Rapid fabrication system for three‐dimensional tissues using cell sheet engineering and centrifugation

Abstract

Three-dimensional (3D) tissues can be reconstructed by cell sheet technology, and various clinical researches using these constructed tissues have already been initiated to regenerate damaged tissues. While 3D tissues can be easily fabricated by layering cell sheets, the attachment period for cell adhesion between a cell sheet and a culture dish, or double-layered cell sheets normally takes 20-30 min. This study proposed a more rapid fabrication system for bioengineered tissue using cell sheet technology and centrifugation. A C2C12 mouse myoblast sheet harvested from a temperature-responsive culture dish will attach tightly to a culture dish or another cell sheet at 37°C after a 20 min-incubation. However, the same cell sheet centrifuged (12-34 × g) for 3 min also attached tightly to a dish or another cell sheet at 37°C after only a 3 min-incubation. The manipulation time was reduced by approximately two-thirds by centrifugation. The rapid attachments were also cross-sectionally confirmed by optical coherence tomography. These rapidly constructed cell sheet-tissues using centrifugation showed active cell metabolism, cell viability, and very high production of vascular endothelial growth factor, like those prepared by the conventional method; indicating complete cell sheet-attachment without any cell damage. This new system will be a powerful tool in the fields of cell sheet-based tissue engineering and regenerative medicine, and accelerate the use of cell sheets in clinical applications.

Three-Dimensional Human Cardiac Tissue Engineered by Centrifugation of Stacked Cell Sheets and Cross-Sectional Observation of Its Synchronous Beatings by Optical Coherence Tomography

“…Importantly, the cell sheet beat spontaneously and robustly, and the transmission was detected as clearly before centrifugation as after centrifugation (Supplementary Movie 2). In a previous report, a C2C12 mouse myoblast sheet attached rapidly by the centrifugation method showed (i) active cell metabolism (glucose consumption and lactate production), which indicates the bioactivity of cells, (ii) the release of lactate dehydrogenase (LDH), which indicates cytotoxicity, and (iii) high production of VEGF, which is generally thought to be a major paracrine factor in the repair of the damaged heart tissue by myoblast sheet therapy [24], like those prepared by the conventional method [9]. Those results suggested cell sheets attached rapidly onto the dish by centrifugation without cell damage.…”

“…After the transfer, the cardiac cells on the dish were spontaneously detached as a cardiac cell sheet within 60 min. Harvested cell sheets were layered on a 35 mm diameter polystyrene culture surface (Corning, NY, USA) [5, 6, 19], and layered cell sheets were centrifuged by a swing-type centrifuge (AllegraTM 21R Centrifuge) (Beckman Coulter, CA, USA) to accelerate their attachment as described in a previous report [9]. The movies of human iPS cell-derived cardiac cells were observed with a phase-contrast microscope, ET300 (Nikon, Tokyo, Japan), and the images were recorded with CCD camera equipment, acA1300-30uc (Basler, Ahrensburg, Germany) and the software StreamPix 6 (NorPix, Inc., Quebec, Canada).…”

“…In this study two OCT systems [Proto 3(DT) (Panasonic Healthcare, Tokyo, Japan) [9, 14, 15, 20] and IVS-2000 (Santec, Aichi, Japan)] were used. Their specifications are summarized in Table 1.…”

“…Cell sheet stacking technology enables the transplantation of enormous numbers of cells in thicker 3D tissues and offers hope for more efficient therapies and a wider range of applications in regenerative medicine. Recently, we reported the rapid fabrication technique of a double-layered cell sheet-tissue by the combination of cell sheet stacking technology and centrifugation [9]. An optimal fabrication technique for thicker 3D tissue is important in tissue engineering and regenerative medicine to speed development of life-saving applications.…”

Optical coherence microscopy of living cells and bioengineered tissue dynamics in high-resolution cross-section

“…NIH3T3 cell sheets and BAEC sheets were fabricated by using a 35‐mm thermoresponsive culture dish (UpCell ® dish) (CellSeed, Tokyo, Japan), and the fabrication of 3‐D tissues by layering the cell sheets was performed as described in previous reports . Recently, we developed a rapid fabrication system for cell sheet‐tissues by centrifugation, which can reduce the fabrication time by approximately two‐thirds compared to the conventional method . Thus, in this study, each cell sheet was rapidly attached by centrifugation (12–34 g ) for 3 min onto a culture surface after reattachment, or onto another cell sheet after layering, as described in the previous report …”