The authors would like to thank Jens Geiling from the Institute of Anatomy I, Jena University Hospital, for producing the schematic drawings of rat liver anatomy.

The housing and all procedures carried out were in accordance with German animal welfare legislation. All gauze, covering clothes, and surgical instruments are autoclaved and prepared before the operation. All procedures are carried out under sterile conditions.
1. Preparation of the Rat for the Surgical Procedure
<ol>
	<li>Place the rat in an induction chamber and anesthetize the rat with 4% vaporized isoflurane and 100% oxygen at 0.5 L/min for about 3 min, until the rat is completely anesthetized.</li>
	<li>Take the rat out of the induction chamber and measure its body weight.</li>
	<li>Shave the fur of the surgical region on the abdomen.</li>
	<li>Place the animal back into the isoflurane chamber for an additional 2 min to deepen anesthesia.</li>
	<li>Place the rat on the operation table in supine position.</li>
	<li>Fix the anesthesia mask to the mouth region of the rat and keep the animal anesthetized with a continuous gas flow of 2% vaporized isoflurane and 100% oxygen at a flow rate of 0.5 L/min.</li>
	<li>Fix the limbs with pieces of tape.</li>
	<li>Apply vet ointment on both eyes to prevent dryness.</li>
	<li>Administer buprenorphine 0.05 mg/kg subcutaneously, to relieve pain during the operation period.</li>
	<li>Disinfect the surgical field of the abdomen with 3 rounds of iodine tincture followed by 2 rounds of 70% alcohol.</li>
	<li>Place sterilized gauze around the area where the incision will be made to only leave the operation field of the abdomen exposed.</li>
	<li>Proceed to perform the operation when the toe-pinch withdrawal reflex of the rat is absent.</li>
</ol>
2. Laparotomy of the Rat
<ol>
	<li>Make a transverse abdominal skin and muscle incision using scissors and an electrical coagulator.</li>
	<li>Fix and pull the xiphoid process toward the head using a 4-0 polypropylene suture. 
	NOTE: Pay attention to lift up the xiphoid process vertically to better expose the liver, but proceed with caution to avoid severe respiratory restriction and suffocation.</li>
	<li>Open the peritoneal cavity by pulling both sides of the abdominal walls towards the head with two subcostal hooks to expose the liver.</li>
	<li>Cover the duodenum and small intestine in the abdominal cavity with a moistened gauze to avoid drying.</li>
	<li>Lift left and right median lobes up by using a moistened gauze and hold them against the thorax to better expose the hilum of the liver.</li>
	<li>Place the rat under a stereomicroscope (8X magnification).</li>
	<li>Drop some warm saline into the abdomen and onto the surface of the liver and intestines every several minutes, to prevent drying during the whole procedure.</li>
</ol>
3. Establishment of a Bypass Passage Within the Left Lateral Lobe
<ol>
	<li>Dissect the left portal vein and ligate it with a 6-0 silk suture at the base (Figure 2A).</li>
	<li>Block the left hepatic artery, the left bile duct along with the left median portal vein, the left median hepatic artery, and the left median bile duct with micro clamps to prevent a flow of the perfusate to the left median lobe (Figure 2B).</li>
	<li>Separate the left lateral lobe by cutting off the surrounding ligaments of the lobe with micro scissors.</li>
	<li>Block the left lateral hepatic vein by clamping at the base of the left lateral lobe with micro clamps (Figure 2C). 
	NOTE: Make sure not to clamp the left portal vein as well by mistake.</li>
	<li>Use mosquito clamps to hold the ligature of the left portal vein and keep the vein with proper tension for later cannulation.</li>
	<li>Carefully make an incision in the front wall of the left portal vein by puncturing it with a 24-G needle-dwelling catheter ( Figure 3A). 
	NOTE: To create a bypass, vascular access points on the left portal vein and the left hepatic vein are needed. For this step, it is preferred to create the vascular access by puncturing the vessels with a needle rather than making a larger incision using scissors. This reduces the risk of bleeding and later stenosis.</li>
	<li>Withdraw the catheter and take the needle out of the catheter to obtain a needle-free 24-G catheter.</li>
	<li>Connect the catheter to a perfusion tube, of which the other endpoint connects to a 20-mL syringe with 15 mL of 40 U/mL heparinized saline on a perfusion pump.</li>
	<li>Turn on the pump for perfusing the tube to expel air out from the tube and the needle-free catheter.</li>
	<li>Turn off the perfusion pump.</li>
	<li>Again, insert the needle-free catheter into the left portal vein via the punctured incision on the vein (Figure 3B). 
	NOTE: Owing to the fact there is very limited space for fixing the catheter, it is not fixed at this point. Therefore, the surgeon should use care to avoid the displacement of the cannulated catheter.</li>
	<li>Carefully make another incision at the margin of the exposed region of the left lateral hepatic vein by puncturing it with a 22- or 24-G needle-dwelling catheter (Figure 3C). 
	NOTE: It is recommended that the catheter be slightly smaller than the vessel.</li>
	<li>Withdraw the catheter and take the needle out of the catheter to obtain a needle-free 22-G catheter.</li>
	<li>Turn on the perfusion pump to perfuse heparinized saline into the left lateral lobe via the 24-G cannulated needle-free catheter of the left portal vein at a flow rate of 0.5 mL/min.</li>
	<li>Use dry gauze to absorb out-flowing waste fluid around the incision area of the left lateral hepatic vein.</li>
	<li>Cannulate the left lateral hepatic vein via the punctured incision of the vein with the 22-G needle-free catheter, to generate a fluid outlet to minimize intra-abdominal contamination (Figure 3D). 
	NOTE: It is technically difficult to fix the cannulated catheter to the liver lobe. Therefore, the surgeon should pay attention to avoid displacement of the catheter. Alternatively, without cannulation of the left lateral hepatic vein with a catheter, waste fluid can also be absorbed at the incision region of the vein only with a dry gauze.</li>
	<li>Keep perfusing the left lateral lobe with heparinized saline for around 20 min (group 1) and then only with saline for 2 h, 3 h, or 4 h (group 2, group 3, and group 4, respectively).</li>
	<li>Turn off the pump to stop the perfusion.</li>
</ol>
4. Physiological Reperfusion of the Left Lateral Lobe
<ol>
	<li>Take off both catheters from the left portal vein and the left lateral hepatic vein.</li>
	<li>Close the incision of the left portal vein with an 11-0 polyamide suture.</li>
	<li>Close the incision of the left lateral hepatic vein with an 11-0 polyamide suture as well.</li>
	<li>Unclamp the left lateral hepatic vein.</li>
	<li>Unclamp the left median portal vein, left bile duct, and left hepatic artery.</li>
	<li>Cut off the ligature on the left portal vein to reopen the vein.</li>
</ol>
5. Closure of the Abdominal Wall
<ol>
	<li>Close the muscle layer of the abdominal wall by interrupted suturing with a 4-0 absorbable polyglactin 910 suture.</li>
	<li>Close the skin layer of the abdominal wall by interrupted suturing with a 4-0 absorbable polydioxanone suture.</li>
	<li>After closing the abdomen, disinfect the skin incision with 70% alcohol.</li>
</ol>
6. Postoperative Treatment of the Rat
<ol>
	<li>Place the animal on a warming pad for resuscitation for about 10 min and then put it into a new cage.</li>
	<li>Administer buprenorphine 0.05 mg/kg subcutaneously 2x a day for a consecutive 3 d postoperatively to release pain.</li>
</ol>

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The authors have nothing to disclose.

By blocking and cannulating the left portal vein with a catheter as a fluid inlet and the left lateral hepatic vein with another catheter as a fluid outlet, we successfully generated an in vivo fluid bypass within the left lateral lobe, indicating that although the technique is highly challenging due to the small size of the vessels for cannulation and a high risk of causing bleeding, it is feasible. Even the rats undergoing a long perfusion period of 4 hours survived at least 1 week, showing that the rats could...

The indications for organ transplantation are constantly expanding. In contrast, organ donation rates and overall quality of organs are declining, leading to an increasing demand for grafts. The number of candidates added to the liver transplant waiting list continued to increase (e.g., in the United States, 11,340 patients were added in 2016, compared with 10,636 in 2015)1. Despite substantial efforts, the number of available organs does not meet clinical needs. Due to the increased incidence of liver disease, many patients with end-stage liver diseases die on the transplant waiting list before a donor organ becomes available. To meet the huge demand for donor liver grafts, alternative approaches using liver tissue engineering principles are being actively pursued2. Nowadays, a newly developed biological technique of liver engineering could potentially overcome this shortage.
Liver engineering consists of two steps: the generation of an acellular scaffold, followed by a repopulation of the scaffold. To obtain a biological acellular liver scaffold, the explanted liver is perfused via the vascular system with ionic or nonionic detergents, which can remove the cellular material from the liver. In most previous studies, a biological acellular liver scaffold was achieved by perfusion of the liver with a combination of sodium dodecyl sulfate and TritonX100. As a result, all cells were removed, whereas the structure of the extracellular matrix was maintained. The organ scaffolds were reseeded with mature cells, hepatocellular, as well as endothelial cell lines, and primary hepatocytes with or without the simultaneous application of endothelial cells or mesenchymal stem cells (MSC). Most researchers focus on ex vivo liver engineering3,4,5,6,7,8,9,10,11,12,13,14. However, in most previous studies, only small pieces of repopulated scaffold cubes were transplanted into different heterotopic implantation sites. In a few studies, partial repopulated scaffolds were transplanted as an auxiliary graft. However, the maximal reported survival time was only 72 h8,14. As far as we know, orthotopic transplantation of a repopulated full liver graft has not yet been performed or published about. The long-term function and transplantation of engineered organs are still in their infancy. Therefore, an alternative approach to ex vivo liver engineering is needed.
In vivo liver engineering may represent an alternative to study hepatic repopulation under physiological conditions. The advantages of in vivo liver engineering compared to ex vivo liver engineering are manifold. The in vivo repopulated partial liver scaffold is subjected to physiological blood perfusion with proper temperature, sufficient oxygen, nutrients, and growth factors in contrast to ex vivo perfusion with artificial culture medium. Furthermore, the remaining partial normal liver maintains the hepatic function, principally allowing long-term survival. Since an implanted ex vivo engineered liver graft is still incapable of sustaining the long-term survival of experimental animals by its liver function8,&#160;we envision that in vivo partial liver engineeringwould ultimately become a promising model to further study the evolution of engineered livers with longer survival observations than ex vivo.
Recently, one research group (Pan and colleagues) presented, for the first time, a technique of in vivo liver engineering15. They achieved the isolated perfusion of the right inferior liver lobe in living rats despite anatomic and technical challenges. They reported the first intraoperative results of in vivo repopulation using a rat primary hepatocyte cell line. However, the in vivo surgical perfusion model of Pan et al. has disadvantages. They achieved single liver lobe perfusion in rats at the expense of completely blocking the portal vein and inferior vena cava, which may cause severe harm to the animal. The experimental rats were sacrificed after only 6 hours of intraoperative observation time. Therefore, the in vivo liver lobe perfusion technique needs further improvement to achieve postoperative survival.
We developed a novel survival model for in vivo liver lobe perfusion, based on previous studies of the hepatic anatomy of rat16, the portal vein cannulation technique for hemodynamic monitoring in mice17, and liver bioengineering18,19. The key steps for the procedure are illustrated in Figure&#160;1A - 1E.
This technique is suitable for those who want to use this experimental in vivo perfusion model for basic research on partial organ treatment by infusion with drugs, in vivo decellularization as a chemical resection for organ diseases (e.g., liver cancer), in vivo cell culture in a decellularized matrix comparing ex vivo two-dimensional and three-dimensional cell culture systems20,21,22,23,24,25,26, and in vivo liver engineering by decellularization and repopulation.

<table border="0" cellpadding="0" cellspacing="0" style="width:605px;" width="606">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Perfusion Pump</td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusor VI</td>
			<td colspan="2">B. Braun, Melsungen</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Catheter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Versatus-W&#160; Catheter</td>
			<td>Terumo</td>
			<td>SR+DU2419PX</td>
			<td>24G, 0.74&#215;19mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Versatus-W&#160; Catheter</td>
			<td>Terumo</td>
			<td>SR+DU2225PX</td>
			<td>22G, 0.9&#215;25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro surgical instrument</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro scissors</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 14058-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro serrefine</td>
			<td>F&#183;S&#183;L</td>
			<td>No.18055-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro clamps applicator</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 18057-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight micro forceps</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 00632-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved micro forceps</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 00649-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro needle-holder</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;">No. 12061-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">general surgical instruments</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">standard sissors</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mosquito clamp</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serrated forcep</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">teethed forcep</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
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a novel surgical technique as a foundation for in vivo partial liver engineering in rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

Twelve male (aged 12 - 13 weeks) Lewis rats were used to assess the effect of selective liver lobe perfusion. The experiment was performed in four groups (n = 3 rats per group). Using different perfusion periods of 20 minutes, 2 hours, 3 hours, and 4 hours, following the steps described above, we successfully achieved in vivo single lobe perfusion.
In Vivo Perfusion of the Left...

Watch this Scientific Journal Video about A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat at JoVE.com

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

a-novel-surgical-technique-as-foundation-for-vivo-partial-liver

Research

JoVE Journal

Bioengineering

8.0K Views.  University Hospital Jena. We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Video: A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

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 ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Viral Nanoparticles for In vivo Tumor Imaging

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

Viral Nanoparticles for In vivo Tumor Imaging

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

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

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

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 ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...