Stacked stem cell sheets enhance cell-matrix interactions

Abstract

Cell sheet engineering has enabled the production of confluent cell sheets stacked together for use as a cardiac patch to increase cell survival rate and engraftment after transplantation, thereby providing a promising strategy for high density stem cell delivery for cardiac repair. One key challenge in using cell sheet technology is the difficulty of cell sheet handling due to its weak mechanical properties. A single-layer cell sheet is generally very fragile and tends to break or clump during harvest. Effective transfer and stacking methods are needed to move cell sheet technology into widespread clinical applications. In this study, we developed a simple and effective micropipette based method to aid cell sheet transfer and stacking. The cell viability after transfer was tested and multi-layer stem cell sheets were fabricated using the developed method. Furthermore, we examined the interactions between stacked stem cell sheets and fibrin matrix. Our results have shown that the preserved ECM associated with the detached cell sheet greatly facilitates its adherence to fibrin matrix and enhances the cell sheet-matrix interactions. Accelerated fibrin degradation caused by attached cell sheets was also observed.

Keywords: cell sheet; cell-matrix interactions; fibrin; mesenchymal stem cells.

Figure 1. Micropipette Transfer Method. (A) The modified micropipette tip is fabricated by cutting the 1ml micropipette tip by the depicted configuration. (B) A cell sheet detached from the thermo-responsive surface is drawn into the modified micropipette tip along with media and dispensed to the desired location. (C) Multiple cell sheets can be transferred and stacked within the tip by drawing them into the tip, allowing them to settle to bottom of tip, and then dispensing on desired substrate.

Figure 2. Cell sheet transfer and cell viability. (A) The morphology of confluent hMSC monolayer growing on the thermoresponsive substrate prior to cell sheet detachment. (B) hMSC sheet morphology after transfer and attachment to the new cell culture substrate using the developed micropipette method. (C) Cell viability of single layer cell sheet after manipulation with a micropipette compared with the same amount of dissociated cells. No significant difference (P = 0.98) is identified between the two groups. (D) H&E staining of multi-layer hMSC sheets on fibrin matrix. Scale bar is 200µm for A and B and 50 µm for D.

Figure 3. Representative confocal images of 3-layer hMSC sheets. (A) Gallery image of sectional view showing the 3 layers of hMSC sheets. Cells at the top and bottom layers are labeled with AlexaFluor Phalloidin Green and cells at the middle layer are labeled with CellTracker Red CMTPX. (B) Cross section view. Scale bar is 100 µm for both A and B.

Figure 4. Cell sheet and fibrin matrix interactions. (A) The effect of fibrinogen concentration on cell proliferation. Cell proliferation was measured using a PrestoBlue cell proliferation assay on the monolayer hMSC sheet 24 h after transferring to fibrin matrices with different fibrinogen concentrations. RFU is the fluorescent emission intensity of samples at 590nm when excited with 560nm light. Results are the means ± SD of 3 samples. Asterisk (*) indicates significant difference (P < 0.05). (B and C) H&E Staining of 4-layer hMSC sheets cultured on fibrin matrix for 1 d and 7 d, respectively. Scale bar is 50 µm (B) and 200µm (C).