This work is supported in part by a grant from the National Institutes of Health (R21NS098129). We would like to acknowledge excellent technical assistance from SueSan Chen, Jonathan Tan, Courtney Cavanaugh, Shi Kai Ng and Feng Yuan Bu, who designed and built a structure to support lights used during high speed imaging experiments described in this manuscript.

<ol>
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The authors have nothing to disclose.

The key to obtaining a consistent, biofidelic phenotype in this model is applying a consistent biofidelic mechanical insult. This model can generate pulse durations as short as 10-15 ms, which are similar to the pulse durations for human head impacts according to cadaveric experiments12,13. The consistency of this insult depends on the alignment of the plate with the indenter block and consistent lubrication of the indenters. When the indenter block is well aligned, there is no trend in the applied strain across rows or columns (Figure 2C). A thin layer of lubricant typically creates less friction than a thick layer, and viscous greases are not recommended because they foul the silicone and obstruct the passage of light during microscopy. The actual stage displacement amplitude can fall substantially short of the prescribed displacement amplitude when many indenters are used, and the prescribed stage displacement amplitude is large (&#62; 3 mm). However, while the actual displacement is less than the prescribed displacement at large amplitudes, it remains repeatable (Figure 2B). Therefore, large, actual displacements amplitudes can be reliably obtained by entering a prescribed value in excess of the desired value. Displacement amplitude matters only because it is an easily recorded proxy for the peak membrane strain, which directly measures the mechanical insult that induces pathology. Therefore, the procedure described for determining membrane strain from stage displacement is critical. This process should be repeated if any major changes are made to the system that affect the interaction between the plate and the indenters, for example if different diameter indenters, different indenter materials or coatings, or different types of silicone bottomed plate are used. The process of realigning the indenter block and determining the zero position should be repeated at the start of each experiment. A schematic of the stretching device is shown in Figure 1. CAD models required to reproduce the device are provided as supplemental materials: &#39;Injury Device - FULL ASSEMBLY - Generic 3D.STEP&#39;; the associated bill of materials provided as &#39;Supplementary Table 1: Custom Built Devices - BOM.xlsx. Also see Supplementary Table 2 96 Well Plate _loader - Pinout Wiring Diagram.xlsx, which describes the cabling connections that connect various components of the systems. &#39;Interconnector_circuit_board.dip&#39; describes a circuit board that interconnects the cables.
If the device is deactivated with the stage near the middle of its travel, the stage will move after the power is cut off because it is spring-loaded. When the power is restored, the feedback loop will detect a large difference between the last known prescribed position and the actual position. This will cause the stage to move suddenly to the position it was in when the device was deactivated. This sudden motion can cause errors in the output of the encoder, so care should be taken to deactivate the device only when it is in its unpowered resting position at the top of its travel.
The fabrication clamp is designed to bring the plate body and silicone bottom together in a manner that allows optimal bonding. To this end, there are three key features in the design presented in the supplemental file &#39;Press Die - Generic 3D.STEP&#39;. First, the clamp plate body holder is parallel to the silicone bottom. If this is properly built, it will require no adjustment after the initial setup. Second, the layer of foam rubber in the clamp provides a small amount of compliance under the plate, as a completely rigid system would theoretically experience a sudden increase from zero clamping force to infinite clamping force when the clamp was closed. The position of the crossbar and set screw of the clamp are adjustable so that the distance between the two sides of the clamp can be fine-tuned.
Every effort should be made to provide a bright, white background behind the dot on the well bottom during strain characterization experiments. The better the contrast in these images, the easier it will be to automate the process of measuring the height and width of the dot, which can become tedious for a human operator analyzing a large experiment. High-speed videography of the bottom of a well in a 96-well plate presents challenges because the walls of the well tend to cast shadows. The use of a dome light or diffuse axial light that can illuminate along the line of sight of the camera without obscuring the image eliminates shadows or specular reflections that would arise with a conventional light source. The brightest available light source should be used because bright illumination allows images to be acquired with a short exposure time. Short exposure times minimize motion blur. Upgrading the light emitting diodes (LEDs) in the diffuse axial light allows shorter exposure times during high-speed video acquisition. The LEDs can be upgraded by opening the diffuse axial light, removing the stock LEDs, mounting 4 high power LED arrays to the back pane using LEDs holders, connecting them to a constant current power supply, and reassembling the diffuse axial light (see Table of Materials for catalog numbers). The disadvantage of upgrading the LEDs is that the passively cooled LEDs cannot be kept on for more than a few seconds due to the risk of overheating. Therefore, a different light is needed for alignment of the post-block and camera adjustment.
The presented method of quantifying membrane strain by measuring the dilation of a dot stamped on to the membrane is relatively crude, but it can scale up to multiple wells in a robust manner. The strain field across the well bottom can be characterized in more detail using digital image correlation. This technique involves spraying a speckled pattern onto the base of the well and then imaging it at high-speed during deformation. Commercial software can then be used to quantify strain at every point in the image by tracking the evolution of the speckled pattern.
This protocol produces a multi-faceted, clinically relevant, stretch injury phenotype in hiPSCNs. Cell death, neurite degeneration, and neurite beading are all well-documented sequelae of TBI in humans and animal models15. The key to success in this model is establishing and maintaining healthy cultures. Generally speaking, a cell culture protocol developed with conventional rigid plates is a worthwhile starting point for stretchable plate culture. However, the possibility that the cells in question may respond differently on silicone must always be considered. This is particularly true of hiPSCNs, which are very sensitive to culture conditions. Some examples of optimizing cell density and laminin concentration are supplied in the Representative Results section (Figure 3, Figure 4). Activation of the silicone with plasma treatment is vital. Silicone is hydrophobic and unreactive; in its natural state, it will not bind to laminin or other molecules used to promote cell attachment. Plasma treatment renders the surface hydrophilic and exposes reactive groups. These changes allow adhesion molecules to bind to the silicone and promote cell attachment. It is important to note that the plasma treatment effect dissipates within minutes unless the surface is submerged in liquid, and so procedures that involve drying the activated surface should be performed as quickly as possible. A simple way to check if the effect of plasma treatment has worn off is to place a droplet of water on the surface. On untreated silicone, the droplet will bead up whereas on plasma treated silicone, it will spread out. With the hiPSCNs that we used (see Table of Materials), the manufacturer recommends adding the laminin with the cell suspension rather than pre-coating. This protocol has incorporated this approach successfully. While segmentation can, in theory, be accomplished with open source software or general-purpose programming languages, a high degree of proficiency with these tools is required to obtain good results. Neurites are frequently difficult to distinguish from background signal because they are so slender. Therefore, we recommend the use of commercial software tools distributed by high content microscopy companies with dedicated modules for segmentation and quantification of neurons, if they are available. Even with commercial software, it is wise to export images of the segmentation to visually verify accuracy.
There are some limitations associated with working in stretchable plates compared to working with conventional, rigid plates. Stretchable plates can be imaged as normal with air objectives. However, imaging with immersion objectives is very difficult. Lens oil may damage the silicone. Additionally, the objective exerts pressure on the silicone membrane as it moves upwards. This pressure displaces the membrane vertically, making it difficult to bring the sample into focus. The silicone membranes currently used in fabricating the plates are approximately 250 &#181;m thick. This thickness exceeds the focal distance of many high power, immersion objectives. Special care must be taken to lay the membranes perfectly flat before clamping to achieve the flatness required for microscopy. Autofocus systems can compensate for deviations in the flatness of the finished plate to some extent. Future versions of the protocol may pre-tension the membrane before it is bonded to the plate top to ensure flatness. The adhesive-free procedure for bonding the silicone membrane to the plate top14 is considered an important strength of the current protocol. It eliminates the risk of neurotoxicity from the adhesive as well as any deviations in flatness due to non-uniform thickness of the adhesive layer.
Multi-electrode arrays are commonly used in experiments with hiPSCNs to assess their maturity and functionality. Unfortunately, these systems are incompatible with this model because the cell culture substrate is rigid. It is possible to create a stretchable multi-electrode array, although this has so far only been demonstrated in a single well format16,17. Note that indenters can be removed individually from the indenter block so that some wells are not indented and can serve as shams. Removing the indenter prevents indentation but does not completely eliminate mechanical loading since there is still inertial motion of the fluid in the wells while the stage is moving. It is worth comparing these wells to wells in plates that were never subject to stage motion to measure any pathological influence of fluid motion. Also, the array of indenters in the block should be bisymmetric (symmetrical from front to back and side to side). This precaution ensures that the plate is evenly loaded during indentation, so that the stage does not tilt sideways and cause the rods to bind in their bearings.
One of the primary challenges to therapeutic innovation in neurotrauma is the complexity and heterogeneity of the condition. Trauma applies multi-modal stress to every cell type in the central nervous system simultaneously. Neurons have been reliably generated from human induced pluripotent stem cells (hiPSCs) and are now widely available from commercial vendors. Innovation is proceeding quickly in this field, and other neural cell types such as astrocytes18 and microglia19 are also being derived from hiPSCs. It may soon be possible to isolate the cell-autonomous responses of each of these cell types to trauma in vitro and then to co-culture different cell types to understand how they communicate after trauma. In this way, it may ultimately be possible to recreate the clinical challenge from the bottom up to thoroughly understand it in a human system. This approach is distinct from the conventional approach relying on rodent models and has the potential to generate novel insights that lead to the first therapies for this common, devastating, and intractable condition.

TBI is a major cause of mortality and morbidity in the United States, causing around 52,000 deaths and 275,000 hospitalizations every year1. More than 30 clinical trials of candidate therapeutics for TBI have been conducted without a single success2. This uniform failure suggests that human-specific processes separate human TBI from the pathophysiology observed in commonly used pre-clinical rodent models.
The advent of hiPSCNs has created an opportunity to study neurotrauma in a human in vitro model. Drug screening with hiPSCN-based models may deliver results that are more predictive of clinical success than models employing rodent cells. Also, hiPSCNs can be genetically manipulated to isolate and study the effect of individual human genetic variants on pathology3.
The method described in this manuscript is designed to bring the unique advantages of hiPSCN-based disease modeling to neurotrauma. In vitro stretch injury models of neurotrauma are well established4,5,6 with primary rodent cells and human neural cancer cell lines. Most of these models generate stretch by pneumatically loading a silicone membrane. This approach is effective in a single well format but has proven difficult to scale up to a multi-well format7. As a result, there has never been a high throughput screen for agents to treat stretch injured neurons.
In this model, the membrane stretches due to indentation from underneath with a rigid indenter. This approach has been shown repeatedly to generate clinically relevant pathology in vitro in single well systems8,9,10. Our recent work has shown that it easily scales up to a 96-well format while maintaining pulse durations on the order of tens of milliseconds11, which is the time domain of closed head impact events12,13.
In summary, the key advantages of this in vitro injury model are the 96-well format, the use of hiPSCNs, and the clinically relevant time domain of the insult.

<table border="0" cellpadding="0" cellspacing="0" width="932">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">.010&#34; Silicone Sheet</td>
			<td style="width:205px;">Specialty Manufacturing, Inc</td>
			<td style="width:344px;">#70P001200010</td>
			<td style="width:167px;">Polydimethylsiloxane (PDMS) sheet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sparkleen</td>
			<td>Fisher Scientific&#160;</td>
			<td>#043204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc 256665</td>
			<td>Fisher Scientific&#160;</td>
			<td>#12-565-600</td>
			<td>Bottomless 96 Well Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kim Wipes</td>
			<td>ULINE</td>
			<td>S-8115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>#PDC-001-HC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(3-Aminopropyl) triethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>#440140</td>
			<td>APTES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parchment Paper</td>
			<td>Reynolds</td>
			<td>N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dome Light</td>
			<td>CCS inc</td>
			<td>LFX2-100SW</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dome Light Power Supply</td>
			<td>CCS inc</td>
			<td>PSB-1024VB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axial Diffuse Lighting Unit&#160; &#160; &#160; &#160; &#160; &#160;&#160;</td>
			<td>Siemens</td>
			<td>Nerlite DOAL-75-LED</td>
			<td>Diffuse axial light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High Power LED Array&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;&#160;</td>
			<td>CREE</td>
			<td>XLamp CXA2540</td>
			<td>High Power LED Array&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED holder</td>
			<td>Molex</td>
			<td>1807200001</td>
			<td>LED Holder&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED power supply</td>
			<td>Mean Well</td>
			<td>HLG-320H-36B</td>
			<td>Constant Current Power Supply&#160; &#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastCam Viewer software&#160;</td>
			<td>Photron</td>
			<td></td>
			<td>camera softeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fastcam Mini UX50</td>
			<td>Photron</td>
			<td>N/A</td>
			<td>High Speed Camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-NIKKOR 105mm f/2.8</td>
			<td>Nikon</td>
			<td>#1455</td>
			<td>High Speed Camera Lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1 mg/mL Poly-L-Ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>#P4597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iCells</td>
			<td>Cellular Dynamics International</td>
			<td>#NRC-100-010-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iCell media</td>
			<td>Cellular Dynamics International</td>
			<td>#NRM-100-121-001&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iCell supplement</td>
			<td>Cellular Dynamics International</td>
			<td>#NRM-100-031-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>#L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>Fisher Scientific&#160;</td>
			<td>#H3570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcein AM</td>
			<td>Fisher Scientific&#160;</td>
			<td>#C3099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">voice coil actuator&#160;</td>
			<td>BEI Kimco</td>
			<td>LA43-67-000A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">optical linear encoder&#160;</td>
			<td>Renishaw</td>
			<td>T1031-30A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">servo drive</td>
			<td>Copley Controls</td>
			<td>Xenus XTL&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Controller</td>
			<td>National Instruments</td>
			<td>cRIO 9024 Real Time PowerPC Controller&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cRIO chassis</td>
			<td>National Instruments</td>
			<td>cRIO 9113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">digital input module</td>
			<td>National Instruments</td>
			<td>NI 9411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">data acquistion chassis</td>
			<td>National Instruments</td>
			<td>NI 9113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LabVIEW</td>
			<td>National Instruments</td>
			<td></td>
			<td>instrument control software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hiPSCNs</td>
			<td colspan="2">Cellular Dynamics International</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for high speed stretch injury of human induced pluripotent stem cell-derived neurons in a 96-well format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

The stretcher device is capable of moving the stage repeatably with pulse durations as short as 10-15 ms depending on the amplitude of the pulse (Figure 2A). The pulse amplitudes are highly repeatable, but the pulse duration varies by approximately 1 ms between repetitions. The actual pulse amplitude diverges from the prescribed pulse amplitude when a large number of wells are loaded, and the prescribed amplitude is high (see Figure 2B). As the amplitude of stage displacement is increased beyond 3 mm, the actual displacement amplitude increasingly falls short of the prescribed displacement amplitude (see Figure 2B). Careful alignment of the post block eliminates any trend in the membrane strain across rows or columns (Figure 2C). At the 3.5 mm prescribed stage displacement amplitude (3.3 mm actual displacement amplitude) with 52 wells indented, the mean Lagrangian strain across all well locations was 0.451 (standard deviation of means for all locations = 0.051, mean of standard deviations for all locations = 0.065, n = 5 measurements per well). These results are presented here for completeness although some of them have already been reported11.
An optimal, uninjured culture will have few if any clumps of more than 5 cells. Neurites will be individual, long, slender, and curved with little or no sign of tension or beading (Figure 3A). Under ideal conditions, the viability of the cultures should closely approach the viability specified in the manufacturer&#39;s data sheet (typically 60-70%) and cultures on silicone should resemble those maintained on conventional rigid culture substrates (Figure 3B). Neurites may or may not be visible on a low power bright field microscope. Laminin concentration and cell density both influence cultures at baseline and after injury. Increasing the cell density increased the number and size of clumps that formed in culture. Increasing the laminin concentration often counteracted this effect (Figure 3A). However, increasing the laminin concentration too much blunted the sensitivity of the cultures to injury (Figure 4). The optimal laminin concentration for uninjured cultures was 50 &#181;g/mL of laminin (Figure 3), but the optimal separation between the sham and stretch injured populations was obtained at 10 &#181;g/mL of laminin (Figure 4). Higher laminin concentrations reduced the sensitivity of the cultures to injury at short time points (Figure 4), but also improved baseline cell viability at longer time points (e.g., 7 days). In summary, it is worthwhile to optimize the laminin concentration for each experimental scenario.
Membrane strain, post-injury imaging time point, laminin concentration, and cell density all exerted a highly statistically significant main effect on the neurite length per cell (ANOVA, p &#60;0.001). The effect of membrane strain on neurite length per cell had highly statistically significant interactions with post-injury imaging time point and laminin concentration (ANOVA, p &#60;0.001) and a statistically significant interaction with cell density (ANOVA, p &#60;0.05). Similarly, membrane strain, post-injury imaging time point, cell density, and laminin concentration all exerted a highly statistically significant main effect on cell viability (ANOVA, p &#60;0.001). The effect of the membrane strain on cell viability had a highly statistically significant interaction with the post-injury imaging time point (ANOVA, p &#60;0.001) and a statistically significant interaction with the cell density (ANOVA, p &#60;0.05). These results prove that timing, cell density, and laminin concentration exert an important influence on the relationship between the applied insult and experimental outcomes, so each should be optimized carefully.
Low cell viability and beaded neurites, along with stunted neurite growth, indicate toxic culture conditions that can arise from improperly prepared silicone. Skipping or shortening the water soak or the oven dry can leave absorbed ethanol or water in the membrane, respectively, which can diffuse into the media and stress the cells. Well-injured cultures will have reduced cell viability, shortened or missing neurites, beaded neurites, and neurites that look taut or tensioned. Injury may induce clumping in cell cultures that were well-dispersed pre-injury. Large clumps can confound the morphological analysis. For morphological analysis, the injury level should be tuned such that noticeable changes occur, but cells are still present with some neurites.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57305/57305fig1.jpg" /> 
Figure 1: A labeled schematic of the injury device. (A) Top view, (B) isometric view, (C) front view, (D) right side view. The scale bar applies to the orthographic views (A, B and D). <a href="/files/ftp_upload/57305/57305fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57305/57305fig2.jpg" /> 
Figure 2: Kinematics of the mechanical insult. (A) The stage displacement histories over 5 pulses at a range of prescribed amplitudes (prescribed amplitudes are listed in the legend) when no wells are loaded. (B) The stage displacement histories over 10 pulses at a range of prescribed amplitudes (prescribed amplitudes are listed in the legend) when 52 wells are loaded. (C) The average strain in each well with stage displacement amplitude of 3.3 mm (n = 5 measurements per well, average standard error per well = 0.029). Note that C4-F4 and C9-F9 are unstretched control wells. This figure has been modified from Sherman et al.11 <a href="/files/ftp_upload/57305/57305fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57305/57305fig3.jpg" /> 
Figure 3: Optimization of culture conditions on silicone. (A) The effect of varying cell density and laminin concentration on hiPSCN cultures on silicone. Clumping increases with increasing cell density and decreasing laminin concentration. Cell density and laminin concentration must be optimized to achieve mono-dispersed cultures. Mono-dispersed cultures are less vulnerable to artifacts during quantification. Note that the dynamic range has been adjusted to optimize visualization of the neurites. As a consequence, the much brighter soma are saturated. This presentation is preferred to the alternative of optimizing the dynamic range with respect to the soma, which renders the much dimmer neurites almost invisible. The condition highlighted by the red square was deemed to be optimal for in vitro stretch injury experiments. (B) Under optimal conditions, cultures on silicone membranes appear similar to cultures on conventional rigid substrates. The left panel shows hiPSCNs cultured at 33,750 cells/cm2 with 3.3 &#181;g/mL of laminin on a conventional, rigid 96-well plate (the cell culture substrate is a tissue culture treated cyclic olefin co-polymer). The right panel reproduces the panel outlined in red from (A). Scale bars = 100 &#181;m. <a href="/files/ftp_upload/57305/57305fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57305/57305fig4.jpg" /> 
Figure 4: The injury phenotype and its dependence on laminin concentration. (A) Healthy culture, using 10 &#181;g/mL laminin and 67,500 cells/cm2. Neurites are long with no beads. There are few dead nuclei, and few clumps. (B) Culture using the same culture conditions, injured with 57% peak strain and imaged after 4 h. Neurites are shortened or missing, and some have beads (indicated by arrows). There are fewer Calcein AM-positive cells and more Calcein AM-negative (i.e., dead) nuclei. Injury has increased clumping among the surviving cells. (C) 4 h after injury, neurite length per cell declines with increasing strain in a manner that depends on the laminin concentration. (D) 4 h after injury, cell viability declines with increasing strain in a manner that depends on the laminin concentration. (E) 24 h after injury, neurite length per cell declines with increasing strain in a manner that depends on the laminin concentration. (F) 24 h after injury, cell viability declines with increasing strain in a manner that depends on the laminin concentration. (n = 4 per bar, error bars are &#177; 1 standard deviation, Scale bars = 100 &#181;m). Strain values are deduced from stage displacement using data from a prior publication by Sherman et al.11 <a href="/files/ftp_upload/57305/57305fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Technical drawing of the indenter. <a href="/files/ftp_upload/57305/Supplementary_Figure_1.pdf" target="_blank">Please click here to download this figure.</a>
Supplementary Table 1: Custom Built Devices. <a href="/files/ftp_upload/57305/Supplementary_Table_1-Custom_Devices-Bill_of_Materials.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Table 2: 96 Well Plate-loader Pinout Wiring Diagram. <a href="/files/ftp_upload/57305/Supplementary_Table_2-96_Well_Plate_Loader.xlsx" target="_blank">Please click here to download this table.</a>
Supplementary Code File 1: Computer-aided design drawings of the injury device. <a href="/files/ftp_upload/57305/Supplementary_Code_File_1-Injury_Device-FULL_ASSEMBLY-Generic_3D.STEP" target="_blank">Please click here to download this file.</a>
Supplementary Code File 2: Computer-aided design drawings of the plate fabrication clamp. <a href="/files/ftp_upload/57305/Supplementary_Code_File_2-Press_Die-Generic_3D.STEP" target="_blank">Please click here to download this file.</a>
Supplementary Code File 3: 3D representation of the stamp geometry, suitable for use with a 3D printer. <a href="/files/ftp_upload/57305/Supplementary_Code_File_3-stamp.STL" target="_blank">Please click here to download this file.</a>
Supplementary Code File 4: SubVI for MuStLiMo_si_initialize.vi, which is a SubVI for motion_control.vi. Converts entries in dialog boxes into parameters for motion. <a href="/files/ftp_upload/57305/Supplementary_Code_File_4-compute_init_param.vi" target="_blank">Please click here to download this file.</a>
Supplementary Code File 5: SubVI for Multiple Straight Line Moves_simplified.vi, which is a SubVI for motion_control.vi. Converts entries in dialog boxes into parameters for motion. <a href="/files/ftp_upload/57305/Supplementary_Code_File_5-compute_parameters.vi" target="_blank">Please click here to download this file.</a>
Supplementary Code File 6: SubVI for position_tracker.vi. Counter tracks displacement input from linear encoder. <a href="/files/ftp_upload/57305/Supplementary_Code_File_6-Counter-Continuous_Output.vi" target="_blank">Please click here to download this file.</a>
Supplementary Code File 7: Base LabVIEW Project. <a href="/files/ftp_upload/57305/Supplementary_Code_File_7-in_vitro_neurotrauma.lvproj" target="_blank">Please click here to download this file.</a>
Supplementary Code File 8: Top level VI that moves the device. <a href="/files/ftp_upload/57305/Supplementary_Code_File_8-motion_control.vi" target="_blank">Please click here to download this file.</a>
Supplementary Code File 9: SubVI for motion_control.vi. Executes the rapid displacement that stretches the plate. <a href="/files/ftp_upload/57305/Supplementary_Code_File_9-Multiple_Straight_Line_Moves_simplified.vi" target="_blank">Please click here to download this file.</a>
Supplementary Code File 10: SubVI for motion_control.vi. Executes the slow displacement that moves the stage. <a href="/files/ftp_upload/57305/Supplementary_Code_File_10-MuStLiMo_si_initialize.vi" target="_blank">Please click here to download this file.</a>
Supplementary Code File 11: SubVI for motion_control.vi. Plots a (usually undersampled) displacement history in the motion_control.vi control panel. <a href="/files/ftp_upload/57305/Supplementary_Code_File_11-plot_displacement.vi" target="_blank">Please click here to download this file.</a>
Supplementary Code File 12: Top level VI that records the displacement history. <a href="/files/ftp_upload/57305/Supplementary_Code_File_12-position_tracker.vi" target="_blank">Please click here to download this file.</a>
Supplementary Code File 13: Contains Variable2, which communicates between motion_control.vi and position_tracker.vi. <a href="/files/ftp_upload/57305/Supplementary_Code_File_13-Untitled_Library_2.lvlib" target="_blank">Please click here to download this file.</a>
Supplementary Code File 14: Schematic for printed circuit board. <a href="/files/ftp_upload/57305/Supplementary_Code_File_14-interconnector_circuit_board.dch" target="_blank">Please click here to download this file.</a>
Supplementary Code File 15: Layout for printed circuit board. <a href="/files/ftp_upload/57305/Supplementary_Code_File_15-interconnector_circuit_board.dip" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format at JoVE.com

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

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6.9K Views.  NorthShore University HealthSystem. Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Video: Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...