The authors acknowledge&#160;the National Cancer Institute of the National Institutes of Health Pathway to Independence Award (K99CA201603).

The neonatal rat cardiomyocytes used in this protocol were isolated from 2-day-old Sprague-Dawley rats following a well-established procedure56 approved by the Institutional Animal Care and Use Committee at the Brigham and Women&#39;s Hospital.
1. Instrumentation of the Bioprinter
<ol>
	<li>Insert a smaller blunt needle (e.g., 27G, 1 inch) as the core into the center of a larger blunt needle (e.g., 18G, &#189; inch) as the sheath to construct the dual-layer, concentric microfluidic printhead; make sure that the core needle is protruding slightly (~1 mm) longer than the outer shell (Figure 1A). The alignment is usually adjusted manually, but if necessary spacers of proper sizes can be temporally sandwiched in between the inner/outer needles at both the tip and the barrel sides to assist concentric alignment. Seal the junction of the barrels with epoxy glue and remove the alignment spacers from the tip side when applicable.</li>
	<li>Insert another needle (23G) in the barrel of the central needle in the reverse direction.&#160; Then generate a hole on the side of the barrel of the outer needle and insert a metal connector of matching size into the hole, followed by sealing with epoxy glue.</li>
	<li>Connect the inlets of the printhead to a dual-channel syringe pump for injection of the bioink and the crosslinking solution, individually, through two PVC tubes. Mount the extruder onto the head of a bioprinter using a plastic holder made of poly(methyl methacrylate) (PMMA). 
	NOTE: Bioprinter selection depends on availability. In our case, we have successfully tested this setup on several commercially available bioprinters. However, any bioprinter that features an x-y-z motorized stage should, in principle, enable integration of this microfluidic printhead.</li>
</ol>
2. Bioprinting the Microfibrous Vascular Bed
<ol>
	<li>Make the bioink using a mixture of alginate (4 w/v%, low viscosity), gelatin methacryloyl (GelMA, 1-2 w/v%)57,58, and photoinitiator Irgacure 2959 (0.2 - 0.5 wt.%) dissolved in 25 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethane sulfonic acid (HEPES buffer, pH 7.4) containing 10 vol.% fetal bovine serum (FBS).</li>
	<li>Make a solution of 0.3-M CaCl2 in HEPES buffer containing 10 vol.% FBS as the crosslinking carrier fluid.</li>
	<li>Immediately before bioprinting, dissociate human umbilical vein endothelial cells (HUVECs) from the flasks using treatment by 0.05 w/v% trypsin for 5 - 10 min, and resuspend the cells in the bioink at a concentration of 5 - 10 &#215; 106 cells/mL. Pipette the suspension slowly 5 to 10 times to ensure homogenous distribution.</li>
	<li>Start the injection of the bioink/crosslinking fluid using a dual-channel syringe pump at the same flow rates of 5 &#181;L/mL. The flows can be allowed to continuously run for up to 1 min until they stabilize. Subsequently, initiate the movement of the printhead by controlling the bioprinter at a deposition speed of approximately 4 mm/s (Figure 1B). These speeds may need fine tuning with each new setup to ensure optimal bioprinting. The bioprinting process is usually conducted at room temperature (21 - 25 &#176;C) but this temperature may be altered. The bioprinting process should allow for fast ionic gelation of the alginate component and deposition of a microfibrous scaffold (Figure 1B).</li>
	<li>After the scaffold is bioprinted, achieve chemical gelation by further photocrosslinking the GelMA component, at approximately 5 - 10 mW/cm2 of UV light (360 - 480 nm) for 20 - 30 s (Figure 1C).</li>
	<li>Following the bioprinting and crosslinking, gently rinse the scaffold with phosphate-buffered saline (PBS) to remove the excess CaCl2. Culture the HUVECs-laden microfibrous scaffold in endothelial cell growth medium (EGM) in an incubator at 37 &#176;C and 5 vol.% CO2 for up to 16 days with medium changed at least every 2 days. Monitor the morphologies of the HUVECs under a microscope during the culture period.</li>
</ol>
3. Constructing the Vascularized Tissues
<ol>
	<li>Once the HUVECs have migrated to the peripheries of the microfibers in the scaffold to form the lumen-like structures (Figure 1D), retrieve the scaffold and gently place it on the surface of a hydrophobic surface (e.g., a slab of polymethylsiloxane [PDMS]). Use a piece of sterile filter paper to carefully remove all the medium from the interstitial space of the scaffold with capillary force.</li>
	<li>Immediately add a drop (approximately 20 - 40 &#181;L) of suspension of a secondary cell type (e.g., cardiomyocytes) in medium at a density of 1 - 10 &#215; 106 cells/mL on top of the scaffold, which should infiltrate the entire interstitial space of the scaffold (Figure 1E). Incubate such a configuration in an incubator (37 &#176;C, 5 vol.% CO2, 95% relative humidity) for 0.5 - 2 h to allow the cells to adhere onto the individual microfibers. Monitor the droplet size over the period to ensure that no noticeable evaporation is observed.</li>
	<li>Gently wash the scaffolds by shaking in a PBS bath to remove any non-adherent cells and culture the construct in relevant medium until the desired vascularized tissue is formed.</li>
</ol>

The authors declare that they have no competing financial interests.

Construction of the co-axial printhead represents a critical step towards successful microfluidic bioprinting to allow for simultaneous delivery of both the bioink from the core and the crosslinking agent from the sheath. While in this protocol an example printhead was created using a 27G needle as the core and an 18G needle as the shell, it may be readily extended to a variety of combinations using different sizes of needles. However, the alteration in the needle sizes, which results in the change in the amount of flow ...

Tissue engineering targets to generate functional tissue substitutes that can be used to replace, restore, or augment those injured or diseased in the human body1,2,3,4, often through a combination of desired cell types, bioactive molecules5,6, and biomaterials7,8,9,10. More recently, tissue engineering technologies have also been increasingly adopted to generate in vitro tissue and organ models that mimic the important functions of their in vivo counterparts, for applications such as drug development, in replacement of the conventional over-simplified planar cell cultures11,12,13,14,15,16,17,18,19. In both situations, the ability to recapitulate the complex microarchitecture and hierarchical structure of the human tissues is critical in enabling functionality of the engineered tissues10, and in particular, ways to integrate a vascular network into the engineered tissues are of demand since vascularization presents one of the greatest challenges to the field20,21,22,23.
To date, a variety of approaches have been developed in this regard in an attempt to build blood vessel structures into engineered tissue constructs with various degrees of success8. For example, self-assembly of endothelial cells allows for generation of microvascular networks24; delivery of angiogenic growth factors induces sustained neovascularization25,26; use of vascular progenitor cells and pericytes facilitates endothelial cell growth and assembly24,27; designing scaffold properties enables precise modulation of vascularization28,29; and cell sheet technology allows for convenient manipulation of vascular layering30. Nevertheless, these strategies do not endow the capability of controlling the spatial patterning of the vasculature, often leading to random distribution of blood vessels within an engineered tissue construct and thus limited reproducibility. During the past few years bioprinting has emerged as a class of enabling technologies towards the solution of such a challenge, due to their unparalleled versatility of depositing complex tissue patterns at high fidelity and reproducibility in an automated or semi-automated manner31,32,33. Sacrificial bioprinting34,35,36,37,38, embedded bioprinting39,40,41, and hollow structure bioprinting/biofabrication42,43,44,45,46,47,48,49,50,51,52,53 have all demonstrated the feasibility of generating vascular or vascularized tissues.
Alternatively, a microfluidic bioprinting strategy to fabricate microfibrous scaffolds have been recently developed, where a hybrid bioink composed of alginate and gelatin methacryloyl (GelMA) was delivered through the core of a concentric printhead and a calcium chloride (CaCl2) solution was carried through the outer sheath flow of the printhead54,55. The co-extrusion of the two flows allowed for immediate physical crosslinking of the alginate component to enable microfiber formation, while subsequent photocrosslinking ensured permanent stabilization of the multi-layer microfibrous scaffold. Of note, endothelial cells encapsulated within the bioprinted microfibers were found to proliferate and migrate towards the peripheries of the microfibers assuming lumen-like structures that mimicked the vascular bed54,55. These bioprinted, endothelialized vascular beds could be subsequently populated with desired secondary cell types to further construct vascularized tissues55. This protocol thus provides a detailed procedure of such a microfluidic bioprinting strategy enabled by the concentric nozzle design, which ensures convenient fabrication of vascularized tissues for potential applications in both tissue engineering and organoid modeling.

<table width="1270">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Alginic acid sodium salt from brown algae</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">A0682</td>
			<td style="width:480px;">BioReagent, plant cell culture tested, low viscosity, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Gelatin type A from porcine skin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">G2500</td>
			<td>Gel strength 300</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Irgacure 2959 (2-Hydroxy-4&#39;-(2-hydroxyethoxy)-2-methylpropiophenone)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">410896</td>
			<td>98%</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">HEPES buffer</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">H0887</td>
			<td>1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Fetal bovine serum&#160;</td>
			<td style="width:160px;">Thermo Fisher Scientific</td>
			<td style="width:163px;">10438026</td>
			<td>Qualified, heat-inactivated, USDA-approved regions</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Calcium chloride dihydrate</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">C5080</td>
			<td>BioXtra, &#8805;99.0%</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Phosphate buffered saline</td>
			<td style="width:160px;">Thermo Fisher Scientific</td>
			<td style="width:163px;">10010023</td>
			<td>pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Human umbilical vein endothelial cells</td>
			<td style="width:160px;">Angio-Proteomie</td>
			<td style="width:163px;">cAP-0001</td>
			<td>Human Umbilical Vein Endothelial Cells (HUVECs)</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">GFP-expressing human umbilical vein endothelial cells</td>
			<td style="width:160px;">Angio-Proteomie</td>
			<td style="width:163px;">cAP-0001GFP</td>
			<td>GFP-Expressing Human Umbilical Vein Endothelial Cells (GFPHUVECs)</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Endothelial cell growth medium</td>
			<td style="width:160px;">Lonza</td>
			<td style="width:163px;">CC-3162</td>
			<td>EGM-2 BulletKit</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Dulbecco&#8217;s Modified Eagle Medium&#160;</td>
			<td style="width:160px;">Thermo Fisher Scientific</td>
			<td style="width:163px;">12430054</td>
			<td>High glucose, HEPES</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:160px;">Ellsworth Adhesives</td>
			<td style="width:163px;">184 SIL ELAST KIT 0.5KG</td>
			<td>Clear 0.5 kg Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">UV curing lamp system</td>
			<td style="width:160px;">Excelitas Technologies</td>
			<td style="width:163px;">OmniCure S2000</td>
			<td>Spot UV Light Curing System with Intelligent UV Sensor</td>
		</tr>
	</tbody>
</table>

microfluidic bioprinting for engineering vascularized tissues and organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

The microfluidic bioprinting strategy allows for direct extrusion bioprinting of microfibrous scaffolds using low-viscosity bioinks54,55. As illustrated in Figure 2A, a scaffold with a size of 6 &#215; 6 &#215; 6 mm3 containing &#62;30 layers of microfibers could be bioprinted within 10 min. The immediate ionic crosslinking of the alginate component with CaCl2 al...

Watch this Scientific Journal Video about Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids at JoVE.com

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

microfluidic-bioprinting-for-engineering-vascularized-tissues

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15.6K Views.  Harvard Medical School. We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Video: Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

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

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

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

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

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

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.

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.

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 ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...