Thickness limitation and cell viability of multi-layered cell sheets and overcoming the diffusion limit by a porous-membrane culture insert

Abstract

A major challenge of tissue engineering is the creation of three-dimensional (3D) and functional tissues. However, the tissue ischemic environments make the production of thicker 3D tissues difficult. For evaluating the thickness limitation of tissue, the cell viability and metabolism of a novel in vitro 3D tissue model, which is composed of multilayered cell sheets, were investigated. Human endometrial-derived mesenchymal cells (EMC) were cultured on temperature-responsive culture dishes. Confluently cultured EMCs were harvested as an intact contiguous cell sheet only by lowering temperature. The obtained cell sheets were successfully layered into 3D cell-dense tissues and cultured in vitro for 7 days. Glucose consumption and lactate production in the culture media increased in accordance with the number of layers (single to triple). Histological analyses and cell viability assays showed that viable tissues were found in single-to triple-layered cell sheets and damaged tissues in over quadruple layers. The results concluded that the thickness limitation of layered cell sheets was approximately 40 mm, which was the thickness of triple-layered cell sheets. Importantly, when multi-layered cell sheets were cultured on porous membranes, the adhesion among cell sheets and cell viability were improved, resulting in successful fabricating thicker tissues (~100 mm) than that on normal culture dishes. The metabolic analyses showed that multi-layered cell sheets rely on their anaerobic metabolism, indicating that supplying nutrients rather than oxygen through both upper and bottom tissue surfaces improved the cell viability in 3D tissues. ` `Citation: Sekine W, Haraguchi Y, Shimizu T, Umezawa A, Okano T (2011) Thickness limitation and cell viability of multi-layered cell sheets and overcoming the diffusion limit by a porous-membrane culture insert. J Biochip Tissue chip S1:007.

Real‐time, noninvasive optical coherence tomography of cross‐sectional living cell‐sheets in vitro and in vivo

“…Although there were many interspaces between the cell sheet and the culture surface just after transfer onto another culture dish, the inter-space disappeared rapidly [Supporting Information Figure 1(C,D)]. On the other hand, because a single-layer cell sheet after detachment consists of two or three cell layers and forms 3-D tissue, 13,14,40,44 the cell sheet become thicker than that of two-dimensional cells cultured on a temperature-responsive culture surface before detachment. OCT observations confirmed previous observations in the authors' laboratory.…”

Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique

“…According to previous reports about the thickness limitations of cell sheets, when neonatal rat cardiomyocyte sheets were implanted subcutaneously, stacking cell sheets over three layers did not grow thicker one month after the transplantation [14]. When human endometrial-derived mesenchymal cell sheets were cultured on a dish, the thickness limitation of layered cell sheets was approximately 40 mm [15]. The five-layer HSMM sheet was approximately 70e80 mm in thickness when observed both in paraffin sections and by confocal microscopy.…”

Optical coherence microscopy of living cells and bioengineered tissue dynamics in high-resolution cross-section

“…Those cells were then seeded on a 35‐mm polystyrene culture dish (Corning, NY) and cultured at 37°C until observation by OCM. NIH3T3 cell sheets and BAEC sheets were fabricated by using a 35‐mm thermoresponsive culture dish (UpCell ® dish) (CellSeed, Tokyo, Japan), and the fabrication of 3‐D tissues by layering the cell sheets was performed as described in previous reports . Recently, we developed a rapid fabrication system for cell sheet‐tissues by centrifugation, which can reduce the fabrication time by approximately two‐thirds compared to the conventional method .…”

“…A microscope is an essential tool in the field of cell biology. The histological observation of three‐dimensional (3‐D) tissues using microscopy provides cross‐sectional information, for example, thickness, cell density, cell viability, and cell differentiation . However, the fabrication of a paraffin section or a cryosection for observation terminates the life of cells and tissues.…”