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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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The in vitro neuronal stretch injury model.(A) The injury device consists of a stage positioned above an array of Teflon-coated, aluminum posts and driven vertically by an electromagnetic voice coil. (B) The silicone-bottomed plate consists of a commercially-distributed plate top covalently bonded to a sheet of silicone. Since no sandwich construction or air tight gaskets are employed, the standard geometry of the 96 well plate is preserved. (C) Schematic depiction of the injury process. The plate and post array are shown in cross section. Initially, the plate is positioned so that the silicone membrane touches the posts. To induce injury, the plate is lowered, stretching the membrane over the rims of the posts. Posts can be omitted from the post array to create unstretched, control wells.
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f6: The in vitro neuronal stretch injury model.(A) The injury device consists of a stage positioned above an array of Teflon-coated, aluminum posts and driven vertically by an electromagnetic voice coil. (B) The silicone-bottomed plate consists of a commercially-distributed plate top covalently bonded to a sheet of silicone. Since no sandwich construction or air tight gaskets are employed, the standard geometry of the 96 well plate is preserved. (C) Schematic depiction of the injury process. The plate and post array are shown in cross section. Initially, the plate is positioned so that the silicone membrane touches the posts. To induce injury, the plate is lowered, stretching the membrane over the rims of the posts. Posts can be omitted from the post array to create unstretched, control wells.

Mentions: In this study, the silicone cell culture membrane was indented with a rigid post to induce strain. This approach has important advantages over applying air pressure to induce biaxial strain44. Cylindrical indentation of the membrane creates a spatially homogeneous, equibiaxial, strain field. This assertion is supported by theory4546 and by the fact that the average Lagrangian strain in the X and Y directions were very similar at all levels of stage displacement (see Fig. 1B). This property of the model means that every cell in culture over the head of the post sees the same mechanical insult, regardless of its position or orientation. In biaxial air-driven systems, the circumferential strain peaks at the center and declines to zero at the edge so the strain environment varies continuously across the well bottom47. Also, the conventional geometry of a 96 well plate was retained because inducing deformation without air pressure eliminated the need for gaskets or other special features on the plate bottom (see Fig. 6B). These plates can therefore be manipulated and imaged using existing machinery for high throughput experiments. The relationship between stage displacement and strain in the silicone membrane was linear across all the displacement values tested except for the lowest value, which exhibited almost no strain. This may indicate imperfect zeroing at the start of the displacement pulse or it may indicate a stiction interaction between the silicone and the posts in which deformation begins only after a finite level of tension has been established.


Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format
The in vitro neuronal stretch injury model.(A) The injury device consists of a stage positioned above an array of Teflon-coated, aluminum posts and driven vertically by an electromagnetic voice coil. (B) The silicone-bottomed plate consists of a commercially-distributed plate top covalently bonded to a sheet of silicone. Since no sandwich construction or air tight gaskets are employed, the standard geometry of the 96 well plate is preserved. (C) Schematic depiction of the injury process. The plate and post array are shown in cross section. Initially, the plate is positioned so that the silicone membrane touches the posts. To induce injury, the plate is lowered, stretching the membrane over the rims of the posts. Posts can be omitted from the post array to create unstretched, control wells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: The in vitro neuronal stretch injury model.(A) The injury device consists of a stage positioned above an array of Teflon-coated, aluminum posts and driven vertically by an electromagnetic voice coil. (B) The silicone-bottomed plate consists of a commercially-distributed plate top covalently bonded to a sheet of silicone. Since no sandwich construction or air tight gaskets are employed, the standard geometry of the 96 well plate is preserved. (C) Schematic depiction of the injury process. The plate and post array are shown in cross section. Initially, the plate is positioned so that the silicone membrane touches the posts. To induce injury, the plate is lowered, stretching the membrane over the rims of the posts. Posts can be omitted from the post array to create unstretched, control wells.
Mentions: In this study, the silicone cell culture membrane was indented with a rigid post to induce strain. This approach has important advantages over applying air pressure to induce biaxial strain44. Cylindrical indentation of the membrane creates a spatially homogeneous, equibiaxial, strain field. This assertion is supported by theory4546 and by the fact that the average Lagrangian strain in the X and Y directions were very similar at all levels of stage displacement (see Fig. 1B). This property of the model means that every cell in culture over the head of the post sees the same mechanical insult, regardless of its position or orientation. In biaxial air-driven systems, the circumferential strain peaks at the center and declines to zero at the edge so the strain environment varies continuously across the well bottom47. Also, the conventional geometry of a 96 well plate was retained because inducing deformation without air pressure eliminated the need for gaskets or other special features on the plate bottom (see Fig. 6B). These plates can therefore be manipulated and imaged using existing machinery for high throughput experiments. The relationship between stage displacement and strain in the silicone membrane was linear across all the displacement values tested except for the lowest value, which exhibited almost no strain. This may indicate imperfect zeroing at the start of the displacement pulse or it may indicate a stiction interaction between the silicone and the posts in which deformation begins only after a finite level of tension has been established.

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