Cell sheet biofabrication by co-administration of mesenchymal stem cells secretome and vitamin C on thermoresponsive polymer

  • Behnaz Banimohammad Shotorbani
  • Helder André
  • Abolfazl Barzegar
  • Nosratollah Zarghami
  • Roya Salehi
  • Effat Alizadeh
Tissue Engineering Constructs and Cell Substrates Original Research
Part of the following topical collections:
  1. Tissue Engineering Constructs and Cell Substrates


Cell sheet technology aims at replacement of artificial extracellular matrix (ECM) or scaffolds, popular in tissue engineering, with natural cell derived ECM. Adipose tissue mesenchymal stem cells (ASCs) have the ability of ECM secretion and presented promising outcomes in clinical trials. As well, different studies found that secretome of ASCs could be suitable for triggering cell free regeneration induction. The aim of this study was to investigate the effect of using two bio-factors: secretome of ASCs (SE) and vitamin C (VC) for cell sheet engineering on a thermosensitive poly N-isopropyl acryl amide-Methacrylic acid (P(NIPAAm-MAA)) hydrogel. The results revealed that using thermosensitive P(NIPAAm-MAA) copolymer as matrix for cell sheet engineering lead to a rapid ON⁄OFF adhesion/deadhesion system by reducing temperature without enzymatic treatment (complete cell sheet release takes just 6 min). In addition, our study showed the potential of SE for inducing ASCs sheet formation. H&E staining exhibited the properties of a well-formed tissue layer with a dense ECM in sheets prepared by both SE and VC factors, as compared to those of VC or SE alone. Functional synergism of SE and VC exhibited statistically significant enhanced functionality regarding up-regulation of stemness genes expression, reduced β-galactosidase associated senescence, and facilitated sheet release. Additionally, alkaline phosphatase activity (ALP), mineralized deposits and osteoblast matrix around cells confirmed a better performance of ostogenic differentiation of ASCs induced by VC and SE. It was concluded that SE of ASCs and VC could be outstanding biofactors applicable for cell sheet technology.



This project was supported financially by Umbilical Cord Stem Cell Research Center, Tabriz University of Medical Science (Grant number: 5/104/1149).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Alizadeh E, Akbarzadeh A, Eslaminejad MB, Barzegar A, Hashemzadeh S, Nejati‐Koshki K, et al. Up regulation of liver‐enriched transcription factors HNF4a and HNF6 and liver‐specific MicroRNA (miR‐122) by inhibition of Let‐7b in mesenchymal stem cells. Chem Biol Drug Des. 2015;85:268–79.CrossRefGoogle Scholar
  2. 2.
    Alizadeh E, Eslaminejad MB, Akbarzadeh A, Sadeghi Z, Abasi M, Herizchi R, et al. Upregulation of MiR‐122 via trichostatin A treatments in hepatocyte‐like cells derived from mesenchymal stem cells. Chem Biol Drug Des. 2016;87:296–305.CrossRefGoogle Scholar
  3. 3.
    Akahane M, Nakamura A, Ohgushi H, Shigematsu H, Dohi Y, Takakura Y. Osteogenic matrix sheet‐cell transplantation using osteoblastic cell sheet resulted in bone formation without scaffold at an ectopic site‐. J Tissue Eng Regen Med. 2008;2:196–201.CrossRefGoogle Scholar
  4. 4.
    Biazar E, Montazeri N, Pourshamsian K, Asadifard F, Ghorbanalinezhad E, Keshel SH. et al. Harvesting epithelial cell sheet based on thermo-sensitive hydrogel. J Paramed Sci. 2010;1:27–33.Google Scholar
  5. 5.
    Harimoto M, Yamato M, Hirose M, Takahashi C, Isoi Y, Kikuchi A, et al. Novel approach for achieving double‐layered cell sheets co‐culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature‐responsive culture dishes. J Biomed Mater Res. 2002;62:464–70.CrossRefGoogle Scholar
  6. 6.
    Villa C, Martello F, Erratico S, Tocchio A, Belicchi M, Lenardi C. et al. P (NIPAAM-co-HEMA) thermoresponsive hydrogels: an alternative approach for muscle cell sheet engineering. J Tissue Eng Regen Med. 2014;11:187–196.CrossRefGoogle Scholar
  7. 7.
    Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez F, et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotech. 2008;26:1276–84.CrossRefGoogle Scholar
  8. 8.
    Amable PR, Teixeira MVT, Carias RBV, Granjeiro JM, Borojevic R. Protein synthesis and secretion in human mesenchymal cells derived from bone marrow, adipose tissue and Wharton’s jelly. Stem Cell Res & Ther. 2014;5:53.CrossRefGoogle Scholar
  9. 9.
    Hamada M, Iwata T, Kato Y, Washio K, Morikawa S, Sakurai H, et al. Xenogeneic transplantation of human adipose-derived stem cell sheets accelerate angiogenesis and the healing of skin wounds in a Zucker Diabetic Fatty rat model of obese diabetes. Regen Ther. 2017;6:65–73.CrossRefGoogle Scholar
  10. 10.
    Kaibuchi N, Iwata T, Yamato M, Okano T, Ando T. Multipotent mesenchymal stromal cell sheet therapy for bisphosphonate-related osteonecrosis of the jaw in a rat model. Acta Biomater. 2016;42:400–10.CrossRefGoogle Scholar
  11. 11.
    Kim SJ, Kim WI, Yamato M, Okano T, Kikuchi A, Kwon OH. Successive grafting of PHEMA and PIPAAm onto cell culture surface enables rapid cell sheet recovery. Tissue Eng Regen Med. 2013;10:139–45.CrossRefGoogle Scholar
  12. 12.
    Elloumi‐Hannachi I, Yamato M, Okano T. Cell sheet engineering: a unique nanotechnology for scaffold‐free tissue reconstruction with clinical applications in regenerative medicine. J Intern Med. 2010;267:54–70.CrossRefGoogle Scholar
  13. 13.
    Yang M, Kang E, wook Shin J, Hong J. Surface engineering for mechanical enhancement of cell sheet by nano-coatings. Sci Rep. 2017;7:4464.CrossRefGoogle Scholar
  14. 14.
    de las Heras Alarcón C, Pennadam S, Alexander C. Stimuli responsive polymers for biomedical applications. Chem Soc Rev. 2005;34:276–85.CrossRefGoogle Scholar
  15. 15.
    Yamato M, Okano T. Cell sheet engineering. Mater Today. 2004;7:42–7.CrossRefGoogle Scholar
  16. 16.
    Kubota K, Fujishige S, Ando I. Single-chain transition of poly (N-isopropylacrylamide) in water. J Phys Chem. 1990;94:5154–8.CrossRefGoogle Scholar
  17. 17.
    Baysal BM, Karasz FE. Coil‐globule collapse in flexible macromolecules. Macromol Theory Simul. 2003;12:627–46.CrossRefGoogle Scholar
  18. 18.
    Patel NG, Cavicchia JP, Zhang G, Newby B-mZ. Rapid cell sheet detachment using spin-coated pNIPAAm films retained on surfaces by an aminopropyltriethoxysilane network. Acta Biomater. 2012;8:2559–67.CrossRefGoogle Scholar
  19. 19.
    Shotorbani BB, Alizadeh E, Salehi R, Barzegar A. Adhesion of mesenchymal stem cells to biomimetic polymers: A review. ‎Mater Sci Eng C. 2016;71:1192–1200.CrossRefGoogle Scholar
  20. 20.
    Salehi R, Arsalani N, Davaran S, Entezami A. Synthesis and characterization of thermosensitive and pH‐sensitive poly (N‐isopropylacrylamide‐acrylamide‐vinylpyrrolidone) for use in controlled release of naltrexone. J Biomed Mater Res A. 2009;89:919–28.CrossRefGoogle Scholar
  21. 21.
    Da Silva RM, Mano JF, Reis RL. Smart thermoresponsive coatings and surfaces for tissue engineering: switching cell-material boundaries. Trends Biotechnol. 2007;25:577–83.CrossRefGoogle Scholar
  22. 22.
    Takahashi H, Matsuzaka N, Nakayama M, Kikuchi A, Yamato M, Okano T. Terminally functionalized thermoresponsive polymer brushes for simultaneously promoting cell adhesion and cell sheet harvest. Biomacromolecules. 2011;13:253–60.CrossRefGoogle Scholar
  23. 23.
    Kwon OH, Kikuchi A, Yamato M, Sakurai Y, Okano T. Rapid cell sheet detachment from Poly (N‐isopropylacrylamide)‐grafted porous cell culture membranes. J Biomed Mater Res. 2000;50:82–9.CrossRefGoogle Scholar
  24. 24.
    Kwon OH, Kikuchi A, Yamato M, Okano T. Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes. Biomaterials. 2003;24:1223–32.CrossRefGoogle Scholar
  25. 25.
    Madathil BK, Kumar A, RajanAsari P, Kumary TV. N-isopropylacrylamide-co-glycidylmethacrylate as a thermoresponsive substrate for corneal endothelial cell sheet engineering. Biomed Res Int. 2014;2014:1–7.CrossRefGoogle Scholar
  26. 26.
    Ebara M, Yamato M, Hirose M, Aoyagi T, Kikuchi A, Sakai K, et al. Copolymerization of 2-carboxyisopropylacrylamide with N-isopropylacrylamide accelerates cell detachment from grafted surfaces by reducing temperature. Biomacromolecules. 2003;4:344–9.CrossRefGoogle Scholar
  27. 27.
    Reed S, Wu B. Sustained growth factor delivery in tissue engineering applications. Ann Biomed Eng. 2014;42:1528–36.CrossRefGoogle Scholar
  28. 28.
    Discher DE, Mooney DJ, Zandstra PW. Growth factors, matrices, and forces combine and control stem cells. Science. 2009;324:1673–7.CrossRefGoogle Scholar
  29. 29.
    Korkmaz A, Kolankaya D. The protective effects of ascorbic acid against renal ischemia-reperfusion injury in male rats. Ren Fail. 2009;31:36–43.CrossRefGoogle Scholar
  30. 30.
    Wei F, Qu C, Song T, Ding G, Fan Z, Liu D, et al. Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity. J Cell Physiol. 2012;227:3216–24.CrossRefGoogle Scholar
  31. 31.
    Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self‐renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997;64:278–94.CrossRefGoogle Scholar
  32. 32.
    Kilroy GE, Foster SJ, Wu X, Ruiz J, Sherwood S, Heifetz A, et al. Cytokine profile of human adipose‐derived stem cells: Expression of angiogenic, hematopoietic, and pro‐inflammatory factors. J Cell Physiol. 2007;212:702–9.CrossRefGoogle Scholar
  33. 33.
    Yu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. Int J Mol Sci. 2014;15:4142–57.CrossRefGoogle Scholar
  34. 34.
    Klingemann H. Methods of use of culture supernatant obtained from mesenchymal stem cells from dogs and cats for treatment of organ dysfunction. Google Patents. 2014.Google Scholar
  35. 35.
    Hu G, Xu J-j, Deng Z-h, Feng J, Jin Y. Supernatant of bone marrow mesenchymal stromal cells induces peripheral blood mononuclear cells possessing mesenchymal features. Int J Biol Sci. 2011;7:364.CrossRefGoogle Scholar
  36. 36.
    Alizadeh E, Zarghami N, Eslaminejad MB, Akbarzadeh A, Barzegar A, Mohammadi SA. The effect of dimethyl sulfoxide on hepatic differentiation of mesenchymal stem cells. Artif Cells, Nanomed, Biotechnol. 2016;44:157–64.CrossRefGoogle Scholar
  37. 37.
    Sevivas N, Teixeira FG, Portugal R, Araújo L, Carriço LF, Ferreira N, et al. Mesenchymal stem cell secretome: a potential tool for the prevention of muscle degenerative changes associated with chronic rotator cuff tears. Am J Sports Med. 2017;45:179–88.CrossRefGoogle Scholar
  38. 38.
    Hoseinzadeh S, Atashi A, Soleimani M, Alizadeh E, Zarghami N. MiR-221-inhibited adipose tissue-derived mesenchymal stem cells bioengineered in a nano-hydroxy apatite scaffold. Vitr Cell & Dev Biol-Anim. 2016;52:479–87.CrossRefGoogle Scholar
  39. 39.
    Yang J, Yamato M, Nishida K, Ohki T, Kanzaki M, Sekine H, et al. Cell delivery in regenerative medicine: the cell sheet engineering approach. J Control Release. 2006;116:193–203.CrossRefGoogle Scholar
  40. 40.
    Pokrywczynska M, Lewandowska MA, Krzyzanowska S, Jundzill A, Rasmus M, Warda K, et al. Transdifferentiation of bone marrow mesenchymal stem cells into the islet-like cells: the role of extracellular matrix proteins. Arch Immunol Ther Exp (Warsz). 2015;63:377–84.CrossRefGoogle Scholar
  41. 41.
    Yamato M, Konno C, Utsumi M, Kikuchi A, Okano T. Thermally responsive polymer-grafted surfaces facilitate patterned cell seeding and co-culture. Biomaterials. 2002;23:561–7.CrossRefGoogle Scholar
  42. 42.
    Fujita H, Shimizu K, Nagamori E. Application of a cell sheet–polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability. Biotechnol Bioeng. 2009;103:370–7.CrossRefGoogle Scholar
  43. 43.
    Canavan HE, Cheng X, Graham DJ, Ratner BD, Castner DG. A plasma‐deposited surface for cell sheet engineering: advantages over mechanical dissociation of cells. Plasma Process Polym. 2006;3:516–23.CrossRefGoogle Scholar
  44. 44.
    Chang D, Okano T. 5.530 - Medical Applications of Cell Sheet Engineering A2. In: Paul Ducheyne, editor. Comprehensive biomaterials. Oxford: Elsevier; 2011. p. 405–19.CrossRefGoogle Scholar
  45. 45.
    Chen G, Qi Y, Niu L, Di T, Zhong J, Fang T, et al. Application of the cell sheet technique in tissue engineering (Review). Biomed Rep. 2015;3:749–57.CrossRefGoogle Scholar
  46. 46.
    Biazar E, Khorasani M, Daliri M. Cell sheet engineering: solvent effect on nanometric grafting of poly-N-isopropylacrylamide onto polystyrene substrate under ultraviolet radiation. Int J Nanomed. 2011;6:295.CrossRefGoogle Scholar
  47. 47.
    Altomare L, Cochis A, Carletta A, Rimondini L, Farè S. Thermo-responsive methylcellulose hydrogels as temporary substrate for cell sheet biofabrication. J Mater Sci: Mater Med. 2016;27:1–13.Google Scholar
  48. 48.
    Haraguchi K, Takehisa T, Ebato M. Control of cell cultivation and cell sheet detachment on the surface of polymer/clay nanocomposite hydrogels. Biomacromolecules. 2006;7:3267–75.CrossRefGoogle Scholar
  49. 49.
    Kwon OH, Kikuchi A, Yamato M, Sakurai Y, Okano T. Rapid cell sheet detachment from poly (N‐isopropylacrylamide)‐grafted porous cell culture membranes. J Biomed Mater Res: Off J Soc Biomater Jpn Soc Biomater. 2000;50:82–9.CrossRefGoogle Scholar
  50. 50.
    Wang T, Liu D, Lian C, Zheng S, Liu X, Wang C, et al. Rapid cell sheet detachment from alginate semi-interpenetrating nanocomposite hydrogels of PNIPAm and hectorite clay. React Funct Polym. 2011;71:447–54.CrossRefGoogle Scholar
  51. 51.
    Takahashi H, Nakayama M, Yamato M, Okano T. Controlled chain length and graft density of thermoresponsive polymer brushes for optimizing cell sheet harvest. Biomacromolecules. 2010;11:1991–9.CrossRefGoogle Scholar
  52. 52.
    Tang Z, Akiyama Y, Yamato M, Okano T. Comb-type grafted poly (N-isopropylacrylamide) gel modified surfaces for rapid detachment of cell sheet. Biomaterials. 2010;31:7435–43.CrossRefGoogle Scholar
  53. 53.
    Sudo Y, Kawai R, Sakai H, Kikuchi R, Nabae Y, Hayakawa T, et al. Star-shaped thermoresponsive polymers with various functional groups for cell sheet engineering. Langmuir. 2018;34:653–62.CrossRefGoogle Scholar
  54. 54.
    Nash ME, Carroll WM, Nikoloskya N, Yang R, Connell CO, Gorelov AV, et al. Straightforward, one-step fabrication of ultrathin thermoresponsive films from commercially available pNIPAm for cell culture and recovery. ACS Appl Mater & Interfaces. 2011;3:1980–90.CrossRefGoogle Scholar
  55. 55.
    Marote A, Teixeira FG, Mendes-Pinheiro B, Salgado AJ. MSCs-derived exosomes: cell-secreted nanovesicles with regenerative potential. Front Pharmacol. 2016;7:231.CrossRefGoogle Scholar
  56. 56.
    Ranganath SH, Levy O, Inamdar MS, Karp JM. Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease. Cell Stem Cell. 2012;10:244–58.CrossRefGoogle Scholar
  57. 57.
    Parekkadan B, van Poll D, Suganuma K, Carter EA, Berthiaume F, Tilles AW, et al. Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure. PLoS One. 2007;2:e941.CrossRefGoogle Scholar
  58. 58.
    Teixeira FG, Carvalho MM, Panchalingam KM, Rodrigues AJ, Mendes-Pinheiro B, Anjo S, et al. Impact of the secretome of human mesenchymal stem cells on brain structure and animal behavior in a rat model of Parkinson’s disease. Stem Cells Transl Med. 2017;6:634–46.CrossRefGoogle Scholar
  59. 59.
    Kusuma GD, Carthew J, Lim R, Frith JE. Effect of the microenvironment on mesenchymal stem cell paracrine signaling: opportunities to engineer the therapeutic effect. Stem Cells Dev. 2017;26:617–31.CrossRefGoogle Scholar
  60. 60.
    Yu HS, Park M-K, Kang SA, Cho K-S, Mun SJ, Roh H-J. Culture supernatant of adipose stem cells can ameliorate allergic airway inflammation via recruitment of CD4+CD25+Foxp3 T cells. Stem Cell Res & Ther. 2017;8:8.CrossRefGoogle Scholar
  61. 61.
    Monsel A, Zhu YG, Gudapati V, Lim H, Lee JW. Mesenchymal stem cell derived secretome and extracellular vesicles for acute lung injury and other inflammatory lung diseases. Expert Opin Biol Ther. 2016;16:859–71.CrossRefGoogle Scholar
  62. 62.
    Wang B, Lee WY, Huang B, Zhang JF, Wu T, Jiang X, et al. Secretome of human fetal mesenchymal stem cell ameliorates replicative senescen. Stem Cells Dev. 2016;25:1755–66.CrossRefGoogle Scholar
  63. 63.
    Katagiri W, Osugi M, Kawai T, Hibi H. First-in-human study and clinical case reports of the alveolar bone regeneration with the secretome from human mesenchymal stem cells. Head Face Med. 2016;12:5.CrossRefGoogle Scholar
  64. 64.
    Yu J, Tu Y-K, Tang Y-B, Cheng N-C. Stemness and transdifferentiation of adipose-derived stem cells using l-ascorbic acid 2-phosphate-induced cell sheet formation. Biomaterials. 2014;35:3516–26.CrossRefGoogle Scholar
  65. 65.
    Tew SR, Murdoch AD, Rauchenberg RP, Hardingham TE. Cellular methods in cartilage research: primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells. Methods. 2008;45:2–9.CrossRefGoogle Scholar
  66. 66.
    Wongin S, Waikakul S, Chotiyarnwong P, Siriwatwechakul W, Viravaidya-Pasuwat K. Effect of cell sheet manipulation techniques on the expression of collagen type II and stress fiber formation in human chondrocyte sheets. Tissue Eng Part A. 2018;24:469–78.CrossRefGoogle Scholar
  67. 67.
    Tang Z, Akiyama Y, Okano T. Temperature-responsive polymer modified surface for cell sheet engineering. Polymers. 2012;4:1478.CrossRefGoogle Scholar
  68. 68.
    Kondo M, Murakami D, Yamato M, Takagi R, Namiki H, Okano T. Serum-dependent epithelial cell sheet shrinkage upon detachment from temperature-responsive culture surfaces. FASEB J. 2009;23:468.5–.5.Google Scholar
  69. 69.
    Sato Y, Wakitani S, Takagi M. Xeno-free and shrinkage-free preparation of scaffold-free cartilage-like disc-shaped cell sheet using human bone marrow mesenchymal stem cells. J Biosci Bioeng. 2013;116:734–9.CrossRefGoogle Scholar
  70. 70.
    Maeda S, Fujitomo T, Okabe T, Wakitani S, Takagi M. Shrinkage-free preparation of scaffold-free cartilage-like disk-shaped cell sheet using human bone marrow mesenchymal stem cells. J Biosci Bioeng. 2011;111:489–92.CrossRefGoogle Scholar

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Authors and Affiliations

  • Behnaz Banimohammad Shotorbani
    • 1
    • 2
  • Helder André
    • 3
  • Abolfazl Barzegar
    • 2
  • Nosratollah Zarghami
    • 1
    • 4
  • Roya Salehi
    • 5
  • Effat Alizadeh
    • 1
    • 4
  1. 1.The Umbilical Cord Stem Cell Research Center (UCSRC)Tabriz University of Medical SciencesTabrizIran
  2. 2.Research Institute for Fundamental Sciences (RIFS)University of TabrizTabrizIran
  3. 3.Department of Clinical NeuroscienceSt. Erik Eye Hospital, Karolinska InstitutetStockholmSweden
  4. 4.Department of Medical Biotechnology, Faculty of Advanced Medical SciencesTabriz University of Medical SciencesTabrizIran
  5. 5.Drug Applied Research Center and Department of Medical Nanotechnology, Faculty of Advanced Medical SciencesTabriz University of Medical SciencesTabrizIran

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