Free Standing Cell Sheet Assembled with Ultrathin Extracellular Matrix as an Innovative Approach for Biomimetic Tissues

Li qingtao (Department of Mechanical Engineering, University of Manitoba, Winnipeg MB R3T 2N2, Canada)
Chen jun (Department of Mechanical Engineering, University of Manitoba, Winnipeg MB R3T 2N2, Canada)


Current artificial tissue-substitutes have limited clinical applications due to unmatched complex combination of cells and extracellular matrix (ECM) as seen in native tissues. Significant efforts have been made to fabricate numerous biomedical materials for this purpose including hydrogels, porous materials and nanoparticles aggregations. A rational approach is urgently needed to fabricate multifunctional materials to match the complicated biomedical uses via architecting materials with hierarchic and organized structures. To meet the above requirements, use of a free-standing cell sheet with layer-by-layer (LbL) ultrathin films as “nano-clothing” on it may provide us a pragmatic approach.Another significant advantage in the use of LbL ultrathin films to build tissue scaffold comes from the bottomup approach. The free-standing planar films are highly amenable to surface modification and engineering to match the various ends in specific tissue compartment.

Materials and Methods

Hydroxylated glass wafers were placed into a 3 wt% toluene solution of tissue culture polystyrene (APTES) and refluxed under nitrogen atmosphere for 24 h. N-isopropylacrylamide (NIPAM) was spin-coated onto the glass wafers at 800 rpm for 30 s. The surface was characterized by contact angle, ATR and AFM. Human C2C12 cells were seeded onto the PNIPAM-grafted slides. LBL assembly of Gelatin-(chitosan-alginate)3 were prepared on a monolayer of Cells. A 0.5% (w/v) type A Gelatin, a 0.1% (w/v) chitosan and a 0.1% (w/v) Alginic acid solution were pre-warmed to 37 °C before being applied to the LbL deposition. After the LbL assembly of gelatin-(chitosan-alginate)3 on a monolayer of cells, the film was washed with cold PBS three times. The film was immersed in a cold PBS solution and kept in 4 °C for 1 h or room temperature for 2–3 h to allow the formation of the free-standing cell–gelatin–(chitosan/ alginate)3. Vitality of the cells was investigated using the LIVE/DEAD Kit. F-actin in the cytoskeleton stained by phallacidin, and nuclei stained by Topro-3. To analyze osteogenic differentiation of BMSCs, the cells were divided into three groups: a tissue culture polystyrene (TCPS) group, a TCPS + BMP2 (10 ng/ml, ProSpec, USA) group, and a cell-gelatin-(chitosan/alginate)3 -BMP2 group. Then quantitative real-time PCR was performed by SYBER Green assays.


We grafted temperature-responsive PNIPAM onto glass slides by a two-step method. First formed by hydrolyzing APTES on a clean glass surface. Then, the graft polymerization of NIPAM was carried out. The cells were firstly cultured on the hydrophobic PNIPAM layer on the petri-dishes. Once 80–90% confluence of the cells was achieved, the LbL process was conducted on the surface of the cell sheet. We employed gelatin as the cell contacting layer and chitosan-alginate as the nano-matrix. Gelatin is a natural biocompatible polyelectrolyte (PE) providing excess charges. Alternate-charged PE layers of chitosan and alginate with 0.1 w/v% were assembled onto the gelatin-coated human myoblast cells C2C12 or mesenchymal stem cells on the PNIPAM-grafted surfaces as sketched in Figure 1. The morphology of a free-standing cell-nanomembrane is shown in Figure 2. CLSM images of the top view and cross sections of the nanomembranes show a homogeneous assembly of gelatin–(chitosan/alginate)3 nanofilms on the surface of the cell sheet ( Figure 3). To investigate the effects of regulator-loaded membranes on the osteogenic differentiation of stem cells, a free-standing membrane composed of BMP2-loaded gelatin-(chitosan/alginate)3 membranes lined with mouse BMSCs was fabricated utilizing electrostatic interactions between the protein and polymer to mediate assembly and characterized. Compared to the TCPS +BMP2 and TCPs groups on day 7, Runx2, COL-I, BSP, and OPN mRNA expression in the cell-gelatin-(chitosan/alginate) 3 -BMP2 group elicited respectively an Ɉ14-fold, Ɉ13-fold, Ɉ3.7- fold, and Ɉ19-fold increase as compared to the group of TCPS + BMP2, and elicited a Ɉ1.5-fold, Ɉ1.3-fold, Ɉ30-fold, and Ɉ1.9-fold increase, respectively, as compared to the TCPS group. This long-term ability of enhancing the osteogenic differentiation of mouse BMSCs and the freestanding soft property make it very suitable for the potential application of tissue regeneration.

Discussion and Conclusion

In summary, a novel strategy to fabricate a free-standing multilayer nanomembrane lined with a cell sheet was developed in the present work. The architecture was well organized, even after the complex was peeled off the substrate. The scaffold mechanically supported the cell sheets and maintained the cell proliferation at the minimal cost of adding extra polymer components on the cells. The gelatin–(chitosan/alginate)3 assemblies exhibited good cell compatibility to meet the fundamental requirement for the consecutive application of the tissue construction.

Figure 1. Schematic illustration of fabrication of free-standing cell-nanomembranes.

Figure 2. A) The contact angles of various surfaces in the process of preparation of the freestanding film. B,C) AFM and SEM (scanning electron microscopy) images of the morphology of a free-standing nanomembrane-cell sheet. D) Photo of free-standing film floating in a PBS buffer.

Figure 3. A–D) CLSM images of C2C12 cell-gelatin-(rh-chitosan/alginate) 3 nanomembranes and E) cross-section scanning images of the free-standing cell nanombranes.


This work was supported by the NSERC Discovery Grant and NSERC RTI Grant, Manitoba Health Research Council Establishment Grant, Dr. Moore House Fellowship, Manitoba Diabetes Foundation, Manitoba Institute of Child Health, National Basic Research Program of China (Grant No. 2012CB619100),China 863 Project (Grant N. 2012AA020504)


1. Alsberg, E., et al. (2002). "Engineering growing tissues." 99(19): 12025-12030. 2. Co, C. C., et al. (2005). "Biocompatible micropatterning of two different cell types." Journal of the American Chemical Society 127(6): 1598-1599.

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