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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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Evolution of injury phenotype with increasing strain.(A) Representative images of neurons stained with calcein AM (green) and Hoechst 333342 (blue) 4 hours after injury at various levels of strain. As strain increases, the neurite network becomes less extensive and the number of calcein-AM negative nuclei increases, indicating cell death. (B) In the control condition, neurites have a large, constant thickness. (C) In injured neurons, neurites are shorter and thinner with beads (see white arrows) distributed along their length.
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f3: Evolution of injury phenotype with increasing strain.(A) Representative images of neurons stained with calcein AM (green) and Hoechst 333342 (blue) 4 hours after injury at various levels of strain. As strain increases, the neurite network becomes less extensive and the number of calcein-AM negative nuclei increases, indicating cell death. (B) In the control condition, neurites have a large, constant thickness. (C) In injured neurons, neurites are shorter and thinner with beads (see white arrows) distributed along their length.

Mentions: The injury phenotype increased with increasing strain (see Fig. 3). At the time of injury, the iCell neurons were well-attached and established extensive neurite networks on the silicone-bottomed plates. 4 hours after injury, there was negligible evidence of injury at or below the 17% strain level. A clear injury phenotype emerged at the 38% strain level and rapidly approached saturation at higher strains. The injury phenotype had three components: cell death, shortening of neurites, and changes in neurite shape. Control neurites were thick and uniform (see Fig. 3B) while injured neurites were thin with round beads distributed along their length (see Fig. 3C).


Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format
Evolution of injury phenotype with increasing strain.(A) Representative images of neurons stained with calcein AM (green) and Hoechst 333342 (blue) 4 hours after injury at various levels of strain. As strain increases, the neurite network becomes less extensive and the number of calcein-AM negative nuclei increases, indicating cell death. (B) In the control condition, neurites have a large, constant thickness. (C) In injured neurons, neurites are shorter and thinner with beads (see white arrows) distributed along their length.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5037451&req=5

f3: Evolution of injury phenotype with increasing strain.(A) Representative images of neurons stained with calcein AM (green) and Hoechst 333342 (blue) 4 hours after injury at various levels of strain. As strain increases, the neurite network becomes less extensive and the number of calcein-AM negative nuclei increases, indicating cell death. (B) In the control condition, neurites have a large, constant thickness. (C) In injured neurons, neurites are shorter and thinner with beads (see white arrows) distributed along their length.
Mentions: The injury phenotype increased with increasing strain (see Fig. 3). At the time of injury, the iCell neurons were well-attached and established extensive neurite networks on the silicone-bottomed plates. 4 hours after injury, there was negligible evidence of injury at or below the 17% strain level. A clear injury phenotype emerged at the 38% strain level and rapidly approached saturation at higher strains. The injury phenotype had three components: cell death, shortening of neurites, and changes in neurite shape. Control neurites were thick and uniform (see Fig. 3B) while injured neurites were thin with round beads distributed along their length (see Fig. 3C).

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