Name: 3d printing of biomolecular models for research and pedagogy
Uploaded: 2017-03-13T14:00:00.000+00:00
Duration: 9 min 17 s
Description: Watch this Scientific Journal Video about 3D Printing of Biomolecular Models for Research and Pedagogy at JoVE.com

The authors are grateful for the support of Deis3D, the Brandeis 3D Printing Club, and members of Brandeis Library/LTS/Makerlab. This work was funded in part by a grant awarded to Pomeranz Krummel by the NSF, Award No. 1157892; an ESIT grant of the BMBF, awarded to the University of T&#252;bingen; and US Federal funds from the National Institutes of Health, Department of Health and Human Services, under Contract No. GS35F0373X. Molecular graphics and analyses were performed with the UCSF Chimera package. Chimera was developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311).

1. Preparing 3D Model Files for Printing
NOTE: 3D model files of biomolecules can be generated through two methods: (1) online using the automated tools of the NIH 3D Print Exchange10, or (2) locally using molecular modelling software. Automatically generated models will use the processes detailed in this protocol to create printable representations, but details of the representation cannot be chosen by the user. In contrast, custom model generation allows the user control over the visual properties of the biomolecule. Individual atoms, residues, and bonds can be displayed, and the scale of ribbons, bonds, and struts can be specified. The NIH 3D Print Exchange automated tools and the protocol below both use UCSF Chimera, a free and open source molecular modelling software package11 that is well suited to exporting 3D files of biomolecules. All 3D files exported by Chimera use angstroms for the distance unit. When these files are imported into a slicing software at 1 mm/distance unit, the models will be scaled at 10 million times magnification.
<ol>
	<li>Automatically generate a 3D printable model with the NIH 3D Print Exchange 
	NOTE: The NIH 3D Print Exchange runs Chimera scripts, which are similar to the steps described in steps 1.2-1.3.
	<ol>
		<li>Locate the molecular data file of the biomolecular structure to print from a database-either the PDB, EMDB, or PubChem (supplement 1.1). Record the accession number for the biomolecule of interest.</li>
		<li>Navigate to the NIH 3D Print Exchange (3dprint.nih.gov) and create a new user account, if a first-time user.</li>
		<li>Navigate to the &#34;Quick Submit&#34; feature, enter the biomolecule accession code, and click submit.</li>
		<li>After generating the model of the biomolecule, navigate to the model page and download the biomolecule STL file in &#34;ribbon&#34; or &#34;surface&#34; representation. Proceed to section 2 of the protocol.</li>
	</ol>
	</li>
	<li>Generate a custom molecular model with UCSF Chimera 
	NOTE: Greater detail on using Chimera for making 3D models, including command line equivalents for many steps, can be found in supplement 1.2.
	<ol>
		<li>Download and install UCSF Chimera (https://www.cgl.ucsf.edu/chimera/download.html).</li>
		<li>Using Chimera, retrieve the molecular data file by doing one of the following:
		<ol>
			<li>Using the toolbar command File &#62; Fetch by ID, enter a Pubchem, PDB, or EMDB accession code to retrieve the file directly from the database.</li>
			<li>Using the toolbar command File &#62; Open, retrieve a local molecular data file; by default, the molecule will display ribbons for protein and nucleic acid, atoms and bonds for ligands, and residues within 5 &#197; of a ligand.</li>
			<li>Using the Chimera command line, accessed by going to Favorites &#62; Command Line, use the Open command and enter the accession code.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparing a 3D-printable representation of a biomolecule 
	NOTE: There are several ways that a biomolecular structure may be displayed or represented. The selection of a particular representation for printing should be made based on how best to provide the greatest insight into the biomolecule structure-function. Commonly used representations include &#34;ribbons,&#34; &#34;surface,&#34; and &#34;atoms/bonds.&#34; However, it is best to explore using a combination of these representations to display select side chains or ligands. In addition, the 3D-printed structure should be robust enough to be printed and to not break when handled. Thus, it is important to consider this when selecting a representation or displaying a side chain. Also consider introducing supporting structures, or &#34;struts.&#34; Finally, when printing the model, it will be important to scale it so that all features will print correctly. Thus, for larger biomolecules, printing entirely in ribbons or atom representations might not be feasible due to the scale at which these would need to be printed.
	<ol>
		<li>Generating a 3D-printable representation in &#34;ribbons&#34; 
		&#8203;Note: More detail can be found in supplement 1.2.1.
		<ol>
			<li>Select the visible &#34;solvent,&#34; which includes ions, by using Select &#62; Structure &#62; solvent.</li>
			<li>Hide the selected &#34;solvent&#34; using Actions &#62; Atoms/Bonds &#62; Hide.</li>
			<li>Thicken the diameter of the ribbon so that it can be successfully printed. Use the Ribbon Style Editor menu under Tools &#62; Depiction. 
			NOTE: Suggested parameters for an initial attempt: under the Scaling tab, change the Height of every item to at least 0.7 and the width of each setting to at least the following: Coil 0.7; Helix 1.4; Sheet:1.4; Arrow (base): 2.1; Arrow (tip) 0.7; Nucleic 1.0.</li>
			<li>If a nucleic acid is present, alter the base representation with Actions &#62; Atoms/Bonds &#62; nucleotide objects &#62; settings. Change the sugar/base display to ladder and the rung radius to 0.6.</li>
			<li>Optional: Proceed to step 1.3.3 to introduce support structures.</li>
		</ol>
		</li>
		<li>Generate a 3D-printable &#34;surface&#34; representation of the molecule 
		NOTE: More detail can be found in supplement 1.2.2.
		<ol>
			<li>Hide all previous representations. Use Actions &#62; Atoms/Bonds &#62; hide, and Actions &#62; Ribbons &#62; hide.</li>
			<li>When rendering atoms as spheres, adjust the radius by selecting the desired atom(s) and going to the Actions &#62; Inspect menu. 
			NOTE: Changing the default atomic radius can make it easier to distinguish different atom types in the printed model.</li>
			<li>Hide any displayed ribbons, atoms, bonds, and pseudobonds by using Actions &#62; Atoms/Bonds &#62; Hide and Actions &#62; Ribbons &#62; Hide.</li>
			<li>If surface detail is desired, add hydrogen atoms so that the surface calculation is more accurate. Use Tools &#62; Structure Editing &#62; AddH.</li>
			<li>Generate a surface by entering surf #0 grid 0.5 in the command line.</li>
		</ol>
		</li>
		<li>Generate a 3D-printable representation in &#34;atoms/bonds.&#34; 
		NOTE: More detail can be found in supplement 1.2.3.
		<ol>
			<li>Hide the solvent. Use Select &#62; Structure &#62; solvent and then Actions &#62; Atoms/Bonds &#62; hide.</li>
			<li>Display specific residues and/or ligands in the representation as atoms and bonds by selecting and showing them with Actions &#62; Atoms/Bonds &#62; show. The way atoms are represented can be altered in the Actions &#62; Atoms/Bonds dropdown by selecting stick, ball &#38; stick, or sphere.</li>
			<li>After making a selection, increase the radius of the stick or ball and stick representations with the Actions &#62; Inspect menu.</li>
			<li>Optional: Proceed to step 1.3.4 to introduce support structures.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Adding structural support to a 3D-printable representation 
	NOTE: More detail can be found in supplements 1.2.4 and 1.2.5. At this stage, struts can be added to the 3D model. It is recommended for alpha-helices and beta-sheet secondary structures to include backbone hydrogen bonds for stability, although small proteins (i.e., less than 50 residues) are often represented as a ribbon with a typical thickness and may print well without such support. However, for larger proteins, even with the addition of hydrogen bonds, many ribbon models are still too delicate to be successfully printed. Struts are physical connections within the model that do not reflect any molecular property but add to the mechanical strength, thus facilitating printing and handling. Chimera offers a quick way to automatically add struts to a model with the strut command via the command line, and individual struts may also be manually displayed using the Distances tool.
	<ol>
		<li>Display hydrogen bonds to prepare a sturdier print. Use the menu Tools &#62; Structure Analysis &#62; FindHBond.</li>
		<li>Use Tools &#62; General Controls &#62; PseudoBond Panel to modify the hydrogen bonds. Select the &#34;hydrogen bond&#34; pseudobonds, click the attributes button and check the &#34;Component PseudoBond Attributes&#34; box. In the bottom panel, change the bond style from wire to stick and the radius value from 0.2 to 0.6.</li>
		<li>Optional: Add support structure(s), or &#34;struts,&#34; using the struts command. To create blue struts with a radius of 1.0 &#197; in the carbon alpha of every 70 residues no further than 8 &#197; apart, use the command: struts @ca length 8 loop 70 color blue rad 1.0 fattenRibbon false.</li>
		<li>Optional: To create individual struts with the Distances tool, select two atoms by shift-ctrl-clicking on each of them, use Tools &#62; Structure Analysis &#62; Distances, and click create to add a pseudobond. Navigate to the PseudoBond Panel, select the &#34;distance monitor&#34; pseudobonds, click the attributes button, and check the &#34;Component PseudoBond Attributes&#34; box. In the bottom panel, change the bond style from wire to stick and the radius value from 0.2 to 0.01.</li>
		<li>Exporting the Chimera rendering as an STL 3D model file
		<ol>
			<li>Once the desired representation has been obtained, use File &#62; Export Scene to export the 3D file. Select STL as the file type and name and save the model. Note: This STL file can be repaired, oriented, and printed as described in section 2 of the protocol.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Process STL Files for Printing
<ol>
	<li>Repair STL files with Autodesk Netfabb 
	NOTE: A model may require repair when it contains multiple overlapping pieces with intersecting geometries, which is generally the case with ribbon models and atomic models. Overlapping geometry may cause errors when the file is read by some slicing software, as intersecting regions can be interpreted as the exterior of the model. More detail can be found in supplement 2.1.
	<ol>
		<li>Download and install the Standard version of the software.</li>
		<li>Open the program and import the STL file to be repaired. If there are problems with the mesh, a warning sign will be displayed.</li>
		<li>Use Extras &#62; Automatic Part Repair, select Extended Repair, and wait while the file is processed; for small models, this will take seconds, but for large models, it may take minutes.</li>
		<li>Right-click on the model and select Export Part &#62; As STL or use Project &#62; Export Project as STL to save the repaired model; the program will add &#34;repaired&#34; to the filename to distinguish it from the original file.</li>
	</ol>
	</li>
	<li>Orient models for printing with Autodesk Meshmixer 
	NOTE: Optimal orientation of a model prior to slicing will reduce the number of overhangs and therefore the number of supports required during the printing process. An optimally oriented model will print faster, use less material, and be less likely to fail during printing. More detail can be found in supplement 2.2.
	<ol>
		<li>Download and install software</li>
		<li>Import the repaired STL file into the program.</li>
		<li>Select Analysis &#62; Orientation.</li>
		<li>Adjust the Strength Weight value to 100, Support Vol Weight value to 0, Support Area Weight to 0, and then update the model. This will rotate the model to minimize the number of overhangs. Accept the resulting orientation.</li>
		<li>Use File &#62; Export and choose binary STL file from the dropdown menu. Save the file.</li>
	</ol>
	</li>
</ol>
3. Slicing and Printing
<ol>
	<li>Select a filament material 
	NOTE: The selection of a printing material should be done before using a slicing software, as printing settings will differ for the selected material. The three materials that are widely used are polylactic acid (PLA), thermoplastic elastomers (TPE), and acrylonitrile butadiene styrene (ABS). PLA is generally the most effective material for printing detailed molecular models, as it cools quickly, adheres well to the build plate, and rarely warps. TPE is a material similar to PLA and can be used to produce flexible models. It is recommended for complementary protein surface models or protein ribbon models. ABS is stronger and more flexible than PLA, but it produces potentially hazardous particulates while printing12. It is generally not recommended for printing molecular models, as the higher material temperature results in less precise production of small features. More detail can be found in supplement 3.1.
	<ol>
		<li>Printing with polylactic acid (PLA).
		<ol>
			<li>Set the nozzle temperature to 210 &#176;C. To ensure the adhesion of the part to the bed, set the bed temperature to 70 &#176;C. If using an unheated bed, cover it with painters&#39; tape. Use active cooling.</li>
		</ol>
		</li>
		<li>Printing with thermoplastic elastomers (TPE)
		<ol>
			<li>Repeat step 3.1.1.1. Set the printing speed to 1,200 mm/min or less.</li>
		</ol>
		</li>
		<li>Printing with acrylonitrile butadiene styrene (ABS)
		<ol>
			<li>Do not use cooling. Set the nozzle temperature to 240 &#176;C. To ensure the adhesion of the part to the bed, set the bed temperature to 110 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Generate G-code 
	NOTE: The model will be imported at 10 million times magnification by default. Ribbon models should be scaled to 20 million times (200%) or greater. Surface models print well at 100% or greater. More detail can be found in supplement 3.2.
	<ol>
		<li>Download and install printing slicing software.</li>
		<li>Use File &#62; Import Model and select the repaired and oriented STL file.</li>
		<li>Scale the model by double-clicking on the model and entering the scaling factor in the window on the right-hand side of the screen.</li>
		<li>Generate support structures for the model. Select the support icon and use normal supports, with a pillar resolution of 1 and a max overhang angle of 50&#176;.</li>
		<li>Click generate all supports. Add or remove support structure features to customize support placement.</li>
		<li>Select a process and click edit process settings.</li>
		<li>Configure the profile for the printer and material that is being used. 
		&#8203;NOTE: A raft and brim should be included, and ribbon models should be printed at 100% infill. Detailed profile settings can be found in supplement 3.2.</li>
		<li>Convert the model into a G-code file that can be read by the printer. Click the &#34;prepare to print&#34; button and select the process containing the printer/material profile. Observe the path of the printer nozzle and inspect it for errors that could cause a print to fail. 
		&#8203;NOTE: Errors that can cause the print to fail include the absence of supports under overhangs, undesired cavities, missing layers, or areas that are too thin to print.</li>
		<li>Save the G-code file to the desktop or directly to an SD card.</li>
	</ol>
	</li>
	<li>Operate the printer 
	NOTE: Each printer make or model is unique, and its preparation and calibration for printing will vary accordingly. Refer to the manual for the printer.
	<ol>
		<li>Ensure that the workstation is connected to the printer or that the SD card with GCODE is in the printer.</li>
		<li>Prepare the printer by loading the filament and ensuring that the bed is level; for instructions on these procedures, refer to the manual of the printer.</li>
		<li>Start the print from the computer or locally from an SD card via the printer menu.</li>
		<li>Watch the print until a first layer has been successfully completed. If there are any errors in the first layer, abort and restart the print.</li>
	</ol>
	</li>
</ol>
4. Post-production Processing
NOTE: Care of course should be taken at this, the final, stage. Supporting structures on the model should be removed. This is generally done manually, although alternative approaches, such as use of a dissolvable support, can be used; see supplement 4.
<ol>
	<li>Detach the print from the build plate by gently pulling it sideways. If the raft adheres strongly to the build plate, separate it by inserting a sharp edge between them.</li>
	<li>Remove the support structures from the model. Many supports can be removed by hand, by breaking them off of the part and the raft. Flexible models can be detached by pulling them away from the part. For supports that are difficult to reach or are connected to delicate structures, use cutting pliers to clip the point where the support connects to the part.</li>
</ol>

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The authors have nothing to disclose.

Physical 3D models of biomolecules provide a powerful complement to more common computer-based methods of visualization. The additional properties of a physical 3D representation contribute to the intuitive understanding of biomolecular structure. The construction of physical 3D models of biomolecules can facilitate their study through the use of a medium that takes advantage of well-developed modes of human sensation. 3D models serve not only as an aid to the researcher, but may be used to facilitate pedagogical activities and can increase the achievement of learning outcomes13,14,15. Magnets can be added to plastic models to allow for assembly and disassembly, as shown with a model of polypeptides16. Also, 3D-printed objects can be used in research, both in the manufacturing of lab equipment17, as well as to make microfluidic devices for cells18 and models of crystals19 or neurons20. The manipulation of physical models can serve to promote collaborative discussions that can inspire new insights.
Recent developments in 3D printing technologies and reductions in the cost of printers enables the creation of complex, physical 3D models of biomolecules by an individual user. Although FFF printing technology is more common and less expensive than other methods, it poses a number of limitations. The 3D printing process is time consuming, and mechanical failures do occur. FFF printers can usually only print one material per part, restricting the display of color information. The resolution of models made on FFF printers is low, around 100 &#181;m per layer. We advise the reader to work with these limitations and to develop an approach for their printer and biomolecule(s) of interest. We have presented the processes required for a user to develop a custom 3D representation of their biomolecule of interest that is accurate, informative, and printable. As with any new technology, there are often &#34;growing pains&#34; that must be overcome during its usage. We provide several examples where problems may be encountered in the process of 3D printing biomolecules (see supplement 6).
Finally, through this article, it is our objective to contribute to the growth of a community of users engaged in the 3D printing of biomolecules. Importantly, the NIH has established a database for the public to share 3D models and the methods used to print them10. We strongly encourage participation in this unique resource (see supplement 7 for instructions on how to upload a 3D model print and background information to the NIH 3D Print Exchange).

A thorough understanding of the function and activity of a biomolecule requires the determination of its three-dimensional (3D) structure. This is routinely achieved using X-ray crystallography, NMR, or electron microscopy. 3D structures can be understood through the perception of models, or accurate objects resembling the structures that they represent1. Historically, the construction of physical 3D models was necessary for investigators to validate, explore, and communicate the resulting hypotheses regarding function of biomolecules. These models, such as Watson-Crick&#39;s DNA double helix and Pauling&#39;s alpha helix, provided unique insight into structure-function relationships and were pivotal to our early understanding of nucleic acid and protein structure-function2,3,4. Although complex protein and nucleic acid models can be created, the time and cost of building a physical model was eventually outweighed by the relative ease of computer-aided molecular visualization.
The development of 3D printing, also known as additive manufacturing, has again enabled the construction of physical models of biomolecules5. 3D printing is the process of fabricating a physical, 3D object from a digital file through the sequential addition of layers of a material(s). A variety of mechanisms are used in this process. Until recently, the machines used to produce physical models of biomolecules were too expensive to be widely used. However, in the last decade, 3D printing technology, fused filament fabrication (FFF) in particular, has advanced significantly, making it accessible for consumer use6. FFF printers are now commonly available in high schools, libraries, universities, and laboratories. The greater affordability and accessibility of 3D printing technology has made it possible to convert digital 3D biomolecular models into accurate, physical 3D biomolecular models7,8,9. Such models include not only simple representations of single biomolecules, but also complex macromolecular assemblies, such as the ribosome and virus capsid structures. However, the process of printing individual biomolecules and macromolecular assemblies poses several challenges, particularly when using thermoplastic extrusion methods. In particular, representations of biomolecules often have complex geometries that are difficult for printers to produce, and creating and processing digital models that will print successfully requires skill with molecular modelling, 3D modelling, and 3D printer software.
The 3D workflow for printing a biomolecule broadly occurs in four steps: (1) preparing a biomolecular model from its coordinate file for 3D printing; (2) importing the biomolecular model into a &#34;slicing&#34; software to segment the model for the printer and to generate a support structure that will physically prop up the biomolecular model; (3) selecting the correct filament and printing the 3D model; and (4) post-production processing steps, including removing support material from the model (Figures 1 and 2). The first step in this process, computationally manipulating the coordinate file of the biomolecule, is critical. At this stage, the user may build model reinforcements in the form of struts, as well as remove structures that are extraneous to what the user chooses to display. In addition, the choice of representation is made at this stage: whether to display all or part of the biomolecule as a surface representation, ribbons, and/or individual atoms. Once the necessary additions and/or subtractions of content are made and the representation is selected, the structure is saved as a 3D model file. Next, the file is opened in a second software program to convert the model into a 3D print file that can be printed, layer by layer, into a plastic replica of the biomolecule.
The goal of our protocol is to make the fabrication of molecular models accessible to the large numbers of users who have access to FFF printers but not to more expensive 3D printing technologies. Here, we provide a guide for the 3D printing of biomolecules from 3D molecular data, with methods that are optimized for FFF printing. We detail how to maximize the printability of complex biomolecular structures and ensure the simple post-processing of physical models. The properties of several common printing materials or filaments are compared, and recommendations on their use to create flexible prints are provided. Finally, we showcase a series of examples of 3D-printed biomolecular models that demonstrate the use of different molecular representations.

<table><tbody><tr><td>&lt;strong&gt;Filament&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PLA 3D Printing Filament (1.0 kg Roll)</td><td>Quantum3D Printing</td><td>http://quantum3dprinting.com/</td><td>Very good quality PLA filament, strongly recomended</td></tr><tr><td>NinjaFlex Flexible 3D Printing Filament</td><td>Ninjatek</td><td>https://ninjatek.com/</td><td>High quality flexible filament</td></tr><tr><td>PLA Filaments PrimaValue &amp;amp; PrimaSelect</td><td>3DPrima</td><td>http://3dprima.com/</td><td>High quality European supplier of filament</td></tr><tr><td>&lt;strong&gt;Printers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prusa I3 MK2 3D Printer</td><td>Prusa Research</td><td>http://www.prusa3d.com/</td><td>A popular 3D printer</td></tr><tr><td>MakerGear M2 Revision E (M2e)</td><td>MakerGear</td><td>http://www.makergear.com/</td><td>Closed source, very high quality printer</td></tr><tr><td>Ultimaker 2</td><td>Ultimaker</td><td>https://ultimaker.com/</td><td>Very reliable, easy to use printer, highest rating on 3Dhubs.com</td></tr><tr><td>Flashforge Creator Pro</td><td>Flashforge</td><td>http://www.flashforge-usa.com</td><td>Reliable, dual extrusion printer, highest rating on 3Dhubs.com</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Simplify3D Slicer</td><td>Simplify3D</td><td>https://www.simplify3d.com/</td><td>Excellent slicing software</td></tr><tr><td>Netfabb</td><td>Autodesk</td><td>http://www.autodesk.com/education/free-software/netfabb</td><td>Mesh repair software, available free of cost for educational purposes</td></tr><tr><td>Chimera</td><td>University of California, San Francisco</td><td>https://www.cgl.ucsf.edu/chimera/</td><td>Chimera molecular vizualizer</td></tr><tr><td>Meshmixer</td><td>Autodesk</td><td>http://www.meshmixer.com/</td><td>Used for orienting models, but has other features</td></tr></tbody></table>

3d printing of biomolecular models for research and pedagogy

The construction of physical three-dimensional (3D) models of biomolecules can uniquely contribute to the study of the structure-function relationship. 3D structures are most often perceived using the two-dimensional and exclusively visual medium of the computer screen. Converting digital 3D molecular data into real objects enables information to be perceived through an expanded range of human senses, including direct stereoscopic vision, touch, and interaction. Such tangible models facilitate new insights, enable hypothesis testing, and serve as psychological or sensory anchors for conceptual information about the functions of biomolecules. Recent advances in consumer 3D printing technology enable, for the first time, the cost-effective fabrication of high-quality and scientifically accurate models of biomolecules in a variety of molecular representations. However, the optimization of the virtual model and its printing parameters is difficult and time consuming without detailed guidance. Here, we provide a guide on the digital design and physical fabrication of biomolecule models for research and pedagogy using open source or low-cost software and low-cost 3D printers that use fused filament fabrication technology.

Stable and informative 3D printable models of biomolecules can be prepared by: (i) thickening bonds to provide stability, (ii) carefully selecting the secondary structure representation type or style that would provide the greatest insight and stability, (iii) printing the biomolecule in more than one molecular representation, (iv) using a filament that will render all or part of a biomolecule flexible, or (v) generating a complex assembly that is modular (i.e., in connectable pieces).
To illustrate how to print such informative and stable models, we focused on the components of chromatin and on producing a hypothetical model of chromatin. Chromatin is a highly complex protein-DNA assembly. The fundamental protein subunit of chromatin is the histone protein. There are four histone proteins, each consisting of a helix-loop-helix (a &#34;histone fold&#34;) followed by an extended alpha helix and a second &#34;histone fold.&#34; Histone protein structure can easily be produced by using a &#34;ribbon&#34; representation (Figure 3A). Alternatively, the histone protein structure can be displayed using only its surface (Figure 3B). There are two copies of each of the four histone proteins, which assemble to form a globular histone octamer. The histone octamer is too big to print entirely as a ribbon or stick representation, due to the larger scale at which these features need to be printed. Thus, such a large protein assembly is best displayed using surface representation (Figure 3C). DNA will chart a path around the histone octamer to form a 10 nm-diameter nucleosome core particle. The path of DNA can best be displayed by printing two separate models and using a flexible filament for the DNA (Figure 3D). Nucleosome core particles stack upon each other to form a higher-order assembly, a 30 nm-diameter &#34;fiber,&#34; a left-handed suprahelical structure. To best illustrate how the 10-nm nucleosome core particles may stack to form a 30-nm chromatin assembly, print individual &#34;di-nucleosome&#34; particles (Figure 3E) and then stack them after printing (Figure 3F).
Once mastered the single extrusion surface and ribbon workflow described above, explore making a range of atomic, molecular, and composite models, as illustrated in Figure 4. For example, combine surface and ribbon representations to set apart different parts of a complex (see DNA polymerase, Figure 4B). Make more instructive and appealing models by using a dual extrusion printer that can melt two filaments simultaneously into a single 3D object (see Figure 4C). Alternatively, paint parts of the models (see guanine and the alpha helix, Figure 4A). Print and assemble the subunits of a protein complex, like the sodium channel, or take it even further by printing distinct parts of a complex and assembling them later into a larger, multi-color model (see the HIV-antibody and ribosomal complexes, Figure 4C). Such composite models are better able to show functional features compared to single-filament prints. Different colors can highlight, for example, glycosylation versus protein (HIV model) or RNA versus protein (see ribosome model, Figure 4C). They also allow for the creation of educational 3D puzzles, like the antibody binding to the HIV surface (see gp120 bound by antibody, Figure 4C), where just one 3D configuration gives a close fit of both parts. Instructions on printing these models can be found in supplement 5. &#160;In addition, we have provided&#160;a supplemental video illustrating the construction of a 3D model of the Fo/F1 proton ATP synthase which was printed in pieces and assembled in such a manner so that it can recapitulate the rotary mechanism that occurs during this enzymes catalytic mechanism.
<img alt="Figure 1" src="/files/ftp_upload/55427/55427fig1.jpg" /> 
Figure 1. Workflow to prepare and print a 3D model. Illustrated are the stages in producing a physical 3D biomolecular print: (i) preparing the model, including selecting the representation; (ii) opening a saved .stl file of the model and processing the file using slicing software; (iii) printing the model and choosing the material or filament; and finally, (iv) performing the post-production steps. <a href="http://cloudflare.jove.com/files/ftp_upload/55427/55427fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55427/55427fig2.jpg" /> 
Figure 2. Visuals of different representations of models at various stages of preparation. Top row: Common representations of two models (ubiquitin (PDB 1UBQ) and arginine) visualized using the program Chimera. Middle row: The printing toolpath generated from the Chimera STL models, colored by the feature type of ubiquitin and arginine (orange: infill pattern; dark blue: outer shell; light blue: inner shell). Bottom row: Final prints of ubiquitin and arginine. Surface and two ribbon models of ubiquitin printed at 300% of the default Chimera STL output (Chimera default is 1 nm in the model and 1 cm in print), while the arginine model was printed at 1,000%. The Chimera default ribbon or stick models are too thin to print properly, but thickened versions will print reliably. <a href="http://cloudflare.jove.com/files/ftp_upload/55427/55427fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55427/55427fig3.jpg" /> 
Figure 3. Nucleosome case study. (A) Single-histone H3 protein rendered by thickening &#34;ribbons,&#34; printed at 300%. (B) Histone H3 protein &#34;surface&#34; representation, printed at 200%. (C) Histone protein octamer printed at 100%. (D) Histone protein octamer (orange) in complex with flexible DNA (white) printed at 100%. (E) Dinucleosome surface model printed with a default probe radius and printed at 100% scale. (F) A model of the chromatin &#34;30-nm fiber&#34; created by manually stacking individually printed models of the &#34;10-nm&#34; dinucleosome, where the surface was rendered with a probe radius of 3 &#197;, printed at 50% and 25% sizes, and held together with Play-Doh. 3D prints were generated from a model of the dinucleosome (PDB 1ZBB). All models are freely available for download at the NIH 3D Print Exchange11. <a href="http://cloudflare.jove.com/files/ftp_upload/55427/55427fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55427/55427fig4.jpg" /> 
Figure 4. Examples of 3D-printed models produced using filament printers. (A) Left, a ball-and-stick model of water molecules in hexagonal ice crystals (dual-filament print). Middle, model of a nucleotide (guanine). Right, a protein alpha helix backbone-only model showing hydrogen bonds (black). Guanine and the alpha helix were colored manually with sharpies. (B) Left, sodium channel, composed of 4 subunits that can be joined together (PDB 3E89). Middle, Plasmodium falciparum L-lactate dehydrogenase (PDB 1T2D) printed as ribbons. Right, model of the DNA polymerase active site (PDB 1KLN), showing DNA as surface and protein as ribbons. (C) Left, HIV lipid envelope with glycoprotein (PDB 5FUU) bound by antibodies (PDB 1IGT), printed at 15%. Middle, detail of the glycoprotein antigen surface at 150%, with the variable region of the antibody shown as ribbons (PDB 5FYJ). Right, models of the bacterial 70S Ribosome (PDB 4V5D) at 40% and 20%. Percentages refer to standard Chimera output, where 100% means 1 nm in the molecule prints as 1 mm. All models are freely available for download at the NIH 3D Print Exchange11. <a href="http://cloudflare.jove.com/files/ftp_upload/55427/55427fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about 3D Printing of Biomolecular Models for Research and Pedagogy at JoVE.com

3D Printing of Biomolecular Models for Research and Pedagogy

Physical models of biomolecules can facilitate an understanding of their structure-function for the researcher, aid in communication between researchers, and serve as an educational tool in pedagogical endeavors. Here, we provide detailed guidance for the 3D printing of accurate models of biomolecules using fused filament fabrication desktop 3D printers.

The construction of physical three-dimensional (3D) models of biomolecules can uniquely contribute to the study of the structure-function relationship. 3D ...

3d-printing-of-biomolecular-models-for-research-and-pedagogy

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

23.6K Views.  Brandeis University. Physical models of biomolecules can facilitate an understanding of their structure-function for the researcher, aid in communication between researchers, and serve as an educational tool in pedagogical endeavors. Here, we provide detailed guidance for the 3D printing of accurate models of biomolecules using fused filament fabrication desktop 3D printers.

Video: 3D Printing of Biomolecular Models for Research and Pedagogy

Multimodal 3D Printing of Phantoms to Simulate Biological Tissue

Biomedical optical imaging is playing an important role in diagnosis and treatment of various diseases. However, the accuracy and the reproducibility of an optical imaging device are greatly affected by the performance characteristics of its components, the test environment, and the operations. Therefore, it is necessary to calibrate these devices by traceable phantom standards. However, most of the currently available phantoms are homogeneous phantoms that cannot simulate multimodal and dynamic characteristics of biological tissue. Here, we show the fabrication of heterogeneous tissue-simulating phantoms using a production line integrating a spin coating module, a polyjet module, a fused deposition modeling (FDM) module, and an automatic control framework. The structural information and the optical parameters of a &#34;digital optical phantom&#34; are defined in a prototype file, imported to the production line, and fabricated layer-by-layer with sequential switch between different printing modalities. Technical capability of such a production line is exemplified by the automatic printing of skin-simulating phantoms that comprise the epidermis, dermis, subcutaneous tissue, and an embedded tumor.

Spin coating, polyjet printing, and fused deposition modeling are integrated to produce multilayered heterogeneous phantoms that simulate structural and functional properties of biological tissue.

Biomedical optical imaging is playing an important role in diagnosis and treatment of various diseases. However, the accuracy and the reproducibility of ...

Interactive Molecular Model Assembly with 3D Printing

With the growth in accessibility of 3D printing, there has been a growing application of and interest in additive manufacturing processes in chemical laboratories and chemical education. Building on the long and successful history of physical modeling of molecular systems, we present select models along with a protocol to facilitate 3D printing of molecular structures that are able to do more than represent shape and connectivity. Models assembled as described incorporate dynamic aspects and degrees of freedom into saturated hydrocarbon structures. As a representative example, cyclohexane was assembled from parts printed and finished using different thermoplastics, and the resulting models retain their functionality at a variety of scales. The resulting structures show configurational space accessibility consistent with calculations and literature, and versions of these structures can be used as aids to illustrate concepts that are difficult to convey in other ways. This exercise enables us to evaluate successful printing protocols, make practical recommendations for assembly, and outline design principles for physical modeling of molecular systems. The provided structures, procedures, and results provide a foundation for individual manufacture and exploration of molecular structure and dynamics with 3D printing.

Physical modeling of microscopic systems helps obtain insights that are difficult to gain by other means. To facilitate the construction of physical molecular models, we demonstrate how 3D printing can be used to assemble functional macroscopic models that capture qualities of molecular systems in a tactile way.

With the growth in accessibility of 3D printing, there has been a growing application of and interest in additive manufacturing processes in chemical ...

Chemistry

Scaled Anatomical Model Creation of Biomedical Tomographic Imaging Data and Associated Labels for Subsequent Sub-surface Laser Engraving (SSLE) of Glass Crystals

Biomedical imaging modalities like computed tomography (CT) and magnetic resonance (MR) provide excellent platforms for collecting three-dimensional data sets of patient or specimen anatomy in clinical or preclinical settings. However, the use of a virtual, on-screen display limits the ability of these tomographic images to fully convey the anatomical information embedded within. One solution is to interface a biomedical imaging data set with 3D printing technology to generate a physical replica. Here we detail a complementary method to visualize tomographic imaging data with a hand-held model: Sub Surface Laser Engraving (SSLE) of crystal glass. SSLE offers several unique benefits including: the facile ability to include anatomical labels, as well as a scale bar; streamlined multipart assembly of complex structures in one medium; high resolution in the X, Y, and Z planes; and semi-transparent shells for visualization of internal anatomical substructures. Here we demonstrate the process of SSLE with CT data sets derived from pre-clinical and clinical sources. This protocol will serve as a powerful and inexpensive new tool with which to visualize complex anatomical structures for scientists and students in a number of educational and research settings.

Scaled Anatomical Model Creation of Biomedical Tomographic Imaging Data and Associated Labels for Subsequent Sub-surface Laser Engraving SSLE of Glass Crystals

A methodology is described herein for representing anatomical imaging data within crystals. We create scaled three-dimensional models of biomedical imaging data for use in Sub-Surface Laser Engraving (SSLE) of crystal glass. This tool offers a useful complement to computational display or three-dimensionally printed models used within clinical or educational settings.

Biomedical imaging modalities like computed tomography (CT) and magnetic resonance (MR) provide excellent platforms for collecting three-dimensional data ...

Bioengineering

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

This article describes a method of transforming a low-cost commercial 3D printer into a bacterial 3D printer that can facilitate printing of patterned biofilms. All necessary aspects of preparing the bioprinter and bio-ink are described, as well as verification methods to assess the formation of biofilms.

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced ...

Voxel Printing Anatomy: Design and Fabrication of Realistic, Presurgical Planning Models through Bitmap Printing

Most applications of 3-dimensional (3D) printing for presurgical planning have been limited to bony structures and simple morphological descriptions of complex organs due to the fundamental limitations in accuracy, quality, and efficiency of the current modeling paradigm. This has largely ignored the soft tissue critical to most surgical specialties where the interior of an object matters and anatomical boundaries transition gradually. Therefore, the needs of the biomedical industry to replicate human tissue, which displays multiple scales of organization and varying material distributions, necessitate new forms of representation. 
Presented here is a novel technique to create 3D models directly from medical images, which are superior in spatial and contrast resolution to current 3D modeling methods and contain previously unachievable spatial fidelity and soft tissue differentiation. Also presented are empirical measurements of novel, additively manufactured composites that span the gamut of material stiffnesses seen in soft biological tissues from MRI and CT. These unique volumetric design and printing methods allow for deterministic and continuous adjustment of material stiffness and color. This capability enables an entirely new application of additive manufacturing to presurgical planning: mechanical realism. As a natural complement to existing models that provide appearance matching, these new models also allow medical professionals to "feel" the spatially varying material properties of a tissue simulant-a critical addition to a field in which tactile sensation plays a key role.

This method demonstrates a voxel-based 3D printing workflow, which prints directly from medical images with exact spatial fidelity and spatial/contrast resolution. This enables the precise, graduated control of material distributions through morphologically complex, graduated materials correlated to radiodensity without loss or alteration of data.

Most applications of 3-dimensional (3D) printing for presurgical planning have been limited to bony structures and simple morphological descriptions of ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Biology

Bioprinting Bacteria in Hydrogel Matrices for the Study of Motility and Growth

3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. However, the majority of previous work has focused on studies of cells in bulk liquids or at flat surfaces, which do not fully recapitulate the complexity of many natural bacterial habitats. Here, this gap in knowledge is addressed by describing the development of a method to 3D-print dense colonies of bacteria into jammed granular hydrogel matrices. These matrices have tunable pore sizes and mechanical properties; they physically confine the cells, thus supporting them in 3D. They are optically transparent, allowing for direct visualization of bacterial spreading through their surroundings using imaging. As a proof of this principle, here, the capability of this protocol is demonstrated by 3D printing and imaging non-motile and motile Vibro cholerae, as well as non-motile Escherichia coli, in jammed granular hydrogel matrices with varying interstitial pore sizes.

Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices

This protocol describes a procedure for three-dimensional (3D) printing of bacterial colonies to study their motility and growth in complex 3D porous hydrogel matrices that are more akin to their natural habitats than conventional liquid cultures or Petri dishes.

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. ...

Design and Validation of a Volumetric-extrusion Bioprinter for Bioprinting of Soluble Basement Membrane Extract for Translational Research

In translational research, 3D in vitro models are essential for advancing scientific understanding and therapeutic development. To this aim, Soluble Basement Membrane Extracts (SBMes), derived from mouse sarcoma cells, play the key role of replicating mechanical and biochemical features of in vivo microenvironment. They are particularly valuable in a variety of research fields, from tissue engineering to drug testing. In particular, they have become fundamental in cancer research as traditional 2D cell cultures, widely used in the field, can provide misleading information since they do not capture the three-dimensional (3D) structure and tumor microenvironment.
In cancer research, effective 3D in vitro models are crucial for better understanding cancer evolution and anticipating challenges, such as drug resistance mechanisms. Organoids have emerged as promising 3D in vitro models, offering a more accurate representation of tumor biology. They grow in SBMes as they create an environment where cells can grow and self-organize into 3D structures. However, SBMe presents significant challenges, including low mechanical properties and complex rheological behavior, preventing its use with commercially available pneumatic-extrusion bioprinting systems. 
To address these limitations, we developed a low-cost, volumetric-controlled bioprinting system and a specific protocol for printing structures with SBMe. This system is based on a volumetric extruder and overcomes the limitations of traditional pneumatic-driven approaches. Once assembled and programmed with a specific g-code, the system can bioprint both pure and diluted SBMe to obtain both single and multi-layer constructs. This approach offers a more reliable and scalable method for producing 3D cell culture models, paving the way for more effective research into novel treatments and drug testing.

Soluble basement membrane extracts are the most widely used biological matrices in cancer research, but their complex rheological behaviour makes their bioprinting difficult with commercially available bioprinting systems. This work presents a customized bioprinting strategy to produce pure matrix constructs with good shape fidelity in both single and multiple layers.

In translational research, 3D in vitro models are essential for advancing scientific understanding and therapeutic development. To this aim, Soluble ...

Applications of 3D-Printing for Micro-Scale Neuronal Cell Culture Devices

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Author Spotlight: Modular Neuronal Networks for Analyzing Brain Functions

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

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Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices