We would like to thank the following institutions for supporting our AV loop research: Staedtler Stiftung, Dr. Fritz Erler Fonds, Else Kr&#246;ner Fesenius Stiftung, Baxter Healthcare GmbH, DFG, IZKF/ELAN/EFI/Office for Gender and Diversity, the Forschungsstiftung Medizin, Friedrich-Alexander University of Erlangen-N&#252;rnberg (FAU), AO Foundation, Manfred Roth Stiftung, Xue Hong, Hans Georg Geis Foundation, Deutscher Akademischer Austauschdienst (DAAD), Germany, and the Ministry of Higher Education and Scientific Research, Iraq. We would like to thank Stefan Fleischer, Marina Milde, Katrin K&#246;hn and Ilse Arnold-Herberth for their excellent technical support.

The Animal Care Committee of the Friedrich-Alexander University of Erlangen-N&#252;rnberg (FAU) and the Government of Middle Franconia, Germany, approved all the experiments. For the experiments, male Lewis rats with a body weight of 300 - 350 g were used.
1. The Arteriovenous Loop Model in the Rat
<ol>
	<li>Implantation Procedure (Figure 2)
	<ol>
		<li>For anesthesia use a special plastic box that is connected via tube to the isoflurane vaporizer and closed by a lid. Turn on supply gas and flow meter between 0.8 - 1.5 L/min.</li>
		<li>Place the rat in the induction plastic box and seal the top. Turn on isoflurane vaporizer to 5%.</li>
		<li>Carefully observe the rat during induction of anesthesia. After 3 - 4 min, the rat will be anesthetized.</li>
		<li>Confirm proper anesthesia: loss of righting reflex, loss of palpebral reflex, withdrawal reflex, positive corneal reflex.</li>
		<li>Remove the rat from the box and check its weight for calculation of drugs. Apply eye ointment to prevent dryness while under anesthesia.</li>
		<li>Administer pain medication and antibiotics (e.g., 7.5 mg/kg enrofloxacin subcutaneously (s. c.), 12.5 mg/kg tramadol and 100 mg/kg metamizole both intravenously (i. v.)). Administer weight-adapted crystalloids during operation (e.g., 30 ml/kg s. c.).</li>
		<li>Place the rat on its back on a warming plate at 37 &#176;C under anesthesia with 1 - 2% isoflurane inhalation administered via mask.</li>
		<li>Monitor anesthesia properly and increase isoflurane if anesthesia level is too low (movement of the rat, response to pain, jaw tone, no loss of reflexes (see 1.1.4.), heart rate increasing). Be careful not to overdose isoflurane (loss of corneal reflexes, high heart rate, decrease in oxygen saturation). Use a special pulse oximetry for small animals for checking the oxygen saturation (95 - 100%) and heart rate (250 - 450/min) of the rat. During the operation monitor temperature of the rat (36 - 40 &#176;C) and adjust temperature of the warming plate if necessary.</li>
		<li>Shave the inner sides of the hind limbs with an electric razor and disinfect the area with antiseptics. Spread the hind limbs and fix them with adhesive tape.</li>
		<li>Lay the rat under a surgical microscope and cover the rat with sterile draping. Ensure that the whole operation procedure is performed under sterile conditions.</li>
		<li>Open the skin in the middle of the left thigh with a longitudinal incision from the upper knee to the groin using a scalpel (No. 10).</li>
		<li>Cut the subcutaneous tissue and fascia in layers of approximately 3 cm in length using dissecting scissors and microforceps until the femoral vascular bundle is exposed from the pelvic artery in the groin to the bifurcation of the femoral artery in the knee.</li>
		<li>Separate the vessels and remove the adventitia using adventitia scissors and microforceps.</li>
		<li>Coagulate the side branches using electric coagulation. Cover the operation field with a damp compress.</li>
		<li>Open the skin on the right side as described for the left side, 1.1.11 - 1.1.14.</li>
		<li>For harvesting the venous graft, ligate the right femoral vein by electric coagulation on the proximal and distal ends at a distance of 1 - 1.5 cm.</li>
		<li>Remove the venous graft with microforceps and flush the venous graft with a heparin solution (50 IU/ml in 0.9% sodium chloride solution) using an irrigation cannula and transfer it to the left thigh. Cover the operation field at the right side with a damp compress.</li>
		<li>Ligate the femoral vein on the left side proximally at the inguinal region with a microvessel clamp. Coagulate the femoral vein distally by electric coagulation, at the upper knee before branching, at a distance of about 2 cm.</li>
		<li>For anastomosis connect the proximal end of the venous graft with the proximal end of the vein by end-to-end anastomosis with an 11-0 suture. Use about 8 interrupted sutures. Begin with placing the first two sutures at the 12 o&#8217;clock and 6 o&#8217;clock positions. Then, put in 2 to 3 more sutures between these points on the front side and then put 2 to 3 more sutures into the back side.</li>
		<li>Ligate the femoral artery in the same way described for the femoral vein (1.1.18.). Make sure the loop vessels are not twisted. Anastomose the distal end of the venous graft with the proximal end of the artery as described for the femoral vein (steps 1.1.19.).</li>
		<li>Administer 25 IU heparin intravenously. Make again sure the loop vessels are not twisted, Remove the clamps and check for leakage and patency of the loop for about 5 min.</li>
		<li>Dribble papaverine (e.g., 4 mg/ml) on the vessels to prevent vascular spasms. If there is patency, the loop expands and the pulse of the artery can be observed.</li>
		<li>Prefill the implantation chamber with the first half (about 500 &#181;l) of the matrix (e.g., a hydrogel or a bone matrix with or without cells). Embed the loop into the implantation chamber.</li>
		<li>Fill the implantation chamber with the second half of the matrix to a total volume of 1,000 &#181;l. Seal the chamber with the chamber lid.</li>
		<li>Fix the implantation chamber on the thigh with a non-absorbable 6-0 suture. Fix the chamber lid with a non-absorbable 6-0 suture. Stop possible bleeding with electric coagulation.</li>
		<li>Close the skin with absorbable 4-0 sutures. Cover the wound with aluminum spray.</li>
		<li>Administer antibiotics and analgesics (e.g., 7.5 mg/kg enrofloxacin s. c., tramadol 12.5 mg/kg per os (p. o.) (through drinking water) for 3 - 5 days and afterwards depending on the behavior of the rat).</li>
		<li>Turn the vaporizer off and allow the rat to breathe supply gas until it begins to awaken.</li>
		<li>Place the rat in a box with thermal support and observe it carefully until fully recovered.</li>
		<li>Do not leave the rat unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
		<li>Do not return the rat to the company of other animals until fully recovered.</li>
	</ol>
	</li>
	<li>Explantation Procedure
	<ol>
		<li>Perform the explantation after a short-time or long-time implantation (according to the study design, for examples please see References 27-32). Anesthetize the rat according to steps 1.1.1.-1.1.9.</li>
		<li>Open the skin of the abdomen with a scalpel (No. 10) and move the intestines aside with a compress. Expose the abdominal aorta and the vena cava caudalis using cotton swabs.</li>
		<li>Cannulate the abdominal aorta (e.g., 24 gauge plastic cannula) and cut the vena cava caudalis with dissecting scissors.</li>
		<li>Flush the abdominal aorta with 0.9% sodium chloride solution containing 100 IU/ml heparin until the leaking fluid is clear. Perfuse the aorta with 30 ml perfusion solution (e.g., contrast agent or India ink).</li>
		<li>Euthanize the rat by injection of a lethal dose of embutramide, mebezonium iodide, tetracaine hydrochloride injectable solution (0.1 - 0.2 ml/kg) in deep anesthesia.</li>
		<li>Ligate the abdominal aorta and vena cava caudalis with a non-absorbable 4-0 suture.</li>
		<li>Close the open wound area with 1 - 2 clamps. Store the rat for 24 hr at 4 &#176;C (curing of the perfusion solution).</li>
		<li>Place the rat on its back. Spread the hind limbs and fix them with adhesive tape.</li>
		<li>Open the skin above the chamber with a longitudinal incision with a scalpel (No. 10).</li>
		<li>Remove the connective tissue from the chamber and the loop pedicle using dissecting scissors and microforceps. Cut the loop pedicle at a distance of 1 cm from the chamber opening with dissecting scissors and remove the chamber.</li>
		<li>Open the lid of the chamber and carefully remove the construct from the chamber with forceps and fix it in 4% buffered formalin solution for 24 hr at room temperature. Afterwards use the construct for 3D micro-computed tomography or paraffin-embedded for histological analysis30.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

For over a decade, we have successfully used the arteriovenous (AV) loop for tissue engineering purposes and studying angiogenesis in vivo in the small animal model. We could demonstrate that this microsurgical model is very well suited for engineering different tissues and that it can also be used for angiogenesis or antiangiogenesis studies.
Significance of the Technique with Respect to Existing/Alternative Methods 
Engineered tissues or organs require a functional blood vessel network to supply the nutrients and oxygen they need for their survival and successful integration after transplantation into the defect site41. A number of different prevascularization strategies have been developed over the past decades, which can be differentiated according to in vitro vs. in vivo and extrinsic vs. intrinsic approaches.
Scaffolds can be fabricated with tubular-shaped structures and seeded with vascular cells such as endothelial cells or progenitor cells in vitro42. On the other hand, for in vivo prevascularization, scaffolds are implanted in a highly vascularized area such as subcutaneous or muscle tissue21. Afterwards, these extrinsically vascularized constructs can be transplanted into the defect site. However, the drawback of these approaches is the lack of microsurgical connection with the recipient&#39;s vessels after transplantation. Particularly in the case of large-scale constructs, immediate connection to the host vasculature is essential for immediate supply of the engineered tissue43. The obvious and most promising solution to this problem lies in the generation of an intrinsically vascularized tissue or organ by a vascular axis, such as the AV loop model.
Besides using the AV loop method as described above, axial vascularization can also be induced by using AV bundles instead44 or only one vessel such as the epigastric artery45. However, in several publications the AV loop model proved to be superior with regard to degree of vascularization and the amount of de novo tissue formation. Tanaka et al. compared both methodological approaches and observed significantly higher tissue formation and a greater degree of developing capillaries in the loop compared to the bundle group46. Dong et al. also conducted a study using the AV loop or AV bundle approaches for bone tissue engineering in a rabbit model, which likewise showed significantly higher vascular density in the loop compared to the bundle group47. We were able to confirm these results as well as show in a previous study that the AV loop model has a higher capacity for angiogenesis48.
To the best of our knowledge, there is no comparable model for analyzing vascularization in vivo in an isolated and well-characterized environment. Therefore, the AV loop model represents a powerful tool for evaluating how different cell types or growth factors contribute to vessel network formation or vascularization processes in different tissues without disturbances from surrounding structures, such as invading cells or growth factors.
Limitations of the Technique 
However, one significant challenge of the proposed model is the high complexity of the surgery. For one, the treatment of defects using the AV loop model requires a two-step procedure - prevascularization of the scaffold and transplantation into the defect site. This means that the patient has to undergo two surgeries. In addition, microsurgical skills are a necessary prerequisite for successful anastomosing submillimeter vessels49. Therefore, the AV bundle is sometimes considered more useful for clinical application since it also offers promising, although less, potential for angiogenesis and tissue generation compared to the AV loop46. However, this operation can be learned step-by-step even by non-surgeons, using small caliber silicon tubes for the training in the beginning and afterwards the vessels of dead animals (e.g., chicken legs) before doing the AV loop operation in a live animal. In contrast, most practiced micro-surgeons can perform this operation with only a short time of training.
Critical Steps within the Protocol 
In general, due to the small caliber of the vessels there is the risk of thrombus formation and closure of the loop vessels. However, in the rat model 80%-100% of the loops on average were patent using only short-time heparin anticoagulation post-surgery28,30,31,34,38,39.
Furthermore, due to the high complexity of the surgery it will take a couple of hours (depending on the expertise of the surgeon). It is essential to check proper anesthesia of animals during the whole operation and to adequately supply infusion for maintaining an adequate blood pressure. During the postoperative period it is of high importance to check the health of the animal several times, to administer analgesics/antibiotics and to check the operation wound. Since in most cases implantation of an isolated chamber is performed, it is possible that infection in the inner of the chamber occurs without noticing. Therefore, it is very important to maintain sterility during the whole operation and administration of antibiotics should carefully be done over a period of 3 - 5 days.
Modifications of the Protocol 
The chamber can be individually adjusted to the size and shape of the defect. Furthermore, also membranes can be used for enclosing the AV loop as performed by Manasseri et al.50. In addition, the scaffold, supplemented cells and growth factors can be chosen according to the different tissue types. Recently, Miomas et al. combined gene therapeutic approaches successfully with the AV loop model and could induce enhancement of vessel growth by transduction with VEGF165 51. Recently, we adjusted the rat AV loop model for muscle tissue engineering purposes. Instead of the femoral vessels, the epigastric vein and saphenous artery were used, which enabled implantation of the obturator nerve in the axially vascularized scaffold for motoric innervation (&#34;EPI loop model&#34;)36. Besides implantation of a motoric nerve, the neurotization of bone tissue engineered constructs with sensory nerves is reported to be beneficial for enhanced osteogenesis and better repair of bone defects52. The AV loop induces minimal donor site morbidity and can be created at various sites of the body52. It would be possible to use superficial vessels at other sites of the body for generation of the AV loop or even to use other animals such as the rabbit or the mouse model.
Future Applications or Directions after Mastering this Technique 
Recently, our working group implanted a well-characterized murine embryonal endothelial progenitor cell (EPC) line (T17b) expressing the guanylate binding protein-1 (GBP-1) - a marker and intracellular inhibitor of endothelial cell functions such as proliferation, migration and invasion - in the rat AV loop model. The antiangiogenic capacity of differentiated GBP-1-EPC could be demonstrated by a significant reduction of blood vessel density in the AV loop constructs. With regard to clinical application, the proinflammatory antiangiogenic GTPase GBP-1 could open up new avenues of antiangiogenic therapies, e.g., for cancer or other diseases53. Based on this study, it is conceivable that the AV loop model can be used for establishing a pathological vascularization network for further analysis and possible modulation. For example, this model provides an optimal opportunity to gain a better understanding of tumor angiogenesis, its influencing factors and the exact role of the different cells involved in tumor vessel network formation such as EPCs, tumor cells and stem cells54. In vivo cancer models are often carried out in genetically modified mice to simulate the processes and growth characteristics of different human cancer types and have proven to be excellent for drug development and preclinical trials55. Furthermore, there are xenograft models for transplanting tumors into experimental animals such as immunocompromised mice56. A more clinically related approach involves transplantation from the patient&#39;s tumor, known as &#34;personalized mouse models&#34; or &#34;patient-derived tumor xenografts models&#34;57. However, these models are not practical for studying the influence of one single cell source or growth factor without effects from the surrounding tissue.
The AV loop model makes it possible to use tissue engineering methods to study tumor biology. This is defined as &#34;tumor engineering&#34; by Ghajar et al. and involves &#34;the construction of complex culture models that recapitulate aspects of the in vivo tumor microenvironment to study the dynamics of tumor development, progression, and therapy on multiple scales&#34;58. A tumor environment can be constructed within the isolated implantation chamber, which allows a precise analysis of cell-cell interactions, angiogenesis, modulation, enhancement and inhibition. Furthermore, the AV loop model may prove beneficial for developing or validating therapies concerning the interruption of neoangiogenesis or the inhibition of tumor growth.
Using this approach for inducing vascularization, it is possible to engineer tissues in a clinically relevant size. In further studies, we were able to generate axially vascularized bone tissue for transplantation with a significant volume of about 15 cm&#179; in a relatively short time of 12 weeks59,60. In order to translate these findings to clinical practice, a proof of principle study using the tibia defect model will be performed in the near future prior for application in humans. As a first step, we could successfully demonstrate in situ bone tissue engineering in a large volume defect in a clinical scenario with long-term stability61. Applying the described AV loop model makes it is possible to provide a therapy tailored to the individual patient&#39;s requirements. Based on our results the idea of the human body itself serving as a living bioreactor still holds great promise for the future.

Most tissues and organs in the human body are dependent on a functional blood vessel network that supplies nutrients, exchanges gases and removes waste products. Malfunction of this system caused by local or systemic vascular problems can lead to a multitude of severe diseases. Moreover, in research areas such as tissue engineering or regenerative medicine, a functional blood vessel network within artificially generated tissues or transplanted organs is indispensable for successful clinical application.
For decades researchers have been investigating the exact mechanisms involved in the growing vasculature to gain deeper insight into pathological situations in order to find novel therapeutic interventions and provide better prevention of vascular disorders. In the first step, basic processes such as cell-cell interactions or the effect of molecules on cells of the vascular system are usually investigated by in vitro 2D or 3D experiments. Traditional 2D models are easy to perform, are well established and have contributed greatly to a better understanding of these processes. For the first time in 1980, Folkman et al. reported in vitro angiogenesis seeding of capillary endothelial cells on gelatin coated plates1. This immediately gave way to publication of a multitude of further 2D angiogenesis experiments on endothelial cell tube formation assay2, migration assay3 and the co-culturing of different cell types4, as well as others. These assays are still used today and accepted as standard in vitro methods.
However, this experimental setup is not always appropriate for the study of in vivo cell behavior since most cell types require a 3D environment to form relevant physiological tissue structures5. It could be shown that the architecture of the 3D matrix is decisive for capillary morphogenesis6 and that cell-extra cellular matrix (ECM) interactions and 3D culture conditions regulate important factors involved in tumor angiogenesis7. The 3D matrix provides complex mechanical inputs, can bind effector proteins and establish tissue-scale solute concentration gradients. Moreover, it is considered necessary in order to imitate in vivo morphogenetic and remodeling steps in complex tissues5. In these systems, both angiogenesis and vasculogenesis can be studied. While angiogenesis describes the sprouting of capillaries from preexisting blood vessels8, vasculogenesis refers to the de novo formation of blood vessels through endothelial cells or their progenitors9,10. Maturation of vessels is described in a process called &#39;arteriogenesis&#39; via recruitment of smooth muscle cells11. A typical angiogenic in vitro model is the sprouting of endothelial cells from existing monolayers seeded as a monolayer on gel surfaces, on surface of microspheres embedded within a gel or by building endothelial cell spheroids12. In vasculogenic models single endothelial cells are entrapped in a 3D gel. They interact with adjacent endothelial cells to form vascular structures and networks de novo, typically in combination with supportive cells12.
However, even complex 3D in vitro models cannot mimic in vivo settings completely given the multitude of cell-cell and cell-ECM interactions13. Substances with high in vitro activity do not automatically show the same effects in vivo and vice versa14. For a comprehensive analysis of vascularization processes there is an urgent need to develop in vivo models that better simulate the situation in the body. A large range of in vivo angiogenesis assays are described in the literature, including the chick chorioallantoic membrane assay (CAM), the zebrafish model, the corneal angiogenesis assay, the dorsal air sac model, the dorsal skinfold chamber, the subcutaneous tumor models14. However, these assays are often associated with limitations, such as rapid morphological changes, problems in distinguishing new capillaries from already existing ones in the CAM assay, or the limited space in the corneal angiogenesis assay15. Furthermore, non-mammalian systems are used (e.g., the zebrafish model16), which leads to problems in xenotransplantation17. In the subcutaneous tumor model, angiogenesis originating only from the tumor itself cannot be analyzed since the adjacent tissue greatly contributes to the vascularization process. Moreover, the surrounding tissue can have a decisive role in shaping the tumor microenvironment18.
Not only for studying angiogenesis or vasculogenesis is there a strong need for a standardized and well-characterized in vivo model but also for studying different vascularization strategies in tissue engineering and regenerative medicine. Today, the generation of complex artificial organs or tissues is possible both in vitro and in vivo. 3D bioprinting provides an on-demand fabrication technique for generating complex 3D functional living tissues19. Furthermore, bioreactors can be used for generating tissues20 or even the own body can be used as bioreactor21. However, the main barrier to successful application of artificially generated tissues is the lack of vascularization within the engineered constructs. Immediate connection to the host&#39;s vasculature after transplantation is a major prerequisite for survival, especially in the case of large-scale artificial tissues or organs.
Different in vitro or in vivo prevascularization strategies were developed to establish a functional microvasculature in constructs prior to implantation22. The implantation of a scaffold with in vitro preformed engineered capillaries onto the dorsal skin of mice led to rapid anastomosis of the mice vasculature within a day23. In contrast, a spheroid co-culture consisting of human mesenchymal stem cells and human umbilical vein endothelial cells assembled into a three-dimensional prevascular network developed further after in vivo implantation. However, anastomosis with the host vasculature was limited24. Above all, in poorly vascularized defects, such as necrotic or irradiated areas, this so-called extrinsic vascularization &#8212; the ingrowth of vessels from the surrounding area into the scaffold &#8212; often fails. Intrinsic vascularization, on the other hand, is based on a vascular axis as a source of new capillaries sprouting into the scaffold25. Using the axial vascularization approach, the engineered tissue can be transplanted with its vascular axis and connected to local vessels at the recipient site. Immediately after transplantation, the tissue is adequately supported by oxygen and nutrients, which creates the right conditions for optimal integration.
Due to the limited availability of models for investigating in vivo angiogenesis and in recognition of the growing importance of generating axially vascularized tissue, we developed the microsurgical approach of Erol and Spira further to generate an arteriovenous (AV) loop in the animal model26. The use of a completely closed implantation chamber makes this method very well suited to study blood vessel formation under &#34;controlled&#34;, well characterized in vivo conditions (Figure 1). This model is not only useful for the investigation of angiogenesis but is also optimally suited for the axial vascularization of scaffolds for tissue engineering purposes.

<table><tbody><tr><td>0.9% sodium chloride</td><td>Berlin-Chemie AG</td><td>34592508</td><td></td></tr><tr><td>11-0 Ethilon / polyamide 6/6</td><td>Ethicon</td><td>EH7438G</td><td></td></tr><tr><td>4-0 Vicryl / polygalactin 910</td><td>Ethicon</td><td>V392H</td><td></td></tr><tr><td>6-0 Prolene / polypropylene</td><td>Ethicon</td><td>8695H</td><td></td></tr><tr><td>aluminium spray</td><td>Pharma Partner Vertriebs-GmbH</td><td>1020</td><td></td></tr><tr><td>antiseptics&amp;nbsp;</td><td>BODE Chemie GmbH&amp;nbsp;</td><td></td><td></td></tr><tr><td>Catheter&amp;nbsp;</td><td>B Braun Meslungen AG</td><td>4251612-02</td><td></td></tr><tr><td>contrast agent</td><td>Flowtech</td><td>&amp;nbsp;MV-122</td><td></td></tr><tr><td>embutramide, mebezonium iodide, tetracaine hydrochloride injectable solution&amp;nbsp;</td><td>Intervet International GmbH</td><td></td><td></td></tr><tr><td>encre de chine intense Indian ink</td><td>Lefranc &amp;amp; Bourgeois</td><td></td><td></td></tr><tr><td>Enrofloxacin</td><td>Bayer AG</td><td></td><td></td></tr><tr><td>eye ointment</td><td>Bayer AG</td><td></td><td></td></tr><tr><td>Formalin 4%</td><td>Carl Roth GmbH &amp;amp; Co. KG</td><td>P087.4</td><td></td></tr><tr><td>Heparin</td><td>Ratiopharm GmbH</td><td></td><td></td></tr><tr><td>isoflurane&amp;nbsp;</td><td>Abbott Laboratories</td><td>6055482</td><td></td></tr><tr><td>Lewis rat, male</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>Metamizol-Natrium&amp;nbsp;</td><td>Ratiopharm GmbH</td><td></td><td></td></tr><tr><td>papaverine / Paveron N</td><td>Linden Arzneimittel-Vertrieb-GmbH</td><td></td><td></td></tr><tr><td>tramadol / Tramal</td><td>Gr&amp;uuml;nenthal GmbH</td><td></td><td></td></tr></tbody></table>

the arteriovenous (av) loop in a small animal model to study angiogenesis and vascularized tissue engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

Tissue Engineering 
For bone tissue engineering purposes, a number of different bone substitutes were implanted in the small animal rat AV loop model27,28,33,34. Vascularization could perfectly be demonstrated by 3D micro-computed tomography (micro-CT) (Figure 3A). Vascularization of a processed bovine cancellous bone (PBCB) matrix was significantly higher in the loop group compared to the group without vascularization. A constantly growing and maturating blood vessel network developed within the implantation chamber over 8 weeks. Between 4 and 8 weeks, continuous growth of the vascularized tissue towards the center of the constructs was observed, whereas no increase was detected in the non-vascularized group33. Prevascularization of the PBCB matrix for 6 weeks in the AV loop model led to superior survival of the injected osteoblasts compared to the control osteoblasts. In contrast to the control groups, expression of bone-specific genes was detected in the AV loop group with implanted osteoblasts28. As a further matrix, sintered bioactive glass together with fibrin gel was implanted in the AV loops of the rats. After 3 weeks, a dense network of newly formed vessels has developed demonstrated by micro-CT and histology27.
The implantation chamber was modified in order to accelerate scaffold vascularization. By using a perforated titanium chamber, intrinsic vascularization was supported by extrinsic vessels from the surrounding tissue. At just 2 weeks after implantation of a &#946;-tricalciumphosphate hydroxyapatite (&#946;-TCP/HA)/fibrin matrix, 83% of the vessels were connected to the AV loop with continuous increase over time and reached 97% connection after 8 weeks34. With the implantation of 5 x 106 bone marrow derived mesenchymal stem cells (MSC) and bone morphogenetic protein 2 (BMP-2), a significant increase in bone formation compared to the BMP-2 or MSC alone groups could be induced. At 6 and 12 weeks, the fibrin matrix was completely degraded and replaced by highly vascularized connective tissue in all groups (Figure 3B 6 week implantation). There was a significant decrease in vessel number in the BMP-2/MSC group between 6 and 12 weeks and after 12 weeks in the other groups. This was probably due to maturation of the vascular network or the compact arrangement of bone structures leading to a limited vascular network formation32.
Besides bone, other tissues such as muscle or liver can also be engineered in the AV loop model.
For engineering axially vascularized muscle tissue, experiments with primary myoblasts in an AV loop fibrin matrix were carried out. After a prevascularization time of 2 weeks for 2, 4, and 8 weeks, 1 x 106 myoblasts were transplanted into the AV loop chamber. Transplanted myoblasts could be redetected even after 8 weeks using carboxyfluorescein diacetate succinimidyl ester (CFDA) labeling. The cells kept their myogenic phenotype within the fibrin matrix and expression of the muscle-specific markers MEF-2 and desmin was positive after 4 weeks. However, myogenic marker gene expression was negative after 8 weeks, which was probably due to the absence of myogenic stimuli and rapid absorption of the fibrin matrix35. To increase myogenic stimulation, a new modification of the rat AV loop was developed using the epigastric vein instead of the saphenous vein in order to achieve a more proximal positioning of the isolation chamber. Hence, additional incorporation of the obturator motor nerve was geometrically facilitated. By using this AV loop modification, which is referred to as the EPI loop, we could show myogenic differentiation of co-implanted myoblasts and MSC36.
For hepatic tissue engineering 4 x 106 pkh-26 labeled fetal liver cells were transplanted within a fibrin matrix in the rat AV loop model for 2 weeks. In the control group, matrices without an AV loop and cell-free matrices were implanted. Functional capillaries arose from the AV loop vessels and highly vascularized neo-tissue was observed within the chamber after 14 days of implantation, as shown by CD31 staining and India ink labeling. There was no difference between the cell-free and hepatocyte AV loop group. The AV loop vascularized the fibrin matrix densely and viable fetal cells could be detected after explantation by positive pkh-26 staining and liver cell-specific cytokeratin 18 (CK-18) immunohistology mainly in the proximity of the major vascular axis. mRNA levels of CK-18 were elevated in the AV loop cell group. In contrast, no CK-18 expression could be detected in constructs without a loop or cells37.
Angiogenesis Studies 
The AV loop consists of three segments: the vein, the arterial graft and the interpositional venous graft (IVG) segment (Figure 1). Three-dimensional evaluation of the vascular system demonstrated that newly formed vessels originated both from the venous and arterial portion as well as from the venous interponate. A great number of newly formed vessels were observed from the IVG33. With in vivo MRA, scanning electron microscopy of corrosion casts and immune histology, the onset of angiogenesis in a fibrin matrix was observed between day 10 and 14. Above all, the venous and IVG segments gave rise to many capillaries and larger vessels. A gradual reduction in luminal caliber as a sign of arterialization of the IVG due to the increase in endovascular pressure and shear stress was detected from day 7 on38. In further studies, it could be confirmed that vascular sprouting mainly takes place at the non-arterial graft39.
The exact analysis of angiogenesis processes and the stimulation and inhibition of blood vessel formation could be visualized in the AV loop implantation chamber. The growth factors vascular endothelial growth factor A (VEGFA) and basic fibroblast growth factor (bFGF) induced a higher absolute and relative vascular density and faster resorption of the fibrin matrix compared to the growth factor-free control group31. Further, remodeling phenomena and maturation of the vascular network within the isolation chamber were visualized over an implantation period of 8 weeks. In AV loop chambers processes of intercapillary interconnection and intussusceptive angiogenesis as well as possible lymphatic growth were identified immunohistologically as parameters of neovascular maturation39. By applying the PHD (prolyl hydroxylase domain) inhibitor DMOG (dimethyloxallyl glycine) systemically in rats, it could be shown that the concentration of the hypoxia-inducible factor alpha (HIF-&#945;) correlates with the growing vascularization in the AV loop and is a stimulus for vessel outgrowth40.
<img alt="Figure 1" src="/files/ftp_upload/54676/54676fig1.jpg" /> 
Figure 1: Scheme of an AV Loop in the Rat Model. The AV loop consists of three segments: the vein (V), the arterial (A) graft and the interpositional venous graft (IVG) segment. The AV loop can be embedded into a closed implantation chamber (C) for induction of intrinsic vascularization. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54676/54676fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54676/54676fig2.jpg" /> 
Figure 2: AV Loop Operation in the Rat. (A): Localization of the femoral bundle on the inner side of the hind limbs of the rat. (B/C): Preparation of the femoral vascular bundle in the left and right groin of the rat. The vessels are separated (D), the interpositional vein graft is harvested from the right side (E) and anastomosed with the femoral vein (F) and femoral artery of the left side into an AV loop (G, arrow indicates the anastomoses). The loop vessels are transferred into the implantation chamber prefilled with a matrix (H) and after complete filling (I) the lid is closed (J). A = femoral artery, V = femoral vein, N = femoral nerve, IVG = interpositional venous graft. Scale bar 5 mm (D-J). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54676/54676fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54676/54676fig3.jpg" /> 
Figure 3: Visualization of Vascularization in the Rat AV Loop Model. (A): Micro-CT after perfusion with a contrast agent (yellow perfused vessels). (B): Hematoxylin-Eosin staining of a &#946;-TCP/HA bone substitute with MSC implanted in the AV loop model rat for 6 weeks. The AV loop vessels are perfused with India ink (black color). Scale bar 1 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54676/54676fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering at JoVE.com

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.5K Views.  University Hospital of Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg (FAU).  We beschrijven een microchirurgische benadering voor het genereren van een arterioveneuze (AV) -lus als model voor het analyseren van vascularisatie in vivo in een geïsoleerd en goed gekarakteriseerd omgeving. Dit model is niet alleen nuttig voor het onderzoek naar angiogenese, maar ook optimaal geschikt voor engineering axiaal gevasculariseerd en transplantable weefsels. 

Video: The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

Developmental Biology

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

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 ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Neuroscience

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Medicine

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

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