Functional aligned mesenchymal stem cell sheets fabricated using micropatterned thermo-responsive cell culture surfaces

Abstract

Mesenchymal stem cells (MSCs) are frequently applied for cell transplantation and regenerative therapy because they secrete diverse therapeutic cytokines that prompt immuno-stimulatory and tissue repair processes. Furthermore, cultured MSC sheets exhibit enhanced cytokine secretion compared to their MSC suspensions, and represent a durable, versatile format for tissue engineering as singular, multi-layered, or multi-cell type sandwiched, transplantable constructs. Tissue engineered implants with various cellular orientations have been reported. In this study, patterned, temperature-responsive culture surfaces were used to prepare oriented MSC sheets. Patterned culture surfaces were fabricated by grafting polyacrylamide (PAAm) onto commercial poly(N-isopropylacrylamide) (PNIPAAm)-modified plastic via photopolymerization using a stripe-patterned photomask. Patterned surfaces were characterized using x-ray photoelectron spectroscopy, fluorescently labelled fibronectin and albumin adsorption assays, wetting (contact angle) measurements, atomic force microscopy, and scanning electron microscopy. Striped grafted patterns of PAAm were fabricated on the PNIPAAm-coated culture substrates, and PAAm polymerized within the PNIPAAm overlayer. Cell-aligned MSC sheets were then produced from MSC culture on this patterned surface, secreting higher amounts of therapeutic cytokines (vascular endothelial growth factor, hepatocyte growth factor, and transforming growth factor-β) than non-aligned MSC control sheets. In addition, aligned MSC sheets maintained enhanced cell multi-potent differentiation capabilities. New, aligned MSC sheets might exhibit improved functional properties for cell sheet transplant therapies.

Keywords: Cell therapy; Immunomodulatory; MSC secretome; Regenerative medicine; Stem cell; Stromal cell; Tissue repair; Transplantation.

Graphical abstract

Fig. 1

Schematic illustration of (A) preparation of patterned cell culture substrates through patterned photopolymerization of acrylamide onto commercial thermo-responsive cell culture plastic, (B) schematic of selective surface pattern modification using acrylamide, and (C) cultured cell alignment yielding oriented cell sheets using patterned culture surfaces.

Fig. 2

High-resolution XPS C1s peak deconvolution for (A) TCPS, (B) PNIPAAm-modified culture substrate, and (C) PAAm-patterned culture substrate (take-off angle 15°).

Fig. 3

Characterization of PAAm-patterned commercial PNIPPAm-modified culture surfaces. (A) fluorescent microscopy images of adsorbed rhodamine-labelled fibronectin and Alexa488-labelled bovine serum albumin in patterns. (B) ATR/FT-IR vibrational spectra. (C) Wetting comparison: aqueous sessile drop contact angle measurements at 37 °C (∗p < 0.05; n.s.: not significant). (D) AFM tapping mode image. (E) SEM surface image.

Fig. 4

Characterization of aligned, versus non-aligned NHDF sheets. (A) phase contrast microscopy comparing aligned and non-aligned NHDFs in sheet cultures. (B) cytokine expression in aligned versus non-aligned NHDF sheets. (C) Images of NHDF sheet detachment from each surface by reducing culture temperature. (D) cell numbers in both types of cell sheets (∗∗p < 0.01; n.s.: not significant).

Fig. 5

Characterization of aligned, oriented versus non-oriented MSC sheets. (A) phase contrast microscopy comparing aligned and non-aligned MSCs in sheet cultures; (B) cytokine expression in aligned versus non-aligned MSC sheets; (C) images of MSC sheet detachment from each surface by reducing culture temperature; (D) cell numbers in both types of cell sheets (∗p < 0.05; ∗∗p < 0.01; n.s.: not significant).

Fig. 6

Two-lineage differentiation of oriented MSC sheets. Osteogenic (left 2 columns) and adipogenic (right 2 columns) differentiation was induced for 14 days and confirmed by Alizarin Red S and Oil Red O staining images, respectively. Scale bars: 100 μm.