144
standard to evaluate engineered cardiac tissues, with conduction velocities and
action potential shapes seen in mature adult tissues established as ideal. These measurements also allow engineers to evaluate uniformity across the tissue and to gauge
the maturity of stem cell-derived cardiomyocytes. However, full electrophysiological characterization of these tissues requires extensive facilities and expertise, and,
as a result, much of the literature relies on stimulated contractile force measurements. Sometimes referred to in the literature as “twitch force,” this assessment is
frequently used as a measure of tissue development. Increased force is believed to
correlate with improved tissue maturity and clinical outcomes. Cell and tissue-level
analyses are also frequently performed on engineered tissue in vitro, as cellular
morphology and tissue organization are important determinants of tissue function.
6.3 Cell Sources
Approaches to engineer cardiac grafts employ the combination of multiple cell
types, typically including cardiomyocytes (electrically excitable cells), endothelial
cells (vessel development), and fibroblasts/mural cells (vasculature stabilization).
Each of these phenotypes may be derived from various sources (see Table 6.1).
6.3.1 Cardiomyocytes
Most commonly, cardiac tissue engineering strategies are studied using neonatal rat
ventricular cardiomyocytes (NRVCMs). NRVCMs are isolated from newborn (day
0–3) rat hearts that have been minced and digested with trypsin and collagenase. To
enrich the cardiomyocyte population, cells are usually plated into flasks and incubated for short periods, to allow for the selective adherence of fibroblasts in order to
purify the NRVCM population. NRVCMs retain the ability to contract ex vivo,
undergo a rapid dedifferentiation-redifferentiation cycle, and can be easily plated
into monolayers. NRVCMs are widely used to study morphological, electrophysiological, and biochemical characteristics of cardiomyocytes. Despite their advantages, NRVCMs have the main disadvantage of being rodent-derived.
Electrophysiologically, NRVCMs have little to no plateau phase when cultured
in vitro. In addition, NRVCMs differ in their excitation-coupling, with a lack of
T-tubule systems relative to adult myocytes, and their ability to maintain cytosolic
Ca
2+
signaling independent of the sarcoplasmic reticulum calcium release [56]. And
while the adult rat myocardium has conduction velocities of 69 ± 6 cm/s in the longitudinal direction and 19 ± 5 cm/s in the transverse direction, there is yet to be a
report of tissue-engineered grafts achieving these values [92]. These cells are also
not able to be cultured for extended periods of time, especially in monolayers [26].
While healthy human cardiomyocytes isolated from adult hearts cannot be
obtained ethically, the use of pluripotent stem cells has provided an avenue to obtain
J. Morrissette-McAlmon et al.
standard to evaluate engineered cardiac tissues, with conduction velocities and
action potential shapes seen in mature adult tissues established as ideal. These measurements also allow engineers to evaluate uniformity across the tissue and to gauge
the maturity of stem cell-derived cardiomyocytes. However, full electrophysiological characterization of these tissues requires extensive facilities and expertise, and,
as a result, much of the literature relies on stimulated contractile force measurements. Sometimes referred to in the literature as “twitch force,” this assessment is
frequently used as a measure of tissue development. Increased force is believed to
correlate with improved tissue maturity and clinical outcomes. Cell and tissue-level
analyses are also frequently performed on engineered tissue in vitro, as cellular
morphology and tissue organization are important determinants of tissue function.
6.3 Cell Sources
Approaches to engineer cardiac grafts employ the combination of multiple cell
types, typically including cardiomyocytes (electrically excitable cells), endothelial
cells (vessel development), and fibroblasts/mural cells (vasculature stabilization).
Each of these phenotypes may be derived from various sources (see Table 6.1).
6.3.1 Cardiomyocytes
Most commonly, cardiac tissue engineering strategies are studied using neonatal rat
ventricular cardiomyocytes (NRVCMs). NRVCMs are isolated from newborn (day
0–3) rat hearts that have been minced and digested with trypsin and collagenase. To
enrich the cardiomyocyte population, cells are usually plated into flasks and incubated for short periods, to allow for the selective adherence of fibroblasts in order to
purify the NRVCM population. NRVCMs retain the ability to contract ex vivo,
undergo a rapid dedifferentiation-redifferentiation cycle, and can be easily plated
into monolayers. NRVCMs are widely used to study morphological, electrophysiological, and biochemical characteristics of cardiomyocytes. Despite their advantages, NRVCMs have the main disadvantage of being rodent-derived.
Electrophysiologically, NRVCMs have little to no plateau phase when cultured
in vitro. In addition, NRVCMs differ in their excitation-coupling, with a lack of
T-tubule systems relative to adult myocytes, and their ability to maintain cytosolic
Ca
2+
signaling independent of the sarcoplasmic reticulum calcium release [56]. And
while the adult rat myocardium has conduction velocities of 69 ± 6 cm/s in the longitudinal direction and 19 ± 5 cm/s in the transverse direction, there is yet to be a
report of tissue-engineered grafts achieving these values [92]. These cells are also
not able to be cultured for extended periods of time, especially in monolayers [26].
While healthy human cardiomyocytes isolated from adult hearts cannot be
obtained ethically, the use of pluripotent stem cells has provided an avenue to obtain
J. Morrissette-McAlmon et al.