Development of a New Assay System for Evaluating the Permeability of Various Substances Through Three-Dimensional Tissue

Abstract

A novel assay system with cell-dense three-dimensional (3D) tissue was developed for measuring the permeability of substances. In this paper, the permeabilities of various molecules containing nutrients, a cytokine, and a chemokine were examined and analyzed. A single-layered cell sheet was approximately 20 mum thick, and as the number of layers of these cell sheets increased, so did the total thickness of the tissue. The diffusion rates of glucose and pyruvic acid were reduced to approximately 30-40% by a single-layered cell sheet compared with the control without the cell sheet, and the diffusion of both substances were completely inhibited by a quadruple-layered cell sheet. The diffusion rate of creatinin was reduced to approximately 50% and 15-20% by a single-layered and by a quintuplet-layered cell sheet, respectively. On the other hand, the diffusion rate of stromal cell-derived factor 1alpha, vascular endothelial growth factor, beta2-microglobulin, and transferrin was reduced to approximately 10%, 5%, 20%, and 10%, by only a single-layered cell sheet, respectively. The diffusion of these substances were completely inhibited by a double-layered cell sheet. These results show that the permeability of substances through 3D tissue significantly decreased with the increase of the molecular weight. Therefore, the system could give a simulated living-tissue condition for measuring the permeability of substances. To our knowledge, this is the first report about measuring the permeability of substances through cell-dense 3D tissues without scaffolds. The assay system is believed to contribute to the progress of physiology, metabology, biochemistry, and pharmacokinetics. Further, the system may give some hints for developing a new dialysis membrane technology for an artificial kidney.

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.…”

“…C2C12 cell sheets were fabricated by the previously reported method. 13,40 Briefly, 6.0 3 10 5 C2C12 cells were plated onto a 35-mm temperature-responsive culture dish and cultured at 37 C. After 3 days, the culture dish was transferred to a CO 2 incubator set at 20 C to harvest a C2C12 cell sheet. After the cell sheet was transferred to a 35-mm conventional culture dish (Becton Dickinson and Company, Franklin Lakes, NJ) warmed on a heat plate at 37 C, adhesions between the cell sheet and the culture surface were observed by OCT.…”

Mammalian cell cultivation using nutrients extracted from microalgae

“…After the heat treatment, neutralization was performed using hydrochloric acid or sodium hydroxide. Glucose and amino acid concentrations were measured by the hexokinase method and liquid chromatography–mass spectrometry, respectively.…”

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

“…Therefore, for explaining the increasing of glucose consumption and lactate production at Day 7, surviving cells were speculated to proliferate while dying cells were found. In the examination of the permeability of glucose across multi-layered cell sheets, almost the complete inhibition of glucose permeability was observed on over quadruple-layered cell sheets [17]. Glucose consumption also might show no increase in accordance with the number of cell sheets due to the insufficient supply of glucose.…”