98
O 2 measurement method needs several properties to be considered superior, including accuracy, sensitivity, repeatability, rapidity, and noninvasiveness. Although
some methods are used more commonly in a broader range of applications, no “gold
standard” exists for all applications, since the method chosen usually depends on
the purpose of the measurement. In vivo O 2 measurement methods can be divided
into two main categories: (1) direct measurements, where the concentration or the
partial pressure of O 2 is directly measured, and (2) indirect measurements, where
levels of O 2 -indicative molecules (e.g., hemoglobin, cytochrome) are detected and
correlated to relative O 2 concentrations.
The most common direct measurements are electrodes, phosphorescent probes,
electron paramagnetic resonance (EPR) oximetry, and nuclear magnetic resonance
(NMR). Some of the indirect measurement methods involve monitoring of hemoglobin/myoglobin, mitochondrial cytochromes, and NADH/FADH [209].
Springett’s paper thoroughly reviews the benefits and limitations of the most recent
methods [209].
In addition, in vitro studies have applied these currently available methods to
monitor O 2 levels quantitatively, such as by measuring O 2 tensions at the cellular
level in 2D monolayer cell cultures or O 2 gradients in 3D gels or scaffolds. Two
major methods used to measure O 2 levels during in vitro cultures are polarographic
and fluorescence quenching techniques. The latter has been shown to surpass the
polarographic technique, which consumes O 2 during the measurements [197]. When
an implemented measurement technique, like the polarographic technique, consumes O 2 , it more likely generates even greater inaccuracies and leads to incorrect
conclusions in low O 2 environments, as occurred in studies investigating the effect
of hypoxia in 3D scaffolds [36, 120, 145]. Fluorescence quenching technology is
available both for invasive applications, using an electrode probe with a very thin
(approximately 5 μm) tip, and for noninvasive applications, using a sensor patch
composed of a ruthenium-based metal complex that can be excited by an external
fluorescent light source.
Modeling Oxygen Transport in Tissues
The limitations of these measurement techniques, caused mostly by the difficulties
in measuring spatial O 2 concentrations in tissues or scaffolds, raise a need for predictive mathematical models. Transport of O 2 in vivo is controlled by several parameters, including blood flow rate, degree of vascularization in the tissue, physiological
distance of the cells from the microvasculature, and, depending on cell type, the
cells’ rate of O 2 consumption. These factors affect O 2 distribution in the tissue, and
some can also have an impact on O 2 transport in 3D in vitro cultures of pluripotent
or vascular cells. Additional factors that in vitro studies should consider are the
geometry of the scaffold, the available surface area for O 2 transport from the environment to the system, and controlled dissolved O 2 levels in the culture media.
In general, fundamental mathematical models estimating O 2 distribution in 3D
constructs can be classified into: (1) static models, where O 2 is only transported via
diffusion, and (2) dynamic models, where convectional transport of O 2 is also incorM. R. Blatchley et al.
O 2 measurement method needs several properties to be considered superior, including accuracy, sensitivity, repeatability, rapidity, and noninvasiveness. Although
some methods are used more commonly in a broader range of applications, no “gold
standard” exists for all applications, since the method chosen usually depends on
the purpose of the measurement. In vivo O 2 measurement methods can be divided
into two main categories: (1) direct measurements, where the concentration or the
partial pressure of O 2 is directly measured, and (2) indirect measurements, where
levels of O 2 -indicative molecules (e.g., hemoglobin, cytochrome) are detected and
correlated to relative O 2 concentrations.
The most common direct measurements are electrodes, phosphorescent probes,
electron paramagnetic resonance (EPR) oximetry, and nuclear magnetic resonance
(NMR). Some of the indirect measurement methods involve monitoring of hemoglobin/myoglobin, mitochondrial cytochromes, and NADH/FADH [209].
Springett’s paper thoroughly reviews the benefits and limitations of the most recent
methods [209].
In addition, in vitro studies have applied these currently available methods to
monitor O 2 levels quantitatively, such as by measuring O 2 tensions at the cellular
level in 2D monolayer cell cultures or O 2 gradients in 3D gels or scaffolds. Two
major methods used to measure O 2 levels during in vitro cultures are polarographic
and fluorescence quenching techniques. The latter has been shown to surpass the
polarographic technique, which consumes O 2 during the measurements [197]. When
an implemented measurement technique, like the polarographic technique, consumes O 2 , it more likely generates even greater inaccuracies and leads to incorrect
conclusions in low O 2 environments, as occurred in studies investigating the effect
of hypoxia in 3D scaffolds [36, 120, 145]. Fluorescence quenching technology is
available both for invasive applications, using an electrode probe with a very thin
(approximately 5 μm) tip, and for noninvasive applications, using a sensor patch
composed of a ruthenium-based metal complex that can be excited by an external
fluorescent light source.
Modeling Oxygen Transport in Tissues
The limitations of these measurement techniques, caused mostly by the difficulties
in measuring spatial O 2 concentrations in tissues or scaffolds, raise a need for predictive mathematical models. Transport of O 2 in vivo is controlled by several parameters, including blood flow rate, degree of vascularization in the tissue, physiological
distance of the cells from the microvasculature, and, depending on cell type, the
cells’ rate of O 2 consumption. These factors affect O 2 distribution in the tissue, and
some can also have an impact on O 2 transport in 3D in vitro cultures of pluripotent
or vascular cells. Additional factors that in vitro studies should consider are the
geometry of the scaffold, the available surface area for O 2 transport from the environment to the system, and controlled dissolved O 2 levels in the culture media.
In general, fundamental mathematical models estimating O 2 distribution in 3D
constructs can be classified into: (1) static models, where O 2 is only transported via
diffusion, and (2) dynamic models, where convectional transport of O 2 is also incorM. R. Blatchley et al.