Saremco Dental Switzerland and Olympus Microsystems France Companies are gratefully acknowledged for their financial support. Saremco Dental is also acknowledged for the generous donation of the dental composites ELS extra low shrinkage. The authors also wish to express their appreciation to Dr Simon Banks from the department of Chemistry (University College London) for his assistance with English revision.

The protocol consists of three major steps: the first step comprises the preparation of composite extract in culture media; the second concerns the Primary Human Gingival Fibroblasts (HGF) cell culture, the contact of cells with the composite extracts and cell staining; the third step consists of confocal imaging and the fluorescent signal analysis. For gingival tissues taking, the protocol was approved in compliance with French legislation, informed consent and local ethical committee.
1. Composite Extracts Preparation
Use Dulbecco&#8217;s modified Eagle medium (DMEM) as the elution medium. Ensure that the ratio between the surface area of the specimens and the volume of extraction medium is at the lowest level of the ISO 10993/12 requirements. The procedure is recently described11 and outlined briefly below:
<ol>
	<li>Form spherical samples of the uncured dental composite using a bespoke holder. The holder size is 2 mm in radius, which is in accordance with generally advised curing depth in the oral clinical treatment.</li>
	<li>Prepare plate wells, each containing 1 ml of culture medium (DMEM), with a 5% mix of penicillin/streptomycin and 1% of amphotericin but without fetal bovine serum (FBS).</li>
	<li>Cure samples within the well containing DMEM medium using a LED light lamp with a 1,070 mW cm-2 intensity (&#955; = 465-475 nm) for 40 sec. Ensure that the distance between the curing tip and the samples is in the range of 0.5 mm. Use this curing mode to simulate the temporary contact between tooth and the uncured dental composite. In clinical situation, the contact occurs when the restorative composite is placed in the oral cavity.</li>
	<li>Incubate samples (composites specimens in DMEM medium) at 37 &#176;C under humidified atmosphere of 5% CO2 in air. Incubate DMEM medium (without composite samples) for control cells in the same conditions.</li>
</ol>
2. HGF Cell Culture, Composite Extracts Testing and Cells Staining
<ol>
	<li>Growth and maintenance of cell cultures 
	Isolate human gingival fibroblasts from healthy gingival tissue biopsies of patients taken during routine orthodontic extractions.
	<ol>
		<li>Cultivate human gingival fibroblasts in DMEM in the presence of 10% FBS, 5% Penicillin/Streptomycin and 1% Amphotericin B. Maintain cells at 37 &#176;C in incubator under a humidified atmosphere of 5% CO2 in air.</li>
		<li>Changed medium every 3 days, and passage cells every 5 days. After reaching confluency, harvest cells by trypsinization.</li>
		<li>Centrifuge cells at 90 x g for 5 min and re-suspend them in the culture medium. Count cell with a scepter handheld automated cell counter.</li>
		<li>Seed cell suspension at a cell density of 2.5 x 104 cells/ml in a chamber slide system. Allow the incubation of cells overnight at 37 &#176;C in a 5% atmosphere.</li>
	</ol>
	</li>
	<li>Addition of composite extracts to the cells in culture
	<ol>
		<li>Replace the media of two wells of the chamber slide by 1 ml of the tested composite extracts (after 24 hr of incubation).</li>
		<li>Maintain the two remaining wells (control cells) in the same conditions.</li>
		<li>Incubate the chamber slide containing the four wells at 37 &#176;C in incubator, under a humidified atmosphere of 5% CO2 in air.</li>
	</ol>
	</li>
	<li>Fluorescent staining 
	Use the Live/Dead cytotoxicity stain for eukaryotic cells according to the manufacturer&#8217;s instructions. 
	NOTE: the kit provides a quick two-color fluorescent assay to determine viability of cells in a population based on plasma membrane integrity and esterase activity. The stain distinguish live from damaged cells by simultaneously labelling with green-fluorescent Calcein-AM to indicate intracellular esterase activity and red-fluorescent Ethidium homodimer-1 (EthD-1) to indicate loss of membrane integrity.
	<ol>
		<li>Prepare a working solution of the two stain reagents containing 2 &#956;M of Calcein AM and 4 &#956;M EthD-1 (final concentration reported to be suitable for fibroblast staining). Add the stain to the cultured adherent cells in the presence or absence of the selected composite extracts.</li>
		<li>Observe directly and rapidly the stained cell populations at the least 15 min after staining and through the time-lapse imaging. Do not centrifuge and do not fixe the stained cells.</li>
	</ol>
	</li>
</ol>
3. Confocal Time Lapse Imaging and Signal Analysis
The system used is equipped with a four diode laser units and a multi-stained image for up to four fluorescent dyes for each specimen. The system can perform a mapping image (10X magnification) that gives an overview of the test sample on which one can move to observe a region of interest (600X magnification).
<ol>
	<li>Time lapse imaging
	<ol>
		<li>Place the specimens (the chamber slide containing the stained samples) on the dark room of the confocal integrated incubator (37 &#176;C, 5% of CO2 and humid atmosphere).</li>
		<li>Select the corresponding lasers. Use the two laser sources 473 nm (15 mW) and 559 nm (18 mW) to excite Calcein and EthD-1.</li>
		<li>Use a two sequential mode to acquire images through line sequences without crosstalk in imaging with the two used fluorescence dyes.</li>
		<li>Use a detector with a newly developed spectrum to automatically set conditions in accordance with the fluorescence dye in scanning unit.</li>
		<li>Optimize the bandwidths of the detected fluorescence for each channel to the maximum. Record all acquisitions sequentially to avoid potential cross-talking.</li>
		<li>Select the most suitable imaging conditions based on the fluorescent dye selection using the acquisition software.</li>
		<li>Acquire a Map image that is automatically created from the center in a spiral pattern, with the area of interest being easily selected for closer examination.</li>
		<li>Select observation mode. Select up to five types of observation modes including time-lapse, Z-stack, and multi-area as needed. Use a combination of Z-stack and time-lapse modes in the present study and complete swiftly the image capturing,</li>
		<li>Change and switch the fluorescent dye. Alternatively, overlay the fluorescent and the light contrast images with each other. Observe live image using the selected point on the left lower map screen.</li>
		<li>Determine the imaging area by using the framing and zooming functions. Optionally, switch between the displays for each type of fluorescence dye. 
		NOTE: The microscope used in this protocol has the same functionality as a high conventional confocal in a compact design. The microscope is equipped with inverted water-immersion objectives which are optimally suited for stable time-lapse imaging of live cells with a simplified built-in incubator.</li>
	</ol>
	</li>
	<li>Signal analysis and quantification
	<ol>
		<li>Use the imaging software to quantify the fluorescence signal. Obtain five map images with the 10X objective for both tested sample and cell control.</li>
		<li>Obtained 1,024 &#215; 1,024 pixel regular images, with a 0.231 &#181;m &#215; 0.231 &#181;m pixel size. Store images as Olympus Image Format (OIF) for signal analysis. This format enables storing various parameter settings and images together. Use different analysis tools (measurement of intensity profile, intensity ratio between the two used channels).</li>
		<li>Store the maximal projection images as 12 bits/pixel TIFF files.</li>
		<li>Preform statistical analyses using the R commander software. Analyze differences using one-way analysis of variance (ANOVA) with a repetition test. Report results as mean standard deviation (&#177;SD) and statistical significance at p&#60;0.05.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests, they have nothing to disclose.

Biocompatibility material behavior is the most prerequisite parameter to be evaluated before medical use. International standards cover medical devices ISO 1099333 and specifically dental materials ISO 740534. The absence of the toxic effects to the surrounding cells is the key norm in the biocompatibility evaluation of such materials. That is why cytotoxicity testing has such importance in the assessment of dental material biocompatibility35-37. The ISO suggested testing methods could be based on metabolic activity or membrane integrity. Selected endpoints should follow specific conditions for ranking adverse effects induced by a test material in order to minimize the time and the cost of the assessment assays. The current method of investigation (3D confocal time lapse imaging) is membrane integrity based. The cytotoxicity evaluation via this endpoint seems to be a valuable marker of cell biocompatibility with tested materials and it is also ISO approved endpoint. In vitro tests are designed to determine how a material sample affects a particular cell type. The colorimetric MTT assay was originally described by Mossman (1983) as a useful method for the measurement of in vitro cytotoxicity and cell proliferation. The MTT test is based on the conversion of the tetrazolium salt into formazan crystals by active cells, which determines mitochondrial activity. This can be translated by viability rate38. Gupta and collaborators reported that the MTT test is the most widely used method for measuring cytotoxicity of composite resins. Succinic dehydrogenase and alkaline phosphatase responses have also been used for the same purpose39. However, the fate cells cannot be followed up, since the MTT assay requires killing the cells at each measurement time point. In order to monitor more closely the behavior of the stained cells in the current study, direct observation of live cells was performed, thanks to time lapse CLSM imaging combined with a short protocol staining, automated image analysis and fluorescent signal quantification.
Dental composites come in close contact with oral tissues, especially gingival and pulpal cells. Fibroblasts would be the target of components released from these restorative composites40- 41. Furthermore, several studies have reported that immortalized cells and primary cells may have different cellular responses in contact with biomaterials. Primary cells were found to be more appropriate to assess biomaterials effects42. This explains the use of primary human gingival fibroblast cells as cell model for cytotoxicity assessment of the tested dental composite in the current study. The toxicity potential of the released substances from dental composite has been widely studied43-44. In order to assess the cytotoxic potential of materials, two contact model cells could be performed. In the direct contact model system the material is directly placed with target cells while in the indirect contact system, target cells are in contact with elutes from biological media. In clinical applications and for restorative procedures, dental composite are placed in indirect contact with gingival tissues. For this reason, the current experimental protocol was focused on the indirect contact system with human gingival fibroblasts (cells in presence of the tested composite extracts). As previously reported by Dahl and collaborators, cytotoxicity responses were ranked as severe (&#60;30%), moderate (30&#8211;60%), slight (60&#8211;90%) or non-cytotoxic (&#62;90%) based on the activity relative to values obtained for the controls45. Therefore, according to this ranking and in the light of the obtained results (Table 1), composite ELS extra low shrinkage could be ranked as very slightly cytotoxic and showed comparable behavior to control cells during the entire time lapse period. Using the precisely same investigation method, the results of this study could be compared to those of a recently published study. The ELS extra low shrinkage composite demonstrated superior biocompatibility compared to the two previously tested composites used for the same clinical application that is the direct restoration11. Further studies are still necessary to establish more complete comparison.
When deciding which cytotoxicity assay to approve for a specific endpoint assessment, it is important to understand the advantages and the limitations of each assay. The main purpose of confocal imaging is to achieve a 3-D architecture of cells and organs. The technique is able to reveal not only a specimen&#8217;s surface, but also its subsurface. The protocol described herein underlines the use of 3D CLSM time lapse confocal imaging, as a sensitive method to assess the in vitro biocompatibility behavior of a dental composite. However, in order to avoid the limitations encountered in this work, Anti-vibration workstations could be used in order to allow more stable and longer time lapse imaging; the tabletop could isolate the used confocal system from the detrimental effects that vibrations in the lab may cause. Furthermore, materials to be used in the oral cavity should ideally be harmless to the gingival tissues and should contain no substances that could cause systemic toxicity. In accordance with the methodology used and the results obtained from the current study it can be concluded that the tested composite (ELS extra low shrinkage) is not cytotoxic in the present experimental conditions. As the CLSM method can sensitively give the initial rate of viable cells, other possibilities could be foreseen to assess more endpoints. In particular, as it has been recently reported that composite monomers affect cells through reactive oxygen species (ROS)46-48, further investigation is needed to assess correlations if any between cytotoxicity signaling networks and ROS production. The future protocol could be designed to simultaneously evaluate ROS production (H2O2 and/or O2- staining) and cytotoxicity ratio (Live/dead staining) using time lapse CLSM imaging.

One of the main standards quoted in the ISO recommendations to define biocompatibility is the absence of material toxicity to cells. Resin-based dental composites may release components from the resinous matrix, which could be initially due to partial polymerization, and/or later due to degradation processes over time1-4. These released components come into contact with oral tissues and may have various drawbacks like urticarial and mucosal reactions5, development of allergy and hypersensitivity reactions in patients6, modifications of gingival fibroblasts morphology and reduction on type I collagen protein7, immunosuppression or immunostimulation on mitogen-driven proliferation of purified T-lymphocytes and spleen cells8 and DNA damage in primary human gingival fibroblasts, which underlines their genotoxic potential9. Many parameters can affect dental composite toxicity such as the shade of the composite, the light curing, the chemical composition of the resin monomer, the filler and the degree of conversion10-13. Since in vitro toxic potential of materials may predict in vivo situations and could be compared to clinical situations by the study of some relevant endpoints. The cytotoxicity of dental composites and their components have been widely evaluated using cell culture systems14-16.
These in vitro systems have been developed to assess dental composites biocompatibility in term of: cell growth, and cell apoptosis using THP-1monocytes like cells17, cytotoxicity in human gingival fibroblasts18, inflammatory potential in HaCaT keratinocytes like cells2, cell functionality odontoblast-like MDPC-23 cells19, reduction of total RNA levels, and significant increase in induction of apoptosis on human gingival keratinocytes20 and DNA damage on gingival and pulp fibroblast cells21.
Different methodologies are suggested to investigate the biocompatibility of dental composites. Colorimetric assays, which involve specific colorants and light adsorption measurements, remain the most commonly used, due to their relative simplicity and low cost22-26. Microscopy analysis by light contrast frequently consumes more time and incurs higher costs. However, these microscopy techniques have some important advantages. The observation of cell structure gives extended information about experienced toxic effects, thus providing more relevant and sensitive data. In particular, the introduction of confocal microscopy27 has allowed for the observation of biological structures with improving axial resolution compared to traditional wide-field imaging fluorescence microscopes. Hence, confocal imaging has become a powerful investigative tool in different fields of medical and science researches. In contrast to electronic microscopy techniques, scanning confocal microscopy offers the ability to visualize distinct components of cells by incorporation of specific fluorescent markers. Most of these specific tracers are stable in an aqueous environment, sensibly measurable, inexpensive and non-toxic28, allowing their use in clinical as well as laboratory research studies. Moreover, the specimen drying and fixation required for conventional electronic microscopy analysis is not necessary for time lapse confocal imaging, thus time-dependent acquisition is also possible on samples. Compared to the two-dimensional imaging, the confocal imaging principle enables a specimen subsurface visualization and the three-dimensional structure reconstruction from the different obtained depth images; which result in, more precise and more structural details29, 30. The present video study is an example of the use of three-dimensional Time-Lapse Confocal Laser Scanning Microscopy (3D-CLSM) combined with Live/Dead fluorescent labeling and signal quantification as an innovative method. The technique presented in this paper has the advantage of enabling sensitive evaluation and time-lapse imaging of live cells without changing the assessed cell structure. No preparation steps are required before confocal analysis. Previously, the same staining was used for assessing the viability of cultured cancellous bone cores31 but without confocal time-lapse following. Similarly the same time-lapse confocal procedure was used to assess erythromycin time-kill activity in bacteria of activated sludge according to their Gram type32 using a specific bacteria Live/dead staining. These methods have been recently adapted and used to compare the toxicity of dental composites based on methacrylate monomers11.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Olympus Fluoview FV10i&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Confocal microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O2/Co2 Incubator</td>
			<td style="width:71px;">Pnasonic</td>
			<td style="width:119px;">MCO 5M</td>
			<td>Cell incubation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Live/Dead&#174; Kit</td>
			<td>Invitrogen&#160;</td>
			<td style="width:119px;">MP 03224</td>
			<td>Stain&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ELS extra low shrinkage&#174;&#160;</td>
			<td style="width:71px;">Saremco&#160;</td>
			<td style="width:119px;"></td>
			<td>Dental composite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM medium</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D 6046</td>
			<td>Culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T1426</td>
			<td>harvesting cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chamber slide system&#160;&#160;</td>
			<td style="width:71px;">PAA</td>
			<td style="width:119px;">14220040X</td>
			<td>Culture chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin&#160;</td>
			<td style="width:71px;">PAA</td>
			<td style="width:119px;">P11-010</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amphotericin B</td>
			<td style="width:71px;">PAA</td>
			<td style="width:119px;">P11-001</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foetal bovine serum&#160;</td>
			<td style="width:71px;">PAA</td>
			<td style="width:119px;">A11-101</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Counter</td>
			<td>Merck Millipore</td>
			<td style="width:119px;">00110230TP1</td>
			<td>Cell counting&#160;</td>
		</tr>
	</tbody>
</table>

confocal time lapse imaging as an efficient method for the cytocompatibility evaluation of dental composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

Figure 1 showed an overview of the Live/Dead stained cells in obtained mapping images (10X objective) for both control and cells exposed to the tested composite extracts (ELS extra low shrinkage). The three dimensional structure of the cells could be visualized at the magnification 600X. Visualization of cells collected in the control chamber and of cells collected in the chamber in the presence of the tested composite extracts is presented in Figure 2. The maximal projection (objective 60X) shows the fate of selected cell populations at the start of the time lapse (at 15 min) and at the end of the time lapse period (at 5 hr). Fifty seven stacks of images (xyzt mod scan, 39 images per stacks, voxel-depth 1.00 &#956;m) were obtained during this period. A slight decrease in the green fluorescent signal and very slight variation in the red fluorescent were obtained in exposed cells. No significant variation was observed for control cells. Composite extracts exposed cells assumed similar spindle morphology to control cells; morphology was not altered in these experimental conditions (despite the green signal decrease and the red signal increase in the presence of the tested composite). Image analysis was performed on five selected different image stacks corresponding to different cell populations from the obtained images for the tested composite as compared to negative control cells for both green and red signal. The intensity profile of stained cells is illustrated in Figure 3; the green and red signal evolution of the cells in contact with the tested composite was comparable to those of control cells. Calcein and EthD-1 fluorescence emission intensities were measured on control cells and on composite exposed cells. The measurement was done at one hour intervals for up to five hr. The green/red fluorescence ratio (based on integrated intensities of the green 495-515 nm and red 620-650 nm signals) was calculated during the time-lapse period. The viability ratios for control and exposed cells to ELS extra low shrinkage extracts are shown in the Table 1. In the presence of the tested composite, the percentage of live cells was found to decrease slightly from 100% to 93.9% after 1 hr of contact. Significant difference was observed between the tested composite and the negative control (cells without any composite extracts) after 5 hr of the indicated time-lapse period.
<img alt="Figure 1" src="/files/ftp_upload/51949/51949fig1highres.jpg" /> 
Figure 1: Overview of mapping image showed stained cells at objective 10X, (A) control cells and (B) cells in presence of composite extracts (ELS extra low shrinkage) in the beginning of the time lapse period (t = 15 min). Green areas are live cells and red areas are damaged cells. In this experiment conditions, no variation (for both green and red fluorescence) was observed for composites exposed cells compared to negative control cells.
<img alt="Figure 2" src="/files/ftp_upload/51949/51949fig2highres.jpg" /> 
Figure 2: CLSM images at objective 60X of a cell population from (A) control chamber and (B) composite extracts chamber in the beginning and the end of the exposure time-lapse period (I: 15 min and II: 5 hr, respectively). Green areas are live cells and red areas are damaged cells. The panel of the images demonstrated slight change in the intensity signal (slight decrease in the green signal and very slight increase in the red one). No cell morphology alterations were observed in the presence of composite extracts; expose cells maintained their spindle-shaped and fibroblastic morphology as control cells.
<img alt="Figure 3" src="/files/ftp_upload/51949/51949fig3highres.jpg" /> 
Figure 3: Line profile of cells observed in time-lapse microscopy. (A) Control cells (B) Composite extracts exposed cells, (I) at 15 min and (II) at 5 hr. No visible variation in the green signal evolution in the presence of tested composite regardless the time contact. However, slight increase of the red signal was observed in the presence of composite after 5 hr of contact.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td rowspan="2">Contact time (hour)</td>
			<td colspan="5">Cell Viability (%)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Control cells</td>
			<td>100</td>
			<td>100</td>
			<td>100</td>
			<td>100</td>
			<td>100</td>
		</tr>
		<tr>
			<td>ELS extra low shrinkage&#174;</td>
			<td>93.9 &#177;7</td>
			<td>91.3&#177;5*</td>
			<td>89.5&#177;3*</td>
			<td>87.6&#177;2*</td>
			<td>87.7&#177;3*</td>
		</tr>
	</tbody>
</table>
Table 1: Rate of live HGF cells evolution after 1, 2, 3, 4 and 5 hr of contact with composite extracts. Cell viability was assayed by staining using the Live/Dead cytotoxicity kit. Data show mean values &#177; SD of five images stacks analysis. Values are significantly different from control cells at p &#60;0.05.

Watch this Scientific Journal Video about Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites at JoVE.com

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

confocal-time-lapse-imaging-as-an-efficient-method-for

Research

JoVE Journal

Medicine

13.5K Views.  UMR CNRS 5615, Université Lyon1. The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

Video: Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...