Monitoring of dissolved oxygen and cellular bioenergetics within a pancreatic substitute

Abstract

This work investigated the use of nuclear magnetic resonance (NMR) spectroscopy in combination with a mathematical model of an encapsulated cell system as a method for rapidly assessing the status of a pancreatic substitute. To validate this method, an in vitro experiment was performed in which the encapsulated cells were perfused in an NMR-compatible system and the dissolved oxygen (DO) concentration of the perfusing medium was lowered from 0.20 to 0.05 mM, then returned to 0.20 mM in a stepwise fashion. The cellular metabolic activity and bioenergetics were evaluated by measuring the oxygen consumption rate (via DO sensors) and nucleotide triphosphate levels (via (31)P NMR). By incorporating a perfluorocarbon emulsion into the alginate beads, the cellular oxygenation state was monitored by measuring the average intrabead DO (AIDO) concentration by (19)F NMR. The in vitro measurements were then compared with model predictions based on the measured external DO concentration and time. Model-predicted cell growth and AIDO closely matched the experimentally acquired data. As the DO concentrations both external to and within the pancreatic substitute are needed to apply this methodology in vivo, the feasibility of measuring the DO concentration from two distinct bead populations implanted in the peritoneal cavity of mice was established. It is concluded that PFC incorporation and (19)F NMR measurements, in combination with a mechanistic model of the encapsulated system, allow the tracking of the state of a pancreatic substitute in vitro and potentially in vivo.

In Vivo Noninvasive Monitoring of Dissolved Oxygen Concentration Within an Implanted Tissue-Engineered Pancreatic Construct

“…The PFC emulsion was prepared following a protocol adapted from Joseph et al 32 and described by Gross et al 26 The emulsion was added to 3% w/v low viscosity, high mannuronic content alginate (LVM; NovaMatrix, Drammen, Norway) at a concentration of 50 mg/mL, and mixed thoroughly. Cells were encapsulated in barium alginate beads as described previously.…”

“…Measurements confirmed the peritoneal cavity of normal mice to be hypoxic, with DO values comparable to those reported in literature. 26,30,35 Equivalent peritoneal DO values were also obtained in diabetic mice within 2 days postimplantation.…”

“…Measuring DO within the experimental implant offers an assessment of the metabolic activity of the cells within the construct. 26 In a previous study, we demonstrated the capability of this method to acquire real-time DO measurements within alginate beads implanted in the peritoneal cavity of normal mice and to acquire correlations between changes in intrabead DO and growth of the encapsulated cells or attachment of host cells to the beads, the latter two evaluated postexplantation. 30 In this study, we expand on our previous work by implementing the dual PFC method to measure DO levels in bTC-tet cell-containing barium alginate beads implanted in the peritoneal cavity of diabetic mice.…”

“…[17][18][19][20] As construct oxygenation significantly affects cell survival and function, monitoring of oxygen levels in pancreatic implants could provide direct information on their functionality. However, most reports on the effect of hypoxia on construct function are limited to in vitro settings 22,[24][25][26][27] ; in vivo evaluation of hypoxia is currently performed through indirect methods postexplantation. 15,16,28 We are therefore pursuing the development of a noninvasive method of monitoring dissolved oxygen concentration (DO) in a tissue-engineered pancreatic construct using 19 F nuclear magnetic resonance (NMR) spectroscopy, with perfluorocarbons (PFCs) as oxygen concentration markers.…”

Development and Validation of Noninvasive Magnetic Resonance Relaxometry for the In Vivo Assessment of Tissue-Engineered Graft Oxygenation

“…Eleven publications reporting information on PFCE R 1 oxygen sensitivity were identified. 28,[30][31][32][33][34][35][36][38][39][40] One was excluded for inconsistent reporting of data 38 and another was excluded for results that differed from other reported data by several standard deviations, 40 leaving eleven reported calibrations 28,[30][31][32][33][34][35][36]39 that were combined with our data for analysis. Oxygen sensitivity decreased (-6.39 · 10 -5 mmHg -1…”