Skip to main content
SpringerLink
Log in

Cryopreservation of amniotic membrane with and without glycerol additive

  • Basic Science
  • Published:
Graefe's Archive for Clinical and Experimental Ophthalmology Aims and scope Submit manuscript

Abstract

Purpose

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).

Methods

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.

Results

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).

Discussion

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Bourne G (1962) The foetal membranes. A review of the anatomy of normal amnion and chorion and some aspects of their function. Postgrad Med J 38:193–201

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Tan EK, Cooke M, Mandrycky C, Mahabole M, He H, O'Connell J, McDevitt TC, Tseng SCG (2014) Structural and biological comparison of cryopreserved and fresh amniotic membrane tissues. J Biomater Tissue Eng 4:379–388. https://doi.org/10.1166/jbt.2014.1180

    Article  CAS  Google Scholar 

  3. de Rotth A (1940) Plastic repair of conjunctival defects with fetal membranes. Arch Ophthalmol 23:522–525. https://doi.org/10.1001/archopht.1940.00860130586006

    Article  Google Scholar 

  4. Sorsby A, Symons HM (1946) Amniotic membrane grafts in caustic burns of the eye (burns of the second degree). Br J Ophthalmol 30:337–345

    Article  PubMed Central  PubMed  Google Scholar 

  5. Kim JC, Tseng SC (1995) Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea 14:473–484

    Article  PubMed  CAS  Google Scholar 

  6. Lee SH, Tseng SC (1997) Amniotic membrane transplantation for persistent epithelial defects with ulceration. Am J Ophthalmol 123:303–312

    Article  PubMed  CAS  Google Scholar 

  7. Tseng SCG, Espana EM, Kawakita T, Di Pascuale MA, Li W, He H, Liu T-S, Cho T-H, Gao Y-Y, Yeh L-K, Liu C-Y (2004) How does amniotic membrane work? Ocul Surf 2:177–187. https://doi.org/10.1016/s1542-0124(12)70059-9

    Article  PubMed  Google Scholar 

  8. Tsubota K, Satake Y, Kaido M, Shinozaki N, Shimmura S, Bissen-Miyajima H, Shimazaki J (1999) Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation. N Engl J Med 340:1697–1703. https://doi.org/10.1056/nejm199906033402201

    Article  PubMed  CAS  Google Scholar 

  9. Akle CA, Adinolfi M, Welsh KI, Leibowitz S, McColl I (1981) Immunogenicity of human amniotic epithelial cells after transplantation into volunteers. Lancet 2:1003–1005

    Article  PubMed  CAS  Google Scholar 

  10. Meller D, Pauklin M, Thomasen H, Westekemper H, Steuhl KP (2011) Amniotic membrane transplantation in the human eye. Dtsch Arztebl Int 108:243–248. https://doi.org/10.3238/arztebl.2011.0243

    Article  PubMed  PubMed Central  Google Scholar 

  11. Liu J, Sheha H, Fu Y, Liang L, Tseng SC (2010) Update on amniotic membrane transplantation. Expert Rev Ophthalmol 5:645–661. https://doi.org/10.1586/eop.10.63

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Hahn A, Thanos M, Reinhard T, Seitz B, Steuhl KP, Meller D (2010) Procedural guidelines. Good practice procedures for acquisition and preparation of cryopreserved human amniotic membranes from donor placentas. Ophthalmologe 107:1020–1031. https://doi.org/10.1007/s00347-010-2269-6

    Article  PubMed  CAS  Google Scholar 

  13. Hennerbichler S, Reichl B, Pleiner D, Gabriel C, Eibl J, Redl H (2007) The influence of various storage conditions on cell viability in amniotic membrane. Cell Tissue Bank 8:1–8. https://doi.org/10.1007/s10561-006-9002-3

    Article  PubMed  CAS  Google Scholar 

  14. Wolbank S, Hildner F, Redl H, van Griensven M, Gabriel C, Hennerbichler S (2009) Impact of human amniotic membrane preparation on release of angiogenic factors. J Tissue Eng Regen Med 3:651–654. https://doi.org/10.1002/term.207

    Article  PubMed  CAS  Google Scholar 

  15. Thomasen H, Pauklin M, Noelle B, Geerling G, Vetter J, Steven P, Steuhl KP, Meller D (2011) The effect of long-term storage on the biological and histological properties of cryopreserved amniotic membrane. Curr Eye Res 36:247–255. https://doi.org/10.3109/02713683.2010.542267

    Article  PubMed  CAS  Google Scholar 

  16. Crouch SP, Kozlowski R, Slater KJ, Fletcher J (1993) The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J Immunol Methods 160:81–88

    Article  PubMed  CAS  Google Scholar 

  17. Azuara-Blanco A, Pillai CT, Dua HS (1999) Amniotic membrane transplantation for ocular surface reconstruction. Br J Ophthalmol 83:399–402

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Espana EM, Prabhasawat P, Grueterich M, Solomon A, Tseng SC (2002) Amniotic membrane transplantation for reconstruction after excision of large ocular surface neoplasias. Br J Ophthalmol 86:640–645

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Kruse FE, Joussen AM, Rohrschneider K, You L, Sinn B, Baumann J, Volcker HE (2000) Cryopreserved human amniotic membrane for ocular surface reconstruction. Graefes Arch Clin Exp Ophthalmol 238:68–75

    Article  PubMed  CAS  Google Scholar 

  20. Gage AA, Baust JM, Baust JG (2009) Experimental cryosurgery investigations in vivo. Cryobiology 59:229–243. https://doi.org/10.1016/j.cryobiol.2009.10.001

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Shepherd JP, Dawber RP (1984) Wound healing and scarring after cryosurgery. Cryobiology 21:157–169

    Article  PubMed  CAS  Google Scholar 

  22. Aupperle H, Doll N, Walther T, Kornherr P, Ullmann C, Schoon HA, Mohr FW (2005) Ablation of atrial fibrillation and esophageal injury: effects of energy source and ablation technique. J Thorac Cardiovasc Surg 130:1549–1554. https://doi.org/10.1016/j.jtcvs.2005.06.052

    Article  PubMed  Google Scholar 

  23. Tadrous PJ (2010) On the concept of objectivity in digital image analysis in pathology. Pathology 42:207–211. https://doi.org/10.3109/00313021003641758

    Article  PubMed  Google Scholar 

  24. Oxlund H, Helmig R, Halaburt JT, Uldbjerg N (1990) Biomechanical analysis of human chorioamniotic membranes. Eur J Obstet Gynecol Reprod Biol 34:247–255

    Article  PubMed  CAS  Google Scholar 

  25. Nakamura T, Yoshitani M, Rigby H, Fullwood NJ, Ito W, Inatomi T, Sotozono C, Nakamura T, Shimizu Y, Kinoshita S (2004) Sterilized, freeze-dried amniotic membrane: a useful substrate for ocular surface reconstruction. Invest Ophthalmol Vis Sci 45:93–99. https://doi.org/10.1167/iovs.03-0752

    Article  PubMed  Google Scholar 

  26. Dumont F, Marechal PA, Gervais P (2004) Cell size and water permeability as determining factors for cell viability after freezing at different cooling rates. Appl Environ Microbiol 70:268–272

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Ahmad S, Singh VA, Hussein SI (2017) Cryopreservation versus fresh frozen meniscal allograft: a biomechanical comparative analysis. J Orthop Surg (Hong Kong) 25:2309499017727946. https://doi.org/10.1177/2309499017727946

    Article  Google Scholar 

  28. Muldrew K, McGann LE (1990) Mechanisms of intracellular ice formation. Biophys J 57:525–532. https://doi.org/10.1016/s0006-3495(90)82568-6

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Elder E, Chen Z, Ensley A, Nerem R, Brockbank K, Song Y (2005) Enhanced tissue strength in cryopreserved, collagen-based blood vessel constructs. Transplant Proc 37:4625–4629. https://doi.org/10.1016/j.transproceed.2005.10.033

    Article  PubMed  CAS  Google Scholar 

  30. Koizumi NJ, Inatomi TJ, Sotozono CJ, Fullwood NJ, Quantock AJ, Kinoshita S (2000) Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res 20:173–177

    Article  PubMed  CAS  Google Scholar 

  31. Tseng SC, Li DQ, Ma X (1999) Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. J Cell Physiol 179:325–335. https://doi.org/10.1002/(sici)1097-4652(199906)179:3<325::aid-jcp10>3.0.co;2-x

    Article  PubMed  CAS  Google Scholar 

  32. Laurent R, Nallet A, Obert L, Nicod L, Gindraux F (2014) Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank. Cell Tissue Bank 15:267–275. https://doi.org/10.1007/s10561-014-9437-x

    Article  PubMed  Google Scholar 

  33. Perepelkin NM, Hayward K, Mokoena T, Bentley MJ, Ross-Rodriguez LU, Marquez-Curtis L, McGann LE, Holovati JL, Elliott JA (2016) Cryopreserved amniotic membrane as transplant allograft: viability and post-transplant outcome. Cell Tissue Bank 17:39–50. https://doi.org/10.1007/s10561-015-9530-9

    Article  PubMed  Google Scholar 

  34. Cui HG, Li HY (2007) Effect of basic fibroblast growth factor (bFGF) on the treatment of exposure of the orbital implants. J Zhejiang Univ Sci B 8:620–625. https://doi.org/10.1631/jzus.2007.B0620

    Article  PubMed  PubMed Central  Google Scholar 

  35. Modaresifar K, Azizian S, Zolghadr M, Moravvej H, Ahmadiani A, Niknejad H (2017) The effect of cryopreservation on anti-cancer activity of human amniotic membrane. Cryobiology 74:61–67. https://doi.org/10.1016/j.cryobiol.2016.12.001

    Article  PubMed  CAS  Google Scholar 

  36. Mahbod M, Shahhoseini S, Khabazkhoob M, Beheshtnejad AH, Bakhshandeh H, Atyabi F, Hashemi H (2014) Amniotic membrane extract preparation: what is the best method? J Ophthalmic Vis Res 9:314–319. https://doi.org/10.4103/2008-322x.143367

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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

Author information

Authors and Affiliations

  1. Department of Ophthalmology, RWTH Aachen University, Pauwelsstraße 30, 52074, Aachen, Germany

    Malina Wagner, Peter Walter, Sabine Salla, Sandra Johnen, Niklas Plange & Matthias Fuest

  2. Department of Electron Microscopy, University Hospital RWTH Aachen, Aachen, Germany

    Stephan Rütten

  3. Department of Gynecology and Obstetrics, RWTH Aachen University, Aachen, Germany

    Tamme W. Goecke

Authors
  1. Malina Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter Walter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sabine Salla
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sandra Johnen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Niklas Plange
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stephan Rütten
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tamme W. Goecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Matthias Fuest
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matthias Fuest.

Ethics declarations

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wagner, M., Walter, P., Salla, S. et al. Cryopreservation of amniotic membrane with and without glycerol additive. Graefes Arch Clin Exp Ophthalmol 256, 1117–1126 (2018). https://doi.org/10.1007/s00417-018-3973-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00417-018-3973-1

Keywords

Search

Navigation