This work was supported by The British Heart Foundation and The Oak Foundation.

Perform all animal experiments according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals.
1. Preparation of Culture Media
<ol>
	<li>Make culture media for human fibroblast cell line CCL-153: F-12K Medium, 10% fetal bovine serum (FBS) and 100 U/ml penicillin and streptomycin.</li>
	<li>Make Reprogramming Media for PiPSC generation: Knockout Dulbecco&#8217;s modified eagle&#8217;s medium (DMEM) containing 20% Knockout Serum Replacement, 0.1 mM &#946;-mercaptoethanol, 0.1 mM Minimum Essential Medium (MEM) Non-Essential Amino Acids, 10 ng/ml basic Fibroblast Growth Factor 2 (bFGF-2) and 100 U/ml penicillin and streptomycin. Add bFGF-2 freshly each time before using the media.</li>
	<li>Make Differentiation Media (DM) to induce smooth muscle cell (SMC) differentiation: &#945;-MEM media containing 10% FBS, 0.1 mM &#946;-mercaptoethanol, 100 U/ml penicillin and streptomycin and 25 ng/ml Platelet Derived Growth Factor (PDGF-&#946;&#946;).</li>
	<li>Make Endothelial Differentiation Media (EC-DM) to induce endothelial cell (EC) differentiation: Endothelial growth media-2 (EGM-2) media containing 50 ng/ml Vascular Endothelial Growth Factor (VEGF) and 100 U/ml penicillin and streptomycin.</li>
</ol>
2. Reprogramming Human Fibroblasts into Partially-induced Pluripotent Stem Cells (PiPSC)
<ol>
	<li>Culture human fibroblast cell line CCL-153 on 0.04% gelatin solution coated flasks in culture medium.</li>
	<li>Passage cells every 3 days at a ratio of 1:6. Use cells before passage 9 for optimal efficiency of PiPSC generation. Cells are ready for transfection upon reaching 80 - 90% confluency.</li>
	<li>Linearize polycistronic plasmid pCAG2LMKOSimO containing 4 reprogramming factors octamer-binding transcription factor 4 (OCT4), SOX2, Kruppel-like factor 4 (KLF4) and C-MYC (pCAG-OSKM). Digest 5 &#181;g of plasmid with 5 units of PvuI restriction enzyme for 3 hr at 37 &#176;C. Purify the plasmid with a commercial kit according to manufacturer&#8217;s protocol.</li>
	<li>Transfect 2 x 106 human fibroblasts with 4 &#181;g of linearized pCAG-OSKM plasmid by electroporation with human dermal fibroblast (NHDF) nucleofection kit according to manufacturer&#8217;s protocol. Gently seed transfected cells onto pre- 0.04% gelatin coated T25 flask containing 5 ml of pre-warmed Reprogramming Media.</li>
	<li>After 24 hr, change medium with supplements of 25 &#181;g/ml neomycin to select a pure population of transfected cells.</li>
	<li>Change medium every other day until day 4. 
	Note: Human fibroblasts become PiPSC after 4 days of reprogramming.</li>
</ol>
3. Differentiation of PiPSC into Endothelial and Smooth Muscle Cells
<ol>
	<li>Seed PiPSC on collagen IV pre-coated dishes containing DM for 4 days to induce differentiation into SMCs. Change medium every other day until day 4.</li>
	<li>Seed PiPSC on collagen IV pre-coated dishes containing endothelial-DM for 6 days to induce differentiation into endothelial cells. Change medium every other day until day 6.</li>
</ol>
4. Decellularized Aorta Graft Preparation
Note: All solutions and equipment should be sterile.
<ol>
	<li>Sacrifice the mouse by cervical dislocation and fix the mouse in a supine position under dissection microscope.</li>
	<li>Cut through the sternum and around the rib cage to open the thoracic cavity. Remove the heart, lungs and esophagus to expose the aorta. Gently remove the peri-aorta fat with forceps.</li>
	<li>Cut the aorta from the anterior end with scissors and gently hold the end with blunt forceps. Detach the aorta from the spine column behind by blunt dissection. Carefully close all the branching arteries from the aorta by ligation with a bipolar electrocoagulator and cutting from the far end of the ligation with scissors.</li>
	<li>Use a 5 ml syringe to flush the aorta lumen with 3 ml of saline solution containing 100 U of heparin to prevent the formation of blood clots.</li>
	<li>Cut the posterior end of the aorta before it branches into the renal. Keeping the integrity of the aorta is very important during the whole procedure.</li>
	<li>Flush the aorta lumen with 5 ml of 0.075% sodium dodecyl sulphate (SDS) solution diluted in phosphate buffered saline (PBS).</li>
	<li>Soak the thoracic aorta in 0.075% SDS solution in a 10 cm Petri dish. Place the Petri dish on an orbital shaker for 2 hr at 150 rpm at RT.</li>
	<li>Flush the aorta lumen with 5 ml of PBS.</li>
	<li>Soak the aorta in PBS in a 10 cm Petri dish. Place the Petri dish on the orbital shaker for 2 hr at 150 rpm at RT. Refresh PBS every 20 min.</li>
	<li>Flush the aorta lumen with 5 ml of PBS.</li>
	<li>Keep decellularized aorta graft in PBS at 4 &#176;C for up to one week.</li>
</ol>
5. Dual Seeding of PiPSC and Bioreactor Conditioning
Note: All solutions and equipment should be sterile. Perform all operations in a tissue culture hood.
<ol>
	<li>Assemble the bioreactor flow circuit as illustrated in Figure 1. Connect incubation chamber, media reservoir, peristaltic pump and compliance chamber with tubing. Minimal volume of medium needed for circulation is 40 ml.</li>
	<li>Pre-condition the decellularized vessel with culture medium by soaking the aorta in DM medium in a 10 cm Petri dish. Place the Petri dish on the orbital shaker for 1 hr at 50 rpm at RT.</li>
	<li>Insert 1 cm length Nylon tubes (OD 0.9 mm, ID 0.75 mm) into both ends of the decellularized vessel under microscope. Tie the vessel and the tubes with 8-0 silk sutures.</li>
	<li>Assemble the decellularized aorta graft in the incubation chamber by connecting the Nylon tubes with the inlet and outlet ports (1/32&#8221; tubing) from the chamber wall. Maintain incubation chamber with 5mls of medium each time.</li>
	<li>Trypsinize PiPSC by adding pre-warmed trypsin to cover the culture dish and gently rock the dish 10 times. Discard the trypsin and leave the dish in incubator for 2 - 3 min. Add pre-warmed culture medium in the dish and mix the medium with the cells.</li>
	<li>Count cell number using a hemocytometer. Centrifuge two aliquots of cell suspensions containing 5 x 105 cells each for 5 min at 300 x g. Aspirate the supernatant completely.</li>
	<li>After aspirating the supernatant completely, resuspend 1 pellet of the PiPS cell pellets in 50 &#181;l of DM. Inject this cell suspension into the decellularized vessel lumen.</li>
	<li>Then, resuspend the other PiPS cell pellet in 100 &#181;l of Matrigel. Carefully pipette the mixture onto the decellularized aorta graft.</li>
	<li>Wait for 10 - 15 min until the mixture turns into a gel-like state and evenly wraps around the vessel external surface. Fill the incubation chamber with DM.</li>
	<li>Place the whole bioreactor setting in a 5% CO2 incubator at 37 &#176;C. Manually rotate the decellularized graft 90&#176; around the longitudinal axis every half an hour in the first 2 hr.</li>
	<li>Keep static culture for 12 hr to enable cell adhesion.</li>
	<li>Deliver DM through the lumen by the peristaltic pump to induce smooth muscle cell differentiation. Start initial flow rate at 5 ml/min and stepwise increase to 20 ml/min over 24 hr.</li>
	<li>Keep the circulating medium flow rate at 20 ml/min for 24 hr.</li>
	<li>Stop the medium flow and move the bioreactor setting out of the incubator.</li>
	<li>Re-seed the lumen of the graft with PiPSC. Trypsinize, count and centrifuge 1 x 106 PiPSC as in steps 5.5 - 5.6. Resuspend the cells in 50 &#181;l of EGM-2 medium containing 50 ng/ml VEGF. Inject the cell mixture into the lumen of the graft.</li>
	<li>Change the medium in the incubation chamber and whole circulation to EGM-2 medium containing 50 ng/ml VEGF to induce endothelial differentiation.</li>
	<li>Move the bioreactor setting back to the incubator. Manually rotate the graft 90&#176; every 30 min in the first 2 hr and then keep a static culture for 12 hr.</li>
	<li>Start circulating flow from 5 ml/min and stepwise increase to 35 ml/min. Keep the flow rate at 35 ml/min for 5 days. Change circulating and chamber medium every other day.</li>
	<li>Harvest the engineered graft for further grafting to mouse.</li>
</ol>
6. Grafting of Double-seeded PiPSC Graft into Mice
<ol>
	<li>Use NOD.CB17-Prkdcscid/NcrCrl male mice as vessel graft recipients. Always ensure that the mice are about 10 weeks old, weigh approximately 25 - 35 g, and are in good health conditions.</li>
	<li>Anesthetize recipient mouse by administering a combination of Hypnorm (25 mg/kg) and Hypnovel (25 mg/kg) intraperitoneally. Apply petrolatum ophthalmic ointment on eyes to prevent dryness while under anesthesia. Confirm efficient anesthetization by ensuring that mice have relaxed muscles and are breathing steadily.
	<ol>
		<li>Fix the mouse in a supine position with its neck shaved and extended. Administer atropine sulfate (1.7 mg/kg) in combination with the anaesthetics to ensure that the mouse respiratory tract remains clear.</li>
	</ol>
	</li>
	<li>Prepare a midline incision from the mandible to sternum of the mouse. Under a dissecting microscope with 5- to 10-fold amplification, lift the right salivary glands laterally and remove the right cleidomastoid muscle to expose the right common carotid artery.</li>
	<li>Gently remove attached tissues to mobilize the right common carotid artery from the distal end toward the proximal end. Ligate the middle portion of the common carotid artery twice with an 8-0 silk suture and dissect between the two ties.</li>
	<li>Pass through a cuff made of autoclavable nylon tube in each vessel end and fix each end with microhemostat clamps.</li>
	<li>Remove suture tie&#160;on one end and apply a drop of heparin (100 U/ml). Immediately after, invert the distal end of the artery to cover the whole cuff body and fix the inverted vessel with an 8-0 silk suture to the cuff.
	<ol>
		<li>Repeat the same procedure to the other portion of the artery. Flush artery with saline solution to remove blood clots.</li>
	</ol>
	</li>
	<li>Implant a decellularized vessel graft between two ends of the carotid artery by sliding the decellularized vessel ends of the over the artery cuffs and fixing it with 8-0 silk sutures. Remove vascular clamps from either ends before evaluating the pulsation of the graft.</li>
	<li>Place the right salivary gland back to its original position. Close the skin on the surgical location with an interrupted suture using a 6-0 polyglactin suture.</li>
	<li>Consistently observe vital signs of mouse after the procedure is completed, until it resumes consciousness. Sacrifice recipient mice by cervical dislocation and harvest vessel grafts either 24 hr or 3 weeks later for further analysis.</li>
</ol>

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The authors have no conflicting financial interests.

The current protocol indicates a sound, fast, simple, efficient and reproducible strategy in which functional tissue engineered vessels can be generated using PiPSC from human fibroblasts. This technique represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell therapy in the near future. Critical steps to ensure the efficacy of the protocol include the preparation of PiPSC, preparation of sterile and fully decellularized aortic grafts, successful seeding and different...

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. To date, graft materials used in surgery include biocompatible synthetic polymers (polytetrafluoroethylene [Teflon], expanded polytetrafluoroethylene [ePTFE; Gore-Tex] or polyethylene terephthalate [Dacron]), allografts, autologous tissue (pericardium or saphenous vein) and xenografts1. Whilst artificial grafts (e.g., Gore-Tex and Dacron) are most commonly used, these materials likely cause numerous short- and long-term complications that include stenosis, calcium deposition, thrombo-embolization and infections. Although patients with biological grafts present with decreased thrombo-embolic events, they still encounter limitations such as secondary graft failure and shortened durability owing to calcification degradation2. Therefore, despite significant improvements in surgical techniques over the years, researchers and clinicians are still burdened with the need for identifying the ideal conduit for vascular diseases. More recently, the research field of vascular tissue engineering has generated a concept in which cells are incorporated into biodegradable scaffolds, with the aim of creating a biomimetic environment that epitomizes a functional vessel for successful grafting1. Fundamentally, the success of the vascular constructs depend on three essential components; cells that comprise the scaffold, i.e., an endothelial cell inner layer and a smooth muscle cell layer, a scaffold containing the appropriate extracellular matrix to provide mechanical properties comparable to the native vasculature, and the molecular/cellular signaling that is required for initiating/regulating repair.
Long term graft patency and sustained development of the neo-tissues are highly dependent on effective cell seeding of scaffolds, thereby rendering the decision of cell type of critical importance. Several reports demonstrate the use of mature endothelial and smooth muscle cells from various sources to develop small diameter conduits3-6. Although promising, the lack of sufficient autologous vessels to obtain mature endothelial and smooth muscle cells remain a considerable burden. More recently, stem cells from various sources have been exploited for vascular tissue engineering applications. Indeed, a variety of stem cell types including embryonic stem cells7, induced pluripotent stem cells (iPSCs)8,9, PiPSC10,11, bone marrow-derived mononuclear cells12, mesenchymal stem cells13, endothelial progenitor cells and adult vessel wall-derived stem cell antigen-1 (Sca-1)+ stem/progenitor cells14,15 have all been demonstrated to be capable of differentiation into either functional endothelial or smooth muscle cells in response to defined media and culture conditions. Furthermore, the unlimited self renewal capacity of the stem cells make them better candidates unlike mature endothelial and smooth muscle cells which can only divide for a finite number of times before undergoing growth arrest and senescence.
The selection of scaffold material to generate successful tissue engineered vessel for grafting depends on several factors such as biocompatibility, biomechanical properties, and rate of biodegradation. Fundamentally, materials used to create scaffolds for the grafts should be biodegradable and will not mount unnecessary recipient immune responses. Additionally, it must encompass a suitable porosity and microstructure for cell attachment and subsequent survival. To date, the most common materials used for scaffolds in vascular tissue engineering include polymers of polyglycolic acid, polylactic acid, and poly &#949;-caprolactone16. More recently, decellularized biological materials have also been applied with some success. Several laboratories have shown that seeding decellularized human, canine or porcine vessels with autologous cells provided a biological graft that resisted clotting and intimal hyperplasia17-19. Other strategies in vascular tissue engineering include extracellular matrix proteins-based vascular grafts e.g., seeding cells in fibrin gel13 and generating cell sheets without scaffold support20, 21.
The current protocol demonstrates the differentiation of human PiPSC into functional endothelial and smooth muscle cells, the generation of a bioreactor consisting of a decellularized vessel scaffold to harbor functional PiPSC-derived vascular cells, and grafting of the tissue engineered vessels into severe combined immunodeficiency (SCID) mice. PiPSC are an optimum cell type to use for tissue engineering of vessel grafts because these cells do not form tumors in mice or raise ethical and allo-immune responses. Furthermore, we have shown that the strategy for generating PiPS-endothelial cells and PiPS-smooth muscle cells is efficient and reproducible10,11. Thereafter, we designed a decellularized vessel for seeding of PiPSC-derived vascular cells to closely mimic the matrix proteins that exists within a native vessel, thus enhancing grafting and survival efficacy. Furthermore, the decellularization of the vessels prior to PiPSC seeding prevents the occurrence of inflammatory responses mounted by immune cell types such as macrophages. More importantly, this protocol does not only represent a methodology to generate promising vascular conduits for translation into humans, but also provides valuable means of studying and understanding the molecular mechanisms that govern vascular tissue regeneration through mouse models.

<table><tbody><tr><td>Human Fibroblasts CCL-153</td><td>ATCC</td><td>CCL-153</td><td>Prenatal human embryonic fibroblasts</td></tr><tr><td>ATCC F-12K Medium (Kaighn&#039;s Modification of Ham&#039;s F-12 Medium)</td><td>ATCC</td><td>30-2004</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Knockout DMEM medium optimized for embryonic stem cells</td><td>Life technologies (Gibco)</td><td>12660-012</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Life technologies (Invitrogen)</td><td>10828-028</td><td></td></tr><tr><td>Human Basic FGF-2&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-093-837</td><td></td></tr><tr><td>alpha-MEM medium</td><td>Life technologies (Invitrogen)</td><td>32571093</td><td></td></tr><tr><td>Human PDGF</td><td>R&amp;amp;D System</td><td>120-HD-001</td><td></td></tr><tr><td>Gelatin Solution 2%</td><td>Sigma</td><td>G1393</td><td></td></tr><tr><td>Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td><td>Addgene</td><td>20866</td><td></td></tr><tr><td>&amp;nbsp;PvuI Restriction Enzyme</td><td>New England Biolabs</td><td>RO150S</td><td></td></tr><tr><td>SureClean Plus</td><td>Bioline</td><td>BIO-37047</td><td></td></tr><tr><td>Nucelofection Kit (NHDF Kit)</td><td>LONZA</td><td>VPD-1001</td><td></td></tr><tr><td>Neomycin</td><td>SIGMA</td><td>G418</td><td></td></tr><tr><td>KL 1500 LCD, Illumination for Stereo Microscopy</td><td>SCHOTT</td><td>KL 1500 LCD</td><td>Cold light illumination for stereo microscopy</td></tr><tr><td>Nikon Zoom Steromicroscope SMZ800</td><td>Nikon</td><td>SMZ800</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>10% SDS Stock Solution Molecular Biology Reagent</td><td>Severn Biotech</td><td>CAS 151-21-3</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Matrigel (10mg/ml)</td><td>BD</td><td>A6661</td><td></td></tr><tr><td>Shaker IKA Vibrax with Shaking platform VX 7&amp;nbsp;</td><td>Jepson Bolton&#039;s, Janke&amp;amp;Kunkel</td><td>S32-102</td><td></td></tr><tr><td>Masterflex L/S Digital Pump Drive</td><td>Cole-Parmer</td><td>WZ-07523-80</td><td></td></tr><tr><td>Masterflex L/S 6-channel, 6-roller cartridge pump head</td><td>Cole-Parmer</td><td>EW-07519-15</td><td></td></tr><tr><td>Masterflex L/S large cartridges for pump head</td><td>Cole-Parmer</td><td>EW-07519-75</td><td></td></tr><tr><td>Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td><td>Cole-Parmer&amp;nbsp;</td><td>WZ-96410-14</td><td>Tubing goes through the peristaltic pump</td></tr><tr><td>0.5mm ID, 0.8 mm OD Silicone Tubing</td><td>SILEX</td><td>N/A</td><td>Tubings connect incubation chamber, media reservoir and compliance chamber&amp;nbsp;</td></tr><tr><td>Fitting Reducer 0.5 to 1.6, natural Polypropyline</td><td>Ibidi</td><td>10829</td><td>Adapter connect above two types of tubings</td></tr><tr><td>1/32&quot; Tubing, ID 0.01&quot; (250&amp;micro;m) Material: PEEK</td><td>LabSmith</td><td>T-132-010P</td><td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td></tr><tr><td>One-Piece Fittings&amp;nbsp;</td><td>LabSmith</td><td>T-132-100</td><td>Fix the above tubings through the incubation chamber wall</td></tr><tr><td>Nylon tubes (OD 0.9mm, ID 0.75mm)&amp;nbsp;</td><td>Smiths Medical</td><td>N/A</td><td>Tubings insert into two ends of the aorta graft</td></tr><tr><td>NOD.CB17-Prkdc&lt;sup&gt;scid&lt;/sup&gt;/NcrCrl mouse</td><td>Charles River</td><td></td><td></td></tr><tr><td>Surgical sutures, 8-0&amp;nbsp; silk</td><td>ETHICON</td><td>W819</td><td></td></tr><tr><td>Hypnorm</td><td>Vetapharm</td><td>Vm21757/4000</td><td>Neuroleptanalgesic for use in mice</td></tr><tr><td>Hypnovel (Midazolam)</td><td>Roche</td><td>59467-70-8</td><td>Induction of anaesthesia</td></tr><tr><td>Dissecting microscope</td><td>Carl Zeiss</td><td>Stemi 2000</td><td></td></tr><tr><td>Nylon Tubing</td><td>Portex LTD</td><td>800/200/100/200</td><td>0.65 mm in diameter and 1 mm in length; to make artery cuff</td></tr><tr><td>Electrocoagulator</td><td>Martin&amp;nbsp;</td><td>SN 54.131</td><td>Ligation of artery branches on aorta</td></tr><tr><td>Bipolar micro hemostat forceps</td><td>Martin</td><td>80-91-12-04</td><td>Fixation of vessel ends</td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>JFX-7</td><td></td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>JFL-3dZ</td><td></td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>D-5aZ</td><td></td></tr><tr><td>Mini applier&amp;nbsp;</td><td>AESCULAP</td><td>FE572K</td><td></td></tr><tr><td>Micro hemostats clips</td><td>AESCULAP</td><td>FE720K</td><td></td></tr><tr><td>Surgical sutures, 6-0 VICRYL</td><td>ETHICON</td><td>V489</td><td></td></tr></tbody></table>

generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

The successful generation of PiPSC was confirmed 4 days after nucleofecting human fibroblasts with a linearized pCAG2LMKOSimO plasmid carrying 4 transcription factors, OCT4, SOX2, KLF4 and c-MYC (OSKM). PiPSC displayed a markedly distinct phenotype when compared to fibroblasts (Figure 2A) and expressed the 4 reprogramming factors at mRNA (Figure 2B) and protein (Figure 2C) levels10. The efficacy of a PiPSC-based vascular graft is highly dependent on the abilit...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

generation-and-grafting-of-tissue-engineered-vessels-in-a-mouse-model

Research

JoVE Journal

Bioengineering

12.0K Views.  King's College London BHF Centre.  Hier presenteren we een protocol om tissue engineered vaartuig enten die functioneel zijn voor het enten in muizen door dubbel zaaien gedeeltelijk geïnduceerde pluripotente stamcellen genereren (PiPSC) - afgeleide gladde spiercellen en PiPSC - afgeleide endotheelcellen op een gedecellulariseerde schip steiger bioreactor. 

Video: Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Medicine

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

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 ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

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