The authors would like to acknowledge the partial funding from the European FP7 Research Project NAMDIATREAM (NMP4-LA-2010-246479, http://www.namdiatream.eu) and the INTERREG IV France-Switzerland NAOMI project.

1. Culture and Expansion of Human ESC
<ol>
	<li>Prepare the cell propagation medium (called PM) containing Knockout DMEM supplemented with 20% Knockout Serum, 1% MEM Non-Essential Amino Acids, 1% L-glutamine 200 mM, 1% penicillin-streptomycin, and 3.5 &#181;l &#946;-mercaptoethanol.</li>
	<li>Thaw human ESC (hESC) in 8 ml PM medium and centrifuge them 5 min at 115 x g to discard DMSO-supplemented freezing medium.</li>
	<li>Add 1 ml of PM medium containing 10 mM Rock inhibitor to prevent apoptosis and improve cell survival upon thawing (24 hr incubation only).</li>
	<li>Add basic fibroblast growth factor (bFGF) to the PM&#160;at the desired concentration (4-10 ng/ml) for maintenance of pluripotency.</li>
	<li>Plate the hESC on irradiated human foreskin feeder cells (&#947;HFF) (Figure&#160;1A), previously plated at a concentration of 2 x 104 cells/cm2.</li>
	<li>Change medium daily and, when necessary, remove portions of colonies starting to differentiate by aspiration using an elongated sharply&#160;cut Pasteur pipette.</li>
	<li>Once a week, passage colonies enzymatically:
	<ol>
		<li>Remove medium and wash colonies with 2 ml PBS.</li>
		<li>Remove PBS and incubate them with 0.5 ml per well of warm collagenase IV at 1 mg/ml for 10 min at 37 &#176;C, 5% CO2.</li>
		<li>Collect colony fragments in 2 ml new PM&#160;and centrifuge at 115&#160;x g for 3 min.</li>
	</ol>
	</li>
	<li>Alternatively, colonies can be passaged mechanically by scraping:
	<ol>
		<li>Remove medium and wash colonies with 2 ml PBS.</li>
		<li>Manually scrape colonies using the StemPro EZPassage roller scraper tool (Figure 1B).</li>
		<li>Collect colony fragments and centrifuge then for 3 min at 115 x g to remove the supernatant.</li>
		<li>Plate fragments on &#947;HFF or process them for differentiation into embryoid bodies (EBs).</li>
	</ol>
	</li>
</ol>
2. Human ESC Differentiation Protocol
<ol>
	<li>Prepare the differentiation medium (DM) using Knockout DMEM supplemented with 2% Hyclone serum, 1.0% MEM Non-Essential Amino Acids, 1.0% L-glutamine 200 mM, 1% penicillin-streptomycin, 3.5 &#181;l &#946;-mercaptoethanol.</li>
	<li>Remove medium and wash colonies with 2 ml PBS.</li>
	<li>EB formation of colony fragments in suspension:
	<ol>
		<li>Remove PBS and incubate them with 0.5 ml per well of warm collagenase IV at 1 mg/ml for 10 min at 37 &#176;C, 5% CO2.</li>
		<li>Collect colony fragments in 2 ml new DM and centrifuge at 115 x g for 3 min.</li>
		<li>Culture them in suspension in ultra-low attachment 6-well&#160;plates (2 ml per well) for 4 days (Figure 1C).</li>
		<li>Collect and plate the newly formed EBs on 0.1% gelatin-coated 24-well plates (about 3 EBs/well) (Figure 1D).</li>
	</ol>
	</li>
	<li>Alternatively, forced aggregation of single cells into EBs using the Aggrewell plate:
	<ol>
		<li>Remove PBS and dissociate colonies into single cells using accutase (0.5 ml/well).</li>
		<li>Centrifuge and resuspend cells in DM&#160;at 1.2 x&#160;106/ml.</li>
		<li>Add 2 ml per Aggrewell chamber for 1 day (Figure 1B&#39;).</li>
		<li>Collect newly formed EBs (Figure 1C&#39;)&#160;and plate them on 0.1% gelatin-coated 24-well plates (about 3 EBs/well).</li>
		<li>Change DM&#160;every 2-3 days until the appearance of beating clusters of cardiomyocytes (15-30 days).</li>
	</ol>
	</li>
</ol>
3. Air-liquid 3D Cultures of Beating Clusters and Labeling with HNPs
<ol>
	<li>Identify and manually dissect clusters of beating cardiomyocytes (Figure&#160;1D), normally appearing after 2-4 weeks of culture, using an elongated sharply&#160;cut Pasteur pipette.</li>
	<li>Deposit 4 PTFE filters per insert that will be placed on a 6-well&#160;plate (Figure&#160;1F).</li>
	<li>Add 1 ml of DM medium on top of each well.</li>
	<li>Add one dissected beating cluster on each PTFE filter (Figure 1E), maximum 4/well, i.e. 24 aggregates per plate&#160;(Figure 1F&#39;).</li>
	<li>Change DM medium every 2-3 days.</li>
	<li>To label the clusters, remove the PTFE filter containing the beating cluster and put it on 3.5 cm glass bottom dish (170 &#181;m thick).</li>
	<li>Add 1 ml of HNP at a concentration of 50 &#181;g/ml for 30 min.</li>
</ol>
4. Non-linear Optical Imaging
<ol>
	<li>Sample preparation.&#160;After HNP labeling (see Step&#160;3.6), transfer either whole differentiating early hEBs or cardiac beating clusters (dissected upon contraction appearance) on PTFE filters over a 170 &#181;m thick glass-bottom dish compatible with the working distance of high numerical aperture objectives. Alternatively, apply a direct coating of beating clusters to the glass-bottom dish pre-coated with 0.1% gelatin.</li>
	<li>Transfer samples to an all-in-one microscope equipped with incubating chamber ensuring stable temperature, humidity and CO2 control over extended periods of time.</li>
	<li>After initial inspection, image the sample via wide-field imaging under white light illumination to identify structures of interest within differentiated EBs or active beating clusters that will be selected for further investigations.</li>
	<li>If cell autofluorescence from NADH and SH from HNPs have to be simultaneously acquired, set a shorter laser excitation wavelength (i.e. 720 nm) to match the molecular absorption. Use one narrow band-pass spectral filter centered at half the excitation wavelength (i.e.,&#160;&#955;&#160;= 360 &#177;&#160;10 nm) to detect SH, and another one to detect autofluorescence.</li>
	<li>Firstly perform a fast, low resolution, three-dimensional scan to rebuild the overall morphology of the cardiac cluster and select an image plane within the cluster volume with several visible HNPs.</li>
	<li>If necessary, change the excitation wavelength freely to best match the sample&#39;s optical properties or to avoid photobleaching, photo damage and to image deeper into tissues with an IR wavelength (i.e., 790 nm) and replace the second harmonic filter accordingly (i.e., 395 nm). Optimize the settings of the laser pulse pre-compressor at the working wavelength of choice by maximizing the second&#160;harmonic signal of HNPs deposited on the sample.</li>
	<li>To monitor in real time the cardiac cluster contractions, set the microscope optical scanner in the fastest mode such as resonant mode, allowing high speed acquisition of two-dimensional images.</li>
	<li>Choose the settings as best compromise between image contrast and acquisition speed, such as:
	<ul type="disc">
		<li>Scan averaging: 4</li>
		<li>Pixel dwell time: 0.1 &#181;sec</li>
		<li>Image rate: 3.75 frames per sec (fps)</li>
	</ul>
	</li>
	<li>Laser power is adjusted according to focal spot size and scanning speed. 50 mW (measured at the entrance of the objective) is a typical value for 0.1 &#181;sec pixel dwell time and Plan APO 20X&#160;N.A. 0.75 objective preserving&#160;cell viability.</li>
</ol>
5. Image Analysis
<ol>
	<li>Axial calibration procedure. Select a single HNP deposited on a bare substrate. Adjust the focus (i.e. the axial position of the nanoparticle) for maximizing the SH intensity. Vary in a controlled way the axial position of the microscope stage to obtain the calibration curve relating SH intensity as a function of the HNP offset from the focal plane. The output of this calibration procedure for the Plan APO 20X&#160;N.A. 0.75 objective is reported in Figure&#160;4C.</li>
	<li>Select HNPs present in all video frames and determine their periodic movements. This procedure can be easily automated, for example by a custom code seeking for signal maximum intensity spots in a defined region of interest and connecting them among different frames (an example code written in Matlab R2009 b using the function &#39;region props&#39;&#160;of the Image Processing Toolbox is presented).</li>
	<li>Assess the quantification of the out-of-plane displacements, associated with non-continuously traceable HNPs moving along the optical axis, by converting their intensity modulations throughout different frames into axial displacement using the calibration curve established at Step&#160;5.1. Note that this procedure gives access to axial movements but cannot be used to determine the absolute direction (upwards or downwards).</li>
</ol>

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The authors have nothing to disclose.

The application of nanotechnology to stem cell research is a relatively new but rapidly expanding field. As pointed out by various review articles on the subject, the use of nanoparticles can be applied to accomplish different research tasks, ranging from cell tracking (both in vitro and&#160;in vivo), to intracellular delivery of proteins and genes, not least the creation of biomimetic cellular environments for preferential stimulation/inhibition of specific differentiation pathways19,20. Th...

Nonlinear microscopy systems, thanks to their inherent three dimensional sectioning capabilities, have increasingly triggered the demand for photo-stable fluorophores with two-photon absorption bands in the near-infrared. Only in the last couple of years, to complement the development of fluorescence-based labels (dyes, quantum dots, up-converting nanoparticles), a different imaging methodology has been exploiting the use of a novel family of inherently nonlinear nanoparticles as labels, i.e. harmonic nanoparticles (HNPs) which have been specifically developed for multi-photon microscopy. These labels, based on inorganic noncentrosymmetric crystals, exert optical contrast generating the SH of the excitation frequency: for example by converting a fraction of near infrared pulsed excitation light (&#955; = 800 nm) into visible blue light (&#955;/2 = 400 nm). Several authors in the recent past have tested different materials, including iron iodate Fe(IO3)3&#160;1, potassium niobate (KNbO3)2, lithium niobate (LiNbO3)3, barium titanate (BaTiO3)4,5, potassium titanyl phosphate (KTiOPO4, KTP)6-8, and zinc oxide (ZnO)5,9,10. Compared to fluorescent probes, HNPs possess a series of attractive properties, such as complete absence of bleaching and blinking, narrow emission bands, excitation-wavelength tunability (from ultraviolet to infrared), orientation retrieval capability, and coherent optical response. These unique properties have been recently explained in two comprehensive review papers11,12. The possibility of working in the infrared spectral region, which increases imaging depth by minimizing scattering and absorption, also drastically limits sample photo degradation13,14. Moreover, the infinitely photo-stable signal guaranteed by HNPs makes them ideal probes for long-term cell tracking, which is particularly appealing for regenerative medicine applications15.
In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with unfunctionalized HNPs. The synthesis and preparation of colloidal suspensions is detailed in a previous publication and in references therein16 and is beyond the scope of this work. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented. Human ESC can be let to differentiate within so called embryoid bodies (EBs) in two different ways,&#160;either by EB formation of colony fragments in suspension or, alternatively, forced aggregation of single cells into EBs using the Aggrewell plate, as illustrated in Figure 1A. Culturing beating clusters of cardiac cells on polytetrafluoroethylene (PTFE) porous filters facilitates their long-term maintenance for further studies (for example electrophysiological measurements of action potentials).
The excitation source of the scanning microscope should be able to deliver ultrashort pulses (with a pulse duration smaller than 300 fsec at the sample) in order to reach the peak power needed to perform second harmonic imaging of HNPs. For instance, the most common fsec-source used for imaging are tunable Ti:Sapphire lasers. Alternatively, other ultrafast lasers can be employed, for instance erbium ion17, chrome forsterite18 or Ti:sapphire pumped infrared optical parametric oscillators. The microscope can be equipped with an objective with preferably a rather high numerical aperture. Very importantly, prior to measurements, and each time the objective is replaced, it is mandatory to minimize the dispersion present in the set-up (lenses) by optimizing the settings of the laser pulse pre-compressor at the working wavelength of choice. This procedure, detailed in the protocol, ensures that the laser pulse is as close as possible to the transform limited duration (i.e. shortest as possible) at the focal plane and maximizes the sample nonlinear response.
The goal of the image analysis described at the end of the protocol is to identify and track in 3D HNPs movements associated with the rhythmic contractions of beating cardiac clusters. Tracking nanoparticles in the image plane is simply realized by identifying their positions in successive movie frames. To extract information on axial movement, a prior calibration of the nonlinear intensity response as a function of axial displacement is mandatory. Note that for long-term measurements, an active interferometric control of sample axial position is required to maintain the validity of the calibration curve in the presence of thermal and/or mechanical drifts.
The HNPs used here to trace beating cells within aggregates are based on potassium niobate oxide (KNbO3), but other available nonlinear nanomaterials are reviewed in detail in the work of Staedler et al16.
The nonlinear optical efficiencies of most of the nanomaterials investigated so far are very comparable. The choice for KNbO3 was essentially motivated by the good stability of the colloidal solution and its good biocompatibility, tested on several human cell lines even at fairly high concentration and long exposition times16.
Given the novelty of the nanomaterial employed for this work, the main characteristics of HNPs as compared to fluorescent/luminescent bio-markers are shown in a short original computer video animation realized by the authors.

<table border="0" cellpadding="0" cellspacing="0" width="1008">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Microscope Incubator</td>
			<td style="width:231px;">OKO LAB</td>
			<td style="width:205px;">UNO package (top stage)</td>
			<td style="width:343px;">&#160;37&#176;C, 5% CO2, moisturized</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="2" style="height:64px;width:231px;">Multiphoton microscope</td>
			<td rowspan="2" style="width:231px;">Nikon</td>
			<td rowspan="2" style="width:205px;">AR1-MP</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:343px;">Fast scanning, four non photomultiplier descanned detectors</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="3" style="height:64px;width:231px;">Filters SHG and autofluorescence</td>
			<td rowspan="3" style="width:231px;">Semrock</td>
			<td style="width:205px;">FF01-360/12-25</td>
			<td rowspan="3" style="width:343px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">FF01-395/11-25&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:205px;">FF02-485/20-25</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="3" style="height:64px;width:231px;">Microscope objectives</td>
			<td rowspan="3" style="width:231px;">Nikon</td>
			<td style="width:205px;">CFI Plan Fluor 10x</td>
			<td style="width:343px;">NA 0.30, WD 16 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">CFI Plan Apo 20x</td>
			<td style="width:343px;">NA 0.75, WD 1.0 mm, VC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:205px;">CFI Apo 40x</td>
			<td style="width:343px;">NA 1.25, WD 0.18mm &#955;S, Nano-Crystal Coat</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Rhock inhibitor&#160;</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:205px;">Y-27632</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Knockout DMEM</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">10829</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Knockout Serum&#160;</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">10828</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">MEM Non-Essential Amino Acids&#160;</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">1140</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">L-glutamine 200mM&#160;</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">25030</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Penicillin-streptomycin</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">15140</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:205px;">M7522</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Collagenase IV&#160;</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:205px;">17104-019</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Roller scraper tool&#160;</td>
			<td style="width:231px;">StemPro EZPassage, Invitrogen</td>
			<td style="width:205px;">23181-010</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">StemPro Accutase&#160;</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:205px;">S11105-01</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Aggrewell system&#160;</td>
			<td style="width:231px;">StemCell Technologies</td>
			<td style="width:205px;">27845</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Hyclone serum&#160;</td>
			<td style="width:231px;">Thermo Scientific</td>
			<td style="width:205px;">SH30070.03</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Gelatin</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:205px;">G9391</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">6-well plates&#160;</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:205px;">353046</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">24-well plates&#160;</td>
			<td style="width:231px;">Nunclon</td>
			<td style="width:205px;">142475</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Polytetrafluoroethylene (PTFE) filters</td>
			<td style="width:231px;">Millipore&#160;</td>
			<td style="width:205px;">NA76/25</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Inserts</td>
			<td style="width:231px;">Millipore</td>
			<td style="width:205px;">PICM03050</td>
			<td style="width:343px;"></td>
		</tr>
	</tbody>
</table>

harmonic nanoparticles for regenerative research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Prior to assessing the beating activity by confocal imaging, a careful characterization of the nonlinear optical response of the PTFE filters was performed, either alone or in the presence of HNPs at high concentration (1 mg/ml). It was ensured that: i) the bare substrate two-photon excited fluorescence is very weak and cannot prevent measuring the relevant biological samples, and ii) the SH emission from isolated HNPs can be easily acquired by imaging through the substrate in epi-detection mode (Figure 2</strong...

Watch this Scientific Journal Video about Harmonic Nanoparticles for Regenerative Research at JoVE.com

Harmonic Nanoparticles for Regenerative Research

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

harmonic-nanoparticles-for-regenerative-research

Research

JoVE Journal

Bioengineering

11.2K Views.  University of Geneva. Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Video: Harmonic Nanoparticles for Regenerative Research

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with turn-key performance and microjoule-level pulse energy is used to seed the regenerative chirped-pulse amplifier. The amplifier is placed in airtight housing. It operates at room temperature and exhibits stable operation at a 5 kHz repetition rate, with a pulse-to-pulse stability less than 1%. By employing a 1.5 mm-thick beta barium borate crystal, the frequency of the laser output is doubled to 515 nm, with an average power of 70 W, which corresponds to an optical-to-optical efficiency of 70%. This superior performance makes the system an attractive pump source for optical parametric chirped-pulse amplifiers in the near-infrared and mid-infrared spectral range. Combining the turn-key performance and the superior stability of the regenerative amplifier, the system facilitates the generation of a broadband, CEP-stable seed. Providing the seed and pump of the optical parametric chirped-pulse amplification (OPCPA) from one laser source eliminates the demand of active temporal synchronization between these pulses. This work presents a detailed guide to set up and operate a Yb:YAG thin-disk regenerative amplifier, based on chirped-pulse amplification (CPA), as a pump source for an optical parametric chirped-pulse amplifier.

A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.