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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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Kinematics of the injury device.(A) Stage displacement histories over 10 pulses at a range of target amplitudes. (B) Average strain in each well in misaligned configuration with displacement of 2.9 mm (n = 5 measurements per well, average standard error per well = 0.056). Note that C4, D4, E4, F4, C9, D9, E9 and F9 are uninjured control wells. (C) The Lagrangian strain in the membrane increased with increasing stage displacement (n = 200–260 wells over 10 plates, bars = standard deviation). (D) Average strain in each well in optimally aligned configuration with displacement of 3.3 mm (n = 5 measurements per well, average standard error per well = 0.029). (E) Distribution of strains in optimally aligned configuration (circle = average strain in a single well location, square = average strain across all well locations, error bars = 1 standard deviation).
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f1: Kinematics of the injury device.(A) Stage displacement histories over 10 pulses at a range of target amplitudes. (B) Average strain in each well in misaligned configuration with displacement of 2.9 mm (n = 5 measurements per well, average standard error per well = 0.056). Note that C4, D4, E4, F4, C9, D9, E9 and F9 are uninjured control wells. (C) The Lagrangian strain in the membrane increased with increasing stage displacement (n = 200–260 wells over 10 plates, bars = standard deviation). (D) Average strain in each well in optimally aligned configuration with displacement of 3.3 mm (n = 5 measurements per well, average standard error per well = 0.029). (E) Distribution of strains in optimally aligned configuration (circle = average strain in a single well location, square = average strain across all well locations, error bars = 1 standard deviation).

Mentions: The injury device generated a rapid, highly repeatable, stage displacement history (see Fig. 1A and Table 1). The post array sits in an aluminum block on the injury device that accommodates set screws for optimal alignment with the stage. When these set screws were omitted, a modest misalignment was introduced that created systematic variation in the membrane strain across the plate, with the highest strains on the right side (see Fig. 1B). This configuration was used for dose response experiments and the associated systematic variation was advantageous because it eliminated gaps in the spectrum of strains that would otherwise emerge between different levels of stage displacement (see Fig. 1C). The strain was negligible for the lowest amplitude of displacement tested but rose in an approximately linear manner at higher levels of displacement (see Fig. 1C). The strains in the x and y direction were indistinguishable, indicating that the strain field was equibiaxial28. Since low variation across the plate is desirable for applications other than dose response studies, membrane strain was also characterized with optimal alignment. This eliminated systematic variation across the plate (see Fig. 1D). The remaining random variation across well locations created a distribution with a mean of 0.45 and a standard deviation of 0.051 (see Fig. 1E). The average standard deviation of strains at a given well location over multiple tests on different plates was 0.065.


Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format
Kinematics of the injury device.(A) Stage displacement histories over 10 pulses at a range of target amplitudes. (B) Average strain in each well in misaligned configuration with displacement of 2.9 mm (n = 5 measurements per well, average standard error per well = 0.056). Note that C4, D4, E4, F4, C9, D9, E9 and F9 are uninjured control wells. (C) The Lagrangian strain in the membrane increased with increasing stage displacement (n = 200–260 wells over 10 plates, bars = standard deviation). (D) Average strain in each well in optimally aligned configuration with displacement of 3.3 mm (n = 5 measurements per well, average standard error per well = 0.029). (E) Distribution of strains in optimally aligned configuration (circle = average strain in a single well location, square = average strain across all well locations, error bars = 1 standard deviation).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Kinematics of the injury device.(A) Stage displacement histories over 10 pulses at a range of target amplitudes. (B) Average strain in each well in misaligned configuration with displacement of 2.9 mm (n = 5 measurements per well, average standard error per well = 0.056). Note that C4, D4, E4, F4, C9, D9, E9 and F9 are uninjured control wells. (C) The Lagrangian strain in the membrane increased with increasing stage displacement (n = 200–260 wells over 10 plates, bars = standard deviation). (D) Average strain in each well in optimally aligned configuration with displacement of 3.3 mm (n = 5 measurements per well, average standard error per well = 0.029). (E) Distribution of strains in optimally aligned configuration (circle = average strain in a single well location, square = average strain across all well locations, error bars = 1 standard deviation).
Mentions: The injury device generated a rapid, highly repeatable, stage displacement history (see Fig. 1A and Table 1). The post array sits in an aluminum block on the injury device that accommodates set screws for optimal alignment with the stage. When these set screws were omitted, a modest misalignment was introduced that created systematic variation in the membrane strain across the plate, with the highest strains on the right side (see Fig. 1B). This configuration was used for dose response experiments and the associated systematic variation was advantageous because it eliminated gaps in the spectrum of strains that would otherwise emerge between different levels of stage displacement (see Fig. 1C). The strain was negligible for the lowest amplitude of displacement tested but rose in an approximately linear manner at higher levels of displacement (see Fig. 1C). The strains in the x and y direction were indistinguishable, indicating that the strain field was equibiaxial28. Since low variation across the plate is desirable for applications other than dose response studies, membrane strain was also characterized with optimal alignment. This eliminated systematic variation across the plate (see Fig. 1D). The remaining random variation across well locations created a distribution with a mean of 0.45 and a standard deviation of 0.051 (see Fig. 1E). The average standard deviation of strains at a given well location over multiple tests on different plates was 0.065.

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