repeated freeze-thaw cycles and nucleases to remove all nuclear and cellular components. The remaining corneal ECM is lyophilized to remove all water and milled into a fine powder. The ECM powder is weighed and dissolved in pepsin solution at a concentration of 20 mg/mL. Hydrogels are formed by neutralizing the pH of the solution and maintaining it at 37 °C until fibrillogenesis has occurred. Corneal stromal cells may be suspended throughout the hydrogel solution prior to gelation to generate a corneal stromal substitute.

Key words

Hydrogel Scaffold Cornea Stroma Collagen Extracellular matrix Keratocyte 

Notes

Acknowledgements

The research is supported by funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 637460) and Science Foundation Ireland and Marie-Curie Action COFUND (grant no. 11/SIRG/B2104).

References

  1. 1.
    Gain P, Jullienne R, He Z, Aldossary M, Acquart S, Cognasse F, Thuret G (2016) Global survey of corneal transplantation and eye banking. JAMA Ophthalmol 134(2):167–173.  https://doi.org/10.1001/jamaophthalmol.2015.4776CrossRefPubMedGoogle Scholar
  2. 2.
    Fernandez-Perez J, Ahearne M (2019) Influence of biochemical cues in human corneal stromal cell phenotype. Curr Eye Res 44(2):135–146.  https://doi.org/10.1080/02713683.2018.1536216CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Petroll WM, Miron-Mendoza M (2015) Mechanical interactions and crosstalk between corneal keratocytes and the extracellular matrix. Exp Eye Res 133:49–57.  https://doi.org/10.1016/j.exer.2014.09.003CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Jester JV, Ho-Chang J (2003) Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines control in vitro contractility and extracellular matrix contraction. Exp Eye Res 77(5):581–592CrossRefGoogle Scholar
  5. 5.
    Lynch AP, O'Sullivan F, Ahearne M (2016) The effect of growth factor supplementation on corneal stromal cell phenotype in vitro using a serum-free media. Exp Eye Res 151:26–37.  https://doi.org/10.1016/j.exer.2016.07.015CrossRefPubMedGoogle Scholar
  6. 6.
    Hassell JR, Birk DE (2010) The molecular basis of corneal transparency. Exp Eye Res 91(3):326–335.  https://doi.org/10.1016/j.exer.2010.06.021CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Massoudi D, Malecaze F, Galiacy SD (2016) Collagens and proteoglycans of the cornea: importance in transparency and visual disorders. Cell Tissue Res 363(2):337–349.  https://doi.org/10.1007/s00441-015-2233-5CrossRefPubMedGoogle Scholar
  8. 8.
    Meek KM (2009) Corneal collagen-its role in maintaining corneal shape and transparency. Biophys Rev 1(2):83–93.  https://doi.org/10.1007/s12551-009-0011-xCrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lynch AP, Wilson SL, Ahearne M (2016) Dextran preserves native corneal structure during decellularization. Tissue Eng Part C Methods 22(6):561–572.  https://doi.org/10.1089/ten.TEC.2016.0017CrossRefPubMedGoogle Scholar
  10. 10.
    Wilson SL, Sidney LE, Dunphy SE, Dua HS, Hopkinson A (2016) Corneal decellularization: a method of recycling unsuitable donor tissue for clinical translation? Curr Eye Res 41(6):769–782.  https://doi.org/10.3109/02713683.2015.1062114CrossRefPubMedGoogle Scholar
  11. 11.
    Ahearne M, Lynch AP (2015) Early observation of extracellular matrix-derived hydrogels for corneal stroma regeneration. Tissue Eng Part C Methods 21(10):1059–1069.  https://doi.org/10.1089/ten.TEC.2015.0008CrossRefPubMedGoogle Scholar
  12. 12.
    Lu Y, Yao QK, Feng B, Yan CX, Zhu MY, Chen JZ, Fu W, Fu Y (2015) Characterization of a hydrogel derived from decellularized corneal extracellular matrix. J Biomater Tiss Eng 5(12):951–960.  https://doi.org/10.1166/jbt.2015.1410CrossRefGoogle Scholar
  13. 13.
    Saldin LT, Cramer MC, Velankar SS, White LJ, Badylak SF (2017) Extracellular matrix hydrogels from decellularized tissues: structure and function. Acta Biomater 49:1–15.  https://doi.org/10.1016/j.actbio.2016.11.068CrossRefPubMedGoogle Scholar
  14. 14.
    Kim H, Park MN, Kim J, Jang J, Kim HK, Cho DW (2019) Characterization of cornea-specific bioink: high transparency, improved in vivo safety. J Tissue Eng 10:2041731418823382.  https://doi.org/10.1177/2041731418823382CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kim H, Jang J, Park J, Lee KP, Lee S, Lee DM, Kim KH, Kim HK, Cho DW (2019) Shear-induced alignment of collagen fibrils using 3d cell printing for corneal stroma tissue engineering. Biofabrication 11(3).  https://doi.org/10.1088/1758-5090/ab1a8b
  16. 16.
    Ahearne M, Coyle A (2016) Application of uva-riboflavin crosslinking to enhance the mechanical properties of extracellular matrix derived hydrogels. J Mech Behav Biomed Mater 54:259–267.  https://doi.org/10.1016/j.jmbbm.2015.09.035CrossRefPubMedGoogle Scholar
  17. 17.
    Fernandez-Perez J, Ahearne M (2019) Decellularization and recellularization of cornea: progress towards a donor alternative. Methods 171:86–96.  https://doi.org/10.1016/j.ymeth.2019.05.009
  18. 18.
    Fernandez-Perez J, Ahearne M (2019) The impact of decellularization methods on extracellular matrix derived hydrogels. Sci Rep 9(1):14933.  https://doi.org/10.1038/s41598-019-49575-2CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Crapo PM, Gilbert TW, Badylak SF (2011) An overview of tissue and whole organ decellularization processes. Biomaterials 32(12):3233–3243.  https://doi.org/10.1016/j.biomaterials.2011.01.057CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ahearne M, Liu KK, El Haj AJ, Then KY, Rauz S, Yang Y (2010) Online monitoring of the mechanical behavior of collagen hydrogels: influence of corneal fibroblasts on elastic modulus. Tissue Eng Part C Methods 16(2):319–327.  https://doi.org/10.1089/ten.TEC.2008.0650CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College DublinThe University of DublinDublinIreland
  2. 2.Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College DublinThe University of DublinDublinIreland

Personalised recommendations