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

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.

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Purity of iCell neurons.(A) Representative image of uninjured iCell neurons stained with MAP2, GFAP, and Hoechst 33342 demonstrating the absence of astrocytes. (B) Rat astrocyte culture stained and imaged with an identical protocol as a positive control.
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f2: Purity of iCell neurons.(A) Representative image of uninjured iCell neurons stained with MAP2, GFAP, and Hoechst 33342 demonstrating the absence of astrocytes. (B) Rat astrocyte culture stained and imaged with an identical protocol as a positive control.

Mentions: There is no evidence of astrocyte contamination in iCell neuron cultures (see Fig. 2). Fixed iCells were immunostained with microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and the nuclear marker Hoechst 33342. 818 MAP2-positive cells were detected across 6 images but no GFAP-positive cells were detected. Separate cultures of rat astrocytes were stained and imaged simultaneously using an identical protocol to control for errors in GFAP labeling (see Fig. 2B).


Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format
Purity of iCell neurons.(A) Representative image of uninjured iCell neurons stained with MAP2, GFAP, and Hoechst 33342 demonstrating the absence of astrocytes. (B) Rat astrocyte culture stained and imaged with an identical protocol as a positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Purity of iCell neurons.(A) Representative image of uninjured iCell neurons stained with MAP2, GFAP, and Hoechst 33342 demonstrating the absence of astrocytes. (B) Rat astrocyte culture stained and imaged with an identical protocol as a positive control.
Mentions: There is no evidence of astrocyte contamination in iCell neuron cultures (see Fig. 2). Fixed iCells were immunostained with microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and the nuclear marker Hoechst 33342. 818 MAP2-positive cells were detected across 6 images but no GFAP-positive cells were detected. Separate cultures of rat astrocytes were stained and imaged simultaneously using an identical protocol to control for errors in GFAP labeling (see Fig. 2B).

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