Name: Harmonic Nanoparticles for Regenerative Research (Video) | JoVE Journal
Uploaded: 2014-05-01T13:45:00.000+00:00
Duration: 9 min 23 s
Description: Learn in vitro labeling of human embryonic stem cells using second harmonic generating nanoparticles for real-time multi-photon microscopy. This method enables monitoring of cardiac cluster contractions and advances cardiac development research.

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5 minutes

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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</ol>

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><tbody><tr><td>Microscope Incubator</td><td>OKO LAB</td><td>UNO package (top stage)</td><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;, moisturized</td></tr><tr><td>Multiphoton microscope</td><td>Nikon</td><td>AR1-MP</td><td></td></tr><tr><td>Fast scanning, four nonphotomultiplier descanned detectors</td><td></td><td></td><td></td></tr><tr><td>Filters SHG and autofluorescence</td><td>Semrock</td><td>FF01-360/12-25</td><td></td></tr><tr><td>FF01-395/11-25&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>FF02-485/20-25</td><td></td><td></td><td></td></tr><tr><td>Microscope objectives</td><td>Nikon</td><td></td><td></td></tr><tr><td>CFI Plan Fluor 10X</td><td></td><td></td><td>NA 0.30, WD 16 mm</td></tr><tr><td>CFI Plan Apo 20X</td><td></td><td></td><td>NA 0.75, WD 1.0 mm, VC</td></tr><tr><td>CFI Apo 40X</td><td></td><td></td><td>NA 1.25, WD 0.18 mm &amp;lambda;S, Nano-Crystal Coat</td></tr><tr><td>Rhock inhibitor&amp;nbsp;</td><td>Sigma</td><td>Y-27632</td><td></td></tr><tr><td>Knockout DMEM</td><td>Invitrogen</td><td>10829</td><td></td></tr><tr><td>Knockout Serum&amp;nbsp;</td><td>Invitrogen</td><td>10828</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids&amp;nbsp;</td><td>Invitrogen</td><td>1140</td><td></td></tr><tr><td>L-glutamine 200 mM&amp;nbsp;</td><td>Invitrogen</td><td>25030</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Invitrogen</td><td>15140</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma</td><td>M7522</td><td></td></tr><tr><td>Collagenase IV&amp;nbsp;</td><td>Gibco</td><td>17104-019</td><td></td></tr><tr><td>Roller scraper tool&amp;nbsp;</td><td>StemPro EZPassage, Invitrogen</td><td>23181-010</td><td></td></tr><tr><td>StemPro Accutase&amp;nbsp;</td><td>Gibco</td><td>S11105-01</td><td></td></tr><tr><td>Aggrewell system&amp;nbsp;</td><td>StemCell Technologies</td><td>27845</td><td></td></tr><tr><td>Hyclone serum&amp;nbsp;</td><td>Thermo Scientific</td><td>SH30070.03</td><td></td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G9391</td><td></td></tr><tr><td>6-well plates&amp;nbsp;</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>24-well plates&amp;nbsp;</td><td>Nunclon</td><td>142475</td><td></td></tr><tr><td>Polytetrafluoroethylene (PTFE) filters</td><td>Millipore</td><td>NA76/25</td><td></td></tr><tr><td>Inserts</td><td>Millipore</td><td>PICM03050</td><td></td></tr></tbody></table>

harmonic nanoparticles for regenerative research

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...

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 ...

The overall goal of the following experiment is in vitro monitoring of cardiac beading clusters derived from human embryonic stem cells in 3D real time and high spatial resolution. This is achieved by first culturing and expanding embryonic stem cells until they form Embry bodies. Next, the beating clusters are dissected and plated on POLYTETRAFLUOROETHYLENE or PTFE porous filters, which allows them to be maintained as long-term air.Liquid cultures then beating clusters are labeled with harmonic nanoparticles in order to perform realtime multi photon microscopy. Results obtained allow the analysis of spatial elongation and frequency of cardiac contractions at the microscopic scale. So the main advantage of these nanoparticles is that you can use any wavelength, especially infrared, so you can go deeper in tissue and they are also very stable over time.So we can monitor our long processes over long timeframes. This stem cell culture method can help to answer key questions in the field of cardiomyocyte functional contraction in three dimensions. In particular, cardiac maturation and tissue engineering.Individuals new disease method should be aware that the labeling procedure with harmonic nanoparticles can only be used for multiphoton microscopy and cannot be transferred to other kind of imaging systems. We first had the idea of using harmonic nanoparticles in 2005. These nanoparticles present advantages with respect to fluorescent dyes and quantum dots.In fact, their signal doesn't fade out over time and they can be used to monitor developing samples over long time periods. Visual demonstration of the sample preparation is critical for the efficiency and the reproducibility of the different steps, in particular when performing drug testing and could also be applied to different tissues or disease. Tissue modeling.After culturing and expanding human embryonic stem cells or ESCs according to the text protocol and incubating them with warm collagenase, four for 10 minutes, collect the colony fragments in two milliliters of fresh differentiation, medium or DM and centrifuge at 115 times gravity for three minutes. Culture, the colony fragments in suspension in ultra low attachments. Six well plates for four days to allow them to form embryo bodies or ebs.Then plate the newly formed EBS on 0.1%Gelatin coated 24 well plates for 15 to 30 days until the appearance of beating clusters. Remove the PBS and using acutus, dissociate the colonies into single cells after centrifuging and resus, suspending the cells in DM medium plate two milliliters per agro. Well chamber and incubate at 37 degrees Celsius for one day.Collect the newly formed EBS and plate three per well on 0.1%gelatin coated 24 well plates after two to four weeks in culture. Identify clusters of beating cardiomyocytes and using an elongated sharply cut paste pipette, manually dissect them. Deposit four PTFE filters per insert and place each in a well of a six well plate.After adding one milliliter of DM into each, well add one dissected beating cluster onto each PTFE filter. To label the clusters, transfer the PTFE filters with the beating clusters into a 3.5 centimeter glass bottom dish. Add one milliliter of second harmonic generation nanoparticles at 50 micrograms per milliliter and incubate for 30 minutes at 37 degrees Celsius to carry out non-linear optical imaging.After transferring labeled HEBs or cardiac beating clusters to an imaging chamber, use wide field imaging under white light illumination to identify structures of interest within differentiated EBS or active beating clusters. 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 second harmonic generation nanoparticles to monitor in real time. The cardiac cluster contractions set the microscope optical scanner in the fastest mode, allowing multi hertz acquisition of two dimensional images.Analyze the images using the MATLAB software according to the text protocol as demonstrated here prior to assessing the beating activity. Characterization of the nonlinear optical response of the PTFE filters is performed to ensure that the bare substrate to photon fluorescence is very weak and cannot prevent measuring the relevant biological samples and the emission from isolated. Second harmonic generation nanoparticles can be easily acquired by imaging through the substrate in epi detection mode.In this figure, second harmonic generation nanoparticles labeled cardiac structures and EBS are displayed blade. The red, yellow and green colors correspond to NADH autofluorescence while the intense blue second harmonic spots stem from isolated second harmonic generation nanoparticles. Note the good optical contrast and the relatively sparse labeling as compared to quantum dots or up conversion nanoparticles.This figure illustrates a slice view of a second harmonic generation nanoparticles labeled cardiac beating cluster. As shown in this movie, the 3D structures are recorded at high speed to monitor the contraction pattern In the second cell. Harmonic generation nanoparticles aggregate to maintain a high acquisition speed rate for resolving the NP motion.The overall acquisition sensitivity was not sufficient to record cell autofluorescence with second harmonic emission from second harmonic generation. Nanoparticles image analysis performed on movie frames indicates that within the same cardiac cluster displacement are on the order of a few micrometers, and the frequency is constant for in and out of plain second harmonic generation nanoparticles motion. When master the cell preparation technique, the betting cluster dissection the air liquid culture and the lambing can be done in 24 to 36 hours if performed properly.Especially with this technique, open air contacts have to be avoided to prevent contaminations. Following this procedure, one can apply other methods like electrophysiological recordings using multiple electrode arrays in order to answer additional questions like correlating action potential profiles with beating frequency in three dimension. Our novel technique based on the detection of the nonlinear signal by harmonic nanoparticles can be further developed for in vivo Studies infect the wavelengths used for imaging allow deeper penetration in tissues.One possible development could be following the integration of stem cells derived structure for regenerative medicine applications.

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JoVE Journal

Bioengineering

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Learn in vitro labeling of human embryonic stem cells using second harmonic generating nanoparticles for real-time multi-photon microscopy. This method enables monitoring of cardiac cluster contractions and advances cardiac development research.

Harmonic Nanoparticles for Regenerative Research

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