Real‐time quantitation of internal metabolic activity of three‐dimensional engineered tissues using an oxygen microelectrode and optical coherence tomography

Abstract

Recent progress in tissue engineering technology has enabled us to develop thick tissue constructs that can then be transplanted in regenerative therapies. In clinical situations, it is vital that the engineered tissues to be implanted are safe and functional before use. However, there is currently a limited number of studies on real-time quality evaluation of thick living tissue constructs. Here we developed a system for quantifying the internal activities of engineered tissues, from which we can evaluate its quality in real-time. The evaluation was achieved by measuring oxygen concentration profiles made along the vertical axis and the thickness of the tissues estimated from cross-sectional images obtained noninvasively by an optical coherence tomography system. Using our novel system, we obtained (i) oxygen concentration just above the tissues, (ii) gradient of oxygen along vertical axis formed above the tissues within culture medium, and (iii) gradient of oxygen formed within the tissues in real-time. Investigating whether these three parameters could be used to evaluate engineered tissues during culturing, we found that only the third parameter was a good candidate. This implies that the activity of living engineered tissues can be monitored in real-time by measuring the oxygen gradient within the tissues. The proposed measuring strategy can be applied to developing more efficient culturing methods to support the fabrication of engineered thick tissues, as well as providing methods to confirm the quality in real-time. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 855-864, 2017.

Thicker three-dimensional tissue from a “symbiotic recycling system” combining mammalian cells and algae

“…1A ), could be cultured and produce O 2 within a culture media and temperature conditions optimized for mammalian cells. The algae were suspended in a medium for mammalian cells, and oxygen concentration in the medium was measured by an analysis system, which was developed recently 27 28 29 , shown schematically in Fig. 1B .…”

“…Recently, we have developed a measurement system for oxygen concentration using an oxygen microelectrode sensor [a Clark-type oxygen microsensor with a 8–12 μm-diameter tip made of fragile glass (OX-10) (Unisense, Denmark)] and a high-precision electronic balance (HTR-220) (Shinko Denshi, Tokyo, Japan), which can detect the position of the tip of the oxygen sensor when it comes into contact with the bottom of the dish 27 28 29 . The measurement was performed in a humidified atmosphere containing 20% oxygen and 5% CO 2 in a glove box hypoxia workstation (INVIVO2 300) (Ruskinn Technology, Mid Glamorgan, UK).…”

Modeling spatial distribution of oxygen in 3d culture of islet beta‐cells

“…It should be noted that the detection of oxygen tension inside 3D engineered tissues, such as myocytes‐laden collagen construct, myoblast sheet, and cartilegious construct, has been performed using microelectrodes with a tip size of a few µm. Using a microelectrode, the relationship between the depth of an engineered tissue construct and the oxygen tension can be measured.…”

“…19 Taken together, the evaluation of oxygen demand in a 3D b-cell culture using mathematical modeling approach allows us to further optimize the culture conditions including cell seeding density and oxygen supply. It should be noted that the detection of oxygen tension inside 3D engineered tissues, such as myocytes-laden collagen construct, 32 myoblast sheet, 33 and cartilegious construct, 34,35 has been performed using microelectrodes with a tip size of a few mm. Using a microelectrode, the relationship between the depth of an engineered tissue construct and the oxygen tension can be measured.…”

Three-Dimensional Human Cardiac Tissue Engineered by Centrifugation of Stacked Cell Sheets and Cross-Sectional Observation of Its Synchronous Beatings by Optical Coherence Tomography

“…In this study two OCT systems [Proto 3(DT) (Panasonic Healthcare, Tokyo, Japan) [ 9 , 14 , 15 , 20 ] and IVS-2000 (Santec, Aichi, Japan)] were used. Their specifications are summarized in Table 1 .…”