Construction of cardiomyoblast sheets for cardiac tissue repair: comparison of three different approaches

  • Gökçe Kaynak Bayrak
  • Menemşe GümüşderelioğluEmail author
Original Article


Recently, cell sheet engineering has emerged as one of the most accentuated approaches of tissue engineering and cardiac tissue is the pioneering application area of cell sheets with clinical use. In this study, we cultured rat cardiomyoblasts (H9C2 cell line) to obtain cell sheets by using three different approaches; using (1) thermo-responsive tissue culture plates, (2) high cell seeding density/high serum content and (3) ascorbic acid treatment. To compare the outcomes of three methods, morphologic examination, immunofluorescent stainings and live/dead cell assay were performed and the effects of serum concentration and ascorbic acid treatment on cardiac gene expressions were examined. The results showed that cardiomyoblast sheets were successfully obtained in all approaches without losing their integrity and viability. Also, the results of RT-PCR analysis showed that the types of tissue culture surface, cell seeding density, serum concentration and ascorbic acid treatment affect cardiac gene expressions of cells in cell sheets. Although three methods were succeeded, ascorbic acid treatment was found as the most rapid and effective method to obtain cell sheets with cardiac characteristics.


H9C2 cell line Cardiac tissue Cell sheet engineering Ascorbic acid Thermo-responsive surface 



This study was financially supported by The Hacettepe University Scientific Research Projects Coordination Unit Project No. FBA-2017-16248. The authors would like to thank Selin Gümüşderelioğlu for English editing of text language.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10616_2019_325_MOESM1_ESM.docx (3.7 mb)
Supplementary material 1 (DOCX 3803 kb)


  1. Bel A, Planat-Bernard V, Saito A, Bonnevie L, Bellamy V et al (2010) Composite cell sheets: a further step toward safe and effective myocardial regeneration by cardiac progenitors derived from embryonic stem cells. Circulation 122:118–123CrossRefGoogle Scholar
  2. Chen CH, Chang Y, Wang CC, Huang CH, Huang CC, Yeh YC, Hwang SM, Sung HW (2007) Construction and characterization of fragmented mesenchymal-stem-cell sheets for intramuscular injection. Biomaterials 28:4643–4651CrossRefGoogle Scholar
  3. Choi YC, Morris GM, Lee FS, Sokoloff L (1980) The effect of serum on monolayer cell culture of mammalian articular chondrocytest. Connect Tissue Res 7:105–112CrossRefGoogle Scholar
  4. Cui H, Liu Y, Cheng Y, Zhang Z, Zhang P, Chen X, Wei Y (2014) In vitro study of electroactive tetraaniline-containing thermosensitive hydrogels for cardiac tissue engineering. Biomacromolecules 15:1115–1123CrossRefGoogle Scholar
  5. Cui H, Miao S, Esworthy T, Zhou X, Lee S-J, Liu C, Yu Z-X, Fisher JP, Mohiuddin M, Zhang LG (2018) 3D bioprinting for cardiovascular regeneration and pharmacology. Adv Drug Deliv Rev 132:252–269CrossRefGoogle Scholar
  6. Dergilev KV, Makarevich PI, Tsokolaeva ZI, Boldyreva MA, Beloglazova IB, Zubkova ES, Menshikov MY, Parfyonova YV (2017) Comparison of cardiac stem cell sheets detached by Versene solution and from thermoresponsive dishes reveals similar properties of constructs. Tissue Cell 49:64–71CrossRefGoogle Scholar
  7. Dergilev K, Tsokolaeva Z, Makarevich P, Beloglazova I, Zubkova E, Boldyreva M, Ratner E, Dyikanov D, Menshikov M, Ovchinnikov A, Ageev F, Parfyonova Y (2018) C-kit cardiac progenitor cell based cell sheet improves vascularization and attenuates cardiac remodeling following myocardial infarction in rats. BioMed Res Int 2018, 3536854. CrossRefGoogle Scholar
  8. Dott W, Mistry P, Wright J, Cain K, Herbert KE (2014) Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity. Redox Biol 2:224–233CrossRefGoogle Scholar
  9. Feridooni T, Mac Donald C, Shao D, Yeung P, Agu RU (2013) Cytoprotective potential of anti-ischemic drugs against chemotherapy-induced cardiotoxicity in H9C2 myoblast cell line. Acta Pharm 63:493–503CrossRefGoogle Scholar
  10. Furuta A, Miyoshi S, Itabashi Y, Shimizu T, Kira S et al (2006) Pulsatile cardiac tissue grafts using a novel three-dimensional cell sheet manipulation technique functionally integrates with the host heart, in vivo. Circ Res 98:705–712CrossRefGoogle Scholar
  11. Grinnell F, Fukamizu H, Pawelek P, Nakagawa S (1989) Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid. Exp Cell Res 181:483–491CrossRefGoogle Scholar
  12. Guo P, Zeng JJ, Zhou N (2015) A novel experimental study on the fabrication and biological characteristics of canine bone marrow mesenchymal stem cells sheet using vitamin C. Scanning 37:42–48CrossRefGoogle Scholar
  13. Hakimi O, Poulson R, Thakkar D, Yapp C, Carr A (2014) Ascorbic acid is essential for significant collagen deposition by human tenocytes in vitro. Oxid Antioxid Med Sci 3:119–127CrossRefGoogle Scholar
  14. Haraguchi Y, Shimizu T, Yamato M, Kikuchi A, Okano T (2006) Electrical coupling of cardiomyocyte sheets occurs rapidly via functional gap junction formation. Biomaterials 27:4765–4774CrossRefGoogle Scholar
  15. Haraguchi Y, Shimizu T, Sasagawa T, Sekine H, Sakaguchi K et al (2012) Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc 7:850–858CrossRefGoogle Scholar
  16. Hata H, Matsumiya G, Miyagawa S, Kondoh H, Kawaguchi N et al (2006) Grafted skeletal myoblast sheets attenuate myocardial remodeling in pacing-induced canine heart failure model. J Thorac Cardiovasc Surg 132:918–924CrossRefGoogle Scholar
  17. Hoashi T, Matsumiya G, Miyagawa S, Ichikawa H, Ueno T et al (2009) Skeletal myoblast sheet transplantation improves the diastolic function of a pressure-overloaded right heart. J Thorac Cardiovasc Surg 138:460–467CrossRefGoogle Scholar
  18. Hong Y, Yu M, Weng W, Cheng K, Wang H, Lin J (2013) Light-induced cell detachment for cell sheet technology. Biomaterials 34:11–18CrossRefGoogle Scholar
  19. Huang CH, Chen HW, Tsai MS, Hsu CY, Peng RH et al (2009) Antiapoptotic cardioprotective effect of hypothermia treatment against oxidative stress injuries. Acad Emerg Med 16:872–880CrossRefGoogle Scholar
  20. Itabashi Y, Miyoshi S, Kawaguchi H, Yuasa S, Tanimoto K et al (2005) A New method for manufacturing cardiac cell sheets using fibrin-coated dishes and its electrophysiological studies by optical mapping. Artif Organs 29:95–103CrossRefGoogle Scholar
  21. Ivanyuk D, Budash G, Zheng Y, Gaspar JA, Chaudhari U et al (2015) Ascorbic acid-induced cardiac differentiation of murine pluripotent stem cells: transcriptional profiling and effect of a small molecule synergist of wnt/β-catenin signaling pathway. Cell Physiol Biochem 36:810–830CrossRefGoogle Scholar
  22. Jadaun P, Yadav D, Bisen PS (2018) Spirulina platensis prevents high glucose-induced oxidative stress mitochondrial damage mediated apoptosis in cardiomyoblasts. Cytotechnology 70:523–536CrossRefGoogle Scholar
  23. Kato S, Shanley JR, Fox JC (1996) Serum stimulation, cell-cell interactions, and extracellular matrix independently influence smooth muscle cell phenotype in vitro. Am J Pathol 149:687–697Google Scholar
  24. Kawamura M, Miyagawa S, Miki K, Saito A, Fukushima S et al (2012) Feasibility, safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte sheets in a porcine ischemic cardiomyopathy model. Circulation 126:29–37CrossRefGoogle Scholar
  25. Kondoh H, Sawa Y, Miyagawa S, Sakakida-Kitagawa S, Memon IA et al (2006) Longer preservation of cardiac performance by sheet-shaped myoblast implantation in dilated cardiomyopathic hamsters. Cardiovasc Res 69:466–475CrossRefGoogle Scholar
  26. Koo MA, Lee MH, Kwon BJ, Seon GM, Kim MS, Kim D, Nam KC, Park JC (2018) Exogenous ROS-induced cell sheet transfer based on hematoporphyrin-polyketone film via a one-step process. Biomaterials 161:47–56CrossRefGoogle Scholar
  27. Law CH, Li JM, Chou HC, Chen YH, Chan HL (2013) Hyaluronic acid-dependent protection in H9C2 cardiomyocytes: a cell model of heart ischemia–reperfusion injury and treatment. Toxicology 303:54–71CrossRefGoogle Scholar
  28. Leung GP, Tse CM, Man RY (2007) Characterization of adenosine transport in H9C2 cardiomyoblasts. Int J Cardiol 116:186–193CrossRefGoogle Scholar
  29. L’Heureux N, Pâquet S, Labbé R, Germain L, Auger FA (1998) A completely biological tissue-engineered human blood vessel. FASEB J 12:47–56CrossRefGoogle Scholar
  30. Martinez EC, Wang J, Gan SU, Singh R, Lee CN, Kofidis T (2010) Ascorbic acid improves embryonic cardiomyoblast cell survival and promotes vascularization in potential myocardial grafts in vivo. Tissue Eng Part A 16:1349–1361CrossRefGoogle Scholar
  31. Matsuda N, Shimizu T, Yamato M, Okano T (2007) Tissue engineering based on cell sheet technology. Adv Mater 19:3089–3099CrossRefGoogle Scholar
  32. Mejia-Alvarez R, Tomaselli CF, Marban E (1994) Simultaneous expression of cardiac and skeletal muscle isoforms of the L-type Ca2+ channel in a rat heart muscle cell line. J Physiol 478:315–329CrossRefGoogle Scholar
  33. Memon IA, Sawa Y, Fukushima N, Matsumiya G, Miyagawa S et al (2005) Repair of impaired myocardium by means of implantation of engineered autologous myoblast sheets. J Thorac Cardiovasc Surg 130:1333–1341CrossRefGoogle Scholar
  34. Menard C, Pupier S, Mornet D, Kitzmann M, Nargeot J, Lory P (1999) Modulation of L-type calcium channel expression during retinoic acid-induced differentiation of H9C2 cardiac cells. J Biol Chem 274:29063–29070CrossRefGoogle Scholar
  35. Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K et al (2006) Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 12:459–465CrossRefGoogle Scholar
  36. Narayanan AS, Page RC, Swanson J (1989) Collagen synthesis by human fibroblasts regulation by transforming growth factor-β in the presence of other inflammatory mediators. Biochem J 260:463–469CrossRefGoogle Scholar
  37. Pereira SL, Ramalho-Santos J, Branco AF, Sardão VA, Oliveira PJ, Carvalho RA (2011) Metabolic remodeling during H9C2 myoblast differentiation: relevance for in vitro toxicity studies. Cardiovasc Toxicol 11:180–190CrossRefGoogle Scholar
  38. Ricotti L, Polini A, Genchi GG, Ciofani G, Iandolo D et al (2012) Proliferation and skeletal myotube formation capability of C2C12 and H9c2 cells on isotropic and anisotropic electrospun nanofibrous PHB scaffolds. Biomed Mater 7, 035010. CrossRefGoogle Scholar
  39. Rodgers BD, Interlichia JP, Garikipati DK, Mamidi R, Chandra M et al (2009) Myostatin represses physiological hypertrophy of the heart and excitation–contraction coupling. J Physiol 587:4873–4886CrossRefGoogle Scholar
  40. Sekine H, Shimizu T, Hobo K, Sekiya S, Yang J et al (2008) Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts. Circulation 118:145–152CrossRefGoogle Scholar
  41. Sekine H, Shimizu T, Dobashi I, Matsuura K, Hagiwara N et al (2011) Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell ınjection. Tissue Eng Part A 17:2973–2980CrossRefGoogle Scholar
  42. Shimizu K, Ito A, Lee JK, Yoshida T, Miwa K et al (2007) Construction of multi-layered cardiomyocyte sheets using magnetite nanoparticles and magnetic force biotechnology and bioengineering. Biotechnol Bioeng 96:803–809CrossRefGoogle Scholar
  43. Tao H, Nuo M, Min S (2018) Sufentanil protects the rat myocardium against ischemia–reperfusion injury via activation of the ERK1/2 pathway. Cytotechnology 70:169–176CrossRefGoogle Scholar
  44. Watkins SJ, Borthwick GM, Arthur HM (2011) The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro. Vitro Cell Dev Biol Anim 47:125–131CrossRefGoogle Scholar
  45. Wei FL, Qu CY, Song TL, Ding G, Fan ZP et al (2012) Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity. J Cell Physiol 227:3216–3224CrossRefGoogle Scholar
  46. Witek P, Korga A, Burdan F, Ostrowska M, Nosowska B et al (2016) The effect of a number of H9C2 rat cardiomyocytes passage on repeatability of cytotoxicity study results. Cytotechnology 68:2407–2415CrossRefGoogle Scholar
  47. Yamato M, Okano T (2004) Cell sheet engineering. Mater Today 7:42–47CrossRefGoogle Scholar
  48. Yeh TS, Fang YHD, Lu CH, Chiu SC, Yeh CL et al (2014) Baculovirus-transduced, VEGF-expressing adipose-derived stem cell sheet for the treatment of myocardium infarction. Biomaterials 35:174–184CrossRefGoogle Scholar
  49. Yu J, Tu YK, Tang YB, Cheng NC (2014) Stemness and transdifferentiation of adipose-derived stem cells using L-ascorbic acid 2-phosphate-induced cell sheet formation. Biomaterials 35:3516–3526CrossRefGoogle Scholar
  50. Zahn R, Thomasson E, Guillaume-Gentil O, Vörös J, Zambelli T (2012) Ion-induced cell sheet detachment from standard cell culture surfaces coated with polyelectrolytes. Biomaterials 33:3421–3427CrossRefGoogle Scholar
  51. Zhang H, Yu N, Zhou Y, Ma H, Wang J et al (2016) Construction and characterization of osteogenic and vascular endothelial cell sheets from rat adipose-derived mesenchymal stem cells. Tissue Cell 48:488–495CrossRefGoogle Scholar
  52. Zhou T, Zhou Z, Zhou S, Huang F (2016) Real-time monitoring of contractile properties of H9C2 cardiomyoblasts by using a quartz crystal microbalance. Anal Methods 8:488–495CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Bioengineering DepartmentHacettepe UniversityAnkaraTurkey
  2. 2.Chemical Engineering DepartmentHacettepe UniversityAnkaraTurkey

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