This project received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 Research and Innovation Programme (grant agreement no. 818808). rhCollMA was generously provided by CollPlant (Rehovot, Israel). The authors thank the Technion&#39;s Pre-clinical Research Authority for the assistance with animal care, as well as Janette Zavin, Galia Ben David, and Idan Redenski.

<ol>
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V., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+tissues+and+organs.">3D bioprinting of tissues and organs.</a> Nature Biotechnology. 32 (8), 773-785 (2014).</li><li>Guillotin, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+assisted+bioprinting+of+engineered+tissue+with+high+cell+density+and+microscale+organization.">Laser assisted bioprinting of engineered tissue with high cell density and microscale organization.</a> Biomaterials. 31 (28), 7250-7256 (2010).</li><li>Catros, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+bioprinting+for+creating+on-demand+patterns+of+human+osteoprogenitor+cells+and+nano-hydroxyapatite.">Laser-assisted bioprinting for creating on-demand patterns of human osteoprogenitor cells and nano-hydroxyapatite.</a> Biofabrication. 3 (2), (2011).</li><li>Ronca, A., Ambrosio, L., Grijpma, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+designed+poly(d,l-lactide)/nanosized+hydroxyapatite+composite+structures+by+stereolithography.">Preparation of designed poly(d,l-lactide)/nanosized hydroxyapatite composite structures by stereolithography.</a> Acta Biomaterialia. 9 (4), 5989-5996 (2013).</li><li>Lan, P. X., Lee, J. W., Seol, Y. J., Cho, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+3D+PPF/DEF+scaffolds+using+micro-stereolithography+and+surface+modification.">Development of 3D PPF/DEF scaffolds using micro-stereolithography and surface modification.</a> Journal of Materials Science: Materials in Medicine. 20 (1), 271-279 (2009).</li><li>Hinton, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+printing+of+complex+biological+structures+by+freeform+reversible+embedding+of+suspended+hydrogels.">Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.</a> Science Advances. 1 (9), (2015).</li><li>Lee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+collagen+to+rebuild+components+of+the+human+heart.">3D bioprinting of collagen to rebuild components of the human heart.</a> Science. 365 (6452), 482-487 (2019).</li><li>Bhattacharjee, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Writing+in+the+granular+gel+medium.">Writing in the granular gel medium.</a> Science Advances. 1 (8), 1500655 (2015).</li><li>Szklanny, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+engineered+tissue+flaps+with+hierarchical+vessel+networks+(VesselNet)+for+direct+host-to-implant+perfusion.">3D bioprinting of engineered tissue flaps with hierarchical vessel networks (VesselNet) for direct host-to-implant perfusion.</a> Advanced Materials. 33 (42), 2102661 (2021).</li><li>Schleimer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Training+a+sophisticated+microsurgical+technique:+interposition+of+external+jugular+vein+graft+in+the+common+carotid+artery+in+rats.">Training a sophisticated microsurgical technique: interposition of external jugular vein graft in the common carotid artery in rats.</a> Journal of Visualized Experiments JoVE. (69), (2012).</li><li>Sucher, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+hind+limb+transplantation:+A+new+composite+tissue+allotransplantation+model+using+nonsuture+supermicrosurgery.">Mouse hind limb transplantation: A new composite tissue allotransplantation model using nonsuture supermicrosurgery.</a> Transplantation. 90 (12), 1374-1380 (2010).</li><li>Fensterer, T. F., Miller, C. J., Perez-Abadia, G., Maldonado, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cuff+design+to+facilitate+anastomosis+of+small+vessels+during+cervical+heterotopic+heart+transplantation+in+rats.">Novel cuff design to facilitate anastomosis of small vessels during cervical heterotopic heart transplantation in rats.</a> Comparative Medicine. 64 (4), (2014).</li><li>Novosel, E. C., Kleinhans, C., Kluger, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+is+the+key+challenge+in+tissue+engineering.">Vascularization is the key challenge in tissue engineering.</a> Advanced Drug Delivery Reviews. 63 (4-5), 300-311 (2011).</li><li>Landau, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropoelastin+coated+PLLA-PLGA+scaffolds+promote+vascular+network+formation.">Tropoelastin coated PLLA-PLGA scaffolds promote vascular network formation.</a> Biomaterials. 122, 72-82 (2017).</li><li>Szklanny, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+scaffold+system+for+studying+the+effect+of+local+geometry+and+topology+on+the+development+and+orientation+of+sprouting+blood+vessels.">High-throughput scaffold system for studying the effect of local geometry and topology on the development and orientation of sprouting blood vessels.</a> Advanced Functional Materials. 1901335, 1-13 (2019).</li><li>Song, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+transferrable+microvascular+meshes+for+subcutaneous+islet+transplantation.">Engineering transferrable microvascular meshes for subcutaneous islet transplantation.</a> Nature Communications. 10 (1), 1-12 (2019).</li><li>Luo, Y., Lode, A., Gelinsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+plotting+of+three-dimensional+hollow+fiber+scaffolds+based+on+concentrated+alginate+pastes+for+tissue+engineering.">Direct plotting of three-dimensional hollow fiber scaffolds based on concentrated alginate pastes for tissue engineering.</a> Advanced Healthcare Materials. 2 (6), 777-783 (2013).</li><li>Ying, G. 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The authors have no conflicts of interest to disclose.

Engineering vascularized tissues is one of the main challenges of tissue engineering20. Current methods for creating engineered vascular tissue focus on creating self-assembled microvasculature21,22,23 or fabricating mesoscale vascular scaffolds24,25 and not on recreating a system of hierarchical vasculature, which can be perfused immediately and directly upon implantation26. In this work, we describe a protocol that makes use of two 3D printing modalities to fabricate a hierarchical vessel network composed of microscale and mesoscale vasculatures. The protocol combines a 3D bioprinted, self-assembled microvascular network with a mesoscale vascular scaffold, achieving an implantable, vascularized flap. Furthermore, this paper presents a protocol for directly anastomosing this flap to a rat&#39;s femoral artery.
3D bioprinting has gained interest in recent years due to its versatility over traditional tissue engineering techniques. While this protocol describes the generation of a microvascular network in rhCollMA bioink, the methods used can be applied with few modifications to many other bioinks from the plethora of studied and novel bioinks and support baths27,28. We chose to use rhCollMA as a bioink due to the abundance of type I collagen in the human ECM, providing a suitable environment for cell attachment. Moreover, it is produced recombinantly in plants and further modified with methacrylate groups, which allows for photopolymerization and the formation of stable 3D hydrogels29,30. Photocrosslinking was achieved by the addition of the photoinitiator LAP, which has been shown to be non-toxic and is activated by exposure to 405 nm blue light, reducing the possible phototoxicity of UV light. However, the use of photosensitive bioinks necessitates the use of a phenol red-free culture medium for the preparation of the bioink and the support material. Furthermore, the protocol describes the use of gelatin support material, which enables the high-fidelity extrusion of bioinks such as rhCollMA. Thus, it is critical to ensure the use of cold medium during its preparation and the cooling of the printer bed. Excessive heating might occur due to the light source used for crosslinking or from elevated ambient temperatures.
An extrusion-based bioprinter has been used here to create the bioprinted microvascular network, and there are currently many commercially available bioprinters that can generate similar constructs. Moreover, the proposed methods may be easily modified and applied to study different geometries, sizes, and infill patterns. In this work, a rectilinear infill pattern was chosen to create interconnected pores, and this can be printed relatively quickly with high fidelity.
Air bubbles introduce a significant challenge in extrusion bioprinting, especially inside support materials. Therefore, it is crucial to minimize the presence and formation of these bubbles by using positive displacement pipettes for the transfer of the support material, the preparation of the bioink-cell suspension, and their transfer to the printing cartridges.
In this work, human adipose-derived endothelial cells and dental pulp stem cells were used as supporting cells due to their relatively easy isolation from patients. Moreover, a total cell concentration of 8 x 106 cells/mL was chosen since this concentration has been shown to establish the most developed vascular networks16. While this protocol can be employed to generate microvasculature using different cell types and sources, as well as different bioinks, a calibration of cell concentration must be done to establish the best conditions for the development of the microvascular network. Furthermore, tissue-specific cells (i.e., myoblasts or osteoblasts) can be incorporated within the bioink to achieve tissue-specific vascularized flaps.
The mold for the porous vascular scaffold was fabricated using 3D printed water-soluble material on a commercially available extrusion 3D printer. This results in a cost-effective technique based on rapid prototyping platforms, such that many different geometries and sizes of vascular scaffolds can be studied and screened rapidly31. Nevertheless, a limitation of this method is the resolution limit of most 3D printers32. However, with the rapidly evolving industry surrounding additive manufacturing, these limits are expected to improve over time. The use of organic solvents for the fabrication process is another limitation of the protocol, as most organic solvents are toxic to cells, preventing the ability to combine the bioprinting procedure with the vascular scaffold fabrication process.
The described method of seeding the lumen of the scaffold using aspiration as opposed to pushing the cell suspension has major effects on the localization of the seeded cells. Using negative pressure allows for endothelization of the inner lumen of the scaffold while minimizing any spilling of the cell suspension through the perforations on the scaffold&#39;s wall16.
The described &#34;cuff&#34; method for microsurgical anastomoses can be easily modified and adapted to different vascular scaffold materials or sizes, as well as to different arteries and veins in a wide scale of animal models. The adaptations to the protocol would include different polyimide tube sizes and suture sizes. This method does not require the perforation of the scaffold wall, which might lead to the development of defects. This work presents a protocol that can be expanded to many applications. The critical aspects of this protocol, which include the fabrication of meso- and microscale vasculature and their assembly and implantation, represent critical aspects of engineered flaps both for reconstructive applications, as well as vascular and other tissue engineering studies.

Severe tissue defects are caused by traumatic injuries, congenital defects, or disease, and the current gold standard for treating these defects is by using autologous grafts, vascularized tissue flaps, and microvascular free flaps as tissue substitutes. However, these options have the drawbacks of limited donor site tissue and donor site morbidity1. Thus, there is a growing demand for alternative tissue substitutes that can be used to correct these defects2. The thickness of the engineered tissue constructs is limited by the diffusion of nutrients and gases toward the cells, and, therefore, a proper vascular network is essential for generating large, thick, and properly nourished scaffolds.
Several approaches have been applied to promote the vascularization of engineered implants3, including the in vivo recruitment of vascular support from the host, the delivery of growth factors and cytokines within the scaffolds, the prevascularization of implants, the generation of a perfusable branching microvessel bed using micropatterning techniques4, the use of sacrificial materials for vascular channel/network formation5, as well as the creation of channels within 3D bioprinted constructs5,6. Vascularization of thick tissues requires the incorporation of a hierarchical vascular network consisting of macro-scale and microcapillary-scale vessels. The macro-scale vessels distribute blood effectively throughout the construct and allow for microsurgical anastomoses with the host blood vessels, while the microcapillary-scale vessels allow for nutrient diffusion.
Bioprinting has gained strong attention in recent years due to the advantages it offers over conventional tissue engineering methods. Tissues and organs are complex and intricate 3D objects with a specific architecture. 3D bioprinting, with its ability to deposit layers of biomaterials in high resolution, enables the capability of creating complex tissue and organ substitutes (e.g., kidney, lung, liver)7. Several printing technologies have been adapted for bioprinting, including extrusion-based, inkjet8, laser-assisted deposition9,10, and stereolithography-based11,12 bioprinting. The extrusion-based technologies rely on extruding the material through a nozzle by applying pressure on the material bulk surface opposite to the nozzle.
Free-form reversible embedding of suspended hydrogels (FRESH) is a bioprinting technique13,14 that uses a granular support material in which the extruded material is deposited and fixed in place by the support bath. The support bath gives mechanical support for the extruded, pre-crosslinked bioink until its crosslinking. The main advantage of this technique is that the support bath allows for extruding low-viscosity materials that cannot maintain their shape after extrusion and before crosslinking15. This expands the pool of available materials that can be used as bioinks.
This paper presents a protocol for the generation of a vascularized flap that combines microscale and mesoscale vasculatures. To achieve this, bioprinted, self-assembling, microvascular networks are generated in recombinant human collagen methacrylate (rhCollMA) hydrogel, which then connects to the interior of a larger, implantable, vascular scaffold, resulting in a fully engineered tissue flap16. To establish rapid and direct perfusion of engineered tissues, a direct microsurgical anastomosis to host vessels is required. The vascular scaffold does not have sufficient suture retention strength to be anastomosed using traditional microsurgical vessel wall suturing. Therefore, we describe a &#34;cuff&#34;17,18,19 method to achieve an anastomosis with a rat&#39;s common femoral artery. In this method, the vessel ends are secured with circumferential sutures, without the need to perforate the vessel wall.
Although the proposed protocol has been prepared to study hierarchical vasculature in the rhCollMA environment, this approach can be expanded and applied to a variety of new applications. The protocol can be applied to bioprinting various tissue-specific cells in different bioinks. Moreover, the geometry and size of the constructs can be easily modified to fit specific requirements, such as large tissue reconstruction or biological studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-Dioxane</td>
			<td>Biosolve Chemical</td>
			<td>42405</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G x 0.5&#34; blunt tip dispensing needles</td>
			<td>CML supply</td>
			<td>901-27-050</td>
			<td></td>
		</tr>
		<tr>
			<td>3cc amber syringe barrel &#38; piston set</td>
			<td>Nordson EFD</td>
			<td>7012085</td>
			<td>Amber syringes used to block light and prevent premature crosslinking</td>
		</tr>
		<tr>
			<td>5-0 AssuCryl PGA absorbale suture</td>
			<td>Assut Sutures</td>
			<td></td>
			<td>Absorbable sutures used for skin wound closure</td>
		</tr>
		<tr>
			<td>6-0 polypropelene sutures</td>
			<td>Assut Sutures</td>
			<td>9351</td>
			<td></td>
		</tr>
		<tr>
			<td>Acland clamps</td>
			<td>S&#38;T</td>
			<td>B-1V</td>
			<td></td>
		</tr>
		<tr>
			<td>Adventitia scissors</td>
			<td>S&#38;T</td>
			<td>SAS-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled no.3 jeweler&#39;s forceps</td>
			<td>S&#38;T</td>
			<td>JFAL-3-18</td>
			<td></td>
		</tr>
		<tr>
			<td>BioAssemblyBot 400 3D Bioprinter</td>
			<td>Advanced Solutions</td>
			<td></td>
			<td>a 6-axis 3D bioprinter</td>
		</tr>
		<tr>
			<td>Bovine albumin serum Probumin</td>
			<td>Millipore</td>
			<td>&#160;82-045-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>vetmarket</td>
			<td>B15100</td>
			<td></td>
		</tr>
		<tr>
			<td>BVOH filament</td>
			<td>Verbatim</td>
			<td>55903</td>
			<td>a water-soluble 1.75 mm diameter filament</td>
		</tr>
		<tr>
			<td>Clamp applying forceps</td>
			<td>S&#38;T</td>
			<td>CAF-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental pulp stem cells</td>
			<td>Lonza</td>
			<td>PT-5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dietrich bulldog clamps</td>
			<td>Fine Science Tools (FST)</td>
			<td>18039-45</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium hydrogen phosphate (Na2HPO4)</td>
			<td>Carlo Erba Reagents</td>
			<td>480141</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>S&#38;T</td>
			<td>18039-45</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose, No Phenol Red</td>
			<td>Sartorius</td>
			<td>01-053-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Duratears</td>
			<td>Alcon</td>
			<td>DJ03</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM media + bullet kit</td>
			<td>Sciencell</td>
			<td>#1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Gadot-Group</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>glutamine substitute</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Cy3 antibody</td>
			<td>Jackson</td>
			<td>115-166-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium&#160; 5,000 I.U./mL</td>
			<td>Panpharma</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Human adipose microvascular cells</td>
			<td>Sciencell</td>
			<td>#7200</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin</td>
			<td>Sigma</td>
			<td>F0895-5MG</td>
			<td>A stock concentration of 1 mg/mL</td>
		</tr>
		<tr>
			<td>Isoflurane, USP Terrell</td>
			<td>Piramal Critical Care</td>
			<td>NDC 66794-011-25</td>
			<td></td>
		</tr>
		<tr>
			<td>LifeSupport</td>
			<td>Advanced Biomatrix</td>
			<td>5244</td>
			<td>a gelatin support slurry for FRESH 3D bioprinting</td>
		</tr>
		<tr>
			<td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
			<td>Sigma-Aldrich</td>
			<td>900889</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-glucose DMEM</td>
			<td>Biological Industries</td>
			<td>01-050-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>MICROMAN E M1000E, 100-1,000 &#181;L</td>
			<td>Gilson</td>
			<td>FD10006</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-SMA antibody</td>
			<td>Dako</td>
			<td>M0851</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Gibco</td>
			<td>11140068</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools (FST)</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>ChemCruz</td>
			<td>&#160;SC-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Nystatin Solution</td>
			<td>Biological Industries</td>
			<td>03-032-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene oxide), M.W. 250,000 to 400,000</td>
			<td>Acros Organics</td>
			<td>178602500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(L-lactic acid), IV 5.0 dl/g (PLLA)</td>
			<td>Polysciencse, Inc.</td>
			<td>18582-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyimide tubing, ID: 0.0249&#34;, OD: 0.0273&#34;</td>
			<td>Cole-Parmer</td>
			<td>95820-05</td>
			<td>A thin-walled tube used to fabricate cuffs for microsurgical anastomoses</td>
		</tr>
		<tr>
			<td>Prusa I3 MK2.5 3D Printer</td>
			<td>Prusa Research</td>
			<td>http://www.prusa3d.com/</td>
			<td>a popular commercial 3D printer</td>
		</tr>
		<tr>
			<td>Resomer&#160;RG 503 H, Poly(D,L-lactide-co-glycolide) (PLGA)</td>
			<td>Evonik Industries</td>
			<td>719870</td>
			<td></td>
		</tr>
		<tr>
			<td>rhCollMA</td>
			<td>CollPlant</td>
			<td>https://collplant.com/</td>
			<td>generously provided by CollPlant (Rehovot, Israel)</td>
		</tr>
		<tr>
			<td>round-handled needle holder</td>
			<td>S&#38;T</td>
			<td>B-15-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle - #3</td>
			<td>Fine Science Tools (FST)</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>small fine straight scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Biosolve Chemical</td>
			<td>19030591</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic (NaH2PO4)</td>
			<td>Riedel-de Haen</td>
			<td>4276</td>
			<td></td>
		</tr>
		<tr>
			<td>Solidworks</td>
			<td>Dassault Systems</td>
			<td></td>
			<td>CAD software</td>
		</tr>
		<tr>
			<td>Straight no.3 jeweler&#39;s forceps</td>
			<td>S&#38;T</td>
			<td>JF-3-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight serrated forceps</td>
			<td>Fine Science Tools (FST)</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scalpel Blade No.15</td>
			<td>Swann-Morton Limited</td>
			<td>305</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>BioLab LTD</td>
			<td>57836</td>
			<td></td>
		</tr>
		<tr>
			<td>TSIM</td>
			<td>Advanced Solutions</td>
			<td></td>
			<td>3D slicing and design software for the BioAssembly Bot</td>
		</tr>
		<tr>
			<td>Vessel dilator</td>
			<td>S&#38;T</td>
			<td>D-5a.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Tivato 700 surgical microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of engineered vascular flaps using 3d printing technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

This protocol describes the fabrication of an engineered flap composed of a vascular scaffold (Figure 1Ai) and a bioprinted microvasculature (Figure 1Aii), which were assembled to achieve mesoscale and microscale vasculatures (Figure 1Aiii). Following the protocol, BVOH molds of the vascular scaffold were designed and 3D printed (Figure 1B,C). The obtained printed structures were visually inspected for small strands of BVOH, which might be found in the empty spaces of the molds (Figure 1D). These strands usually indicate incorrect material settings or that the BVOH has absorbed moisture. These strands should be removed as they might lead to incomplete filling of the mold and structural defects in the resulting vascular scaffold. Next, the molds were filled with PLLA:PLGA solution, followed by the freeze-drying process and the washing steps, as described in the protocol. The obtained PLLA:PLGA vascular scaffold was visually inspected to verify vessel wall integrity and lumen patency (Figure 1E).
A neutralized rhCollMA bioink at a concentration of 10 mg/mL was prepared and combined in a 1:1 ratio with the PEO:LAP solution. Human adipose microvascular endothelial cells labeled with Zs-Green1 and dental pulp stem cells were resuspended with the rhCollMA bioink, and the solution was loaded into a printing cartridge and onto the printer. Box shapes with a central channel with a rectilinear pattern (Figure 1D) were bioprinted inside the gelatin support bath. Following printing, the constructs were crosslinked, and the support bath was dissolved and washed. After 4 days of culture, the constructs were live-imaged to check for microvascular network self-assembly. Figure 1D shows an example of a highly developed HAMEC-ZsGreen1 microvascular network in the bioprinted construct.
Next, a fibronectin-coated vascular scaffold was inserted into the central channel of the printed construct (Figure 2A). The assembled constructs were cultured for 2 days, during which the cells contract the gel, attaching it firmly to the vascular scaffold. Then, the vascular scaffold was lined with HAMECs expressing tdTomato, according to the protocol. After 7 days of culture, the constructs were fixed and imaged. Figure 2B shows a side view of the assembled constructs where the endothelial cells in the bioprinted microvasculature are depicted in green, while the endothelial lining of the vascular scaffold is depicted in red. The image shows a green microvascular self-assembly in the bioprinted gel, while the vascular scaffold is lined with red endothelial cells. With higher magnification, sprouts originating from the red endothelial lining are seen sprouting and anastomosing with the bioprinted microvascular network (Figure 2C). Next, the constructs were stained for &#945;-smooth muscle actin (SMA) as a marker for the dental pulp stem cells. Following the immunostaining, the constructs were imaged using a laser-scanning confocal microscope (Figure 2D).
Lastly, after 7 days of culture, the engineered flaps were microsurgically anastomosed to a rat&#39;s femoral artery, as described in the protocol. A video of a representative procedure can be seen in Supplemental Video S1. Figure 2E shows a representative image of a completed anastomoses prior to clamp removal, and Figure 2F shows a representative image of the anastomoses site after clamp removal and hemostasis. There should be no bleeding visible prior to wound closure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63920/63920fig01.jpg" /> 
Figure 1: Representative images of the fabricated meso- and microscale vessels. (A) Schematic overview of the protocol&#39;s steps. Reproduced with permission16. (B) CAD design for the sacrificial mold of the vascular scaffold. (C) Side view of a representative 3D printed sacrificial mold (scale bar = 0.5 mm). (D) Top view of sacrificial mold (scale bar = 0.5 mm) (E) Representative vascular scaffold fabricated using the describe protocol (scale bar = 5 mm). (F) CAD design for the 3D bioprinted rhCollMA microvascular network. Grid lines = 1 mm. (G) Representative image of a highly developed bioprinted vascular network showing HAMEC-ZsGreen1 in green. Scale bar = 200 &#181;m. Abbreviations: CAD = computer-aided design; rhCollMA = recombinant human collagen methacrylate; HAMEC = human adipose microvascular endothelial cells; PLLA = poly-L-lactic acid; PLGA = polylactic-co-glycolic acid. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63920/63920fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63920/63920fig02v2.jpg" /> 
Figure 2: Representative images of the assembled vascularized flap. (A) A photograph of a representative assembly of bioprinted microvasculature and the vascular scaffold. Scale bar = 1 mm. (B) Side view of a representative engineered flap imaged 4 days after endothelial lining of the vascular scaffold. The bioprinted microvasculature is shown in green (HAMEC-ZsGreen1), while the endothelial lining is shown in red (HAMEC-tdTomato). Scale bar = 1 mm. (C) Representative image of anastomoses between the bioprinted vasculature in green and the endothelial lining in red. Scale bar = 200 &#181;m. (D) Representative image of immunostaining for smooth muscle actin (red), nuclei (blue), and endothelial cells (green) after 7 days of incubation. Scale bar = 0.1 mm. (E) Representative image of a completed anastomoses of the engineered flap with a rat&#39;s femoral artery before clamp removal and (F) after clamp removal. Abbreviations: HAMEC = human adipose microvascular endothelial cells; SMA = smooth muscle actin; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63920/63920fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Video S1: Representative microsurgical procedure for anastomosing the vascular scaffold to a rat&#39;s femoral artery. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63920/supplementary video 1 (1).mp4" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies at JoVE.com

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

fabrication-engineered-vascular-flaps-using-3d-printing

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

3.7K Views.  Technion-Israel Institute of Technology. Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

Video: Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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, ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk stratification in various cardiac disorders. Recently, 3D echocardiography became available in routine cardiology practice; however, high-quality image acquisition and subsequent analysis have a steep learning curve. The present article aims to guide the reader through a detailed 3D protocol by presenting tips and tricks and also by highlighting the potential pitfalls to facilitate the widespread but technically sound use of this important technique concerning the LV. First and foremost, we show the acquisition of a high-quality 3D dataset with optimal spatial and temporal resolution. Then, we present the analytical steps toward a detailed quantification of the LV by using one of the most widely applied built-in software. We will quantify LV volumes, sphericity, mass and also systolic function by measuring ejection fraction and myocardial deformation (longitudinal and circumferential strain). We will discuss and provide clinical examples about the essential scenarios where the transition from a conventional echocardiographic approach to a 3D-based quantification is highly recommended.

In this article, we provide a step-by-step acquisition and analysis protocol for the volumetric assessment and speckle-tracking analysis of the left ventricle by 3D echocardiography, particularly focusing on practical aspects that maximize the feasibility of this technique.

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

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3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting