Harvesting prevascularized smooth muscle cell sheets from common polystyrene culture dishes

Abstract

Cell sheet engineering has recently emerged as a promising strategy for scaffold-free tissue engineering. However, the primary method of harvesting cell sheets using temperature-responsive dishes has potential limitations. Here we report a novel cell sheet technology based on a coculture system in which SMCs are cocultured with EPCs on common polystyrene dishes. We found that an intact and highly viable cell sheet could be harvested using mechanical methods when SMCs and EPCs were cocultured on common polystyrene dishes at a ratio of 6:1 for 5 to 6 days; the method is simple, cost-effective and highly repeatable. Moreover, the cocultured cell sheet contained capillary-like networks and could secrete a variety of angiogenic factors. Finally, in vivo studies proved that the cocultured cell sheets were more favorable for the fabrication of vascularized smooth muscle tissues compared to single SMC sheets. This study provides a promising avenue for smooth muscle tissue engineering.

Fig 1. Characteristics of cultured SMCs and EPCs.

(A) Phase contrast image of bladder SMCs at passage 3. (B) Phase contrast images of EPCs at passages 0 and 3, respectively. (C) Tubular network formation of EPCs on Matrigel. (D) EPCs were identified by ac-LDL endocytosis and UEA-1 binding. Nuclei were stained with DAPI. (E) Immunofluorescence images showing CD34 and VEGFR2 expression in EPCs. Nuclei were stained with DAPI. The scale bars show 100 μm in (A), 200 μm in (B, C), and 50 μm in (D, E).

Fig 2. Network formation by EPCs in the coculture.

SMCs were stained with CM-DiI, and EPCs were stained with CMFDA. The scale bars show 200 μm.

Fig 3. Characteristics of SMC-EPC cocultured cell sheets.

(A) Cocultured cells before and during harvest. (B) Macroscopic image of a cocultured cell sheet. (C) H&E staining of a cocultured cell sheet. (D) Live/dead staining of a cell sheet before and after harvest. (E) Live/dead staining of the cells killed by methanol. (F) Quantitative comparison of the cell viability before and after harvest (n = 6). (G) Microscopic images of a cocultured cell sheet adhered to a new culture dish after 2 h, 8 h, 24 h, and 48 h. (H) SEM image of a cocultured cell sheet. (I) TEM image of a cocultured cell sheet. (J) Angiogenic factor secretion of the SMC, EPC and coculture groups was measured by ELISA (n = 9). Results are expressed as means ± SD. **p < 0.01; n.s.: not significant. The scale bars show 200 μm in (A, D, E), 50 μm in (C), 100 μm in (G), 10 μm in (H), and 500 nm in (I).

Fig 4. Results of the cell sheets transplanted in vivo.

(A) Gross appearance, H&E staining, and immunostaining of α-SMA and CD31 of the single SMC sheet grafts and cocultured cell sheet grafts. (B, C) Quantitative comparisons of the tissue thickness and vessel density, respectively. Results are expressed as means ± SD (n = 8). **p < 0.01. The scale bars show 200 μm.