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.

Rapid creation system of morphologically and functionally communicative three‐dimensional cell‐dense tissue by centrifugation

“…A PDMS device for clamping a 60 mm temperature‐responsive culture dish was created by using the lid of 12‐well plate (Corning, NY, USA) and a PDMS (sylpot 184 silicone elastomer kit, Dow Corning Toray, Tokyo, Japan) as shown in Fig. . C2C12 cells or human iPS cell‐derived cardiac cells were seeded into the wells of 24‐well plates or a polydimethylsiloxane (PDMS) ring device (square with 12 mm on a side) set on a 60‐mm temperature‐responsive culture surface.…”

“…The cell metabolism and cell damage after the centrifugation were assessed by the glucose consumption of the cells and lactate dehydrogenase (LDH) release from the cells, respectively. Glucose consumption and LDH release were quantified as shown elsewhere . The medium without the cells or the twice freeze–thawed sample (−80 °C/37°C) of the cells was used as background or the positive control of cell death in LDH release assay, respectively.…”

“…1. 26,27 C2C12 cells or human iPS cell-derived cardiac cells were seeded into the wells of 24-well plates or a polydimethylsiloxane (PDMS) ring device (square with 12 mm on a side) set on a 60-mm temperature-responsive culture surface. Another PDMS (KE-1308, Shin-Etsu Chemical, Tokyo, Japan) was used for making the square-type PDMS device.…”

“…Glucose consumption and LDH release were quantified as shown elsewhere. 27 The medium without the cells or the twice freeze-thawed sample (−80 C/37 C) of the cells was used as background or the positive control of cell death in LDH release assay, respectively.…”

Rapid fabrication of detachable three‐dimensional tissues by layering of cell sheets with heating centrifuge

“…The establishment of a rapid fabrication system for thicker 3‐D tissue is important in tissue engineering and regenerative medicine to speed development of life‐saving applications. Recently, we have shortened the manipulation time successfully by a method which uses a conventional plate centrifuge . The centrifuge did not have a heating function and could not keep the temperature above 32°C; therefore, a temperature‐responsive culture dish could not be used as a culture surface where cell sheets are multilayered.…”