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

Abstract

This study reports a rapid fabrication system of a morphologically and functionally communicative three-dimensional (3D) cell-dense tissue without scaffolds by centrifugation. The tight adhesion between C2C12 myoblasts and culture surface was accelerated without significant cell damage by centrifugation (80 x g, 37 °C, 30 min). A thicker tissue created on a temperature-responsive culture surface was harvested by decreasing temperature. The 3D myoblast tissues having approximately 200 μm-thickness were created at 1.5 h [centrifugation (80 x g, 37 °C) for 30 min and tissue harvest for 1 h]. However, in the case of without centrifugation, the myoblast tissues had fragile parts even at 7.5 h after the incubation. Additionally, electrically/functionally communicative and thicker human induced pluripotent stem (iPS) cell-derived cardiac tissues were created rapidly by the centrifugation and cultivation at 37 °C. We report a centrifugation system that significantly shortens the creation time of 3D tissues. We envision that this procedure will contribute to the field of tissue engineering and regenerative medicine.

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

“…First, the angle Φ in Figure a is determined by solving Equation (1). As this equation cannot be solved analytically, we numerically solved it for R = 0.15 m and Λ = 0.045 m by using a combination of Newton–Raphson and bisection methods . The results are shown in Figure S4, where the angle Φ (in degrees) are plotted against the rotational speed ω (in rpm).…”

“…Centrifugation is a very useful method to apply forces without contact in tissue engineering. We have used this for the purpose of the rapid adhering of a cell sheet onto the surface of a culture dish or another cell sheet, and the rapid fabrication of 3D cell‐dense tissue without scaffolds . This also has been used for fabricating biological tissues or biomaterials for tissue engineering.…”

“…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.…”

“…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 …”

Mesenchymal Stem Cell Sheet Centrifuge-Assisted Layering Augments Pro-Regenerative Cytokine Production

“…Centrifugal force has previously been applied to myocardial sheets to facilitate immediate adherence between layers and stimulate cellular communications related to cardiac function, and, similarly, centrifugal force is commonly used for MSC scaffold-free chondrogenesis to imitate the condensation process and promote critical cellular and matrix interactions [ 34 , 35 , 36 , 37 ]. While cell sheet centrifugation has been extensively employed to construct multilayered functional tissues in vitro to replace diseased tissue in vivo [ 38 , 39 , 40 , 41 , 42 ], centrifuge manipulation of 3D cellular interactions to stimulate intrinsic MSC cytokine production in cell sheets has not been previously investigated. This study presents a comparison of centrifuged and conventional MSC sheets, both as single- and multi-layer 3D MSC sheets, to distinguish impact on 3D structure relationships to cell physical and biochemical interactions.…”