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Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format

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ABSTRACT

Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology.

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Injury phenotypes increase with increasing strain.(A) Mean neurite length per cell declines with increasing strain. (B) Cell viability declines with increasing strain. (C) Processes/cell declines with increasing strain. (D) Branches/cell declines with increasing strain. (E) Viable cells/image declines with increasing strain. (F) Dead cells/image increases with increasing strain. Injury metrics are plotting against well-specific strain. For phenotype measurements, n = 160 wells over 5 plates. For strain measurements, n = 800 wells over 50 plates (i.e. each point represents the average of 5 measurements (average standard error = 0.056). In both cases, the fit line represents a generalized logistic regression of the data (see Methods). Estimates for the coefficients of each fit along with confidence intervals and R2 values are presented in Table 2.
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f4: Injury phenotypes increase with increasing strain.(A) Mean neurite length per cell declines with increasing strain. (B) Cell viability declines with increasing strain. (C) Processes/cell declines with increasing strain. (D) Branches/cell declines with increasing strain. (E) Viable cells/image declines with increasing strain. (F) Dead cells/image increases with increasing strain. Injury metrics are plotting against well-specific strain. For phenotype measurements, n = 160 wells over 5 plates. For strain measurements, n = 800 wells over 50 plates (i.e. each point represents the average of 5 measurements (average standard error = 0.056). In both cases, the fit line represents a generalized logistic regression of the data (see Methods). Estimates for the coefficients of each fit along with confidence intervals and R2 values are presented in Table 2.

Mentions: Neurite length and cell viability declined with increasing strain and are distributed in a sigmoidal fashion with respect to strain (see Fig. 4A,B). In the low strain domain (<0.2), there is no obvious increase in injury with increasing strain. In the intermediate strain domain (0.2 < strain < 0.4), injury increases sharply with increasing strain and in the high strain domain (>0.4), the level of injury saturates and becomes strain insensitive again.


Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format
Injury phenotypes increase with increasing strain.(A) Mean neurite length per cell declines with increasing strain. (B) Cell viability declines with increasing strain. (C) Processes/cell declines with increasing strain. (D) Branches/cell declines with increasing strain. (E) Viable cells/image declines with increasing strain. (F) Dead cells/image increases with increasing strain. Injury metrics are plotting against well-specific strain. For phenotype measurements, n = 160 wells over 5 plates. For strain measurements, n = 800 wells over 50 plates (i.e. each point represents the average of 5 measurements (average standard error = 0.056). In both cases, the fit line represents a generalized logistic regression of the data (see Methods). Estimates for the coefficients of each fit along with confidence intervals and R2 values are presented in Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037451&req=5

f4: Injury phenotypes increase with increasing strain.(A) Mean neurite length per cell declines with increasing strain. (B) Cell viability declines with increasing strain. (C) Processes/cell declines with increasing strain. (D) Branches/cell declines with increasing strain. (E) Viable cells/image declines with increasing strain. (F) Dead cells/image increases with increasing strain. Injury metrics are plotting against well-specific strain. For phenotype measurements, n = 160 wells over 5 plates. For strain measurements, n = 800 wells over 50 plates (i.e. each point represents the average of 5 measurements (average standard error = 0.056). In both cases, the fit line represents a generalized logistic regression of the data (see Methods). Estimates for the coefficients of each fit along with confidence intervals and R2 values are presented in Table 2.
Mentions: Neurite length and cell viability declined with increasing strain and are distributed in a sigmoidal fashion with respect to strain (see Fig. 4A,B). In the low strain domain (<0.2), there is no obvious increase in injury with increasing strain. In the intermediate strain domain (0.2 < strain < 0.4), injury increases sharply with increasing strain and in the high strain domain (>0.4), the level of injury saturates and becomes strain insensitive again.

View Article: PubMed Central - PubMed

ABSTRACT

Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology.

No MeSH data available.


Related in: MedlinePlus