The work was supported by grants 1K25MH089851, 1P01DA028555-01A1, 2R01 NS034239, 2R37 NS36126, P01 NS31492, P20RR 15635, P01MH64570, and P01 NS43985 from the National Institutes of Health. The authors thank Ms. Robin Taylor for critical reading of the manuscript and outstanding graphic and literary support. The authors would also like to thank Erin McIntyre, Melissa Mellon, and Lindsay Rice for their technical support.

1. 	Introduction
The selective delivery of drugs and therapeutic macromolecules (peptides, proteins and nucleic acids) to cellular and tissue sites of active disease and ongoing microbial infections will improve pharmaceutical responses during disease1-3. One particular cellular site is the macrophage that is both highly mobile and immune engaging and is a consistent principal target for the human immunodeficiency virus (HIV).4 Importantly, macrophage engaged inflammation also underlies a broad range of disorders that include degenerative, inflammatory, infectious and cancerous diseases; and the cell's mobility to disease sites underlies progression of tissue injuries5-9. Importantly, the use of blood borne macrophages as drug, macromolecule, and signal carriers has gained recent attention for its translational potential. However, a significant obstruction in realizing therapeutic potentials is the blood brain barrier (BBB) amongst other tissue barriers that are impermeable to a spectrum of macromolecules and proteins. These, barriers, nonetheless, do permit cell passage. All together it is projected that in the natural course of disease peripheral macrophages that bypass barriers can carry formulated drugs, markers, and peptides to sites of infection or inflammation. Nonetheless, such technologies remain only in development. It is through our works that cell-mediated delivery can be developed for diagnostic and therapeutic applications and such applications are supported by laboratory and animal models of human disease10-12.
2. Nanomaterial Preparations
Preparation of nanomaterials for drug delivery and biodistribution studies is the topic of a parallel manuscript in this issue (reference parallel manuscript). All procedures for crystalline nanoparticle manufacture are carried out in a laminar flow hood. All surfaces are disinfected prior to use with 70% alcohol. This includes working surface, the exterior of gloves and any spills. All are covered with solution of replicate 70% alcohol immediately with wipes. Gloves are discarded after use and are not worn when entering any other laboratory area. Excipient, drug, sterile water with/containing any/all reagents for manufacture of drug-laden particles are only brought into work areas when needed for procedures. Sterile wrapped pipettes are used only and discarded after use into a biohazard waste container. The wet willing apparatus is disinfected with alcohol prior to and following use. Work area is cleaned immediately before and after with 70% alcohol. Nanoparticle solution is tested for pyrogen in accordance with FDA guidelines to assess the absence of bacterial endotoxin in drug particle solutions used for animals. Briefly,
<ol>
<li>Candidate nanoformulations for in-vivo use are replicated by replacing the drug core or droplet with an identical sized particle or milled piece of superparamagnetic iron oxide (SPIO) before coating with the appropriate surfactant. </li>
<li>This is followed by measures of size, charge, shape, and cytotoxicity to determine whether the SPIO model system has the same properties as the candidate nanoformulated drug. </li>
<li>Finally, cell loading assays are performed by incubation with the candidate SPIO model nanoformultation in order to determine the relaxivity within the cells using phantoms composed of labeled cells suspended in agar gel. Phantoms are prepared in triplicate and are prepared at a series of concentrations in order to quantify the relaxivity due to the SPIO uptake in cells. This provides an index of sensitivity and determines whether the nanoformulations may affect the oxidation state, and hence the visibility of the SPIO in magnetic resonance imaging (MRI) scans.</li>
</ol>
3. 	Methods and Procedures: Animal Preparation
<ol>
<li>Injections/Catheters.	Depending on time of interest, the injections may require use of a catheter to inject the animal within the MRI. Catheters are prepared using a non-magnetic needle and a tubing extension with minimum diameter to minimize dead space in the injection line. The catheter should be prefilled with either the solution containing the nanomaterial to be injected or saline, depending on the dead space and the total acceptable volume of the injection. If possible, the injection may be followed with a physiological saline flush. If acute times are not of extreme importance, a pre-scan can be performed, and the injection may be done outside of the magnet at a predetermined time before the follow up scans for biodistribution measures. Catheters are typically inserted into the tail vein for I.V. injections.</li>
<li>Anesthesia and monitoring. Prior to scanning, the animal is placed into a chamber to induce anesthesia. This chamber is prefilled with the 1.5% isoflurane in 70% nitrous oxide and 30% oxygen in order to speed the onset of anesthesia in the animal and minimize the amount of time required to ensure that the animal will not wake up upon removal from the chamber. Once the animal is fully anesthetized, the animal is removed from the chamber and placed into the stereotactic holder equipped to monitor the breath rate and temperature of the animal while continuing to supply isoflurane during set-up and scanning.</li>
<li>Animal Holders and Adjustment Considerations: Set-up includes eye lubricant to protect against corneal ulcers. The animal is lightly wrapped with gauze and the gauze is taped in place to minimize heat loss during scanning and to provide a positive pressure against the breathing monitor. Animal holders are equipped with adjustable tooth bars, allowing for vertical and horizontal alignment of the head. This is particularly important for high field MRI, as angulation of the head in the caudal-rostral direction will cause additional difficulties with magnetic field inhomogeneity due to magnetic susceptibility. Magnetic field inhomogeneity is deleterious to high quality T2* MRI as well as 1H magnetic resonance spectroscopy (1H MRS) and magnetic resonance diffusion tensor imaging (DTI). In addition to proper positioning of the head angle in the caudal-rostral direction, rotations of the head should be avoided to the degree that is feasible. Allowing for rotation of the animal holder in the magnet will provide compensation for minor rotations, which may occur from animal to animal. This may be further minimized by careful placement of the head and attention to the angulation prior to inserting the animal into the MRI system.</li>
<li>Calibration and Shimming: Once the animal is in the holder and the surface coil is properly placed on the head, the initial position of the animal is determined by a real-time one-dimensional readout in the caudal-rostral direction. Signal is restricted to the area around the surface coil used for reception, limiting the need for interpretation of the observed wave forms. Once the initial position is determined, a 3-plane localizer image is taken to determine the precise position of the animal in the scanner and to allow for movement to the precise location required for the scan(s) of interest. This is followed by adjustment of the homogeneity of the magnetic field or "shimming" the magnet. This is done by mapping the field distribution and calculation of a precisely determined spatial correction based on measured responses of a series of electromagnets or "shim coils" within the system designed to adjust the field homogeneity. Shimming is accomplished using a multi-gradient echo sequence and mapping software developed by Dr. Hetherington13. Regions of homogeneity are matched to the region examined by each individual imaging method. Once shimming is complete, we can acquire the scan(s) of interest from the animal.</li>
</ol>
4. Data Acquisition
<ol>
<li>High Resolution T2* weighted MRI. Biodistribution of SPIO containing nanoparticles can be determined by detecting regions of signal loss in high resolution 3D T2* weighted MRI. The region of the brain is determined from the localizer scans and prescribed on the localizer scans or additional scans as needed. A T2* weighted MRI scan with 150 micron isotropic resolution is then acquired. A high resolution 3D gradient recalled echo MRI scan of the mouse head is acquired using a 25 mm birdcage volume coil with acquisition parameters of echo time= 5 ms, repetition time = 50 ms, 30% echo, flip angle = 35 degrees, averages = 2, field of view = 20 x 20 x 20 mm with a resolution of 128 x 128 x 128 (voxel size = 150 x 150 x 150 &mu;m3), total acquisition time = 30 min. </li>
<li>Diffusion Tensor Imaging (DTI): Diffusion tensor images are quantitative measures of the direction and magnitude of diffusion of water within the cells of the tissue. As a result, the phase of the signal is extremely susceptible to motion, as the scans are sensitized or "weighted" for microscopic water motion. As a result, single shot acquisitions are desired to prevent phase shifts between acquisitions from causing signal smearing; and respiratory gating is required to prevent gross motion during signal acquisition. Therefore, a respiratory gated spin-echo diffusion weighted echo planar imaging (EPI) MR sequence is employed. Again, shimming the region of the scans is very important, as off-resonance effects during the evolution of the signal causes misregistration of the signal frequency, and hence position, in the plane of the image. EPI acquisition parameters included 14 slices, 200 KHz bandwidth, 96 x 96 in plane acquisition zero-filled to 256 x 256, and a 0.5 mm slice thickness. The diffusion encoding used was a balanced, rotationally-invariant and alternating polarity icosahedral scheme (12 directions) 14,15. The encoding scheme was designed to reduce background-diffusion gradient couplings 16. Diffusion weighting b-factor = 800 s mm-2, &delta;=4 ms, &Delta;=15 ms, Gdmax= 40 G/cm, 200 &mu;s rise time, 7 averages for b=0 acquisition, 3 averages for each b=800 encoding direction, for a total acquisition time of 20-40 min, depending upon respiration rate.</li>
<li>Localized 1H Magnetic Resonance Spectroscopy (1H MRS): 1H MRS can be obtained from brain regions prescribed on images acquired during the same imaging session. Anatomical locations are found on images to prescribe the region of interest for acquiring spectra. Once the region is identified, shimming is performed on a region matching the volume of acquisition, checked using a localized water spectrum. Then, the power of the water suppression pulses are optimized, water frequency is measured to ensure on-resonance water signal, and a short test spectrum is acquired to provide quality control. If spectra are of insufficient quality, system settings, including radiofrequency (RF) power and shim settings, are checked. Finally, if quality is still insufficient, a second 3-plane localizer is run to ensure that the animal has not moved from the initial scans. In our experience, this provides a very high degree of reproducibility and accuracy for spectroscopic acquisitions. Finally, the spectra are acquired in short blocks with resetting of the system frequency between acquisitions to eliminate effects of magnetic field drift and to ensure reproducibility and quality of the final scans. At the end of the acquisition, a single pulse water spectrum at a predefined preamplifier gain is used as a quantitative signal amplitude reference.</li>
<li>Histology and Blockface Imaging: After the final MRI scanning session in the time series of experiments, the mouse is perfused, the brain is removed and embedded into a block of OCT, compound which has been darkened using a drop of India ink. The block is placed into a cryostat for slicing and histological analysis. Blockface images are acquired using a digital camera (Canon EOS Digital Rebel 300D with a Canon Ultrasonic EFS 60mm f/2.8 Macro USM lens) mounted to the front of the cryostat with a custom mount and triggered by a remote switch. Digital images are acquired every 50 micrometers through the entire brain volume. Slices are numbered to allow registration within the volume after histological processing and staining. Individual blockface slices were aligned to reconstruct the 3D volume using the block outlines to account for jitter in the position of the cryostat head. The brain volume is then automatically segmented using seed based region growing algorithm in the Analyze software package (AnalyzeDirect, Lexena, KS).</li>
</ol>
5. Data Analyses
<ol>
<li>SPIO detection using MRI: SPIO causes signal loss in T2* weighted MRI; and, as such, the MRI signal void is a sensitive, but not specific marker for SPIO presence in tissue. The sensitivity is dependent on the spatial resolution of the MRI scan and the size of the SPIO particle, with a single micron sized particle detected with 100 micron isotropic resolution. In these works, 150 micron isotropic resolution with 200 nanometer sized SPIO particles are used. To provide both sensitivity and specificity for the presence of SPIO in brain, mice were scanned prior to injection of the SPIO labeled cells to allow subtraction images to be used for positive identification of the cells in the brain at later time points. 3D MRI scans were subimaged using the constrained level set method developed in our laboratory as previously described17. Subimaged brain volumes were then coregistered, signal intensity normalized, and the volumes subtracted to detect regions within the brain volume with signal loss (presence of SPIO), which was not along the edges to eliminate positive false signal from any subpixel registration errors.</li>
<li>Coregistration of Histology and MRI: Coregistration between histology and MRI was accomplished by using the blockface image as a common reference. This approach reduces the complexity of the major problem of correcting the asymmetrical shrinkage of tissue slices during preparation and staining to a two dimensional problem as described in our previous works18,19. Here, MRI and histology detection of SPIO containing macrophages in brain have shown excellent spatial correlation, with an expected overestimation of the volume by the loss in MRI signal and greater sensitivity to few cells demonstrated by histology12. Accurate co-localization of these two signals provides one measure of the accuracy of coregistration and warping of histology back to the original shapes of the slices.</li>
<li>Region of Interest (ROI) Analyses of DTI: DTI scans are typically analyzed by selection of an anatomical ROI to determine the average diffusion properties of the tissue in a particular anatomical substructure. 
Analyses of the diffusion-weighted data are performed using custom programs written in IDL (Interactive Data Language, ITT Visual Information Solutions, Boulder, CO) as previously described 15,20. Analyses produce maps of the tensor diffusivities (&lambda;1, &lambda;2, &lambda;3), average diffusivity (Dav) where: Dav = (&lambda;1+&lambda;2+&lambda;3)/3 and fractional anisotropy (FA), where: 
<img src="/files/ftp_upload/2459/2459eq1.jpg" alt="Equation 1" /> 
Transverse (&#955;&perp;=(&#955;2+&#955;3)/2) and longitudinal (&#955;ll=&#955;1) components of the diffusion tensor were obtained as described elsewhere 21. Once the maps are constructed, ROIs are drawn on T2 weighted MRI overlaid with color encoded &#955;1 directionality maps. Examples of the regions chosen for analysis in the HIV mouse model are displayed in Figure 1.</li>
<li>Spectroscopic Analysis: Quantitation of the metabolic compounds contributing to the peaks of brain 1H MRS is determined using one of a variety of curve fitting methods. A variety of curve fitting techniques have been developed. In our laboratory, we employ a time domain fitting method (QUEST)22 in the jMRUI23 signal processing package that is a linear combination of individual metabolite spectra contributing to the final spectrum. We use a basis set of 22 individual metabolites as potential contributing factors. The basis spectra are simulated and checked using spectra of solutions of individual metabolites. An example of a curve fit result from a single spectrum is shown in Figure 2.</li>
</ol>
6. Representative Results
Examples of DTI and 1HMRS are shown in figures 1 and 2. Additional examples of 1H MRS24-26 and DTI27 results can be seen in our previous publications. Examples of preinjection T2* weighted MRI with an overlay of the location of labeled cells in yellow is shown in Figure 3. The mouse had labeled monocytes derived macrophages injected into the tail vein. Five days later, T2* weighted MRI was acquired and processed as described, above. The mouse was prepared by injection of HIV infected human macrophages into the brain, which is seen as a line of detected mouse monocytes derived macrophages. Further examples of both detection of labeled cells and coregistration with histology can be seen in our previous publications10,12.
<img src="/files/ftp_upload/2459/2459fig1.jpg" alt="Figure 1" /> 
Figure 1. Depiction of regions analyzed for DTI metrics.
<img src="/files/ftp_upload/2459/2459fig2.jpg" alt="Figure 2" /> 
Figure 2. Spectroscopic fitting using QUEST in the jMRUI signal processing suite.
<img src="/files/ftp_upload/2459/2459fig3.jpg" alt="Figure 3" /> 
Figure 3. Detection of SPIO labeled cells migrating from the peripheral blood into a brain region with focal encephalitis. Cell positions (yellow) overlay representative slices from a T2* weighted MRI acquisition as detailed in the text.

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D., Schar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+contrast+visualization+of+iron+oxide-labeled+stem+cells+using+inversion-recovery+with+ON-resonant+water+suppression+(IRON).">Positive contrast visualization of iron oxide-labeled stem cells using inversion-recovery with ON-resonant water suppression (IRON).</a> Magn Reson Med. 58, 1072-1077 (2007).</li><li>Suzuki, Y., Cunningham, C. H., Noguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+serial+evaluation+of+superparamagnetic+iron-oxide+labeled+stem+cells+by+off-resonance+positive+contrast.">In vivo serial evaluation of superparamagnetic iron-oxide labeled stem cells by off-resonance positive contrast.</a> Magn Reson Med. 60, 1269-1275 (2008).</li><li>Kim, Y. B., Bae, K. H., Yoo, S. S., Park, T. G., H, P. a. r. k. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+contrast+visualization+for+cellular+magnetic+resonance+imaging+using+susceptibility-weighted+echo-time+encoding.">Positive contrast visualization for cellular magnetic resonance imaging using susceptibility-weighted echo-time encoding.</a> Magn Reson Imaging. 27, 601-610 (2009).</li><li>Zhou, R., Idiyatullin, D., Moeller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWIFT+detection+of+SPIO-labeled+stem+cells+grafted+in+the+myocardium.">SWIFT detection of SPIO-labeled stem cells grafted in the myocardium.</a> Magn Reson Med. 63, 1154-1161 .</li></ol>

No conflicts of interest declared.

The accurate registration of histology with in-vivo imaging results is a critical step in the development of imaging biomarkers for detection and staging of neuronal disease. Some imaging metrics are likely to be correlated with gross morphological changes including changes in magnetic relaxation properties of tissue used for detecting the presence of white matter disease and cancers. Other more subtle methods, such as DTI, are likely to detect early cellular changes that may not be detectable as histological changes caused by the disease do not appear until later the stages of disease. Still other markers, such as spectroscopic markers, may be indicators of early and reversible changes, which precede even the subtlest cellular alterations.
Biodistribution can be determined non-invasively using a variety of methods. The primary non-invasive methods are positron emission tomography (PET), single photon emission computed tomography (SPECT), optical imaging, and MRI. Nuclear medicine based imaging (PET and SPECT) have been used over the years for many biodistribution, but these methods are limited by the half life of the radiotracers used for labeling the compounds or nanomaterials, especially for PET tracers. Optical imaging can be used for small rodents but cannot be translated to human use except for regions easily accessible such as surface tumors due to light absorption and light scattering. In addition, it is difficult to quantify the optical signals for these same reasons. MRI uses persistent tags such as SPIO that can be tracked in the body over a period of weeks. This, too, must be used with caution, as the label can be transferred to different cells or be reabsorbed by the body.
Detection specificity for SPIO in MRI can be provided by a variety of methods. Detection methods, which provide positive as well as negative signals, are used for improving specificity of the MRI for detecting the presence of SPIO in tissue. The subtraction method used in this work has been used by others, as well28. Other approaches include off resonance detection29-31, phase sensitive imaging that produces a particular pattern near SPIO voids32, and zero echo time image that uses T1 weighting to produce a positive signal intensity in the region of SPIO33. The advancement of these methods for improving quantitation of label, sensitivity and specificity is an area of active research today.

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registered bioimaging of nanomaterials for diagnostic and therapeutic monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Watch this Scientific Journal Video about Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring at JoVE.com

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

registered-bioimaging-nanomaterials-for-diagnostic-therapeutic

Research

JoVE Journal

Bioengineering

10.3K Views.  University of Nebraska Medical Center. Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Video: Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...