The authors would like to thank the funding sources: Army-UNL Center for Trauma Mechanics (DoD/ARO, #W911NF-11-1-0033, PI: Dr. Namas Chandra), UNL Layman Award (26-1110-0033-001, PI: Lim).

1.	Neuronal Cell Culture
<ol>
<li>Brain cells including neuronal cells, astrocytes, and their co-culture may be used as a cell model. As a feasibility demonstration, impulsive pressurization of cell-line neuronal cells is presented.</li>
<li>SH-SY5Y human neuroblastoma cells (ATCC, CRL-2266) are cultured on 18 mm diameter glass coverslips. Cells are seeded at a density of 3&times;103 cells/cm2 using the growth media composed of DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cell cultured glass slides are kept in a 5% CO2 humidified incubator at 37&deg;C.</li>
<li>To obtain the neuronal cell differentiation of SH-SY5Y cells, cells are treated with media further supplemented with 10 &mu;M retinoic acid (RA) for 7 days with media changed every two days. On day 7, cells are ready to be pressurized.</li>
</ol>
2.	Pressurization Equipment: Kolsky Bar
<ol>
<li>The Kolsky bar, developed by Kolsky1 in 1949, has been used to measure the mechanical property of a material at a very high loading rate. The apparatus consists of two bars with a sample placed in contact between the bars. A stress wave, created on the incident bar, propagates to the sample where the wave splits into a reflected and transmitted wave. The stress in the sample is proportional to the transmitted wave. In this study, we utilize a cell pressurization chamber to be placed between the two bars of the Kolsky set-up.</li>
<li>The two aluminum alloy bars (each 6-m long) are suspended by aligned brass bearings, and an in vitro cell pressurization chamber is sandwiched between the bars. The upstream bar is the incident bar with a 130 lb mass clamped onto one end. A friction clamp is placed at a position that will give the desired pulse duration. By engaging the clamp and tightening a scissors jack between the mass and a loading support, the clamp-mass section of the incident bar is pre-compressed to the desired level. Forcing a notched bolt that locks the clamp to a sudden break with a hydraulic system releases the pre-stored compression rapidly and thus generates a pressure pulse. This propagates through the incident bar, drives the test chamber piston, and pressurizes the fluid and cells within the chamber impulsively. The chamber pressurization in turn initiates a pressure pulse propagating in the transmitted bar downstream. The strains associated with the pressure pulses in the bars are measured with compound high resistance strain gauges excited to 45 volts. The gauge signals are recorded with a digital oscilloscope at 1 MHz frequency as a loading history.</li>
<li>The in vitro cell pressurization chamber is composed of a piston-cylinder. The cylinder has 2.6 cm inner diameter and 3.8 cm outer diameter, and has a small hole tapped at the base of the cavity. The hole serves as an air and excess fluid vent during cell sample installation. The piston is 7.5 cm long and is made from the same bar material. The piston is wrapped with two to three layers of plumber's (Teflon) tape that serve as low friction seal. The end cap is 32 mm long and has two small stainless steel screws that secure the glass coverslips (on which cells are cultured) during the loading.</li>
</ol>
3.	Impulsive Pressurization of Neuronal Cells
<ol>
<li>All pressurization chamber parts are sterilized using the autoclave and are kept under UV light. The assembly of the chamber is performed inside the cell culture hood. The vent screw in the chamber is loosely engaged in the air vent at the base of the cavity. Pre-warmed (37&deg;C) fresh growth media is pipetted into the chamber without making bubbles. Then, a cell-cultured glass slide is picked up, and is placed on the cap of the piston (cells facing outside). The small holding screws are tightened down onto the slide to hold it in place. The piston with a cell-cultured glass slide is inserted a little into the cavity of the chamber, and the assembly is tilted with the vent being at the highest point. The vent screw is removed and the piston is pushed into the cavity first forcing out air bubbles, then the excess fluid. Either a reference mark or a jig is used as a guide to ensure the same culture media volume for every test. The axial dimension of the media in the chamber is about 6 mm. Sanitize the vent screw and replace it to make the chamber water tight. The chamber should not leak under this static load, if it does, the Teflon wrap needs to be replaced or reinforced.</li>
<li>At this point, the Kolsky bar system is reset. The heavy mass is moved back to the original position and a new locking bolt replaces the used (broken) one. Engage the new locking bolt with the hydraulics to about 200 psi. Use the scissors jack to compress the pre-loading section of the incident bar up to a pressure little higher than the desired value, and then back it off to engage the full friction of the jack's screw. The data acquisition is now armed.</li>
<li>The assembled cell pressurization chamber is mounted into the system. It is placed in a V block supported by small lab scissors jack and aligned with the two bars. Grease each interface with a light grease layer and rub the butting surfaces together to eliminate any air gaps in between.</li>
<li>The test is now ready to proceed. The clamp locking bolt is forced to break by quickly pumping the hydraulic clamp driver. The clamp will separate and the data acquisition should display the results. The cell pressurization history is determined from the measurements of the transmitted bar gauge. If the transmitted bar used is long enough, this should only be from the first disturbance to the point where the measurements show a negative pressure. The duration of the transmitted pulse may be shorter than the incident pulse if a bubble is trapped. The magnitude may not reach that of the incident pulse, but it should have a sufficiently long plateau before unloading. Otherwise, either a large air bubble in the sealed chamber or a misalignment between the assembly and a bar might have occurred.</li>
<li>The cell pressurization chamber is then removed and disassembled inside the cell culture hood. The vent screw is removed and the piston is pulled free of the chamber. The cell-cultured glass slide is taken from the end of the piston.</li>
</ol>
4.	Assessing Pressurized Cell Behavior
<ol>
<li>After pressurization, the cells may be inspected immediately or further incubated for later examination. With proper aseptic operation processes, longer-term post-incubation is possible.</li>
<li>Pressurized cells can be examined by all molecular and cellular biology techniques. Specifically for neuronal cell pressurization, to investigate the cellular and molecular physiology in TBI conditions, assays assessing pressure-induced changes in neurite outgrowth, microtubule cytoskeletal change, neuronal gene expression, apoptosis, etc., can be performed. As control cell samples, cells that are cultured the same, kept inside the pressurization chamber for the same time period, but not pressurized are used (so called, chamber control).</li>
<li>For assessing the changes in neurite outgrowth, pressurized and chamber control cells are examined by optical microscope immediately after pressurization and 1 and 24 h of post-incubation. An example of neuronal cell images are shown in the 'Representative Results' section (Figure 2). The neurite length change can be quantified by actin immunofluorescent staining and image analysis. Cells are fixed with a 4% w/v paraformaldehyde solution, rinsed with a 0.05% v/v Tween-20 wash buffer, and permeabilized with a 0.1% v/v Triton X-100 solution. After blocking with a 1% w/v bovine serum albumin solution, cells are incubated with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin. Immunofluorescent images of the cells are taken using a fluorescent microscope and the lengths of neurites for pressurized and control cells are quantified using the ImageJ software (NIH).</li>
<li>The neurites can be identified as axons or dendrites. To assess the morphological changes in axons or dendrites, the experiments described above can be repeated by using antibodies detecting each structure, i.e., neurofilament (NF) antibody for axons and microtubule-associated protein (MAP2) antibody for dendrites.</li>
<li>Microtubules are one of the key cytoskeletal components of neurons, and the damage to microtubules has been used as a marker of neuronal injury.2 Microtubule can be visualized immunofluorescently using the &beta;-tubulin antibody. Cells are fixed after 0 and 24 h of post-pressurization, stained with &beta;-tubulin antibody, and observed by the fluorescent microscope.</li>
<li>To assess the effect of impulsive pressurization on neuronal and apoptotic gene expression, total RNA is extracted from pressurized and control cells. Gene expression can be examined by performing quantitative RT-PCR or real-time RT-PCR, similar to our published protocols.3</li>
</ol>
5.	Representative Results:
<img src="/files/ftp_upload/2723/2723fig1.jpg" alt="Figure 1"/> Figure 1. An example of pressure profile applied to the cells within the pressurization chamber. The Kolsky bar apparatus successfully generates 2 MPa level, single-pulse type impulsive pressurization with a duration of about 0.7 ms.
<img src="/files/ftp_upload/2723/2723fig2.jpg" alt="Figure 2"/> Figure 2. An example of neuronal cell response to impulsive pressurization. SH-SY5Y cells exposed to impulsive pressurization at 2 MPa show distinct neurite/axon breakdown relative to chamber control cells.
6.	Difficulties and Solutions
<ol>
<li>Chamber sealing is one of the major obstacles to be overcome. It is found that wide disks with o-rings have a problem of gouging and increased friction. In order to remove the friction effects, as well as prevent gouging, a simple method of piston taping with Teflon plumber's tape is used. This solves the problems and produces desired pressure level and duration.</li>
<li>Pressurized neuronal cell behavior, especially when assessing secondary TBI mechanism or longer term effects, should be examined after a long post-incubation time. The cell-containing chamber is exposed to potentially unsterile conditions while moving to the Kolsky bar, pressurizing, and moving back to the cell culture hood. With pre-sterilization of the chamber parts and proper operation in assembly/disassembly of the cell-cultured slide and chamber inside the cell culture hood, longer-term post-incubation is possible up to several weeks.</li>
<li>The reproducibility of the data is an important parameter in cellular mechanical stimulation experiments. For our device, the reproducibility in neuronal cell response is determined how desired impulsive pressurization profile, in both pressure level and duration, is repeatedly obtained. Since we record the obtained pressure profile for each pressurization, the cell response data from unwanted pressure profile can be manually excluded afterwards.</li>
<li>The entire pressurization steps, including chamber assembly, transfer and mount in the Kolsky bar, pressurization, transfer-back, and chamber disassembly, take less than 10 min. The next cell cultured slide can be pressurized immediately after the previous pressurization. Thus, sufficient number of pressurization experiments can be completed efficiently. Our set-up utilizes 18 mm diameter glass coverslips. One pressurization experiment does not provide enough proteins for western immunoblotting due to substrate size (and also partly because some of the pressurized cells are dead). About three repeated pressurization experiments provide protein amount required for immunoblotting. Again, the reproducibility is checked each time with the pressure profile.</li>
</ol>

<ol>
	<li>Kolsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+mechanical+properties+of+materials+at+very+high+rates+of+loading.">An investigation of mechanical properties of materials at very high rates of loading.</a> Prec. Phys. Soc. London. 62, 676-700 (1949).</li><li>Tang-Schomer, M. D., Patel, A. R., Baas, P. W., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+breaking+of+microtubules+in+axons+during+dynamic+stretch+injury+underlies+delayed+elasticity,+microtubule+disassembly,+and+axon+degeneration.">Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration.</a> FASEB. J. 24, 1401-1410 (2010).</li><li>Lim, J. Y., Taylor, A. F., Li, Z., Vogler, E. A., Donahue, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+expression+and+osteopontin+regulation+in+human+fetal+osteoblastic+cells+mediated+by+substratum+surface+characteristics.">Integrin expression and osteopontin regulation in human fetal osteoblastic cells mediated by substratum surface characteristics.</a> Tissue. Eng. 11, 19-29 (2005).</li><li>Chen, Y. C., Smith, D. H., Meaney, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+approaches+for+studying+blast-induced+traumatic+brain+injury.">In vitro approaches for studying blast-induced traumatic brain injury.</a> J. Neurotrauma. 26, 861-876 (2009).</li><li>Ling, G., Bandak, F., Armonda, R., Grant, G., Ecklund, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explosive+blast+neurotrauma.">Explosive blast neurotrauma.</a> J. Neurotrauma. 26, 815-825 (2009).</li><li>Shepard, S. R., Ghajar, J. B. G., Giannuzzi, R., Kupferman, S., Hariri, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+percussion+barotrauma+chamber:+a+new+in+vitro+model+for+traumatic+brain+injury.">Fluid percussion barotrauma chamber: a new in vitro model for traumatic brain injury.</a> J. Surg. Res. 51, 417-424 (1991).</li><li>VandeVord, P. J., Leung, L. Y., Hardy, W., Mason, M., Yang, K. H., King, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+reactivity+and+survival+genes+in+astrocytes+after+exposure+to+short+duration+overpressure.">Up-regulation of reactivity and survival genes in astrocytes after exposure to short duration overpressure.</a> Neurosci. Lett. 434, 247-252 (2008).</li><li>Huang, H., Feng, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+dynamic+tribological+response+of+closed+fracture+surface+pairs+by+Kolsky-bar+compression-shear+experiment.">A study of the dynamic tribological response of closed fracture surface pairs by Kolsky-bar compression-shear experiment.</a> Int. J. Solids. Struct. 41, 2821-2835 (2004).</li><li>Song, B., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+Hopkinson+pressure+bar+techniques+for+characterizing+soft+materials.">Split Hopkinson pressure bar techniques for characterizing soft materials.</a> Lat. Am. J. Solids. Struct. 2, 113-152 (2005).</li></ol>

No conflicts of interest declared.

Many in vitro experimental techniques have been attempted for studying brain cell damage in TBI conditions. These include neuron/astrocyte stretching, flow-induced shear stress, weight drop, stylus laceration, laser transection, lithotripsy, etc.4,5 It has been assumed that short-duration overpressure is a dominating physical factor for TBI.6,7 Especially, the shockwave of the blast TBI has a pulse duration of an order of milliseconds.4 Based on this assumption and observation, barometer chamber capable of transient pressurization has been developed and used for assessing brain cell behavior under TBI conditions. For example, in the fluid percussion barotrauma chamber a pressure pulse with 20-30 ms long duration and a peak pressure of up to 0.5 MPa was used to examine the human glial cell behavior under TBI.6 Recently, VandeVord et al.7 used metal ball striking barometer chamber to examine the astrocyte cell behavior under TBI but the metal ball striking produced multiple pressure pulses. In this study, we have developed a novel cell pressurization device that generates single-pulse type impulsive pressurization. The device was developed based on the Kolsky bar set-up, which has been used as a material testing apparatus in our8 and other's studies.1,9 The Kolsky bar's material testing capability at very high loading rate has been modified to be able to apply single-pulse type overpressure to the cultured cells within the pressurization chamber (Figure 1). We could successfully control the magnitude and duration of the impulsive overpressure. As a representative cell response, we demonstrated that at 2 MPa pressure SH-SY5Y neuronal cells display significant neurite and axonal loss as compared to unpressurized chamber control cells.
In conclusion, we developed a novel impulsive cell pressurization apparatus using a specially designed Kolsky bar device and demonstrated its potential usage in investigating neuronal cell response in TBI conditions. We also note that this technique is very versatile in that any type of cells can be exposed to impulsive pressures at various level and duration. Therefore, the device can be used to investigate the impulsive pressure-induced cellular mechanotransduction behavior, not only for injuring the cells but also for positively stimulating the cells in their function and fate determination.

<ul>
	<li>SH-SY5Y (ATCC, CRL-2266) human neuroblastoma cells</li>
	<li>18 mm diameter glass coverslips</li>
	<li>Cell culture media: DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin</li>
	<li>Retinoic acid (10 &mu;M) for inducing neurogenesis</li>
	<li>Kolsky bar impulsive pressurization device</li>
	<li>Piston-cylinder cell pressurization chamber</li>
	<li>Stainless steel screws for securing cell cultured coverslip on the piston</li>
	<li>Grease for interfaces between cell chamber and Kolsky bar</li>
	<li>General molecular biology supplies for assessing cell response to pressurization (fixative, antibody, fluorescent dye, PCR supplies, etc.)</li>
</ul>

impulsive pressurization of neuronal cells for traumatic brain injury study

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). We demonstrate in this video article how blast TBI-relevant impulsive pressurization is applied to the neuronal cells in vitro. This is achieved by using well-controlled pressure pulse created by a specialized Kolsky bar device, with complete pressure history within the cell pressurization chamber recorded. Pressurized neuronal cells are inspected immediately after pressurization, or further incubated to examine the long-term effects of impulsive pressurization on neurite/axonal outgrowth, neuronal gene expression, apoptosis, etc. We observed that impulsive pressurization at about 2 MPa induces distinct neurite loss relative to unpressurized cells. Our technique provides a novel method to investigate the molecular/cellular mechanisms of blast TBI, via impulsive pressurization of brain cells at well-controlled pressure magnitude and duration.

Watch this Scientific Journal Video about Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study at JoVE.com

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). ...

impulsive-pressurization-neuronal-cells-for-traumatic-brain-injury

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JoVE Journal

Bioengineering

10.2K Views.  University of Nebraska-Lincoln. Роман импульсивный эксперимент давления клетки была разработана с использованием устройства Кольский бар, чтобы исследовать молекулярные / клеточных механизмов доменных индуцированной черепно-мозговой травмой.

Video: Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Training Persons with Spinal Cord Injury to Ambulate Using a Powered Exoskeleton

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper extremity function and are able to maintain upright balance using forearm crutches. To ambulate in an exoskeleton, the user must acquire the ability to maintain balance while standing, sitting and appropriate weight shifting with each step. This can be a challenging task for those with deficits in sensation and proprioception in their lower extremities. This manuscript describes screening criteria and a training program developed at the James J. Peters VA Medical Center, Bronx, NY to teach users the skills needed to utilize these devices in institutional, home or community environments. Before training can begin, potential users are screened for appropriate range of motion of the hip, knee and ankle joints. Persons with SCI are at an increased risk of sustaining lower extremity fractures, even with minimal strain or trauma, therefore a bone mineral density assessment is performed to reduce the risk of fracture. Also, as part of screening, a physical examination is performed in order to identify additional health-related contraindications. 
Once the person has successfully passed all screening requirements, they are cleared to begin the training program. The device is properly adjusted to fit the user. A series of static and dynamic balance tasks are taught and performed by the user before learning to walk. The person is taught to ambulate in various environments ranging from indoor level surfaces to outdoors over uneven or changing surfaces. Once skilled enough to be a candidate for home use with the exoskeleton, the user is then required to designate a companion-walker who will train alongside them. Together, the pair must demonstrate the ability to perform various advanced tasks in order to be permitted to use the exoskeleton in their home/community environment.

Training a person with paralysis to ambulate using a powered exoskeleton may present challenges. The goals are to present the candidate selection criteria and the training procedures for exoskeletal-assisted walking and other mobility skills that can be progressed as the participant's skill level improves.

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...