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

Abstract

Confluent cultured cells on a temperature-responsive culture dish can be harvested as an intact cell sheet by decreasing temperature below 32°C. A three-dimensional (3-D) tissue can be fabricated by the layering of cell sheets. A resulting 3-D multilayered cell sheet-tissue on a temperature-responsive culture dish can be also harvested without any damage by only temperature decreasing. For shortening the fabrication time of the 3-D multilayered constructs, we attempted to layer cell sheets on a temperature-responsive culture dish with centrifugation. However, when a cell sheet was attached to the culture surface with a conventional centrifuge at 22-23°C, the cell sheet hardly adhere to the surface due to its noncell adhesiveness. Therefore, in this study, we have developed a heating centrifuge. In centrifugation (55g) at 36-37°C, the cell sheet adhered tightly within 5 min to the dish without significant cell damage. Additionally, centrifugation accelerated the cell sheet-layering process. The heating centrifugation shortened the fabrication time by one-fifth compared to a multilayer tissue fabrication without centrifugation. Furthermore, the multilayered constructs were finally detached from the dishes by decreasing temperature. This rapid tissue-fabrication method will be used as a valuable tool in the field of tissue engineering and regenerative therapy. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:692-701, 2018.

Analysis of force vector field during centrifugation for optimizing cell sheet adhesion

“…C2C12 mouse myoblasts (ATCC® CRL‐1772™) were maintained in a 5% CO 2 incubator (PHC Corporation, Tokyo, Japan) at 37 °C using Dulbecco's Modified Eagle Medium (DMEM; Sigma‐Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Japan Bio Serum, Nagoya, Japan) and 1% penicillin/streptomycin (P/S, Invitrogen, Carlsbad, CA). A C2C12 myoblast sheet was prepared by using a temperature‐responsive culture dish (UpCell® dish, CellSeed, Tokyo, Japan) as described in a previous report . After the medium was removed, the dish or a polystyrene culture dish (Becton, Dickinson and Company, Franklin Lakes, NJ) with the detached cell sheet was centrifuged at 37 °C using a polydimethylsiloxane (PDMS) mold plate, which was fabricated by using PDMS (sylpot 184 silicone elastomer kit, Dow Corning Toray, Tokyo, Japan) a lid of a 6‐well or 24‐well cell culture plate (Becton, Dickinson and Company), and a swing‐type heating plate centrifuge, as shown in the previous studies .…”

“…A C2C12 myoblast sheet was prepared by using a temperature‐responsive culture dish (UpCell® dish, CellSeed, Tokyo, Japan) as described in a previous report . After the medium was removed, the dish or a polystyrene culture dish (Becton, Dickinson and Company, Franklin Lakes, NJ) with the detached cell sheet was centrifuged at 37 °C using a polydimethylsiloxane (PDMS) mold plate, which was fabricated by using PDMS (sylpot 184 silicone elastomer kit, Dow Corning Toray, Tokyo, Japan) a lid of a 6‐well or 24‐well cell culture plate (Becton, Dickinson and Company), and a swing‐type heating plate centrifuge, as shown in the previous studies . In this study, weight was used to adjust the inclination angle made by the bottom face of the PDMS mold plate against the horizontal plane when the centrifuge is stopped.…”

“…The photographs of the cell sheets were taken by a digital camera (GR DIGITAL III, Ricoh, Tokyo, Japan). Cell sheets were layered onto a temperature‐responsive culture dish, as shown in previous reports . A cell sheet or multilayered cell sheets on a temperature‐responsive culture surface was observed cross‐sectionally by optical coherence tomography (OCT) as described in previous reports …”

“…Recently, we have developed a heating plate centrifuge, with which we can maintain the temperature at 37 °C during centrifugation and succeeded in the fabrication of 3D tissues on a temperature‐responsive culture surface . On this surface, cells adhere at 37 °C, which is the temperature above the lower critical solution temperature of PIPAAm, 32 °C, and thus, the fabricated tissues could be harvested only by decreasing the temperature.…”

“…In this experiment, we applied centrifugal force of three different strengths, 20 g (rotational speed: 300 rpm), 55 g (500 rpm), and 220 g (1,000 rpm), and found that (a) the attachment between a cell sheet and a culture surface was too weak with the lowest strength of 20 g and (b) negative effects like sliding or significant deformation of cell sheets were observed with the highest strength of 220 g . Therefore, the middle strength of 55 g was judged to be an optimal strength . However, if we could overcome the negative effects occurring under 220 g , we would be able to further reduce the time required for the adhesion between two cell sheets, or cell sheet and culture surface.…”