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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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Cell segmentation.(A) Neurons injured with 38% strain and imaged 4 hours post injury with calcein AM (green) and Hoechst 33342 staining (blue) (B) Automated segmentation of the image into cell bodies and neurites. Note that beads on neurites are rejected as cell bodies based on their size and the absence of Hoechst-positive nuclei. Extracellular nuclei are rejected as cell bodies based on the absence of calcein AM staining.
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f7: Cell segmentation.(A) Neurons injured with 38% strain and imaged 4 hours post injury with calcein AM (green) and Hoechst 33342 staining (blue) (B) Automated segmentation of the image into cell bodies and neurites. Note that beads on neurites are rejected as cell bodies based on their size and the absence of Hoechst-positive nuclei. Extracellular nuclei are rejected as cell bodies based on the absence of calcein AM staining.

Mentions: Live cell images were preprocessed with CellProfiler software59. A 3 × 3 median filter was applied to the raw images and uneven, background illumination was corrected. MetaXpress software (Molecular Devices) was used to segment individual cells and compute outcome measures including viable cells/image, dead cells/image, neurite length, process number, and branch number (see Fig. 7).


Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format
Cell segmentation.(A) Neurons injured with 38% strain and imaged 4 hours post injury with calcein AM (green) and Hoechst 33342 staining (blue) (B) Automated segmentation of the image into cell bodies and neurites. Note that beads on neurites are rejected as cell bodies based on their size and the absence of Hoechst-positive nuclei. Extracellular nuclei are rejected as cell bodies based on the absence of calcein AM staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Cell segmentation.(A) Neurons injured with 38% strain and imaged 4 hours post injury with calcein AM (green) and Hoechst 33342 staining (blue) (B) Automated segmentation of the image into cell bodies and neurites. Note that beads on neurites are rejected as cell bodies based on their size and the absence of Hoechst-positive nuclei. Extracellular nuclei are rejected as cell bodies based on the absence of calcein AM staining.
Mentions: Live cell images were preprocessed with CellProfiler software59. A 3 × 3 median filter was applied to the raw images and uneven, background illumination was corrected. MetaXpress software (Molecular Devices) was used to segment individual cells and compute outcome measures including viable cells/image, dead cells/image, neurite length, process number, and branch number (see Fig. 7).

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