The authors would like to thank Dr. Peiman Hemmati (Department of Medicine, University of Wisconsin-Madison) for generously providing the H1 MSCs, and Dr. Tim Hacker, Dr. Gouqing Song and Ms. Jill Koch of the University of Wisconsin Cardiovascular Physiology Core Facility for performing mouse surgeries. This work was supported by the National Science Foundation through a Graduate Research Fellowship to Brian Freeman and NIH R21 HL089679.

1. Donor Cell Transfection
<ol>
	<li>Harvest mesenchymal stem cells (MSCs, derived from H1 embryonic stem cells kindly donated by Dr. Peiman Hematti; alternatively, any cell type of any species hypothesized to fuse in vivo could be employed) when 70 - 80% confluent with 1X trypsin (Mediatech, Manassas VA) for 5 min. Inactivate trypsin with &alpha;-MEM complete medium (antibiotic free, Invitrogen, Carlsbad CA)18. Centrifuge at 300 x g for 5 min.</li>
	<li>Carefully aspirate supernatant and re-suspend pellet in 1 mL of 1X PBS and count cells using a hemacytometer.</li>
	<li>Transfer 1.5 x 106 cells to a 1.5 mL centrifuge tube. Centrifuge at 300 x g for 5 min.</li>
	<li>Carefully aspirate supernatant. Resuspend pellet in 300 &mu;L of R Buffer (Neon Transfection System, Invitrogen) and 6 &mu;g (2 &mu;g / 5.0 x 105 cells) of p231 pCMVe-betaAc-STOP-luc (Addgene, Cambridge, MA). Place 3 mL of E Buffer (Invitrogen) into the electroporation docking port per manufacturer's protocol (Neon Transfection System, Invitrogen).</li>
	<li>Transfer cell-plasmid solution to a 100 &mu;L Neon pipet tip and electroporate with a pulse duration of 20 ms and a magnitude of 1500 volts. Place electroporated cells into a 15 mL conical tube containing 9.7 mL &alpha;-MEM complete medium.</li>
	<li>Repeat step 1.5 two additional times and pool transfected cells to yield a total volume of 10 mL. Add the 10 mL cell suspension (1.5 x 106 cells) to a T175 flask containing 10 mL &alpha;-MEM complete medium. Cell viability after electroporation is approximately 30%, to yield approximately 4.5 x 105 viable cells per T175.</li>
	<li>Change &alpha;-MEM complete medium 24 hours following transfection.</li>
	<li>Harvest transfected cells when 70 - 80% confluent (~2 - 3 days after electroporation). Perform cell count using a hemacytometer and resuspend the cells at a concentration of 1.0 x 106 cells/50 &mu;L of &alpha;-MEM complete medium. Minimize time cells spend in suspension to reduce cell death prior to injection.</li>
</ol>
2. Intramyocardial Injection
<ol>
	<li>Induce anesthesia by isoflurane (Phoenix Pharmaceuticals, Inc., St. Joseph, MO) on transgenic mice engineered to constitutively express Cre recombinase in every cell (B6.C-Tg(CMV-cre)1Cgn/J, Jackson Laboratory, Bar Harbor, ME).</li>
	<li>Remove hair from chest region using hair clippers or chemical hair remover.</li>
	<li>Intubate with an 18 gauge catheter (Becton Dickinson &amp; Co, Franklin Lakes NJ) and place on mouse ventilator at 120 - 130 breaths per minute with a stroke volume of 150 &mu;L.</li>
	<li>Make lateral incision across the fourth intercostal space thereby producing a thoracotomy.</li>
	<li>Visualizing the heart, make two 25 &mu;L injections of transfected cell suspension using a 1 mL syringe (Temuro Medical Corporation, Somerset, NJ) and 28 gauge needle (Becton Dickinson &amp; Co, Franklin Lakes NJ). To ease the intramyocardial injection and prevent excessive damage to the organ, bend the needle head ~90 degrees.</li>
	<li>Following injection, use absorbable sutures (e.g., vicryl) to close the ribs and muscle layers. Suture skin closed using 4-0 nylon or silk.</li>
	<li>Allow mouse to recover from anesthesia and extubate.</li>
	<li>Control groups should include Cre mice receiving medium injections only, Cre mice receiving the same concentration of untransfected cells and wild type mice receiving transfected cells (alternatively, Cre mice receiving transfected cells not prone to fuse). </li>
</ol>
3. Biophotonic Imaging
<ol>
	<li>Five to fifteen minutes before imaging, intraperitoneally (IP) inject 10 &mu;L per gram of mouse body weight of 15 mg/mL D-Luciferin (Caliper Life Sciences, Hopkinton, MA).</li>
	<li>Induce anesthesia on mice via isoflurane at 4% for induction and 1 - 2% for maintenance.</li>
	<li>Place mice supine in imaging box with facemask administering 1 - 2% isoflurane for maintenance anesthesia (Xenogen Biophotonic Imaging System, Hopkinton, MA). Several mice can be imaged concurrently. Image sham control mouse with experimental mice for easy comparison of luminescent signal.</li>
	<li>Using Living Image software (Xenogen), set appropriate exposure time (typically 60 sec, see Results). Set area of image to fit mice and keep area consistent throughout imaging to prevent changes in sensitivity. Set subject height at 4.5 cm.</li>
	<li>Acquire luminescence intensity signal corresponding to mouse or mice within the view field and save unmodified image files. Process images to remove background signal corresponding to a control or unmanipulated mouse. Intensity values above background correspond to fused cells within the animal. Intensity analysis can be conducted using Living Image software (Xenogen) or open source image analysis software. Typically, a region of interest (ROI) is selected to compare intensity data between animals and experiments.</li>
</ol>
4. Representative Results
To determine the sensitivity of the Xenogen Biophotonic Imaging System, a cell line which constitutively expresses luciferase (231-LUC-D3H1, Xenogen) was delivered to the myocardium of C57/Bl6 mice (Jackson Laboratory). Cells were injected at concentrations of 1 x 106, 1 x 103, or 1 x 101 cells. Six hours after cell delivery, mice were injected intraperitoneally with luciferin and imaged using the Xenogen system. A specific signal could be detected with 1,000 cells (2 of 6 mice imaged, Figure 2), but detection was more reliable with 10,000 cells (6 of 6 mice imaged). Importantly, this study also served to establish a rough correlation between number of luciferase-expressing cells and signal intensity.
To demonstrate the utility of the described protocol in detecting and tracking cell fusion, MSCs were transfected with the LoxP-Stop-LoxP-Luciferase plasmid (Addgene) and delivered to the myocardium of Cre-expressing mice. Approximately one week after cell delivery, mice were first imaged using the Xenogen system without D-luciferin injection. As expected, without the enzymatic substrate, no intensity signal was detected (Figure 3). Next, D-luciferin was injected intraperitoneally and a signal corresponding to luciferase intensity and thus cell fusion was detected in two of four mice tested. A similar signal was detected one week later (Figure 3), suggesting MSC-coupled fusion products can be maintained in vivo. In this case, the study was terminated to allow for evaluation of heart and surrounding tissue, but one could envision longer-term analyses and more frequent imaging to track the maintenance, proliferation and perhaps migration of fusion products in mice.
<img alt="Figure 1" src="/files/ftp_upload/3581/3581fig1.jpg" /> 
Figure 1. Schematic of Technique to Detect Cell Fusion In Vivo. If fusion between Cre-expressing mouse cells and transplanted cells expressing a floxed luciferase plasmid occurs, luciferase will be expressed. Luciferase can be detected by injecting the enzymatic substrate, D-luciferin, into the mouse and then imaging the mouse using a Xenogen Biophotonic Imaging System (Adapted from 19)
<img alt="Figure 2" src="/files/ftp_upload/3581/3581fig2.jpg" /> 
Figure 2. Sensitivity of detection of luciferase-expressing cells in cardiac tissue with biophotonic imaging . A cell line which constitutively expresses luciferase (231-LUC-D3H1, Xenogen) was delivered to the intramyocardial space of C57/Bl6 mice at various total cell numbers. Representative images of mice receiving 1 x 106, 1 x 103 and 1 x 101 cells (left to right) are shown, imaging was conducted approximately 6 hours after injection.
<img alt="Figure 3" src="/files/ftp_upload/3581/3581fig3.jpg" /> 
Figure 3. Quantification of In Vivo Luminescence Indicative of Cell Fusion. MSCs were transfected with the LoxP-Stop-LoxP-Luciferase plasmid and delivered to the myocardium of Cre-expressing mice. Approximately one week and two weeks after cell delivery, Cre mice were imaged using the Xenogen Biophotonic Imaging System to measure the intensity of luminescence indicative of cell fusion. (A) Overlay of photograph and intensity of luminescence of sham and mice 1-4 (left to right) 17 days after cell delivery. (B) Intensity of luminescence of sham and mice 1-4 (left to right) 17 days after cell delivery. A region of interest was selected (yellow) corresponding to the injection site and intensity levels were determined using ImageJ (free source) software20. (C) Intensity of luminescence was normalized to the same region of interest on sham mouse for all experimental conditions. At one week, mice 3 and 4 showed positive luminescence signal suggesting spontaneous fusion of a mouse cell and transplanted MSC. The signal persisted in mouse 3 at two weeks. To determine organ-specific localization of the signal corresponding to mouse 3, the thoracic cavity was exposed and primary organs excised and imaged. (D) Overlay of photograph and intensity of luminescence of mouse 3. Note localization of intensity signal in the small intestine. (E) Intensity of luminescence of mouse 3. (F) Overlay of photograph and intensity of luminescence of sham mouse. (G) Intensity of luminescence of sham mouse.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+fusion+of+cells+between+species+yields+transdifferentiation+and+retroviral+transfer+in+vivo.">Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo.</a> FASEB. J. 18, 548-550 (2004).</li><li>Nygren, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+hematopoietic+cells+generate+cardiomyocytes+at+a+low+frequency+through+cell+fusion,+but+not+transdifferentiation.">Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.</a> Nat. Med. 10, 494-501 (2004).</li><li>Nygren, J. 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No conflicts of interest declared.

The method described here allows, for the first time, discrete identification and temporal analysis of cell fusion in organisms, including small animals. The approach combines Cre-LoxP recombination with subsequent biophotonic image analysis. The approach is amenable to tracking not only cell-cell fusion, but also virus-cell fusion and so could prove useful for tracking viral infections. Image analysis is rapid and it is possible to image multiple small animals simultaneously. Detection of fusion is limited b...

<table border="1">
<tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Neon Transfection System</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>MPK5000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Neon 100 &mu;L Kit</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>MPK10025</td>
 <td>Contains R and E Buffer</td>
 </tr>
 <tr>
 <td>a-MEM powder</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>12000-022</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>Hyclone, Logan UT</td>
 <td>SH30070.03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>B6.C-Tg(CMV-cre)1Cgn/J</td>
 <td>Jackson Laboratory, Bar Harbor, ME</td>
 <td>006054</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin 10X</td>
 <td>Fisher Scientific, Forest Lawn, NJ</td>
 <td>MT-25-054-Cl</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Fisher Scientific, Forest Lawn, NJ</td>
 <td>25030-081</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-Luciferin</td>
 <td>Caliper Life Sciences, Hopkinton, MA</td>
 <td>122796</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Xenogen Biophotonic Imaging System</td>
 <td>Caliper Life Sciences, Hopkinton, MA</td>
 <td>IVIS Spectrum </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Biocarbonate</td>
 <td>Sigma Aldrich, St. Louis, MO</td>
 <td>S6014-500G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Non-essential Amino Acids</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>11140-050</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

a-cre-lox-p-recombination-approach-for-detection-cell-fusion

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JoVE Journal

Bioengineering

18.7K ビュー.  University of Wisconsin-Madison. 時間の経過とともに生物の細胞融合を追跡する方法について説明する。アプローチは、Creを利用しつのloxP再結合は、細胞融合によりルシフェラーゼ発現を誘導する。生成された発光シグナルは、末梢組織で〜1,000細胞の検出の感度バイオフォトニックイメージングシステムを用いて生体内で検出することができます。

ビデオ: A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...