Strategies for the hypothermic preservation of cell sheets of human adipose stem cells

Autoři: Sara Freitas-Ribeiro aff001;  Andreia Filipa Carvalho aff001;  Marina Costa aff001;  Mariana Teixeira Cerqueira aff001;  Alexandra Pinto Marques aff001;  Rui Luís Reis aff001;  Rogério Pedro Pirraco aff001
Působiště autorů: 3B's Research Group, I3Bs–Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal aff001;  ICVS/3B’s–PT Government Associate Laboratory, Braga/Guimarães, Portugal aff002;  The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Guimarães, Portugal aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


Cell Sheet (CS) Engineering is a regenerative medicine strategy proposed for the treatment of injured or diseased organs and tissues. In fact, several clinical trials are underway using CS-based methodologies. However, the clinical application of such cell-based methodologies poses several challenges related with the preservation of CS structure and function from the fabrication site to the bedside. Pausing cells at hypothermic temperatures has been suggested as a valuable method for short-term cell preservation. In this study, we tested the efficiency of two preservation strategies, one using culture medium supplementation with Rokepie and the other using the preservation solution Hypothermosol, in preserving human adipose stromal/stem cells (hASC) CS-like confluent cultures at 4°C, during 3 and 7 days. Both preservation strategies demonstrated excellent ability to preserve cell function during the first 3 days in hypothermia, as demonstrated by metabolic activity results and assessment of extracellular matrix integrity and differentiation potential. At the end of the 7th day of hypothermic incubation, the decrease in cell metabolic activity was more evident for all conditions. Nonetheless, hASC incubated with Rokepie and Hypothermosol retained a higher metabolic activity and extracellular matrix integrity in comparison with unsupplemented cells. Differentiation results for the later time point showed that supplementation with both Rokepie and Hypothermosol rescued adipogenic differentiation potential but only Rokepie was able to preserve hASC osteogenic potential.

Klíčová slova:

Alizarin staining – Apoptosis – Cell differentiation – Cell metabolism – Cell staining – Collagens – Flow cytometry – Necrotic cell death


1. Israni AK, Zaun D, Rosendale JD, Schaffhausen C, Snyder JJ, Kasiske BL. OPTN/SRTR 2016 Annual Data Report: Deceased Organ Donation. Am J Transplant. 2018 Jan;18:434–63.

2. Pirraco RP, Reis RL. Tissue Engineering: New Tools for Old Problems. Stem Cell Rev Reports. 2015 Jun;11(3):373–5.

3. Hofmann M. Monitoring of Bone Marrow Cell Homing Into the Infarcted Human Myocardium. Circulation. 2005 May;111(17):2198–202. doi: 10.1161/01.CIR.0000163546.27639.AA 15851598

4. Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, et al. Cell sheet engineering: Recreating tissues without biodegradable scaffolds. Biomaterials. 2005 Nov;26(33):6415–22. doi: 10.1016/j.biomaterials.2005.04.061 16011847

5. Pirraco RP, Obokata H, Iwata T, Marques AP, Tsuneda S, Yamato M, et al. Development of Osteogenic Cell Sheets for Bone Tissue Engineering Applications. Tissue Eng Part A. 2011 Jun;17(11–12):1507–15. doi: 10.1089/ten.TEA.2010.0470 21275832

6. Cerqueira MT, Pirraco RP, Santos TC, Rodrigues DB, Frias AM, Martins AR, et al. Human Adipose Stem Cells Cell Sheet Constructs Impact Epidermal Morphogenesis in Full-Thickness Excisional Wounds. Biomacromolecules. 2013 Nov;14(11):3997–4008. doi: 10.1021/bm4011062 24093541

7. Cerqueira MT, Pirraco RP, Martins AR, Santos TC, Reis RL, Marques AP. Cell sheet technology-driven re-epithelialization and neovascularization of skin wounds. Acta Biomater. 2014 Jul;10(7):3145–55. doi: 10.1016/j.actbio.2014.03.006 24650971

8. Matsuda N, Shimizu T, Yamato M, Okano T. Tissue engineering based on cell sheet technology. Adv Mater. 2007 Oct;19(20):3089–99.

9. Burillon C, Huot L, Justin V, Nataf S, Chapuis F, Decullier E, et al. Cultured Autologous Oral Mucosal Epithelial Cell Sheet (CAOMECS) Transplantation for the Treatment of Corneal Limbal Epithelial Stem Cell Deficiency. Investig Opthalmology Vis Sci. 2012 Mar;53(3):1325.

10. Sawa Y, Miyagawa S, Sakaguchi T, Fujita T, Matsuyama A, Saito A, et al. Tissue engineered myoblast sheets improved cardiac function sufficiently to discontinue LVAS in a patient with DCM: report of a case. Surg Today. 2012 Feb;42(2):181–4. doi: 10.1007/s00595-011-0106-4 22200756

11. Kokubo M, Sato M, Yamato M, Mitani G, Kutsuna T, Ebihara G, et al. Characterization of chondrocyte sheets prepared using a co-culture method with temperature-responsive culture inserts. J Tissue Eng Regen Med. 2016 Jun;10(6):486–95. doi: 10.1002/term.1764 23868865

12. Mavilio F, Pellegrini G, Ferrari S, Di Nunzio F, Di Iorio E, Recchia A, et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med. 2006 Dec 19;12(12):1397–402. doi: 10.1038/nm1504 17115047

13. Melanoma—Overview—MatTek Corporation [Internet]. [cited 2019 Jan 19]. Available from:

14. EPISKIN FAQ: Frequently asked questions [Internet]. [cited 2019 Jan 19]. Available from:

15. 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 Jun 7;35(6):530–42. doi: 10.1038/nbt.3889 28591112

16. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol Physiol. 1984 Sep;247(3):C125–42.

17. Hunt L, Hacker DL, Grosjean F, De Jesus M, Uebersax L, Jordan M, et al. Low-temperature pausing of cultivated mammalian cells. Biotechnol Bioeng. 2005 Jan 20;89(2):157–63. doi: 10.1002/bit.20320 15584025

18. Robinson NJ, Picken A, Coopman K. Low temperature cell pausing: an alternative short-term preservation method for use in cell therapies including stem cell applications. Biotechnol Lett. 2014 Feb 24;36(2):201–9. doi: 10.1007/s10529-013-1349-5 24062136

19. Wise H, Abel PW, Cawkill D. Use of Reduced Temperature Cell Pausing to Enhance Flexibility of Cell-Based Assays. J Biomol Screen. 2009 Jul 1;14(6):716–22. doi: 10.1177/1087057109335748 19470715

20. Hajmousa G, Vogelaar P, Brouwer LA, van der Graaf AC, Henning RH, Krenning G. The 6-chromanol derivate SUL-109 enables prolonged hypothermic storage of adipose tissue-derived stem cells. Biomaterials. 2017 Mar;119:43–52. doi: 10.1016/j.biomaterials.2016.12.008 28006657

21. Roobol A, Carden MJ, Newsam RJ, Smales CM. Biochemical insights into the mechanisms central to the response of mammalian cells to cold stress and subsequent rewarming. FEBS J. 2009 Jan;276(1):286–302. doi: 10.1111/j.1742-4658.2008.06781.x 19054067

22. Nishiyama H, Higashitsuji H, Yokoi H, Itoh K, Danno S, Matsuda T, et al. Cloning and characterization of human CIRP (cold-inducible RNA-binding protein) cDNA and chromosomal assignment of the gene. Gene. 1997 Dec;204(1–2):115–20. doi: 10.1016/s0378-1119(97)00530-1 9434172

23. Pogozhykh D, Prokopyuk V, Pogozhykh O, Mueller T, Prokopyuk O. Influence of factors of cryopreservation and hypothermic storage on survival and functional parameters of multipotent stromal cells of placental origin. Almeida-Porada GD, editor. PLoS One. 2015 Oct 2;10(10):e0139834. doi: 10.1371/journal.pone.0139834 26431528

24. Mathew AJ, Baust JM, Van Buskirk RG, Baust JG. Cell Preservation in Reparative and Regenerative Medicine: Evolution of Individualized Solution Composition. Tissue Eng. 2004 Nov;10(11–12):1662–71. doi: 10.1089/ten.2004.10.1662 15684675

25. Rampersad SN. Multiple applications of alamar blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Switzerland). 2012 Sep 10;12(9):12347–60.

26. Chiarella E, Aloisio A, Codispoti B, Nappo G, Scicchitano S, Lucchino V, et al. ZNF521 Has an Inhibitory Effect on the Adipogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells. Stem Cell Rev Reports. 2018 Dec 25;14(6):901–14.

27. Mahler S, Desille M, Frémond B, Chesné C, Guillouzo A, Campion J-P, et al. Hypothermic Storage and Cryopreservation of Hepatocytes: The Protective Effect of Alginate Gel against Cell Damages. Cell Transplant. 2003 Sep 22;12(6):579–92. doi: 10.3727/000000003108747181 14579926

28. Swioklo S, Constantinescu A, Connon CJ. Alginate-Encapsulation for the Improved Hypothermic Preservation of Human Adipose-Derived Stem Cells. Stem Cells Transl Med. 2016 Mar;5(3):339–49. doi: 10.5966/sctm.2015-0131 26826163

29. Rinkes IHMB, Toner M, Sheehan SJ, Tompkins RG, Yarmush ML. Long-Term Functional Recovery of Hepatocytes after Cryopreservation in a Three-Dimensional Culture Configuration. Cell Transplant. 1992 Jul 22;1(4):281–92. doi: 10.1177/096368979200100405 1344301

30. Dunn JCY, Yarmush ML, Koebe HG, Tompkins RG. Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J. 1989 Feb;3(2):174–7. doi: 10.1096/fasebj.3.2.2914628 2914628

31. Tatsumi K, Okano T. Hepatocyte Transplantation: Cell Sheet Technology for Liver Cell Transplantation. Curr Transplant Reports. 2017 Sep 8;4(3):184–92.

32. Kushida A, Yamato M, Konno C, Kikuchi A, Sakurai Y, Okano T. Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces. J Biomed Mater Res. 1999 Jun 15;45(4):355–62. doi: 10.1002/(sici)1097-4636(19990615)45:4<355::aid-jbm10>;2-7 10321708

33. Taylor MJ, Bailes JE, Elrifai AM, Shih SR, Teeple E, Leavitt ML, et al. A new solution for life without blood. Asanguineous low-flow perfusion of a whole-body perfusate during 3 hours of cardiac arrest and profound hypothermia. Circulation. 1995 Jan;91(2):431–44. doi: 10.1161/01.cir.91.2.431 7805248

34. Morel P, Moss A, Schlumpf R, Nakhleh R, Lloveras JK, Field MJ, et al. 72-hour preservation of the canine pancreas: successful replacement of hydroxyethylstarch by dextran-40 in UW solution. Transplant Proc. 1992 Jun;24(3):791–4. 1376525

35. Mathew AJ, Van Buskirk RG, Baust JG. Improved Hypothermic Preservation of Human Renal Cells Through Suppression of Both Apoptosis and Necrosis. Cell Preserv Technol. 2002 Dec;1(4):239–53.

36. Correia C, Koshkin A, Carido M, Espinha N, Šarić T, Lima PA, et al. Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing. Stem Cells Transl Med. 2016 May;5(5):658–69. doi: 10.5966/sctm.2015-0238 27025693

37. Ginis I, Grinblat B, Shirvan MH. Evaluation of bone marrow-derived mesenchymal stem cells after cryopreservation and hypothermic storage in clinically safe medium. Tissue Eng—Part C Methods. 2012 Jun;18(6):453–63. doi: 10.1089/ten.TEC.2011.0395 22196031

38. Boutilier RG. Mechanisms of cell survival in hypoxia and hypothermia. J Exp Biol. 2001 Sep;204(Pt 18):3171–81. 11581331

39. Weisenberg RC. Microtubule Formation in vitro in Solutions Containing Low Calcium Concentrations. Science (80-). 1972 Sep;177(4054):1104–5. doi: 10.1126/science.177.4054.1104 4626639

40. Rubinsky B. Principles of low temperature cell preservation. Vol. 8, Heart Failure Reviews. Kluwer Academic Publishers; 2003. p. 277–84. 12878837

41. Abrahamse SL, Van Runnard Heimel P, Hartman RJ, Chamuleau RAFM, Van Gulik TM. Induction of Necrosis and DNA Fragmentation during Hypothermic Preservation of Hepatocytes in UW, HTK, and Celsior Solutions. Cell Transplant. 2003 Jan 22;12(1):59–68. doi: 10.3727/000000003783985160 12693665

42. Corwin WL, Baust JM, Baust JG, Van Buskirk RG. Characterization and modulation of human mesenchymal stem cell stress pathway response following hypothermic storage. Cryobiology. 2014 Apr;68(2):215–26. doi: 10.1016/j.cryobiol.2014.01.014 24508650

43. Rauen U, Petrat F, Li T. Hypothermia injury/cold-induced apoptosis-Evidenve of an increase in chelatable iron causing oxidative injury in spite of low O2-/H2O2 formation Quantification, distribution and toxicity of chelatable iron in living cells View project Identification of mechanisms leading to outer hair cell death after antibiotica or chemotherapy View project. Artic FASEB J. 2000;

44. Campbell LH, Taylor MJ, Brockbank KGM. Survey of Apoptosis After Hypothermic Storage of a Pancreatic β-Cell Line. Biopreserv Biobank. 2016 Aug;14(4):271–8. doi: 10.1089/bio.2015.0060 26937946

45. Salahudeen AK, Huang H, Joshi M, Moore NA, Jenkins JK. Involvement of the Mitochondrial Pathway in Cold Storage and Rewarming-Associated Apoptosis of Human Renal Proximal Tubular Cells. Am J Transplant. 2003 Mar 1;3(3):273–80. 12614281

46. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 2018 Mar 23;25(3):486–541. doi: 10.1038/s41418-017-0012-4 29362479

47. Kushida A, Yamato M, Konno C, Kikuchi A, Sakurai Y, Okano T. Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces. J Biomed Mater Res. 1999 Jun 15;45(4):355–62. doi: 10.1002/(sici)1097-4636(19990615)45:4<355::aid-jbm10>;2-7 10321708

48. Katori R, Hayashi R, Kobayashi Y, Kobayashi E, Nishida K. Ebselen Preserves Tissue-Engineered Cell Sheets and their Stem Cells in Hypothermic Conditions. Sci Rep. 2016 Dec;6(1):38987.

49. Tremain N, Korkko J, Ibberson D, Kopen GC, DiGirolamo C, Phinney DG. MicroSAGE Analysis of 2,353 Expressed Genes in a Single Cell-Derived Colony of Undifferentiated Human Mesenchymal Stem Cells Reveals mRNAs of Multiple Cell Lineages. Stem Cells. 2001 Sep;19(5):408–18. doi: 10.1634/stemcells.19-5-408 11553849

50. Baddoo M, Hill K, Wilkinson R, Gaupp D, Hughes C, Kopen GC, et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J Cell Biochem. 2003 Aug 15;89(6):1235–49. doi: 10.1002/jcb.10594 12898521

51. Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci. 2000 Apr 113;7(7):1161–6.

52. Okamoto T, Aoyama T, Nakayama T, Nakamata T, Hosaka T, Nishijo K, et al. Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells. Biochem Biophys Res Commun. 2002 Jul;295(2):354–61. doi: 10.1016/s0006-291x(02)00661-7 12150956

53. Zuk PA, Zhu M, Ashjian P, Ugarte DA De, Huang JI, Mizuno Het al. Human Adipose Tissue Is a Source of Multipotent. Raff M, editor. Mol Biol Cell. 2002 Dec;13:4279–95. doi: 10.1091/mbc.E02-02-0105 12475952

54. Falah M, Rayan A, Srouji S. Storage effect on viability and biofunctionality of human adipose tissue-derived stromal cells. Cytotherapy. 2015 Sep;17(9):1220–9. doi: 10.1016/j.jcyt.2015.05.014 26276005

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