Current Stem Cell Reports

, Volume 4, Issue 2, pp 138–148 | Cite as

Preservation Strategies that Support the Scale-up and Automation of Tissue Biomanufacturing

  • Shangping Wang
  • Gloria D. ElliottEmail author
Artificial Tissues (A Atala and JG Hunsberger, Section Editors)
Part of the following topical collections:
  1. Topical Collection on Artificial Tissues


Purpose of Review

Tissue engineering strategies to repair or replace tissues and organs that have been damaged by disease, trauma, or congenital issues usually require many weeks of production during which time patients are incapacitated or reliant on temporary devices. In order to fully meet the rising clinical demand of transplantable tissues/organs, various preservation technologies need to be implemented to create “off-the-shelf” availability of biological components and products. This review will focus on the preservation methods used for biological resources (cells, growth factors, and biological scaffolds) and also for the finished tissue-engineered constructs.

Recent Findings

Recent studies have demonstrated that conventional cryopreservation and vitrification preservation methods can maintain functionality and properties of cells and cell-seeded scaffolds during long-term storage. Lyophilization can also be used as an alternative strategy for engineered tissues that are devoid of cells. Additionally, fabrication technologies combined with freezing/thawing processes will likely emerge as the preferred strategy to better control the physical and biological properties of engineered tissues while simultaneously providing a shelf life for the product.


The development of preservation methodologies for tissue engineering would minimize the shortage of tissues/organs and offer an effective and commercialized strategy for improved automation of tissue biomanufacturing.


Cryopreservation CPA Dry preservation Tissue engineering Scaffolds Biomanufacturing 


Funding Information

This work was supported in part by grant #5RO1GM101796 from the National Institutes of Health (NIH) to GDE.

Compliance with Ethical Standards

Conflict of Interest

Shangping Wang and Gloria D. Elliott declare that they have no conflict of interest.

Human and Animal Rights

This article does not contain any studies with human or animal subjects performed by any of the authors.


Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance

  1. 1.
    Bártolo PJ, Chua CK, Almeida HA, Chou SM, Lim ASC. Biomanufacturing for tissue engineering: present and future trends. Virtual and Physical Prototyping. 2009;4(4):203–16. Scholar
  2. 2.
    Lee K, Silva EA, Mooney DJ. Growth factor delivery-based tissue engineering: general approaches and a review of recent developments. J R Soc Interface. 2011;8(55):153–70. Scholar
  3. 3.
    Teven C, Fisher S, Ameer G, He T-C, Reid R. Biomimetic approaches to complex craniofacial defects. Annals of Maxillofacial Surgery. 2015;5(1):4–13. Scholar
  4. 4.
    Bentley TS. 2014 U.S. organ and tissue transplant cost estimates and discussion. Milliman Research Report. 2014.Google Scholar
  5. 5.
    Organ preservation market by technique, organ type and preservation solution—global industry analysis and forecast to 2023. Crystal Market Research. 2017.Google Scholar
  6. 6.
    Giwa S, Lewis JK, Alvarez L, Langer R, Roth AE, Church GM, et al. The promise of organ and tissue preservation to transform medicine. Nat Biotechnol. 2017;35(6):530–42. Scholar
  7. 7.
    Neves LS, Rodrigues MT, Reis RL, Gomes ME. Current approaches and future perspectives on strategies for the development of personalized tissue engineering therapies. Expert Review of Precision Medicine and Drug Development. 2016;1(1):93–108. Scholar
  8. 8.
    Howard D, Buttery LD, Shakesheff KM, Roberts SJ. Tissue engineering: strategies, stem cells and scaffolds. J Anat. 2008;213(1):66–72. Scholar
  9. 9.
    Berz D, McCormack EM, Winer ES, Colvin GA, Quesenberry PJ. Cryopreservation of hematopoietic stem cells. Am J Hematol. 2007;82(6):463–72. Scholar
  10. 10.
    Hunt CJ. Cryopreservation of human stem cells for clinical application: a review. Transfus Med Hemother. 2011;38:107–23. Scholar
  11. 11.
    Woods EJ, Mullen SF. Organ preservation: cryobiology and beyond. Current Stem Cell Reports. 2016;2(2):104–17. Scholar
  12. 12.
    Katkov II, Kan NG, Cimadamore F, Nelson B, Snyder EY, Terskikh AV. DMSO-free programmed cryopreservation of fully dissociated and adherent human induced pluripotent stem cells. Stem Cells Int. 2011;8:1–8. Scholar
  13. 13.
    Grein TA, Freimark D, Weber C, Hudel K, Wallrapp C, Czermak P. Alternatives to dimethylsulfoxide for serum-free cryopreservation of human mesenchymal stem cells. The International Journal of Artificial Organs. 2010;33(6):370–80. CrossRefPubMedGoogle Scholar
  14. 14.
    Thirumala S, Wu X, Gimble JM, Devireddy RV. Evaluation of polyvinylpyrrolidone as a cryoprotectant for adipose tissue-derived adult stem cells. Tissue Eng Part C Methods. 2010;16(4):783–92. Scholar
  15. 15.
    Petrenko YA, Rogulska OY, Mutsenko VV, Petrenko AY. A sugar pretreatment as a new approach to the Me2SO- and xeno-free cryopreservation of human mesenchymal stromal cells. Cryo Letters. 2014;35(3):239–46.PubMedGoogle Scholar
  16. 16.
    Pasley S, Zylberberg C, Matosevic S. Natural killer-92 cells maintain cytotoxic activity after long-term cryopreservation in novel DMSO-free media. Immunol Lett. 2017;192:35–41. CrossRefPubMedGoogle Scholar
  17. 17.
    Li M, Feng C, Gu X, He Q, Wei F. Effect of cryopreservation on proliferation and differentiation of periodontal ligament stem cell sheets. Stem Cell Research & Therapy. 2017;8(1):77. Scholar
  18. 18.
    Roy I, Gupta MN. Freeze-drying of proteins: some emerging concerns. Biotechnol Appl Biochem. 2004;39(Pt 2):165–77. CrossRefPubMedGoogle Scholar
  19. 19.
    Arakawa T, Prestrelski SJ, Kenney WC, Carpenter JF. Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev. 2001;46(1–3):307–26.CrossRefPubMedGoogle Scholar
  20. 20.
    Dhandayuthapani B, Yoshida Y, Maekawa T, Kumar DS. Polymeric scaffolds in tissue engineering application: a review. International Journal of Polymer Science. 2011;2011:1–19. Scholar
  21. 21.
    El-Sherbiny IM, Yacoub MH. Hydrogel scaffolds for tissue engineering: progress and challenges. Global Cardiology Science & Practice. 2013;2013(3):316–42. Scholar
  22. 22.
    Baiguera S, Del Gaudio C, Jaus MO, Polizzi L, Gonfiotti A, Comin CE, et al. Long-term changes to in vitro preserved bioengineered human trachea and their implications for decellularized tissues. Biomaterials. 2012;33(14):3662–72. Scholar
  23. 23.
    Gallo M, Bonetti A, Poser H, Naso F, Bottio T, Bianco R, et al. Decellularized aortic conduits: could their cryopreservation affect post-implantation outcomes? A morpho-functional study on porcine homografts. Heart Vessel. 2016;31(11):1862–73. Scholar
  24. 24.
    Poornejad N, Frost TS, Scott DR, Elton BB, Reynolds PR, Roeder BL, et al. Freezing/thawing without cryoprotectant damages native but not decellularized porcine renal tissue. Organ. 2015;11(1):30–45. Scholar
  25. 25.
    Wolfinbarger L, Brockbank KGM, Hopkins RA. Application of cryopreservation to heart valves. In: Hopkins RA, editor. Cardiac reconstructions with allograft tissues. New York: Springer New York; 2005. p. 133–60.CrossRefGoogle Scholar
  26. 26.
    Liu Y, Luo D, Wang T. Hierarchical structures of bone and bioinspired bone tissue engineering. Small. 2016;12(34):4611–32. Scholar
  27. 27.
    Elliott GD, Wang S, Fuller BJ. Cryoprotectants: a review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology. 2017;76:74–91. Scholar
  28. 28.
    Tuan-Mu H-Y, Yu C-H, Hu J-J. On the decellularization of fresh or frozen human umbilical arteries: implications for small-diameter tissue engineered vascular grafts. Ann Biomed Eng. 2014;42(6):1305–18. Scholar
  29. 29.
    Creech O, De Bakey ME, Cooley DA, Self MM. Preparation and use of freeze-dried arterial homografts. Ann Surg. 1954;140(1):35–43.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Erskine JM. Freeze-dried arterial homografts: details of technique of processing and analysis of vessels used over a two-year period. AMA Archives of Surgery. 1958;77(6):947–53. Scholar
  31. 31.
    Curtil A, Pegg DE, Wilson A. Freeze drying of cardiac valves in preparation for cellular repopulation. Cryobiology. 1997;34(1):13–22. CrossRefPubMedGoogle Scholar
  32. 32.
    Wang S, Goecke T, Meixner C, Haverich A, Hilfiker A, Wolkers WF. Freeze-dried heart valve scaffolds. Tissue Eng Part C Methods. 2012;18(7):517–25. Scholar
  33. 33.
    Wang S, Oldenhof H, Goecke T, Ramm R, Harder M, Haverich A, et al. Sucrose diffusion in decellularized heart valves for freeze-drying. Tissue Engineering Part C: Methods. 2015;21(9):922–31. Scholar
  34. 34.
    Reves BT, Bumgardner JD, Cole JA, Yang Y, Haggard WO. Lyophilization to improve drug delivery for chitosan-calcium phosphate bone scaffold construct: a preliminary investigation. J Biomed Mater Res B Appl Biomater. 2009;90B(1):1–10. Scholar
  35. 35.
    Ruhe PQ, Kroese-Deutman HC, Wolke JG, Spauwen PH, Jansen JA. Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials. 2004;25(11):2123–32.CrossRefPubMedGoogle Scholar
  36. 36.
    Jansen JA, Vehof JW, Ruhe PQ, Kroeze-Deutman H, Kuboki Y, Takita H, et al. Growth factor-loaded scaffolds for bone engineering. J Control Release. 2005;101(1–3):127–36. Scholar
  37. 37.
    Zhao J, Wang S, Bao J, Sun X, Zhang X, Zhang X, et al. Trehalose maintains bioactivity and promotes sustained release of BMP-2 from lyophilized CDHA scaffolds for enhanced osteogenesis in vitro and in vivo. PLoS One. 2013;8(1):e54645. Scholar
  38. 38.
    Mistry AS, Mikos AG. Tissue engineering strategies for bone regeneration. Adv Biochem Eng Biotechnol. 2005;94:1–22.PubMedGoogle Scholar
  39. 39.
    Chun SY, Lee HJ, Choi YA, Kim KM, Baek SH, Park HS, et al. Analysis of the soluble human tooth proteome and its ability to induce dentin/tooth regeneration. Tissue Eng A. 2011;17(1–2):181–91. Scholar
  40. 40.
    Jiao L, Xie L, Yang B, Yu M, Jiang Z, Feng L, et al. Cryopreserved dentin matrix as a scaffold material for dentin-pulp tissue regeneration. Biomaterials. 2014;35(18):4929–39. CrossRefPubMedGoogle Scholar
  41. 41.
    •• Petrenko YA, Petrenko AY, Martin I, Wendt D. Perfusion bioreactor-based cryopreservation of 3D human mesenchymal stromal cell tissue grafts. Cryobiology. 2017;76:150–3. This study used a perfusion bioreactor-based method to load and remove CPA under perfused flow and demonstrated this technology significantly increased the viability of cryopreserved tissue constructs as compared to using conventional diffusion-based methods.
  42. 42.
    Bakhach J. The cryopreservation of composite tissues: principles and recent advancement on cryopreservation of different type of tissues. Organ. 2009;5(3):119–26.Google Scholar
  43. 43.
    Wang S, Elliott GD. Synergistic development of biochips and cell preservation methodologies: a tale of converging technologies. Curr Stem Cell Rep. 2017;3(1):45–53. Scholar
  44. 44.
    Iwatani M, Ikegami K, Kremenska Y, Hattori N, Tanaka S, Yagi S, et al. Dimethyl sulfoxide has an impact on epigenetic profile in mouse embryoid body. Stem Cells. 2006;24(11):2549–56. Scholar
  45. 45.
    Bissoyi A, Pramanik K, Panda NN, Sarangi SK. Cryopreservation of hMSCs seeded silk nanofibers based tissue engineered constructs. Cryobiology. 2014;68(3):332–42. Scholar
  46. 46.
    Spees JL, Gregory CA, Singh H, Tucker HA, Peister A, Lynch PJ, et al. Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy. Mol Ther. 2004;9(5):747–56. CrossRefPubMedGoogle Scholar
  47. 47.
    Dahl JA, Duggal S, Coulston N, Millar D, Melki J, Shahdadfar A, et al. Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum. The International Journal of Developmental Biology. 2008;52(8):1033–42. Scholar
  48. 48.
    Zhang P, Policha A, Tulenko T, DiMuzio P. Autologous human plasma in stem cell culture and cryopreservation in the creation of a tissue-engineered vascular graft. J Vasc Surg. 2016;63(3):805–14. Scholar
  49. 49.
    • Jain M, Rajan R, Hyon SH, Matsumura K. Hydrogelation of dextran-based polyampholytes with cryoprotective properties via click chemistry. Biomater Sci-Uk. 2014;2(3):308–17. This study reported a dextran-based hydrogel that itself shows cryoprotective properties can serve as scaffolds with cryoprotective properties and also provide structural integrity to tissue constructs.
  50. 50.
    Dahl SL, Chen Z, Solan AK, Brockbank KG, Niklason LE, Song YC. Feasibility of vitrification as a storage method for tissue-engineered blood vessels. Tissue Eng. 2006;12(2):291–300. Scholar
  51. 51.
    Sun M, Jiang M, Cui J, Liu W, Yin L, Xu C et al. A novel approach for the cryodesiccated preservation of tissue-engineered skin substitutes with trehalose. Materials Science and Engineering: C 2016;60(Supplement C):60–6 doi:, 2016.
  52. 52.
    Popa EG, Rodrigues MT, Coutinho DF, Oliveira MB, Mano JF, Reis RL, et al. Cryopreservation of cell laden natural origin hydrogels for cartilage regeneration strategies. Soft Matter. 2013;9(3):875–85. Scholar
  53. 53.
    Wu Y, Wen F, Gouk SS, Lee EH, Kuleshova L. Cryopreservation strategy for tissue engineering constructs consisting of human mesenhymal stem cells and hydrogel biomaterials. Cryo letters. 2015;36(5):325–35.PubMedGoogle Scholar
  54. 54.
    Trufanova NA, Zaikov VS, Zinchenko AV, Petrenko AY, Petrenko YA. Closed vitrification system as a platform for cryopreservation of tissue engineered constructs. Cryo letters. 2016;37(6):440–7.PubMedGoogle Scholar
  55. 55.
    Stephens B, Azimi P, El Orch Z, Ramos T. Ultrafine particle emissions from desktop 3D printers. Atmos Environ. 2013;79:334–9. Scholar
  56. 56.
    •• Adamkiewicz M, Rubinsky B. Cryogenic 3D printing for tissue engineering. Cryobiology. 2015;71(3):518–21. This study described a new cryogenic 3D printing technology for freezing hydrogels and present the potential of the use of freezing/thawing for automation of tissue biomanufacturing.
  57. 57.
    Wang C, Zhao QL, Wang M. Cryogenic 3D printing for producing hierarchical porous and rhBMP-2loaded Ca-P/PLLA nanocomposite scaffolds for bone tissue engineering. Biofabrication. 2017;9(2). ARTN 025031

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Mechanical Engineering and Engineering SciencesUniversity of North Carolina at CharlotteCharlotteUSA

Personalised recommendations