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

Abstract

Three-dimensional (3D) tissues are engineered by stacking cell sheets, and these tissues have been applied in clinical regenerative therapies. The optimal fabrication technique of 3D human tissues and the real-time observation system for these tissues are important in tissue engineering, regenerative medicine, cardiac physiology, and the safety testing of candidate chemicals. In this study, for aiming the clinical application, 3D human cardiac tissues were rapidly fabricated by human induced pluripotent stem (iPS) cell-derived cardiac cell sheets with centrifugation, and the structures and beatings in the cardiac tissues were observed cross-sectionally and noninvasively by two optical coherence tomography (OCT) systems. The fabrication time was reduced to approximately one-quarter by centrifugation. The cross-sectional observation showed that multilayered cardiac cell sheets adhered tightly just after centrifugation. Additionally, the cross-sectional transmissions of beatings within multilayered human cardiac tissues were clearly detected by OCT. The observation showed the synchronous beatings of the thicker 3D human cardiac tissues, which were fabricated rapidly by cell sheet technology and centrifugation. The rapid tissue-fabrication technique and OCT technology will show a powerful potential in cardiac tissue engineering, regenerative medicine, and drug discovery research.

Figure 1

Fabrication and observation of layered human iPS cell-derived cardiac cell sheet-tissues using centrifugation. A detached human iPS cell-derived cardiac cell sheet was transferred onto a polystyrene culture dish (a). The culture dish with the cell sheet was centrifuged (23 ×g, 3 min), and the cross-sectional observation was performed by optical coherence tomography (OCT) (b and c). After incubation (37°C, 5 min), another cardiac cell sheet was transferred onto the first cardiac cell sheet (d). Multilayered cardiac cell sheets were centrifuged (12 ×g for 1 sec and again after removal of the extra medium 34 ×g for 3 min); then the cross-sectional observation of the cell sheets was performed (e and f).

Figure 2

Observation of double-layered human iPS cell-derived cardiac cell sheets by optical coherence tomography (OCT). Just after the transfer of a human iPS cell-derived cardiac cell sheet onto a polystyrene culture dish, there were numerous spaces between the surfaces (a-1). After centrifugation, the spaces were almost entirely eliminated (a-2). Just after the transfer of a second cell sheet onto the first cell sheet, again there were numerous spaces between the surfaces (b-1). After centrifugation, the spaces were almost entirely eliminated (b-2). Three experiments were performed independently and all of them showed similar results. An OCT system [Proto 3(DT)] was used in this study.

Figure 3

Optical coherence tomography (OCT) observation of double-layered human iPS cell-derived cardiac cell sheets fabricated by a conventional method. Just after the layering of a human iPS cell-derived cardiac cell sheet onto first cardiac cell sheet, there were significant spaces between the double-layered cell sheets (a). In the conventional method without centrifugation, some spaces between the double-layered cardiac cell sheets were detected even after the 15 min incubation (b), and spaces were hardly detected after the 30 min incubation (c). Three experiments were performed independently and all of them showed similar results. An OCT system (IVS-2000) was used in this study.

Figure 4

Observation of double-layered human iPS cell-derived cardiac cell sheets by optical coherence tomography (OCT). At 15 min cultivation after centrifugation, while the double-layered cardiac cell sheets did not continuously beat synchronously ((a-1) and (a-3)), the occasional synchronous beating was detected in the cell sheets (a-2). However, within 45 min cultivation, the cardiac cell sheets beat synchronously ((b-1)–(b-6)). Beating areas within the cell sheet are shown with green colors. The time-intervals between (a-1) and (a-3) or (b-1) and (b-6) were 4 s or 1 s, respectively. Three experiments were performed independently and all of them showed similar results. The other two double-layered cardiac cell sheets also synchronized within 45 min after centrifugation. An OCT system (IVS-2000) was used in this study.

Figure 5

Observation of five-layered human iPS cell-derived cardiac cell sheets by optical coherence tomography (OCT). Just after layering of a fifth cardiac cell sheet onto the four-layered cardiac cell sheets and subsequent centrifugation, the lower cell sheets beat synchronously (a). The beating of the lower cardiac cell sheets was also occasionally transmitted to the upper cardiac cell sheets (b). Within 60 min after centrifugation, the five-layered cardiac cell sheets showed synchronous beatings (c). The time-intervals between (a-1) and (a-4), (b-1) and (b-6), or (c-1) and (c-5) were 1.6 s, 1 s, or 1 s, respectively. Beating areas within the cell sheet are shown with green colors. (c) The white arrowheads indicate the initiation sites of the beatings within each section of the tissue. In (c-1) the multilayered cardiac sheets are outlined with dashed lines. An OCT system (IVS-2000) was used in this study.