The authors would like to acknowledge Lina DeLuca, Fakhar Singhera, Hannah Williams, Lynn Deng, Osinachi Nwosu and Sarah Wachtman for their assistance in testing the M.A.P.L.E. platform.

1. Semiautomated &#34;plate to plate&#34; sample transfer preparation
<ol>
	<li>Generate a CSV file as shown in Figure 1 containing source and destination plates using a spreadsheet editing application. The CSV file that is generated must have the following header columns in the order listed: Source_barcode; Destination_barcode; Source_well; Dest_well; Transfer_volume. &#160;&#160;</li>
	<li>Under the header columns, make sure to include one row in the CSV file for each desired pipetting operation (i.e., sample transfer) with the following information:
	<ol>
		<li>Source_barcode: Alphanumeric barcode of the source microplate, e.g., S1007372; leave blank if no barcode is associated.</li>
		<li>Destination_barcode: Alphanumeric barcode of the destination microplate, e.g., D0573282; leave blank if no barcode is associated.</li>
		<li>Source_well: Alphanumeric row and column identifier for well to be pipetted out of from source plate, e.g., H10 for row H (8th row), column 10 (ANSI/SLAS standard well designations, e.g., A1, C10&#8230;).</li>
		<li>Dest_well: Alphanumeric row and column identifier for well to be pipetted out of from destination plate, e.g., A3 for row A (1st row), column 3 (ANSI/SLAS standard well designations, e.g., A1, C10&#8230;).</li>
		<li>Transfer_volume: Volume to be transferred from source_well in source_barcode to dest_well in destination_barcode (numeric and unitless: typically in &#181;L).</li>
	</ol>
	</li>
	<li>Open the Microplate Assistive Pipetting Light Emitter Plate to plate GUI application, shown in Figure 2, by opening the Light Guide program (Maple-LightGuide.exe).</li>
	<li>Click the Select cherrypick file button in the upper left corner of the GUI.</li>
	<li>Use the file browser window, shown in Figure 3, to navigate to the CSV file generated in steps 1.1 and 1.2 above and click the Open button. The application will parse the first row of the CSV file and illuminate the corresponding wells in the source and destination plates.</li>
	<li>Use the Previous well and Next well buttons, in the upper right corner of the GUI, shown in Figure 4, to traverse the CSV file as desired. The GUI will highlight in grey any rows which have been previously illuminated and highlight in brown the currently active row.</li>
	<li>Perform pipetting operations as needed to transfer samples between source well of source plate to destination well of destination plate. An example of a M.A.P.L.E. unassisted hand pipetting operation can be seen in Figure 5, with a comparison of the current user pipetting view seen in Figure 4 and Figure 6. In addition to the user GUI, plate barcodes can be verified via the LCD displays attached to the illumination panels.</li>
	<li>Continue until the end of the CSV file is reached via the Next well button. To load a new CSV file, the Select cherrypick file may be clicked at any time. To exit the program the red X at the top right corner of the GUI can be clicked.</li>
</ol>
2. Multi-well illuminations for parallel transfers and serial dilutions
<ol>
	<li>Open the Microplate Assistive Pipetting Light Emitter &#39;Serial dilution&#39;&#160;application by opening the serial dilution program (Maple-SerialDilution.exe).</li>
	<li>Use the GUI, shown in Figure 7 and Figure 8, to specify the desired titration mode (column or row), plate density and start row(s) or column(s). The GUI also allows users to specify a column or row mask to control which LEDs in a given row or column are illuminated. This allows a subset of LEDs in a row or column to be illuminated instead of illuminating the entire row or column.</li>
	<li>Use the Next and Previous buttons to step through the rows or columns in sequence from the initial start row or column to the last row or column in the plate. Each time the Next or Previous button is clicked, the light panel will illuminate the corresponding LEDs of the microplate.</li>
	<li>Continue until the end of the titration sequence is reached. To exit the program, click the red X at the top right corner of the GUI.</li>
</ol>
3. Laboratory training: assay development and screening format techniques
<ol>
	<li>Place a 96- or 384-well microplate into the portable light guide. The portable light guide contains a battery and all electronics necessary to be used independently of a computer. This allows the portable lightguide to be used in a handheld mode which can be controlled with built-in pushbuttons to toggle between demonstration modes.</li>
	<li>Use the power toggle switch on the portable light guide enclosure to power the system on.</li>
	<li>Determine the mode for the portable light guide to be in. By default, the portable light guide will load into the default HTS demo mode which provides users with a visual representation of a typical assay plate as seen in Figure 9. In this mode, one can use the right pushbutton switch at the top of the portable light guide to toggle through the following sample illumination patterns.
	<ol>
		<li>All wells illuminated with red color to simulate the reagent dispense of an assay, e.g., (suspended cells in media).</li>
		<li>All wells illuminated with a yellow color to simulate dye reagent addition.</li>
		<li>First column and last column of wells illuminated green, remaining middle &#39;sample field&#39;&#160;columns illuminated blue to indicate plate being read on microplate reader. Random wells in the sample field will also have green color of varying intensity to represent hits.</li>
	</ol>
	</li>
	<li>To toggle the light guide between HTS demo mode and Titration demo mode, push the left pushbutton switch. Doing so will switch the portable light guide to the Titration demo mode which provides a visual guide to users to understand how titrations can be performed in compound plates. When the light guide enters the Titration demo mode, the following will occur.
	<ol>
		<li>All wells in column 3 and 13 are illuminated with yellow color.</li>
		<li>Subsequent presses of the rightmost pushbutton switch illuminates columns in sequence, e.g., (4 and 14, 5 and 15, etc.).</li>
		<li>When the pushbutton is pressed after columns 12 and 22 are reached, wells in columns 4-12 and 13-22 are illuminated in decreasing intensity of yellow to represent the titration.</li>
	</ol>
	</li>
	<li>To modify the default behavior of the light guide, plug the portable light guide to a computer via a USB cable and follow the detailed instructions for updating the default firmware via the Arduino IDE which can be found on the project GitHub page8. By updating the firmware, you can modify these modes to display other sequences or sets of LEDs.</li>
</ol>
4. Illumination of artifacts in microplates
<ol>
	<li>Place a 96- or 384-well microplate into the portable light guide.</li>
	<li>Switch the light guide to Illumination mode by pressing the leftmost pushbutton switch two times. 
	NOTE: An example of the practical use of this mode can be seen in Figure 10&#160;and&#160;Figure 11, where compounds have precipitated out of solution and can be observed on the bottom of the microplates. Without backlit illumination, most of the precipitate is invisible to the naked eye, but M.A.P.L.E. backlighting reveals precipitate for user inspection and photographic documentation.</li>
	<li>Use the right pushbutton to toggle between a set of predefined colors as needed for the application. The light panel will turn all LEDs on to the following colors in sequence with each push of the right button: red, blue, green, orange, white, violet, yellow and indigo.</li>
	<li>As an optional step, use a camera or smartphone to photograph the illuminated plate for recordkeeping or documenting the work.</li>
</ol>

<ol>
	<li>Baillargeon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+Microplate-Compatible+Illumination+Panels+for+a+Semiautomated+Benchtop+Pipetting+System.">Design of Microplate-Compatible Illumination Panels for a Semiautomated Benchtop Pipetting System.</a> SLAS TECHNOLOGY: Translating Life Sciences Innovation. , (2019).</li><li>Baillargeon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Scripps+Molecular+Screening+Center+and+Translational+Research+Institute.">The Scripps Molecular Screening Center and Translational Research Institute.</a> SLAS DISCOVERY: Advancing Life Sciences R&amp;D. 24 (3), 386-397 (2019).</li><li>. Pipetting Aid PlatR Available from: <a target="_blank" href="https://biosistemika.com/products/pipetting-platr/">https://biosistemika.com/products/pipetting-platr/</a> (2019)</li><li>Gilson Trackman Pipetting Tracker. Daigger Scientific Available from: <a target="_blank" href="https://www.daigger.com/gilson-trackma-pipetting-tracker-i-gsnf70301">https://www.daigger.com/gilson-trackma-pipetting-tracker-i-gsnf70301</a> (2019)</li><li>TRACKMAN Connected US. Gilson Available from: <a target="_blank" href="https://www.gilson.com/default/systemm-trackman-connected-us.html">https://www.gilson.com/default/systemm-trackman-connected-us.html</a> (2019)</li><li>96 well plate pipette light guide. qit vision Available from: <a target="_blank" href="https://www.qitvision.com/projects/#Plate">https://www.qitvision.com/projects/#Plate</a> (2019)</li><li>. Microplate Assistive Pipetting Light Emitter GitHub repository Available from: <a target="_blank" href="https://github.com/pierrebaillargeon/Microplate-Assistive-Pipetting-Light-Emitter">https://github.com/pierrebaillargeon/Microplate-Assistive-Pipetting-Light-Emitter</a> (2019)</li><li>Hawker, C. D., Schlank, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Standards+for+Laboratory+Automation.">Development of Standards for Laboratory Automation.</a> Clinical Chemistry. 46, 746-750 (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Laboratory+Techniques.+An+Introduction+to+the+Micropipettor.">General Laboratory Techniques. An Introduction to the Micropipettor.</a> JoVE Science Education Database. , (2019).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Laboratory+Techniques.+Introduction+to+the+Spectrophotometer.">General Laboratory Techniques. Introduction to the Spectrophotometer.</a> JoVE Science Education Database. , (2019).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Laboratory+Techniques.+Introduction+to+the+Microplate+Reader.">General Laboratory Techniques. Introduction to the Microplate Reader.</a> JoVE Science Education Database. , (2019).</li></ol>

Authors have no financial interests or conflict of interest with any of the manufactured components suggested in the construction of the M.A.P.L.E. device. Sources presented are strictly for the convenience of the user and any compatible components from alternative sources can be used as needed.&#160;

By releasing M.A.P.L.E. as an open-source platform, we have introduced a laboratory tool that provides utility, but can also be easily extended to meet the evolving needs of the end user. Benchtop microplate sample preparation is a common task that is performed in a wide variety of laboratory environments and this task can be demonstrably improved with a technology such as M.A.P.L.E.
The M.A.P.L.E. platform has been specifically engineered with adaptability to future applications in mind. Each...

As recently published1, the Lead Identification laboratory at Scripps Research2 has developed and released an open-source illumination panel for microplate preparation referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). Manual preparation of microplates, whether they are made for compound management or bio-assay needs, can be prone to human errors that drastically increase as well density of the microplate increases. In addition, proper recordkeeping and data-logging of microplate content/format is also prone to manual entry errors. In high throughput screening (HTS) automation facilities these issues are mitigated through the use of computer-driven robotic workstations that are integrated with automated database recordkeeping; minimizing manual manipulations and reducing the potential of formatting and data recording errors. However, there remain many instances where automation-based instrumentation is simply not feasible or compatible with microplate formatting needs, requiring manual intervention. Moreover, there is also a need to support small-scale laboratory operations that require compact and cost-effective semi-automated devices to improve their throughput, accuracy and automate data-recordkeeping of microplate preparation.&#160;&#160; &#160;
While other microplate illumination systems exist, they are proprietary commercial solutions3,4,5,6,7 limited to select microplate formats and their proprietary closed-source nature prevents user-driven modifications that would allow the adaptation these devices for specialized operations. &#160;M.A.P.L.E. was designed to be an inexpensive open-source device, with source code and all design files available for free online8. Users with knowledge of surface mount soldering techniques can assemble their own M.A.P.L.E. devices with the code and design files available on GitHub, or they can modify the provided printed circuit boards (PCBs) designs, 3D print enclosure computer-aided design (CAD) models and code to meet their specific needs. A full list of parts needed to fabricate the light guide PCBs can be found in Supplementary Tables 1 and 2 and further details regarding the design and implementation of the light panels can be found in recently published documentation1. Users who wish to purchase pre-assembled light guide PCBs based off the open-source files can find them listed online9.
M.A.P.L.E. provides the user with an easily controllable illumination panel which has a microplate-based footprint and LED-to-LED spacing matched to Society for Biomolecular Screening (SBS) specifications for microplates10. M.A.P.L.E. was developed to support 96- and 384-well density microplates and allow users to illuminate wells in any desired configuration, color and intensity. These light panels can be used to illuminate microplates for pipetting operations11, to simulate laboratory formatting operations or instruments such as a microplate reader12,13 for educational and demonstration purposes. The open source nature of the project allows users to easily modify the panels, firmware or graphical user interface (GUI) software to support any new desired functionality. Guidance and data recordkeeping are computer-driven and can be integrated with spreadsheets or ported to a database system. Because M.A.P.L.E. is designed to work with plaintext comma delimited files, any spreadsheet or database software that is able to import or export CSV formatted files can be easily extended to work with M.A.P.L.E. Further, the project enclosure which has been designed for this system tilts the microplate towards the user during pipetting operations, increasing ergonomics by providing a more natural posture for the user while at the lab bench. Specific operational features to the M.A.P.L.E. system include: (i) Facilitating compound management efforts in preparing customized plates by illuminating single source well and destination well across microplates for manual pipetting guidance; assisted through a computer script that can be saved as an electronic record post completion. (ii) M.A.P.L.E. can illuminate any number of wells across microplate rows or columns; which is ideally suited for rapid serial dilution guidance or placement of select replicate controls. (iii) M.A.P.L.E. can be used in a demonstration mode to facilitate laboratory training needs or highlight formatting requirements with respect to sample and control placements or dedicated well usage (e.g., edge-effect barrier gap). (iv) M.A.P.L.E. can backlight transparent/translucent wells to allow visualization of artifacts such as precipitation/crystallization, bubbles, well heterogeneity, empty wells; which also allows end-user to easily photograph plate images for documentation needs

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	<tbody>
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			<td height="21" style="height:21px;width:297px;">96 or 384 well microplate</td>
			<td style="width:121px;"></td>
			<td style="width:161px;"></td>
			<td style="width:167px;">https://en.wikipedia.org/wiki/Microplate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microplate Assistive Pipetting Light Emitter</td>
			<td style="width:121px;">Open source</td>
			<td style="width:161px;"></td>
			<td>https://github.com/pierrebaillargeon/Microplate-Assistive-Pipetting-Light-Emitter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Pipettor</td>
			<td style="width:121px;"></td>
			<td style="width:161px;"></td>
			<td>https://www.jove.com/science-education/5033/an-introduction-to-the-micropipettor</td>
		</tr>
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			<td height="21" style="height:21px;">Spectrometer</td>
			<td>Ocean Optics</td>
			<td style="width:161px;">USB-650 Red Tide</td>
			<td></td>
		</tr>
	</tbody>
</table>

applications for open source microplate-compatible illumination panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

The M.A.P.L.E. platform is capable of illuminating wells in 96- and 384-well microplates in a variety of user configurable ways, allowing straightforward and independent control of color and light intensity in each well. By helping to reduce opportunities for error in manual pipetting operations, M.A.P.L.E. helps users prepare microplates with increased confidence that each well contains the desired contents. The transfer of samples between plates and the preparation of serial dilution plates, such as the examples seen i...

Watch this Scientific Journal Video about Applications for Open Source Microplate-Compatible Illumination Panels at JoVE.com

Applications for Open Source Microplate-Compatible Illumination Panels

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

applications-for-open-source-microplate-compatible-illumination-panels

Research

JoVE Journal

Bioengineering

7.4K Views.  Scripps Florida. Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.  M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping.  In addition, it can assist with examining microplate quality or aid in the detection of errors.   

Video: Applications for Open Source Microplate-Compatible Illumination Panels

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

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

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

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

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...