JoVE Logo
Faculty Resource Center

Sign In





Representative Results






Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Published: October 20th, 2018



1Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, 2Department of Developmental and Regenerative Biology, Jinan University, 3Nansha College Preparatory Academy, 4Department of Cardiology, First Affiliated Hospital of Jinan University
* These authors contributed equally

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

Stem cell therapy has been reported as an effective treatment for many diseases; however, low cell retention and poor survival within the target zone remain critical issues following traditional stem cell injection. To solve this problem, tissue engineering scientists developed the cell sheet technique. A monolayered cell sheet with intact extracellular matrix was firstly prepared by using the temperature-response culture dish1, and its follow-up studies reported the significant improvements of stem cell retention and survival within the infarcted area2,3. Among the methods, constructin....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

All stem cell and animal experiment procedures were conducted according to the ethical guidelines of the National Guide for the Care and Use of Laboratory Animals and approved by the Jinan University Animal Care and Use Committee (Guangzhou, China).

1. Preparation of the DPP Scaffold with the PLA2 Decellularization Method14

Note: See Figure 1A for a schematic of the PLA2 decellularization meth.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The schematic of the multilayered stem cell sheet construction is shown in Figure 1. Preparing the cell sheet scaffold by the PLA2 decellularization method is the first step. Based on the scaffold, a temporary 3D cell structure is constructed by mixing the stem cells with the RAD16-1 peptide hydrogel. In order to obtain a multilayered cell sheet with favorable stem cell bioactivity and optimal mechanical strength, the cell sheet is cultured in a dy.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The present protocol reports an efficient method for constructing a multilayered MSC sheet. This cell sheet exhibits optimal mechanical strength, high cell seeding density, and favorable stem cell bioactivity. Using BMSCs as an example, the 3D cell structure is quickly constructed with RAD16-I peptide hydrogel. After being cultured in the dynamic perfusion system, the multilayered BMSC sheet is successfully obtained and the BMSCs maintain a high expression of stem cell markers.

Constructing th.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by the National Natural Science Foundation of China (grant number 31771064); the Science and Technology Planning Project of Guangdong Province (grant numbers 2013B010404030, 2014A010105029, and 2016A020214012); the Science and Technology Planning Project of Guangzhou (grant number 201607010063); and the Undergraduate Innovation and Entrepreneurship Training Program (grant number 201610559028); the National Science Foundation for Young Scientists of China (grant number 31800819).


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Phospholipase A2 Sigma-Aldrich P6534
Sodium deoxycholate Sigma-Aldrich D6750-100G
Phosphate buffer Gibco BRL 89033
Penicillin streptomycin / amphotericin  Gibco BRL 15640055
Buffer bicarbonate Sigma-Aldrich C3041
Table concentrator Changzhou Aohua Instrument Co. KT20183
Dulbecco's Modified Eagle Medium(DMEM) Corning Cellgro 10-014-CVR
South American fetal bovine serum  Gibco BRL 10270-106/P30-3302
L-Glutamine Corning Cellgro 25-005-CI
0.25% Trypsin/2.21 mM EDTA Corning Cellgro 25-053-CI
Biosafety cabinet Esco,Singapore AC2-2S1
Constant temperature incubator  Esco,Singapore CLS-170B-8
Centrifuge tube  Corning 430790
EP tube Axygen 31617934
Centrifugal machine TOMOS 1-16R 
Sucrose Sigma-Aldrich S9378-500G
Pura Matrix  BD 354250
Dynamic perfusion culture system Minucells and Minutissue D-93077
Peristaltic pump Ismatec IPC N8
Pump tubing Ismatec Nr.1306
MINUSHEET 1300  Regensburg tissue carrier components 
MINUSHEET Regensburg dynamic perfusion system 
MINUSHEET 0006 Regensburg gas exchange equipment 
MINUSHEET 0002 Regensburg 500 mL glass bottle 
MINUSHEET 1301 perfusion culture container 

  1. Miyahara, Y., et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nature Medicine. 12 (4), 459-465 (2006).
  2. Narita, T., et al. The use of cell-sheet technique eliminates arrhythmogenicity of skeletal myoblast-based therapy to the heart with enhanced therapeutic effects. International Journal of Cardiology. 168 (1), 261-269 (2013).
  3. Narita, T., et al. The Use of Scaffold-free Cell Sheet Technique to Refine Mesenchymal Stromal Cell-based Therapy for Heart Failure. Molecular Therapy. 21 (4), 860-867 (2013).
  4. Matsuo, T., et al. Efficiently Piled-Up Cardiac Tissue-Like Sheets With Pluripotent Stem Cell-Derived Cells Robustly Promotes Cell Engraftment and Ameliorates Cardiac Dysfunction After Myocardial Infarction. Circulation. 128 (22), (2013).
  5. Alshammary, S., et al. Impact of cardiac stem cell sheet transplantation on myocardial infarction. Surgery Today. 43 (9), 970-976 (2013).
  6. Chen, G. P., et al. The use of a novel PLGA fiber/collagen composite web as a scaffold for engineering of articular cartilage tissue with adjustable thickness. Journal of Biomedical Materials Research Part A. 67a (4), 1170-1180 (2003).
  7. Cerqueira, M. T., et al. Human Adipose Stem Cells Cell Sheet Constructs Impact Epidermal Morphogenesis in Full-Thickness Excisional Wounds. Biomacromolecules. 14 (11), 3997-4008 (2013).
  8. Sasagawa, T., Shimizu, T., Sekiya, S., Yamato, M., Okano, T. Comparison of angiogenic potential between prevascular and non-prevascular layered adipose-derived stem cell-sheets in early post-transplanted period. Journal of Biomedical Materials Research Part A. 102 (2), 358-365 (2014).
  9. Ishii, M., et al. Multilayered adipose-derived regenerative cell sheets created by a novel magnetite tissue engineering method for myocardial infarction. International Journal of Cardiology. 175 (3), 545-553 (2014).
  10. Godier-Furnemont, A. F., et al. Composite scaffold provides a cell delivery platform for cardiovascular repair. Proceedings of the National Academy of Sciences of the United States of America. 108 (19), 7974-7979 (2011).
  11. Liu, Y., et al. Electrospun nanofibrous sheets of collagen/elastin/polycaprolactone improve cardiac repair after myocardial infarction. American Journal of Translational Research. 8 (4), 1678-1694 (2016).
  12. Arana, M., et al. Epicardial delivery of collagen patches with adipose-derived stem cells in rat and minipig models of chronic myocardial infarction. Biomaterials. 35 (1), 143-151 (2014).
  13. Wang, Y., et al. Preparation of high bioactivity multilayered bone-marrow mesenchymal stem cell sheets for myocardial infarction using a 3D-dynamic system. Acta Biomaterialia. 72, 182-195 (2018).
  14. Wu, Z., et al. The use of phospholipase A(2) to prepare acellular porcine corneal stroma as a tissue engineering scaffold. Biomaterials. 30 (21), 3513-3522 (2009).
  15. Degano, I. R., et al. The effect of self-assembling peptide nanofiber scaffolds on mouse embryonic fibroblast implantation and proliferation. Biomaterials. 30 (6), 1156-1165 (2009).
  16. Lampe, K. J., Heilshorn, S. C. Building stem cell niches from the molecule up through engineered peptide materials. Neuroscience Letters. 519 (2), 138-146 (2012).
  17. Cui, X. J., et al. Transplantation of Mesenchymal Stem Cells with Self-Assembling Polypeptide Scaffolds Is Conducive to Treating Myocardial Infarction in Rats. Tohoku Journal of Experimental Medicine. 222 (4), 281-289 (2010).
  18. Jun, I., et al. Spatially Assembled Bilayer Cell Sheets of Stem Cells and Endothelial Cells Using Thermosensitive Hydrogels for Therapeutic Angiogenesis. Advanced Healthcare Materials. 6 (9), (2017).
  19. Chen, C. H., et al. Porous tissue grafts sandwiched with multilayered mesenchymal stromal cell sheets induce tissue regeneration for cardiac repair. Cardiovascular Research. 80 (1), 88-95 (2008).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved