3D structures made of collagen using mold design and fabrication Matrix proteins are an important aspect of maintaining signaling cascades necessary for growth and homeostasis within 3D cellular environments. However, native matrix materials such as collagen contain properties that make it difficult to extrude and subsequently maintain shape fidelity during the 3D bioprinting process. The ability to place materials that are difficult to print within a mold that is fabricated makes it possible to generate complex 3D structures consisting of native matrix materials such as collagen. TSIM® contains a tool which creates a mold around a structure of interest, and properties regarding the mold are sent to the BioAssemblyBot® for 3D fabrication. The Process A structure of interest is created within TSIM® based upon experiment design requirements. The mold tool is then used to create a mold around the structure. This mold can then be modified to limit the material required to generate the structure. Material parameters are then assigned, followed by 3D printing of the mold structure using the BioAssemblyBot®. After the final desired product is achieved, the desired material is then delivered to the mold. After the entire structure is placed within solution, the mold is dissolved away, leaving behind a structure made of native matrix material. Creating a mold around a structure of interest In this example, a structure is created de novo using the different shape tools within TSIM®. The shapes are then brought together through Boolean operations (Combine two objects tool) creating the final structure of interest. Once this structure is selected, the Create a mold tool is used to generate a mold around the newly created objected. It will create a square mold around the object, after the mold is generated, it will behave as an independent object, and can be manipulated as desired. After the desired location and structure is finalized, a material is then assigned to the structure including necessary print parameters for the successful fabrication of the design. 3D fabrication of a structure using native matrix material The design and print parameters created within TSIM® are sent to the BioAssemblyBot® for 3D fabrication. In this example, the mold structure is created using a pluronic hydrogel printed with a petri dish. Type I collagen was then poured into the structure quickly to ensure the collagen remained cold and therefore not yet gelled while placing it within the mold. The petri dish was then placed in an incubator for 2 hours to gel the collagen within the pluronic mold. Hank’s balanced salt solution was then poured into the petri dish which was then placed in the incubator overnight to dissolve away the mold. What was then left behind was only the final structure made of collagen. The utilization of the mold functionality within TSIM® enables 3D structure creation using native matrix materials that are difficult to utilize in the 3D bioprinting process of fabrication. Cell populations can then be placed and grown within these structures, thereby providing the ability to create defined 3D regions of cell populations to localize different cell types for studies in cell-cell interactions, cell migration, growth, or to precisely place different cell populations for whole tissue generation. To learn about the BioAssemblyBot 400, click here.

The Integrated biofabrication platform that is TSIM® and the BioAssemblyBot® 400 enables the intuitive fabrication of microfluidic systems Generating microfluidic devices commonly involves forming polydimethylsiloxane (PDMS) molds, of a specific channel design, from soft photolithography. This is often time-intensive, making iterations of a channel design effort less efficient. In Advanced Solutions Life Sciences’ integrated biofabrication platform, numerous different channel systems can be digitally prototyped in the TSIM® software environment, and automatically fabricated with the BioAssemblyBot® via sacrificial 3D printing, providing an easy and fast way to fabricate microfluidic systems optimized for a particular application. The Process Within TSIM®, you can utilize sketching tools to create a channel design of interest within each well of a multi-well plate or a custom device. Each component of the design is then assigned a sacrificial material in a printing cartridge fitted with different caliber nozzles. The platform will begin printing the sacrificial material in the channel network pattern, automatically switching between the different tools as needed to complete the network. After the material of the device (e.g. acrylates, collagen, etc.) is flooded and cured around the printed network, the sacrificial material is flushed away leaving behind patent channels within the matrix material. These channel systems are then connected, via integrated inflow and outflow ports, to a pump/fluids management platform to provide perfusion throughout a 3D tissue construct. Designing channel structures within TSIM® Within the software program, TSIM®, the different channel elements that make-up a microfluidic network are created de novo based using the sketch features. A single network can be created (shown below) or multiple networks created with each one being placed in each well of a multi-well plate. TSIM® enables the import of 3D model files of standard multi-well plate formats for this purpose. Of course, a different network design can be created for each well, if desired. After the overall channel design and organization is determined, each component of the structure is assigned a material/tool which includes the relevant nozzle size and printing parameters. In TSIM®, each of the microfluidic elements assigned with a specific material/tool is indicated with a different color (Figure 1). Importantly, network designs can be exported by the TSIM® software program for estimating hemodynamic parameters using computational fluid dynamics programs. Printing the microfluidics platform using the BioAssemblyBot® 400 Next, the digital model of the microfluidic network is sent from TSIM® to the BioAssemblyBot® 400 for fabrication. Once the requisite tools are loaded, the BioAssemblyBot® then executes the fabrication run, seamlessly changing out each tool, as required. Although the same material, in this case, Pluronic hydrogel, is being used to fabricate the structure, the tools are being interchanged to automatically incorporate different nozzle diameters to achieve the targeted channel diameter. The last step of this fabrication process includes the tool pathing capability of the BioAssemblyBot® 400 to generate a structure in a single upward motion to fabricate the inlet and outlet ports that will directly contact with the fluidics management device (Figure 2). After each component of the network is printed, the system is then covered with the desired matrix material such as uncured acrylate or non-gelled collagen (Figure 3). After the network is printed, the surrounding matrix is gelled (or cured), and the device is connected to the fluidics management or pump system, the printed material is flushed out, leaving behind patent channels for experiments on flow characteristics or cellularization facilitating perfusion of 3D tissue models/constructs. To learn about the BioAssemblyBot 400, click here.

Dr. Vahid Serpooshan, at Emory/Georgia Tech University, recently published work on the biofabrication of bacteriostatic bone constructs with in vivo regenerative capacity. In this publication, featured on the cover of Polymers (pictured above), the BioAssemblyBot® platform was used to print a disc shape (3.5mm x 0.9mm) and a cylinder shape (3.5mm x 9mm) for in vitro and in vivo analysis, respectively. The constructs were fabricated using hyperelastic bone (produced by Dimension Inx), where material characteristics and functional assessment was compared between superparamagnetic iron oxide nanoparticle (SPION) loaded or SPOIN-free material. SPION-loaded scaffolds demonstrated a smoother surface structure, retained a high degree of elasticity, significantly increased the compressive modulus in the cylinder shape, and demonstrated higher viability of murine and human seeded cells in vitro compared with SPION-free scaffold. The SPION-loaded HB scaffold also demonstrated adequate engraftment in vivo while importantly showing no signs of infection. Read more: https://www.mdpi.com/2073-4360/13/7/1099 Learn more about the BioAssemblyBot® 400 here. Learn more about DimensionInx Hyperelastic Bone™:

View the published paper here: https://slas-discovery.org/article/S2472-5552(22)13680-4/fulltext In vitro models of human liver tissue and function are proving invaluable in liver disease modelling, hepatotoxicity screening for drugs, predictive drug targeting, and more. Models that recapitulate more of the native liver tissue environment would improve discovery efforts and enable investigations into liver health and disease that is currently challenging to perform with other, less complex models, such as tumor-liver tissue dynamics and parasite infections. This includes the presence of native microvasculature, including the variety of vascular and perivascular niche cells intrinsic to the native microvessel. To this end, ASLS has developed a vascularized liver tissue called MVP liver™ tissue modules. The MVP liver™ tissues are built using the BioAssembly™ biofabrication platform and uses Angiomics® human adipose-derived, intact microvessels (haMVs) as the means to vascularize the liver tissue bed. In fabricating the MVP liver™ tissue modules, primary hepatocytes, primary non-parenchymal cells, and Angiomics® haMVs were combined around a printed tissue frame within wells of a multi-well plate. This forms a structured, thick (~ 1 cm), liver tissue containing healthy hepatocytes and native microvessels (Figure 1). The tissue architecture that is established eliminates necrotic tissue zones, even with large tissue volumes (Figure 1). Furthermore, the biofabrication strategy employed is such that MVP liver™ tissues can be configured in a variety of formats and platforms (including perfusion). MVP™-liver tissues are stable, as measured by LDH release, for 2 or more weeks and produce urea constitutively over the entire culture period (Figure 2). Additionally, the presence of haMVs in the tissue promoted expression of the MRP2 drug transporter and P450 responses, measured as increased expression of the Cyp3A4 gene, to the drug irritant rifampicin (Figure 2). Furthermore, albumin expression was upregulated, and stellate cell (a-actin positive cells) and macrophage (CD45+ cells) numbers were elevated when haMVs were included (data not shown). Via ASLS’ BioApp™ program, a workflow software management system, and the robotic BioAssembly™ Platform, MVP liver™ tissues were automatically biofabricated (Figure 3). The BioAssembly™ Platform consists of a 6-axis robotic arm, capable of multiple automatic tool exchanges, a modular tissue culture incubator called the BioStorageBot™, and a confocal high content analysis scanner all uniquely integrated for automated of tissue and organ manufacturing. In automatically fabricating the MVP liver™ tissue modules, the operator loaded the BioAssembly™ Platform with all required components, calls up the liver tissue BioApp™ and presses “go”. The Platform automatically executed the tissue fabrication, which occurred over 5 days without human intervention, in an enclosed, clean environment. MVP liver™ tissues fabricated with the automated workflow exhibited functional readouts equivalent to liver tissue modules fabricated by hand, including urea production and select gene expression (data not shown). ASLS has developed a human vascularized, thick liver tissue, called MVP liver™ tissue modules, to model liver tissue biology in vitro using a simple, robust, and automated approach. Importantly, the tissue includes isolated, native microvessel fragments (haMVs) that promote liver tissue functionality in the model including enhanced gene expression and drug responses. The modules are readily re-formatted for specific application and study needs. The liver tissue environment is such that the dynamics of focal disease elements, for example tumors or parasite forms, can be examined in the context of healthy liver tissue cells and stroma enabling a breadth of studies and discoveries. Angiomics Human Adipose Microvessels are available for purchase at: https://www.advancedsolutions.com/microvessels BioApps™ and MVPliver™tissue kits are available for purchase at https://www.advancedsolutions.com/bioapps The BioAssembly™ Platform is available at https://www.advancedsolutions.com/

Dr. Vahid Serpooshan, at Emory/Georgia Tech University, has published a review on the state of field of bioprinting for neural tissues. In this review, he discusses the capability requirements for bioprinting technology, biomaterials to achieve biomimetic neural tissue, as well as the variety of applications for which bioprinted neural tissue either can or has been used for. The review also highlights the BioAssemblyBot® system as the tool for these different applications in Figure 1 (shown above). Click on the link below to read more: https://onlinelibrary.wiley.com/doi/10.1002/adhm.202001600

Organoids are invaluable tools for disease modeling and high throughput screening. The majority of organoids contain one or two cell types, that aggregate to form a homogenous spheroid structure. Rarely in the body, however, are tissues so homogenous. In addition to specialized cells, tissues contain a vasculature and a spectrum of other cell types. Not only does the vasculature provide perfusion, but the additional vessel-associated cell types engage in complex signaling with other tissue components and aid in overall tissue function. Thus, the more of these varied cell types in a tissue model or organoid, the better the model biology matches the native tissue. At Advanced Solutions, we utilized whole, intact, microvessel fragments, isolated from human lipoaspirates (Angiomics® haMVs), to vascularize MSC-derived adipose organoids to enhance model adipose tissue function. haMVs were combined with either MSCs or MSC-derived preadipocytes (for adipose organoids) and cultured alone or embedded in 3D matrix. When embedded, new vessels grew out of the organoids and into the surrounding matrix within 2 days (Fig 1). Further, in adipose organoids, neovascular networks appear more mature, with branching and connecting vessels (Fig 1B). Adipose organoids accumulated large lipid droplets (Fig 2A) and expressed adipocyte markers adiponectin and PPARγ (Fig 2B), demonstrating differentiation into mature adipocytes. Highlighting the influence of the haMVs, insulin receptor expression by the adipocytes was upregulated in organoids with microvessels, compared to control organoids without microvessels (Fig 3A-C). ASLS has developed a vascularized, human adipose organoid (vAO) to model adipose tissue biology in vitro using a simple and robust approach involving isolated, intact microvessel fragments (haMVs). VAOs exhibit typical adipose tissue functionality, including responses to inflammatory mediators and active insulin receptor expression that would facilitate insulin resistance studies. Additionally, the vascular component of the vAO retains angiogenic potential suggesting, that if implanted, they will engraft with the surrounding tissue. While the ASLS vascularization approach was demonstrated with MSC and adipose organoids, the fabrication procedure is broadly applicable to other tissue model systems as well. Full manuscript available at https://iopscience.iop.org/article/10.1088/1758-5090/abe187/meta Microvessels are available for purchase at https://www.advancedsolutions.com/microvessels This product is for RESEARCH USE ONLY.

Advanced Solutions’ Chief Scientist, Dr. Jay Hoying, will be presenting at the Olympus® Discovery Summit at the end of this month. The virtual event will analyze the future of research in a post-pandemic workplace. Speaking on April 29th, Dr. Hoying will be discussing “Complex Human Vascularized Tissue Models.” Registration for the event is required. Learn more and register at: https://www.olympus-ims.com/en/news/olympus-discovery-summit-virtually-connects-microscopy-community/

Advanced Solutions’ groundbreaking research into vascularization is featured in the April edition of SME’s SMART Manufacturing. “Vascularization—the development of blood vessels in tissues and organs—is a major impediment to manufacturing substitute tissue and organs. Advanced Solutions, one of the first members of ARMI, believes it has the answer: a product line called Angiomics®.” The article, featured on page 56 of the publication, discusses the process Angiomics® uses to harvest blood vessels from fat. It goes on to discuss how the BioAssemblyBot® “’makes’ the biology” while artificial intelligence performs quality controls. The article is part of a larger feature on ARMI or the Advanced Regenerative Manufacturing Institute (which Advanced Solutions was a founding member). ARMI is led by executive director Dean Kamen, inventor of the insulin pump and personal transport device, Segway. ARMI is a 170-member institute that is working to produce manufactured transplantable human organs. Smart Manufacturing focuses on advanced manufacturing technologies and tools that are driven or enhanced by integrated information technology. Smart Manufacturing is available free of charge to qualified subscribers in the US. Read the article here: http://www.qgdigitalpublishing.com/publication/?m=55884&i=700142&p=58 Smart Manufacturing is a monthly magazine published by SME. SME is a nonprofit association of professionals, educators and students committed to promoting and supporting the manufacturing industry.

Build complex tissue models with one click using Advanced Solutions BioApps™. BioApps unite 3D modeling software, TSIM, with 3D bioprinting hardware, BioAssemblyBots 200 & 400 and living cells & biomaterials like Angiomics Microvessels. Let BioApps build your tissue models giving you more freedom and time to work on other items. BioApps make the biomanufacturing process easy… 1) Simply choose your BioApp and a tissue kit containing all the components necessary will be sent to you. 2) Follow the simple prep steps once the kit arrives. 3) Then the BioApp does the rest, telling your BioAssemblyBot to autonomously perform all steps or tasks specific to that tissue model. The ever-growing selection of available BioApps include manufacturing VAO well plates and 3D Liver constructs, with more coming soon. The BioApps Market is a makerspace of community and Advanced Solutions made applications. You can automate your own workflows by creating your own BioApp with BioApps Maker. Your BioApps can include BioAssemblyBot® 200 or BioAssemblyBot® 400, the BioStorageBot, various BioAssemblyTools, and 3D models from TSIM. Additionally, BioApps can integrate with the Cytiva IN Cell Analyzer with additional integrations coming soon. Learn more and start creating your own BioApps here.

The BioAssemblyBot® Platform can be used to fabricate mesh or weave-like patterns to generate constructs with varying porosity 3D tissue scaffolds that contain precise pore structures are necessary for a myriad of tissue engineering applications. Many engineered tissue scaffolds are porous to enable both the necessary biological interactions inherent in tissues to occur while also maintaining structure fidelity or strength through the connections contained within mesh-like materials. This also recapitulates the porous nature of native scaffolds within a variety of different tissues while simultaneously providing avenues for cell growth to occur. These mesh-like materials are also used for additional applications outside of the direct tissue engineering realm, such as being used as filters, screens, masks and patches. The BioAssemblyBot® technology platform provides the precision required to generate such porous constructs that can be made from a variety of biocompatible of materials through the tool-sets available in the platform. The Process To generate porous or mesh-like scaffold materials, the design of the structure can first be created within the fully-integrated software program, TSIM®, including the appropriate material controls for each component of the construct. In this case study, we demonstrate the ability of the platform to generate structures of varying porosity with two distinct material types. The first example assumes that the scaffold will need to maintain shape over extended periods of time. Therefore, the heat-controlled tool is utilized to extrude biodegradable materials, such polycaprolactone, or PCL. Due to the more rigid nature of PCL, this material requires a melt-extrusion process. To achieve this, PCL pellets are loaded into the heat-controlled syringe. This syringe can maintain temperature control beginning at the storage bay, which allows the tool and material to get up to a temperature that would enable extrusion. This temperature can then continue to be maintained as the tool is being utilized for its designed task as well as when it is returned to the storage bay. To maintain the structure generated with PCL, the print will need to cool down to ambient temperatures. The second example below utilizes hydrogel materials that can be extruded under ambient conditions. Porous Construct Made of PCL After the desired parameters for the utilization of the tool are determined based on the construct as well as desired pore size of interest, the scaffold can then be printed on the BioAssemblyBot® platform as shown in the image below (Figure 1). This type of structure and fabrication strategy is also used to generate mimics of woven material. Varying Porosities Made with Extreme Precision Different tissue types and applications will require scaffolds that contain pores of varying size and density. Through the design work that can be done within TSIM® along with the precision enabled through using the 6-axis arm, the BioAssemblyBot® can generate structures with varying porosities. As seen in (Figure 2) below, different materials as well as design parameters resulted in unique pore dimensions that were repeated throughout the scaffold. The top image shown above contains a magnified view of the PCL structure shown in Figure 1. This illustrates how precisely the pattern was generated using a melt-extrusion process while also exhibiting how this pattern mimics that found in woven fabrics and constructs, which are often used as a supporting material for a variety of regenerative medicine-based applications. The next image illustrates the size of each pore of a construct made of pluronic hydrogel. This material can be extruded under ambient conditions and this structure includes unique porosity dimensions compared to the PCL example. Therefore, altogether these examples demonstrate the flexibility and precision that the BioAssemblyBot® technology contains to fabricate 3D structures with varying porosity.

The enhanced scanning capabilities and associated TSIM® workflows enable the precise printing of structures onto contoured surfaces 3D bioprinting technology platforms typically use gantry axes (X, Y, and Z) as their method of motion control. This enables additive manufacturing, a layer by layer approach to 3D printing, where new constructs are fabricated from the bottom-up in the Z direction. However, to fabricate complex biological constructs and eventually larger organ systems, more freedom of movement is required to meet all fabrication tasks. The BioAssemblyBot® platform, with its 6-axis robotic arm at the center of the platform, has the needed flexibility in print-path motion and directional access; with the added ability to execute a print task from a variety of angles. Further enabling such a workflow, the system visualizes surface features of complex objects within the fabrication envelope, incorporating such features in the 3D digital model. Through unique tool sets that are included with the BioAssemblyBot®, as well as a built-in workflow within the TSIM® software program, this workflow is now achieved. The Process The TSIM® design software when coupled with a BioAssemblyBot® facilitates the capture of high-resolution surface data from objects placed on the BioAssemblyBot® printing surface. Utilizing a laser measurement sensor with submillimeter repeatable accuracy, the surface height is measured across a chosen area and then saved on a local storage device in a simple point cloud format. Any network connected PC running the TSIM® design software can request the point cloud data from the BioAssemblyBot®. After receiving the point cloud, TSIM® automatically filters the data and reconstructs the surface as a triangulated mesh within the TSIM® solid modeling environment. In this form, the surface can be used as a projection target for custom non-planar printing tool paths enabled by the BioAssemblyBot® six-axis robotic manipulator. They’re also highly useful as a visual reference for a variety of bioprinting design and assembly tasks. Workflow example using a patient-derived heart model In this example, a patch is being printed onto the complex surface of a printed three-dimension model of a heart, generated from patient data. Step 1: Scanned surface The first step of this workflow includes the utilization of 3D Laser Scan BioAssemblyTool to scan the surface of the object and import that data into the TSIM® Software Program. In this example, a section of the surface of the heart is the focus of the scan area. Step 2: Cardiac patch sketch Using the sketching tools within the TSIM® software program, two example patterns are created de novo. Step 3: Patch generated into 3D model After the sketch is completed, the 3D model is generated and ready to be placed on the appropriate location on the model. Step 4: Patch is projected onto surface of model The appropriate material is assigned to the design which then instructs the system on how to complete the print the task. Now you are ready to print! Step 5: Print The image below represents the completed print after being printed on the BioAssemblyBot 400 using the 3D Syringe BioAssemblyTool. Watch the process:

Join Dr. Jay Hoying, Advanced Solutions Chief Scientist, at the virtual 10th Annual Laboratory Animal Sciences conference this Wednesday, February 10th. Dr. Hoying’s presentation, “Human In Vitro Vascularized Tissue Models,” will be available on-demand throughout the event. Presentation Abstract: Tissue models, including organoids, are proving valuable in modeling human tissue health and disease for a variety of applications. This is due, in part, to the dynamic cell-cell interactions important in tissue function fostered within the 3D tissue-like space. To this end, the more these models can recapitulate the different interactions found in native tissue, such as that between parenchyma and the microvasculature, the better the fidelity of the model. The microvasculature, which is comprised of a spectrum of cell types and matrix proteins, provides not only perfusion in its support of tissue health, but also cellular interactions and biochemical dynamics important in tissue phenotype and function. We have developed a generalized strategy for creating human, vascularized tissue models for research and therapeutic applications using isolated, intact human microvessel fragments to generate a neovasculature within the fabricated tissue bed. By isolating intact microvessels, instead of dissociated single vascular cells, we have retained the full complement of vascular and vascular-related cells intrinsic to the native microvasculature. With this strategy, we have fabricated vascularized thick tissue constructs and organoids useful in research, pharmaceutical and autologous therapeutic applications and demonstrate that the integrated microvasculature imparts new functionality to the constructs and organoids. Learning Objectives: 1. Define an effective in vitro tissue model 2. Identify the features of a microvasculature 3. Explain the approaches for building in vitro tissue models You can register to attend the on-demand presentation here: https://www.labroots.com/virtual-event/laboratory-animal-sciences-2021

FOR IMMEDIATE RELEASE 2/2/2021 Advanced Solutions Expands the BioAssemblyBot® Platform with the BioAssemblyBot® 200, an Industry Unique Four-Axis Robotic 3D Bioprinter LOUISVILLE, KY, USA – Hello, versatility. Advanced Solutions announces their newest patented, cGMP and UL certified bioprinter, the BioAssemblyBot® 200. BAB200, as its affectionately known, joins the award winning BioAssemblyBot® 400 (BAB400) as an automated multi-tasking bioprinter. BioAssemblyBot® 200 uses a four-axis robotic arm to bioprint and automate the science workflow. “BAB200 provides our customers with a powerful benchtop alternative to develop and translate research ideas into clinical reality.” said Michael Golway, P.E., the Lead Inventor of the BioAssembly platform and CEO of Advanced Solutions. BioAssemblyBot® 200 is built on the foundation of over 50,000+ hours of research and development that created the BioAssemblyBot® 400 platform. Innovations like interchangeable BioAssemblyTools™, vision tip detect, variable print stage, and the design/make software programs of TSIM® and BioApps™, respectively, are just a few of the features from the BAB400 platform that are now available in BAB200. Breaking the mold from other traditional bioprinters, BioAssemblyBot® 200 combines four-axis robotics with three unique forms to meet your needs. Whether in its enclosed form on the lab benchtop, enclosed with HEPA filtration system, or in a biosafety cabinet, BAB200 is a powerhouse at 3D biology efficiency and capability. BioAssemblyBot® 200 is sleek and fits right in with your existing lab equipment. BAB200 is easy-to-use and control with the included touch screen interface. “The BioAssemblyBot 200 is the most versatile bioprinter I have used in my 30+ years of work. My team readily transitions from HEPA-controlled prints on the benchtop to fully sterile work in a biosafety cabinet, performing a spectrum of different printing tasks with the same machine” said Dr. Jay Hoying, Chief Scientist of Advanced Solutions BioAssemblyBot® 200 is more than a 3D bioprinter. With bays for up to five active BioAssemblyTools™, BAB200 offers flexibility in the bioprinting workflow to deliver a rich library of automated science outcomes ensuring a return on investment that will supercharge research results. You can learn more about BAB200 at advancedsolutions.com/bioassemblybot-200. Advanced Solutions is dedicated to the discovery, design, and development of integrated software and hardware solutions for the fields of science that involve living organisms, molecular biology, and biotechnology. Advanced Solutions is based in Louisville, KY, USA.

Tissue Constructs using PDMS Polydimethylsiloxane (PDMS) is a well-known and widely used viscoelastic biomaterial with applications across the tissue engineering and biomedical fields. In general, its use thus far has been limited to applications wherein casting or molding are possible. At Advanced Solutions, we’re using 3D printing of PDMS to create complex shapes potentially useful in a variety of applications. 1) PDMS can be printed with high shape fidelity on the BioAssemblyBot 2) PDMS is capable of self-support during printing 3) Hollow PDMS shapes can be created. These constructs can be used to pattern cell and tissue constructs PDMS as a Biomaterial Silicone-based polymers have been used in tissue constructs since the middle of the 20th century. Their early use as a replacement bile duct and urethra exhibited the material’s useful qualities: high bio-compatibility and elasticity. Subsequent tissue constructs applications throughout the intervening 80 years have shown that silicone-based polymers, among which PDMS is the most common, are safe, broadly applicable, and economical. PDMS is characterized by its low surface tension, low modulus (contributing to its biological consistency), hydrophobicity, thermal and electrical stability. Today, PDMS is commonly used in catheter components, pacemaker leads, and contact lenses. Printing PDMS Due to the limited range of possible configurations of cast and molded shapes, it is desirable to 3-D print with PDMS. A PDMS printing ink is achieved by blending siloxane polymers SE 1700 and Sylgard 184. SE 1700 is a shear-thinning, high viscosity polymer, which gives the ink the shape fidelity needed after printing. Sylgard 184 is lower viscosity and is blended with SE 1700 to enhance the ability of the ink to run through deposition needles under pressure in 3-D printing applications. To make up the printing ink, polymer base and curing agent were combined in a 10:1 ratio. Then SE 1700 and Sylgard 184 were combined in a 4:1 ratio. PDMS blends were loaded into the syringe barrel and centrifuged at 3000g for 5 min to de-gas the material. After printing, constructs were placed in an oven overnight to cure the printed construct (1). PDMS structures were printed using Advanced Solutions Life Sciences’ BioAssemblyBot®.A 22GA needle was used to deposit the PDMS ink at 70PSI.Printed shapes included objects designed to test the capacity of the PDMS ink to span empty spaces, create pore spaces in solid blocks, as well as test shape fidelity after curing. Printed Models As seen in the photos of the printed constructs in Figure 1, printed PDMS adheres to well plates and coheres, supporting the printing of complex shapes and structures. Samples were examined pre- and post- curing and minimal shape deformation was seen during the curing process. Microscopic analysis of a small sample revealed that the lines in these constructs average 380 +/- 50 µm after PDMS was cured in an oven at 60ºC overnight. Hollow structures, such as those shown in Figure 2 demonstrate the ability of printed wells to be created within a PDMS print structure. These hollow structures can be used to pattern cell and tissue constructs. Lattice structures shown in Figure 3 were used to test the ability of the PDMS ink to span longer distances (1 - 2 mm) over empty space. These were largely successful, highlighting the ability of the PDMS bio-ink to cohere and maintain shape while unsupported from below. These designs illustrate that well-supported filaments of the PDMS bio-ink can easily span 2 mm gaps over empty space without sagging. Summary PDMS structures were printed with the Advanced Solutions BioAssemblyBot® using the preparation procedures and print parameters given above. Sample prints showed that printed lines were less than 400 mm in diameter and that inter-line spacing was as large as 1.5 – 2 mm. The utility of PDMS and other silicones now combined with the ability to form complex shapes and structures via 3D printing enables a wide variety of applications and opportunities. 1. Ozbolat, V, Dey, M, Ayan, B, Povilianskas, A, Demirel, MC, and Ozbolat, I. 2017. 3D Printing of PDMS Improves its Mechanical and Cell Adhesion Properties. ACS Biomater. Sci. Eng. Accessed via web 12/22/2017. URL: http://pubs.acs.org

In this article we examine printing a gelatin/alginate blend with incorporated cells using the BioBot Basic. What are Gelatin/Alginate Blends? Gelatin and alginate are used for many biomedical applications, including as scaffolds for tissue engineered constructs (1). Gelatin is a naturally occurring, water soluble protein derived from collagen, and is widely used for 3D bioprinting. Gelatin forms a gel at room temperature, but melts in a 37°C incubator, making its printability temperature dependent. Therefore, gelatin is often combined with other biomaterials, such as silk, agarose, or alginate, to enhance its printability and stability in culture. Alginate is a polysaccharide naturally found in seaweed, which is also frequently blended with other hydrogels for bioprinting applications. Alginate, prior to crosslinking, has poor mechanical properties and does not hold its shape when printed. Additionally, alginate is not cell-adhesive. For these reasons, neither gelatin nor alginate alone is optimal for biological printing. However, when blended, gelatin/alginate can be readily printed, and provides an environment conducive to 3D cell culture (2). The gelatin component enables the material to hold its shape after printing at room temperature. Alginate improves gelatin printability by altering its viscosity, creating a smoother print. After printing the alginate is crosslinked, which allows the construct to maintain its shape after the majority of gelatin has dissolved in a 37°C incubator. Residual gelatin in the construct also improves cellular adhesion. Printing Gelatin/Alginate Gelatin/alginate blends are a well-established bioink used for printing cells in a 3D environment. Concentrations ranging from 4% gelatin/3% alginate to 10% gelatin/9% alginate have been reported for various applications (1,2). Here, we used a 6% gelatin and 5% alginate blend in phosphate buffered saline (PBS). Gelatin was first dissolved in hot PBS, then sterile filtered. Alginate was added to the sterile solution in a biosafety cabinet. Once dissolved, the material was brought to 37°C before combining with human mesenchymal stem cells (Rooster Bio) at 400K cells/ml (3). Prints were performed on a BioBot Basic inside a biosafety cabinet with a 22GA conical needle, at a pressure of 25 PSI and speed of 15 mm/sec (pressure and speed may vary slightly between batches). Structures printed included a cube, a tube, and a rectangular box (Fig 1). After printing, constructs were submerged in a 0.75% calcium chloride solution to crosslink the alginate. After crosslinking the structures were rinsed thoroughly with PBS, flooded with culture medium containing DMEM, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin, and placed in the incubator for two days. To measure cell viability, a Calcein-AM (Thermo Fisher) stain, which stains live cells, and a Hoechst stain (Invitrogen) for total nuclei. Results & Discussion Printed constructs ranged from between 4mm – 8mm in height, width, and depth, with a wall thickness of ~1mm. This demonstrates the printer’s ability to print fine, stable constructs and the bioinks ability to adequately adhere to itself and maintain its structure. Calcein and Hoechst staining indicated an 80% cell viability two days after printing. The small footprint and utility of the BioBot™ Basic is ideal for use in a tissue culture hood for aseptic bioprinting of tissue constructs. Here, we demonstrate using the BioBot™ Basic to print a commonly used gelatin/alginate bioink containing live cells resulting in a viable cell culture. Notes: Other needle sizes and types may be used, although when using non-conical needles, the material printability becomes more temperature sensitive. The optimal printing temperature for non-conical needles is 28°C. Repeated heating cycles should be avoided. Autoclaving gelatin blends should be avoided, as this degrades the gelatin and reduces printability. Coating cell culture plates with alginate or other ECM proteins can be used to prevent constructs from lifting off plates during culture, if needed. Axpe, E.; Oyen, M.L. “Applications of Alginate-Based Bioinks in 3D Bioprinting. Int. J. Mol. Sci. 2016, 17, 1976 Gao, Teng, et al. “Optimization of Gelatin–Alginate Composite Bioink Printability Using Rheological Parameters: a Systematic Approach.” Biofabrication, 2018, 10, p. 034106.

The BioAssemblyBot® together with BioApps™ automates experiments such as an ELISA protocol Automated workflow continues to be a rationale behind incorporating the utility of a 6-axis robotic arm into the field of biofabrication. The latest in software innovations from the Advanced Solutions Life Sciences (ASLS) team has made this a reality. BioApps™ allows users to create automated workflows such that an entire experimental procedure can be completed from start to finish automatically thereby increasing experimental throughput while maintaining high reproducibility. Uniquely, this system can interface with external analytical instruments so that after the assays are set-up, the multi-well plates can be moved into plate readers, PCR machines, and microscopy platforms for assaying. In this example, we are illustrating an automated ELISA-based protocol for detecting COVID-19 related antibodies in patient serum reconfigured for use in a 384-well format whereby the entire workflow for a 384-well plate-based procedure is designed in BioApps™ and automatically completed by the BioAssemblyBot® (Figure 1). The Process The process begins by creating the desired workflow within the BioApps™ interface. Within BioApps™, users build the workflow by assembling different operational tasks, represented by task icons in the software, via workflow pathway connectors, instructing the BioAssemblyBot® as to which tools and steps to execute while progressing through the workflow. The user can also define decision points that can then be integrated into the instruction sets and tied to external data readouts from analytical systems. In this example ELISA, the BioAssemblyBot® then utilizes the latest automation tools and operational features developed by the Advanced Solutions Life Sciences team to execute the workflow. The multi-well plate is moved by the BioAssemblyBot® and automated tool changing steps lead. For this experiment, antigen, blocking and reaction buffers, 2°Ab, and patient samples are all deposited into wells of the plate, with automated tip change, tip disposal, and waste disposal. With BioApps™ and the BioAssemblyBot®, experiments are expedited without personnel intervention. An example of the overall timeframe to complete this type of workflow is provided in the table below (Table 1). Plate Movement and Incubation The Pick & Place BioAssemblyTool™ is used to de-lid and lid the multi-well plate for the incubation and storage steps of this process. Specifically, the stage is heated to 37°C to affix the antigen to the bottom of the wells. Conversely the plate could be transferred to a refrigerator or chilled on-stage, limiting the need to move the plate or for personnel intervention. As previously mentioned, the BioAssemblyBot® can also move the plate to peripheral instrumentation to perform measurements or additional assays depending on the procedure type (such as a thermocycler for PCR-based experiments). Variable Volume Dispensing and Automated Aspiration A key feature to the automated workflow is that the ASLS Pipette tool can dispense a variety of volumes from as low as 0.5 ml to as high as 1000 ml. In this example, the entire volume of antigen needed to place 2 ml of antigen solution per well 384 well plate is loaded into the tip and dispensed across the plate. Similar pipetting steps are performed with different dispensing volumes as needed during the entire procedure. Any wash steps also utilize the Pipette tool for automated solution pipetting, tip change, and waste disposal. Conversely, the plate can be transferred to an automatic washing station and brought back into the work area for subsequent processing steps. The Pipette tool can also aspirate reagents and buffers during the workflow (as shown in Figure 2). Aspirates are disposed-of at a receptacle located at the back of the enclosure and repeated across the entire plate "Automate the Science" - ELISA Testing for COVID-19 Utilizing the latest in Advanced Solutions Life Sciences automation technology, the BioAssemblyBot® automates assays by conducting the preparation, processing, and execution required for an experiment. This provides the throughput necessary for rapid result generation, such as what is needed to combat the current COVID-19 pandemic.

Fabrication of a continuous gradient between two hydrogel materials A major limitation in the field of 3D bioprinting to date has been the ability to create and utilize complex biomaterials. This also relates to the concentration or level of different materials and components across an area or tissue. These concentrations or gradients are important for recapitulating native gradients of signaling molecules as well as varying levels of matrix stiffness that occurs in tissues. A new fabrication methodology developed by the team at Advanced Solutions Life Sciences enables the incorporation of materials that can be mixed at the point of extrusion. Thereby providing a method for a new set of materials to be incorporated into 3D tissue structures by specifically fabricating 3D structures that contain a continuous gradient mixture between two materials. The Process A structure of interest is created using sketching tools within TSIM®. After a structure with the desired parameters are created, a gradient is generated using the “set material gradient” tool. The ratios and the length of the gradient can then be altered based upon the design requirements of the structure or the properties of the materials that are to be mixed. After a gradient is set, a priming process occurs on the mixing tool to ensure that the 3D structure fabricated contains the appropriate gradient that is designated in the digital design. This process illustrates the functionality within TSIM® that has been designed around the 3D Dual Syringe | Mix BioAssemblyTool™. Creating a continuous gradient around a sketch in TSIM® In this example workflow, a structure is created de novo using the “create a sketch” functionality within TSIM®. The number of dots within the line will serve as gradient starting points once the gradient is created, as indicated below in the left panel. After the sketch is complete, a tube is created around the line at the desired dimensions for this experiment as shown in the right panel below. Then, with the tube highlighted, the “set material gradient” is selected, and it is here that the percentage of the primary material across the gradient is designated, where the initiation of the gradient is each point along the sketch (pick a sketch point on TSIM®). In this example, a gradient of 100% primary material, to 0% primary material, back to 100% primary material will be fabricated. The gradient between the two materials is also visible in TSIM® as shown in the image below. After the parameters in the gradient are finalized, this information is sent to the BioAssemblyBot® to complete utilizing the 3D Dual Syringe | Mix BioAssemblyTool™. Creating a gradient using the 3D Dual Syringe | Mix on the BioAssemblyBot The workflow at the BioAssemblyBot® to create a continuous gradient starts with priming the 3D Dual Syringe | Mix, where the primary material (right hand side material) and the secondary material (left-hand side) are pushed through the mixing chamber. This priming step is included in the print workflow as it is necessary for the ability to ensure that the entire mixing chamber is filled so that once the print begins, the desired percentage gradient between the materials is present at the start of the print. After priming, the continuous gradient is printed as a single fabrication step. The 3D Dual Syringe | Mix seen in Figure 3 is a preproduction version of the tool.

Cells can be used as an ‘ink’ for the BioAssemblyBot® platform to generate precise 3D constructs One of the major differentiating factors between other types of additive manufacturing and bioprinting is the incorporation of biocompatible material and/or cells to create 3D tissues and tissue models. Therefore, when utilizing a technology platform in the context of biofabrication, it is imperative that the platform can successfully print cell-laden materials or cell preparations alone precisely and without harm to the cells. The BioAssemblyBot® platform achieves both. The Process Isolated cells are first loaded into syringes within the sterile environment of a biosafety cabinet or hood. The syringes, while capped, are then loaded into the appropriate tool body based on the fabrication task at hand, which are then placed into the associated storage bay within the BioAssemblyBot® enclosure as designated by the integrated TSIM® software program. To maintain a sterile environment within the enclosure of the BioAssemblyBot® platform, the HEPA filtration system will need to be kept on, the switch for which is located on the touch-screen interface attached to the platform. Below are examples of either a heterogeneous primary cell-isolate or cultured cells printed using the BioAssemblyBot® platform. Cells Printed with the BioAssemblyBot® Platform Exhibit High Levels of Viability Cells of the human adipose stromal fraction (ASF) were printed under ambient conditions in the BioAssemblyBot® platform and were suspended in standard growth medium. The percent viability of cells that had been printed were compared to those that had been hand-pipetted into wells of a multi-well plate. After 7 days of culturing, there was not a significant difference in percent viability between these two cell populations (Figure 1). Cell Populations Are Precisely Localized Within 3D Constructs When Printing with the BioAssemblyBot® Human Umbilical Vein Endothelial Cells, or HUVECs, were suspended within 3mg/ml of cold, pH neutral type I collagen at a concentration of 2 million cells/ml and loaded into the BioAssemblyBot® cold tool to prevent gelation. This cell/collagen suspension was then printed inside of 400 μl of un-gelled collagen within a well of a 48-well plate with the print stage temperature set to 25°C during the print. The cell/collagen mix was printed in a 3D line as shown in the schematic and accompanying images. Blue dye was added to the cells/collagen to visualize the printed path. (Figure 2). The ability of the BioAssemblyBot® platform to fabricate 3D constructs out of primary and cultured human cells while maintaining viability and spatial control is key towards the utilization of the system to create complex tissue structures. From design to fabrication, the BioAssemblyBot® enables physiologically relevant tissue generation in a biocompatible manner.

The Advanced Solutions team in Manchester, NH, lead by Dr. Jay Hoying, have been studying the use of Angiomics™ microvessels into Matrigel-based models and assays to improve the biology in a model. Here are their results: Matrigel Matrigel is a solubilized basement membrane matrix derived from EHS mouse sarcomas that is rich in growth factors and signaling molecules. It is commonly used in a variety of applications in biomedical research, acting as a platform for complex signaling and as a support for cells and tissues. For example, it is often used in tumor spheroid models to support cellular hyperproliferation, differentiation, and invasion, key features of tumors (Benton, et al., Adv Drug Deliv Rev, 2014. 79-80: p. 3-18). Matrigel stimulates endothelial adhesion and migration, enhance protease activity, and enables cord formation. As such, it is frequently used in endothelial cell assays serving as simplified models for studying angiogenic activity (Arnaoutova and Kleinman, Nat Protoc, 2010. 5(4): p. 628-35). Matrigel can be used by itself, or combined with collagen, to coat cell culture plates or used as a 3D matrix to directly encapsulate cells or spheroids in 3D culture. Angiomics™ Microvessels and Matrigel Vascularization is a key for the survival and function of nearly every tissue type and is important to include in tissue models. The Angiomics™ microvessel system is a tissue vascularization system utilizing intact fragments of human adipose microvasculatures. These microvessels, when embedded in extracellular matrix, spontaneously sprout, grow, and form a robust neovascular network via bona fide sprouting angiogenesis. As the uses for microvessels continue to expand, we evaluated the angiogenic potential of the Angiomics™ microvessels in Matrigel. Here, we cultured microvessels in a 3mg/ml collagen matrix (our standard reference condition), a pure Matrigel matrix, and a 50:50 collagen:Matrigel matrix (final collagen concentration of 1.5mg/ml). Microvessels are used at a concentration of 100k vessels per ml in all groups. After gelling, angiogenic medium containing RPMI, B27, and 50ng/ml VEGF was added to constructs. After 7 days of culture, phase contrast images were analyzed using Advanced Solutions BioSegment™ Software. This software utilizes machine learning to identify vessel structures and automatically measures vascular-related parameters such vessel length density, a measure of vascularity. Results and Discussion Unlike the extent of angiogenesis by Angiomics™ microvessels cultured in 3D collagen, those cultured in only Matrigel displayed little to no angiogenesis with microvessels dissociating into single cells or single cell clumps (Figure 1). We have previously observed that angiogenic microvessels prefer some amount of fibrillar matrix in order to grow and maintain their structure. While stromal collagen provides this structure for the microvessels, Matrigel alone does not as it represents more of a basement membrane-like composition. In contrast, a blend of collagen and Matrigel promoted robust angiogenesis to much higher total neovessel growth than controls with collagen only (Figure 2). This may be due to growth factors and other signaling molecules present within the Matrigel. Overall, while Matrigel alone may not support microvessel growth, blending Matrigel with other stromal matrices such as collagen may prove advantageous. Thus, incorporating Angiomics™ microvessels into Matrigel-based models and assays is an effective means of improving the biology of the model. Advanced Solutions has a research lab based in Manchester, NH. You can learn more about hour Tissue & Assay Services here.

In a recent review published in Frontiers in Mechanical Engineering, Advanced Solutions’ scientists explore how cell behavior, matrix composition, and tissue forces are critical in producing functional tissues. The team also provides perspectives on the control and manipulation of mechanical forces through designed boundary conditions created using 3D bioprinting-based approaches. Read the full review on Frontiers in Mechanical Engineering.

You’re invited to an exclusive panel discussion this Thursday, November 12th! Advanced Solutions and Cytiva will be hosting a panel discussion about advances in 3D biology with Advanced Solutions President and CEO, Michael Golway, P.E.; Chief Scientist, Dr. Jay Hoying; and Director of Strategic Accounts, Derek Mathers; along with Cytiva’s Cell Analysis Product Specialist Dr. Amy Jablonski. The webinar will highlight innovative techniques in robotics, biofabrication, 3D imaging, and machine learning. When: Thursday, November 12, 2020 at 11:00 Eastern Standard Time Where: Webinar Learn more and register for this free webinar here. Advanced Solutions partners with Cytiva (formerly GE Healthcare Life Sciences) on strategic research & development and distribution. Read more about the partnership here.

The 2nd Australian Bioprinting Workshop for Tissue Engineering and Regenerative Medicine was a great success. University of Technology Sydney’s Department of Biomedical Engineering at the event, in partnership with 3D HEALS, curated an engaging and thought-provoking event with an attendance of over 500 people from leading local and international biotechnology companies, industry, academia and clinician representatives with widely regarded experts in the field. A key highlight of the conference was when the UTS team welcomed Advanced Solutions’ Derek Mathers and Cytiva’s Dr. Alex Rowland to give a joint presentation on their partnership. They shared how BioAssemblyBot® and Cytiva IN Cell Analyzer can be used together to rapidly accelerate innovation of 3D bioprinted constructs, enabling teams to control their tissue models over time. Watch the presentation here (10 minutes). Advanced Solutions partners with Cytiva (formerly GE Healthcare Life Sciences) on strategic research & development and distribution. Read more about the partnership here.

Advanced Solutions CEO and the lead inventor of the BioAssemblyBot®, Michael W. Golway, has been named the 2020 Alumni Fellow of the J.B. Speed School of Engineering at the University of Louisville. To be considered for the honor, one must have displayed outstanding career performance in engineering, exceptional ability in the planning and direction of significant and important projects in technical engineering, as well as fostered professional development of young engineering college students, and made exceptional contributions to technical engineering knowledge. University leadership have bestowed this honor on Michael. Michael received his Bachelor of Science in Engineering Science in 1992 and his Master of Engineering with a specialization in Industrial Engineering in 1993 from the J.B. Speed School of Engineering at the University of Louisville. Michael then earned his Kentucky professional engineering license in 2000. He serves on several boards at the University of Louisville including the engineering school’s Industrial Board of Advisors and as an advisor for both the Bioengineering and Industrial Engineering Departments. Michael is a season ticket holder for Cardinal athletic events and is proud to be a Cardinal. “Speed School taught me how to think and solve problems that put me in a position to not only compete on a global stage, but thrive in a global highly competitive stage,” Michael said while recalling his time at Speed School. “I think of the thousands of my fellow alumni who walked the halls of J.B. Speed and the great things they have accomplished and am humbled to be considered for this honor,” Michael said. Michael previously received the 2015 Professional Award in Industrial Engineering by the Industrial Engineering Department at Speed School. “I am so thankful for my time at Speed School and the University of Louisville. I look back fondly on my college days and how they set me down a path of success,” said Michael. In addition to the 2020 Alumni Fellow, the J.B. Speed School of Engineering awarded honors to an Outstanding Young Bioengineer, and honors to alumni in each of the other six academic departments. You can learn more about those recipients here. You can learn more about the 2020 Alumni Fellow honor and Michael here. Michael Golway, P.E. is the President & CEO of ASI, based in Louisville, KY. The J.B Speed School of Engineering at the University of Louisville which seeks to “serve the University, the Commonwealth of Kentucky, and the engineering profession by providing high quality educational programs to all students; engaging in research and scholarship that will extend knowledge; and assisting the economic development of the regional, state and national economies through technology transfer.” Videos courtesy of the J.B. Speed School of Engineering and the University of Louisville.

With COVID-19 cases continuing to rise across the nation, the U.S. Department of Veterans Affairs is putting their 3D printing network including the BioAssemblyBot® to fight the virus. Dr. Ryan Vega of the VA said “I can take that image, send it to a 3D printer and take it for a patient’s anatomy. This technology allowed us to manufacture necessary equipment, PPE, even testing devices during the pandemic.” Read more about how the VA’s Innovation Ecosystem is using 3D printers to fight COVID-19 on FOX 43.

One of the biggest challenges facing the field of tissue engineering is building a stable yet adaptable microcirculation outside of the body. Advanced Solutions is pioneering the use of human-derived, isolated microvessel fragments in building vasculatures in ex-vivo tissues and tissue models. A recent article published in Frontiers in Physiology, conducted in partnership with the University of Utah’s Scientific Computing and Imaging Institute, demonstrated how stromal cells and chemical cues can be deployed in 3D models to help guide new vessel growth between tissue boundaries and compartments. Angiogenesis - the development of new blood vessels – involves the formation of new vasculatures during development, tissue healing, tumor formation, and tissue grafting. A largely under-studied aspect of angiogenesis is the process by which growing vessels navigate through complex tissue structures and stromal compartments in adult humans. To further explore this important vascular biology, the Advanced Solutions’ team created a novel in-vitro 3D model of a tissue interface consisting of a high-density collagen layer formed between two lower density collagen compartments. In this experiment, the team combined this 3D tissue model system with Angiomics™ microvessels to evaluate how neovessels growing from the parent microvessel fragments across tissue boundaries as they form an expanded neovascular network. The team observed that, unexpectedly, angiogenic neovessels are unable to spontaneously cross the dense collagen interface. The evidence suggested that this was due to the condensed nature of the fibril network of the 3D collagen at the interface, which impeded the neovessel growth across the interface. Years of work by the collaborative team has shown that collagen fibril density, as well as the alignment of the fibrils, provide guidance cues for growing neovessels resulting in differences in neovessel growth rates, branching, and direction. Informed by in vivo implant studies involving the microvessels, further experimentation revealed that stromal cells promoted neovessel invasion through the interface into the neighboring collagen compartment and that this involved biochemical cues from the stromal cells. Additional findings suggest that spatiotemporal cues, perhaps as angiogenic factors, generated by the stromal cells overrode the mechanical cues of the deflective interface to promote neovessel growth into neighboring tissue compartments. In the absence of stromal cells, these factor gradients could be designed into the 3D matrix architecture (3D bioprinting for porosity, fibril layout, solute diffusion) and/or ligand distribution. Better understanding the rules of angiogenesis is helping the Advanced Solutions’ team become the market leader in biofabrication of vascularized, biologically relevant tissue models on their mission to deliver curative solutions. Learn how to use Angiomics™ microvessels to vascularize your 3D Cell Culture. Questions? Get in touch with our Vascularization team