Cryopreservation of amniotic membrane with and without glycerol additive

  • Malina Wagner
  • Peter Walter
  • Sabine Salla
  • Sandra Johnen
  • Niklas Plange
  • Stephan Rütten
  • Tamme W. Goecke
  • Matthias Fuest
Basic Science



Amniotic membrane (AM) is an essential tool in ocular surface reconstruction. In this study, we analyzed the differential effects of glycerol and straight storage at − 80 °C for up to 6 months on the structural, biological, and mechanical properties of amniotic membrane (AM).


Human placentae of 11 different subjects were analyzed. AMs were stored at − 80 °C, either with a 1:1 mixture of Dulbecco’s modified Eagle medium and glycerol (glycerol) or without any medium or additives (straight). Histological image analysis, tensile strength, cell viability, and basic fibroblast growth factor (bFGF) secretion were evaluated at 0.5, 1, 3, and 6 months.


Histologically, neither glycerol nor straight storage significantly altered the epithelial or stromal structure of the AM. However, the cell number of the stroma was significantly reduced during the freezing process, independently of the storage method (p = 0.05–0.001). Tensile strength and Young’s modulus were not influenced by the storage method, but longer storage periods significantly increased the tensile strength of the AMs (p = 0.028). Cell viability was higher in glycerol rather than straight AM samples for up to 3 months of storage (p = 0.047–0.03). Secretion of bFGF at 3 months of storage was significantly higher in glycerol versus straight frozen AM samples (p = 0.04).


Glycerol led to higher cell viability and higher bFGF secretion for up to 3 months of AM storage. However, no significant differences between the two methods were observed at 6 months of storage at − 80 °C.


Amniotic membrane Ocular surface Wound healing Storage 



This work was supported by the Cornea Bank Aachen. We thank Anna Dobias and Claudia Werner for their assistance.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee (Medical Ethical Review Board, University RWTH Aachen (EK 178/15)) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of OphthalmologyRWTH Aachen UniversityAachenGermany
  2. 2.Department of Electron MicroscopyUniversity Hospital RWTH AachenAachenGermany
  3. 3.Department of Gynecology and ObstetricsRWTH Aachen UniversityAachenGermany

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