Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research (NIDCR) and the National Cancer Institute (NCI) of the National Institutes of Health under Award Number R56 DE025098, UG3 DE027695, and F30 CA206296. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the NSF DMR 1206219 and the IADR Innovation in Oral Care Award (2016). 
We would like to thank Jayne Gavrity for her assistance in performing IVIS experiments. We would like to thank Karen Bentley for her input and assistance in performing EM. We would like to thank Pei-Lun Weng for his assistance with IHC. We would like to thank Matthew Ingalls for his assistance in figure preparation. We would like to thank Dr. Elaine Smolock and Emily Wu for critical reading of this manuscript.

All in vivo procedures outlined below were approved by the University Committee on Animal Resources at the University of Rochester, Rochester,&#160;NY.
1. Preparation
<ol>
	<li>Using 32G intracranial catheter tubing with wire inset, cut 3 cm of the tubing to form a beveled end, approximately 45&#176; to the long axis. Confirm that the wire is at least 1 cm longer than the tubing.</li>
	<li>Load 50 &#181;L of PSMA nanoparticle solution (Figure 1), or other injection material, into a Hamilton syringe. To reduce the probability of barotrauma during injection, attach the catheter tubing, with the stylet removed, to the syringe and expel dead volume.</li>
	<li>Inspect the injection solution to ensure the nanoparticle is fully solvated to prevent ductal obstruction following the administration.</li>
	<li>Prepare atropine solution at 0.1 mg/mL. 
	NOTE: Because atropine is light sensitive and degrades over time, this solution should be made the day of injection, and protected from light until administered.</li>
</ol>
2. Accessing and Visualizing Ductal Entry Point
<ol>
	<li>Weigh C57/BL6 mice using an analytical balance.</li>
	<li>Using a 0.5 mL syringe with 29G x &#189;&#34; needle, anesthetize mice with an intraperitoneally injected sterile saline solution of 100 mg/kg ketamine and 10 mg/kg xylazine. Proceed to the following step when the mouse no longer responds to stimuli, which generally occurs within 5 to 10 min following the injection. 
	NOTE: This procedure may also be performed under isoflurane, but will require a custom nose cone that permits access to the oral cavity.</li>
	<li>To prevent dryness during the procedure, apply lubricant to eyes and place the mouse in a prone position on a custom stage. 
	NOTE: To maintain appropriate conditions for intra-oral procedure, tools should be disinfected or sterilized prior to each use.</li>
	<li>Open the oral cavity by securing the maxillary incisors over a metal beam, and use an elastic band to apply downward tension behind the mandibular incisors (Figure 2A).</li>
	<li>Align the mouse beneath the dissecting microscope such that the base of the jaw is visualized.</li>
	<li>To widen the mouth, use a custom, curved steel retractor to apply tension bilaterally to the buccal mucosa.</li>
	<li>To visualize the submandibular papillae, grasp and gently lift the tongue from the floor of the mouth using blunt forceps. 
	NOTE: The papillae will appear as two pale protrusions beneath the tongue (Figure 2B).</li>
	<li>To ease the visualization and further manipulation within the oral cavity, place cotton between the tongue and the buccal mucosa.</li>
</ol>
3. Ductal Cannulation and Line Placement
<ol>
	<li>Using fine, curved forceps, grasp catheter tubing with the wire inset. For optimum manual control during cannulation, align the tubing with the curvature of the forceps (Figure 3A).</li>
	<li>Using the dissecting microscope, move the forceps and wire into the field of view. 
	NOTE: The wire should be protruding from the tubing.</li>
	<li>Gently apply pressure into the base of one submandibular papilla using the wire inset to produce a small, superficial, mucosal puncture (0.076 mm diameter) that will facilitate later entry of the catheter tubing (0.25 mm diameter). If resistance is encountered, cut fresh beveled tips on both the tubing and wire inset with sharp dissecting scissors.</li>
	<li>Following the entry, withdraw the stylet and, using the dissecting microscope, confirm the presence of saliva at the puncture site. Avoid forceful or sudden movement (either withdrawal or insertion) of the stylet that may cause bleeding or compromise ductal integrity.</li>
	<li>Retract the stylet within the tubing (Figure 3B).</li>
	<li>To ensure that injection tubing will fit into Wharton&#39;s duct opening, insert tubing containing the stylet as a rigid guide into the previously made puncture (Figure 2C). 
	NOTE: If not performed quickly, local swelling may prevent re-insertion.</li>
	<li>To prevent back pressure from prolonged ductal obstruction, withdraw the tubing. Inspect to verify that an opening, visible under microscopy, can be seen in the submandibular papilla. If visible bleeding occurs, remove the stylet and reattempt from step 3.2 on the opposing submandibular papillae.</li>
	<li>Without moving the mouse, administer intraperitoneal injection of 1 mg/kg atropine solution, to reduce salivation during the procedure. Wait 5 - 10 min.</li>
	<li>Grasp the end of the syringe tubing, and insert into the orifice using the dissecting microscope (Figure 3C). If resistance is encountered, cut a fresh beveled end to the tubing and reattempt.</li>
	<li>Once the tubing is in place within the submandibular papilla, slowly advance 3 - 5 mm into the duct. Release the tubing from the forceps.</li>
	<li>To improve the seal between the tubing and the submandibular papilla, dry the interface by gently blotting with gauze for 1 min.</li>
	<li>Inspect to confirm that the position of the tubing has not shifted during drying.</li>
</ol>
4. Injection
<ol>
	<li>Inject material at a rate of 10 &#181;L/min. Inspect to confirm that the mouse remains sedated and is not showing signs of distress (Figure 2D). 
	NOTE: Injections of 15 - 50 &#181;L are well tolerated. Injection of larger volumes can result in barotrauma.</li>
	<li>Following the injection, maintain syringe pressure for 5 min to improve the retention of material within Wharton&#39;s duct and SMG (Figure 4). Inspect the submandibular papilla periodically to ensure that tubing does not exit the ductal orifice.</li>
	<li>Using fine forceps, grasp and gently withdraw the tubing from the submandibular papillae. 
	NOTE: It is normal to observe some fluid egress from the papillae.</li>
	<li>Remove the retractor and cotton from the oral cavity before moving the mouse from the stage. 
	NOTE: The animal should not be left unattended until it has regained sufficient consciousness to maintain sternal recumbency. Furthermore, ensure that the mouse is not housed with other mice until fully recovered.</li>
</ol>
5. Verification and Analysis
Note: An in vivo Imaging System (IVIS) can be used to assess retention of fluorescently labeled nanoparticles following injection (as shown 1 h and 24 h after injection in Figure 5).
<ol>
	<li>To better visualize fluorescent signal within the SMG through the skin, remove the ventral fur overlying the SMGs either by shaving or using a chemical depilatory. 
	Note: Following euthanasia, SMG tissue can also be harvested, fixed (overnight in 4% paraformaldehyde), and stained using immunohistochemistry to confirm persistence of fluorescently labeled NP one day following injection (Figure 6).</li>
</ol>

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The authors have nothing to disclose.

Retroductal injection is critical for localized drug delivery to the salivary gland. This technique has applications in screening therapeutic agents for conditions including Sjogren's syndrome and RIH9,10,28. Direct drug delivery into the SMG via retroductal injection provides a key advantage over systemic administration in its potential to reduce off-target effects, including immune activation11. The abi...

Salivary gland dysfunction has many etiologies, including Sj&#246;gren&#39;s syndrome, an autoimmune mediated loss of functional secretory tissue, and radiation induced hyposalivation (RIH), a common sequella of head and neck cancer radiotherapy1. Loss of salivary function due to either condition predisposes individuals to oral and systemic infection, tooth decay, digestive and swallowing dysfunction, speech impairment, and major depression1,2,3. As a result, quality of life significantly suffers, with interventions limited to palliation of symptoms rather than cure4. To investigate novel therapies in vivo, it is of interest to administer bioactive compounds directly to the salivary gland.
Retroductal injection is a valuable method to deliver bioactive compounds directly to the salivary glands and test the efficacy in disease, injury, or under normal tissue homeostasis. The three major salivary glands are the parotid (PG), the submandibular (SMG), and the sublingual (SLG), all of which empty into the oral cavity through excretory ducts. The anatomy of the murine SMG permits direct access through cannulation of Wharton&#39;s duct, located in the floor of the mouth beneath the tongue5. Following the cannulation, solvated drugs can be administered directly to the SMG. Following retroductal delivery, extra-glandular diffusion is restricted by the surrounding tissue capsule which regulates the exchange of material with surrounding structures6. The SMG and its duct are similarly structured in humans, and are routinely accessed during SMG surgery and sialoendoscopy7. In humans and mice, the PG is likewise accessible via Stensen&#39;s duct in the buccal mucosa8.
In murine models of RIH, SMG retroductal injection has been used to deliver therapeutics including growth factors, primary cells, adenoviral vectors, cytokines, and antioxidant compounds to modulate the cellular response to injury, and reduce the resulting tissue damage5,9,10,11,12,13,14,15,16. The most notable clinical success of retroductal injection is the administration of adenoviral vector to direct expression of a water channel (Aquaporin 1; AQP1) in patients following the radiation for head and neck cancer17.
Previously, we have developed and shown the efficacy of a retroductally injected polymeric nanoparticle-siRNA system to protect salivary gland function from RIH11,18,19,20. As an extension of our past work, here, we demonstrate our protocol for retroductal SMG injection using a fluorescently labeled nanoparticle (NP) capable of loading and delivering otherwise poorly soluble drugs21,22,23.
We have synthesized the NP from a diblock copolymer comprised of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA) through reversible addition chain fragmentation (RAFT) polymerization, as described previously21. Through solvent exchange, these polymers spontaneously self-assemble into micelle NP structures with a hydrophobic interior and hydrophilic exterior21. The NPs are labeled with Texas-Red fluorophore to permit the verification of NP delivery into the glands without sacrificing the animal. Live animal imaging and SMG immunohistochemistry is shown at 1 h and 1 day following the injection.
This updated and reproducible cannulation protocol should enable others to achieve retroductal injection. We expect that this refined technique will become critical for in vivo studies and therapeutic development24,25.

<table border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Pilocarpine hydrochloride</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P6503</td>
			<td style="width:225px;">Pilocarpine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Student Vannas Spring Scissors</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">91500-9</td>
			<td>Spring Scissors for Tracheostomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Sterile Saline Solution</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">RDI30296H</td>
			<td>Saline</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:219px;">Dumont #7 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
			<td>Curved Forceps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:219px;">Dumont #5 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
			<td>Straight Forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:219px;">Standard Pattern Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11000-12</td>
			<td>Blunt Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Fine Scissors- Tungsten Carbide</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">14568-09</td>
			<td>Dissection Scissors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Microhematocrit Heparinized Capillary Tubes</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:119px;">22362566</td>
			<td>Capillary tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Lubricant Eye Ointment</td>
			<td style="width:128px;">Refresh</td>
			<td style="width:119px;">N/A</td>
			<td>Refresh Lacri-Lube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Goat polyclonal anti-Nkcc1</td>
			<td style="width:128px;">Santa Cruz Biotech</td>
			<td style="width:119px;">SC-21545</td>
			<td>Nkcc1 Antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">GraphPad Prism</td>
			<td style="width:128px;">GraphPad</td>
			<td style="width:119px;">ver6.0</td>
			<td>Statistical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Cotton tipped applicator</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td>Applicator for eye ointment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">0.5cc Insulin Syringe, 29G x 1/2&#34;</td>
			<td style="width:128px;">BD</td>
			<td style="width:119px;">7629</td>
			<td>Syringe for intraperitoneal injection</td>
		</tr>
	</tbody>
</table>

retroductal nanoparticle injection to the murine submandibular gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Retroductal injection can be used to administer NPs to the murine SMG (Figure 1). Here, we deliver 50 &#181;g PSMA NPs labeled with Texas Red fluorophore.
Proper placement of the mouse allows facile access and visualization of the floor of the mouth (Figure 2A-B). The submandibular papillae are identified as two fleshy protrusions beneath the tongue. Fo...

Watch this Scientific Journal Video about Retroductal Nanoparticle Injection to the Murine Submandibular Gland at JoVE.com

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

retroductal-nanoparticle-injection-to-the-murine-submandibular-gland

Research

JoVE Journal

Bioengineering

9.9K Views.  University of Rochester. Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Video: Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Summary

Explore More Videos

Chapters in this video

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Tissue Engineering of the Intestine in a Murine Model

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Intraductal Delivery to the Rabbit Mammary Gland

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Optical Imaging of Isolated Murine Ventricular Myocytes