Protein-Engineered Large Area Adipose-derived Stem Cell Sheets for Wound Healing

Abstract

Human adipose-derived stem cells (hADSCs) formed robust cell sheets by engineering the cells with soluble cell adhesive molecules (CAMs), which enabled unique approaches to harvest large area hADSC sheets. As a soluble CAM, fibronectin (FN) (100 pg/ml) enhanced the cell proliferation rate and control both cell-to-cell and cell-to-substrate interactions. Through this engineering of FN, a transferrable hADSC sheet was obtained as a free-stranding sheet (122.6 mm²) by a photothermal method. During the harvesting of hADSC sheets by the photothermal method, a collagen layer in-between cells and conductive polymer film (CP) was dissociated, to protect cells from direct exposure to a near infrared (NIR) source. The hADSC sheets were applied to chronic wound of genetically diabetic db/db mice in vivo, to accelerate 30% faster wound closure with a high closure effect (ε_wc) than that of control groups. These results indicated that the engineering of CAM and collagens allow hADSC sheet harvesting, which could be extended to engineer various stem cell sheets for efficient therapies.

Figure 1

Culture conditions on CPP-PEDOT substrate with soluble fibronectin factor for hADSC sheet formation. Photothermal images of PP-PEDOT (a) and SP-PEDOT (b) in the same conditions (I_pw = 2.7 W/cm², t_nir = 3 min, RD = 41.4J). (c) UV/Vis/NIR spectrum of PP-PEDOT (red) and SP-PEDOT (black) substrate (red arrow means 808 nm). After the cells were cultured for 1 day (cell seeding number = 100,000 cells/dish), optical images of the adherent hADSCs on the CPP-PEDOT substrate (d) without soluble fibronectin and (e) with soluble fibronectin (red arrows mean areas of concentrated cells). (f) Effect of harvesting efficiency (ε_d) and sheet area (A_hcs) at different fibronectin concentrations (1 μg/ml to 1 pg/ml, P_sf means the point of sheet formation). (g) Schematic image of the function of soluble fibronectin in hADSC culture conditions. (h–j) Culture conditions on CSP-PEDOT substrate for hADSC sheet formation. (h) Optical image of the adherent hADSCs on the CPP-PEDOT substrate at 1 day. (i) Optical image of the detached sheet fragments from the CPP-PEDOT substrate at 1 day. (j) Effect of the cell adhesion rate and the thickness of collagen layer.

Figure 2

E-cadherin expression of the adherent hADSCs on the CPP-PEDOT substrate and intercellular distance of the harvested cell sheets at different cell concentrations. With 10⁻⁸% (100 pg/ml) soluble fibronectin molecules, DAPI/E-cadherin images of the adherent hADSCs on the substrate at different cell concentrations; cell seeding numbers were (a,d) 1.8 × 10⁵ cells/dish, (b,e) 2.7 × 10⁵ cells/dish, and (c,f) 3.7 × 10⁵ cells/dish. DAPI-stained images of the harvested sheets with different cell seeding numbers: 400,000 cells/dish (g), 800,000 cells/dish (h), and 1,200,000 cells/dish (i). Graphs of the calculated intercellular distance from the DAPI data at 400,000 cells/dish (j), 800,000 cells/dish (k), and 1,200,000 cells/dish (l). The cell groups are randomly selected cell groups in the same area of the cell sheet. Each cell groups in x-axis are consisting of two cells to measure intercellular distance between the two cells(y-axis).

Figure 3

Sheet harvesting process of the hADSCs from the CPP-PEDOT substrate. (a) Effect of the harvesting efficiency (ε_d) and sheet area (A_hcs) at different cell concentrations. Optical microscope images of the detached sheets at 2 min (b), 5 min (c), and 10 min (d). (e) Digital camera image of the lifted sheet using the tiny tweezer. (f) Optical microscope image of the harvested sheet from the CPP-PEDOT substrate (1.3-cm circle pattern). (g) Fluorescent images of E-cadherin expression and live/dead assay (inset) of the harvested sheet. Optical microscope images of large cell sheet detachment with optimized conditions after NIR laser for (h) 2 min and (i) 5 min. (j) The cross-cut images by FE-SEM to identify the morphology of the cells in the harvested hADSCs sheet. The image from confocal microscopy with fluorescein isothiocyanate (FITC) as a fluorophore to trace the collagen (λ_exi = 495 nm, λ_emi = 519 nm) without (k) and with (l) NIR irradiation at different times. The light green region indicates a high concentration of collagen-FITC. (Scale bar: 50 μm) (Inset: magnified image, scale bar: 10 μm).

Figure 4

In vivo wound-healing process. A 50.2-mm² size skin defect was created on the dorsum of diabetic db/db mice. (a) Representative photographs of wound healing with a 16-mm circular template after transplantations of the cell sheet at 0, 5, 7, 9, 11, and 13 days compared with the control. (b) Plot of the wound closure over time after hADSC sheet treatment (black) compared with the control (open). The LMM (linear mixed model) method was used to determine the wound size for confound symmetry covariance within mouse P-value with bonferroni correction. Each value represents the mean ± SD (n = 10). *P < 0.05 compared with the control.