Noninvasive cross-sectional observation of three-dimensional cell sheet-tissue-fabrication by optical coherence tomography

Abstract

Cell sheet engineering allows investigators/clinicians to prepare cell-dense three-dimensional (3-D) tissues, and various clinical trials with these fabricated tissues have already been performed for regenerating damaged tissues. Cell sheets are easily manipulated and 3-D tissues can be rapidly fabricated by layering the cell sheets. This study used optical coherence tomography (OCT) to noninvasively analyze the following processes: (1) adhesions between layered cell sheets, and (2) the beating and functional interaction of cardiac cell sheet-tissues for fabricating functional thicker 3-D tissues. The tight adhesions and functional couplings between layered cell sheets could be observed cross-sectionally and in real time. Importantly, the noninvasive and cross-sectional analyses of OCT make possible to fabricate 3-D tissues by confirming the adherence and functional couplings between layered cell sheets. OCT technology would contribute to cell sheet engineering and regenerative medicine.

Keywords: Cell adhesion; Cell sheet engineering; Noninvasive observation; Optical coherence tomography; Three-dimensional tissue fabrication.

Fig. 1

Observation of an NIH3T3 cell sheet onto a polystyrene culture dish by OCT. Upper and lower left pictures are the merged images of (1) the top-view observations of an NIH3T3 cell sheet onto the dish at 0 (A) and 24 min (B) after transfer, respectively; and (2) the red-colored space images between the cell sheet and the dish, and the right panels are cross-sectional observations of the cell sheet. Green lines in the left pictures show the cutting sites in the right panels.

Fig. 2

Observation of two C2C12 cell sheets by OCT. Upper and lower left pictures are merged images of (1) the top-view observations of two C2C12 cell sheets at 0 (A) and 23 min (B) after layering, respectively; and (2) the red-colored space images between the cell sheets, and the right panels are the cross-sectional observations of the cell sheet. Green lines in the left pictures show the cutting sites in the right panels.

Fig. 3

Observations of multi-layered C2C12 cell sheets by OCT. Three of the pictures on the left show the merged images of (1) the top-view of a triple-layered (A), quadruple-layered (C), and quintuple-layered (E) C2C12 cell sheets just after layering, and (2) the red-colored space images between layered cell sheets. The three other pictures on the left show the merged images of (1) the top-view of a triple-layered (B), quadruple-layered (D), and quintuple-layered (F) C2C12 cell sheets at each time after layering (B: 26 min; D: 23 min; F: 16 min), and (2) the red-colored space images between layered cell sheets. The panels on the right show these same cross-sectional observations. Thicknesses shown in the three photographs (B, D, F) were calculated at the points indicated. Green lines in the left pictures show the cutting sites in the right panels.

Fig. 4

Observation of the beating of a rat cardiac cell sheet by OCT. Beating areas within the cell sheet just after transfer onto a polystyrene culture dish are indicated with green colored markers, at which the correlation of the OCT signals for a short interval (90 ms) was lower than the predetermined level. The transmission of beating areas (arrows) is shown clearly.