This work was supported by the Key Program of Chongqing Natural Science Foundation (No. cstc2020jcyj-zdxmX0027) and the Chinese National Natural Science Foundation Project (No. 31670936, 82041045).

1. Plasmid construction
<ol>
	<li>Insert DNA encoding SpyCatcher sequence (Supplementary File 1) into an ampicillin-resistant pThioHisA-ClyA plasmid (see Table of Materials) between the BamH I and Sal I sites to construct the plasmid pThioHisA ClyA-SC following a previously published report20.</li>
	<li>Ligate the synthesized SpyTag-RBD-Histag fusion gene (Supplementary File 1) into a pcDNA3.1 plasmid (see Table of Materials) between the BamH I and EcoR I sites to construct the plasmid pcDNA3.1 RBD-ST following a previously published report19.</li>
</ol>
2. OMV-SC preparation
<ol>
	<li>Perform ClyA-SC transformation following the steps below.
	<ol>
		<li>Add 5 &#181;L of pThioHisA ClyA-SC plasmid solution (50 ng/&#181;L) to 50 &#181;L of BL21 competent strain, gently blow, and let cool on ice for 30 min.</li>
		<li>Place the solution for 90 s in the water bath at 42 &#176;C, and then immediately put the mixed solution on ice for 3 min.</li>
		<li>Add 500 &#181;L of LB medium to the bacterial suspension and, after mixing, culture at 220 rpm at 37 &#176;C for 1 h.</li>
		<li>Plate all the transformation onto an LB agar plate containing ampicillin (100 &#181;g/mL, see Table of Materials) and culture overnight at 37 &#176;C. 
		NOTE: In subsequent experiments, the ampicillin was kept at the same concentration in the medium. If not, this may cause loss of plasmids in the bacteria.</li>
	</ol>
	</li>
	<li>For OMV-SC production, perform the steps below.
	<ol>
		<li>Isolate a single colony from the plate (step 2.1.4.) to 20 mL of LB (ampicillin-resistant) medium and culture overnight at 220 rpm at 37 &#176;C.</li>
		<li>Inoculate the bacterial solution (from step 2.2.1.) into 2 L medium, culture at 220 rpm, 37 &#176;C for 5 h until the logarithmic growth stage (the OD600nm is between 0.6 to 0.8).</li>
		<li>When the OD at 600 nm of the bacterial solution reaches 0.6-0.8, add isopropyl beta-D-thiogalactopyranoside (IPTG, see Table of Materials) to make the final concentration of the bacterial solution to be 0.5 mM, and then culture overnight at 220 rpm at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Perform OMV-SC purification.
	<ol>
		<li>Centrifuge the bacterial solution at 7,000 x g at 4 &#176;C for 30 min.</li>
		<li>Filter the supernatant with a 0.22 &#181;m membrane filter, then concentrate it using a 100 kD ultrafiltration membrane or hollow fiber column (see Table of Materials).</li>
		<li>Filter the concentrate through a 0.22 &#181;m membrane filter, then centrifuge it at 150,000 x g at 4 &#176;C for 2 h using an ultracentrifuge (see Table of Materials), and discard the supernatant with a pipette.</li>
		<li>Resuspend the precipitation with PBS and store it at &#8722;80 &#176;C. The solution can maintain long-term stability at &#8722;80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. RBD-ST preparation
<ol>
	<li>Perform RBD-ST transfection following the steps below.
	<ol>
		<li>Select an appropriate eukaryotic expression system (e.g., HEK293F) and culture the cells overnight at 130 rpm at 37 &#176;C after recovery.</li>
		<li>Add 20 &#181;L of HEK293F cell solution into the automated cell counter (see Table of Materials), record the number of cells, adjust the concentration to 1 x 106 cells/mL, and then culture the cells at 130 rpm at 37 &#176;C for 4 h.</li>
		<li>Filter RBD-ST plasmid through a 0.22 &#181;m membrane filter, and add 300 &#181;g of plasmids into the cell culture medium (see Table of Materials) until the final volume is 10 mL; shake for 10 s.</li>
		<li>Heat PEI (1 mg/mL, see Table of Materials) to 65 &#176;C in the water bath, mix 0.7 mL of PEI with 9.3 mL of cell culture medium, and shake intermittently for 10 s. 
		NOTE: Do not shake the solution vigorously. Otherwise, the resulting bubbles may affect the transfection efficiency.</li>
		<li>Add plasmid solution to the PEI solution, shake the mixture intermittently for 10 s, and incubate it at 37 &#176;C for 15 min.</li>
		<li>Add the mixture to 280 mL of cell culture medium and culture at 130 rpm at 37 &#176;C for 5 days.</li>
	</ol>
	</li>
	<li>Perform RBD-ST purification.
	<ol>
		<li>Centrifuge the cells at 6,000 x g at 25 &#176;C for 20 min and use a pipette to collect the supernatant.</li>
		<li>Fill the column with 2 mL of Ni-NTA agarose (see Table of Materials) and wash it 3x with 3x PBS.</li>
		<li>Add imidazole (see Table of Materials) into the cell supernatant to make the final concentration of 20 mM, and load the cell supernatant 2x.</li>
		<li>Add 3 column volumes (CV) of PBS containing 20 mM of imidazole for washing, and collect the washing fraction.</li>
		<li>Gradient elute with 3 CV of PBS containing low to high concentrations (e.g., 0.3 M, 0.4 M, 0.5 M) of imidazole; elute 2x for each concentration.</li>
		<li>Use SDS-PAGE21 to identify the RBD-ST in different concentration gradients.</li>
	</ol>
	</li>
</ol>
4. OMV-RBD bioconjugation and purification
<ol>
	<li>Determine the protein concentration by the BCA method (see Table of Materials).
	<ol>
		<li>Serially dilute the standard BSA protein solution from 2 mg/mL to 0.0625 mg/mL, dilute the purified OMV-SC and RBD-ST 10x, then mix BCA working solutions A and B (provided in the assay kit) at a ratio of 50:1 (v/v).</li>
		<li>Add diluted protein solution (25 &#181;L/well) and mix with BCA working solution (200 &#181;L/well); incubate at 37 &#176;C for 30 min.</li>
		<li>Measure the absorbance (OD) at 562 nm of each well and calculate the protein concentration from the standard curve22.</li>
	</ol>
	</li>
	<li>Perform bioconjugation of OMV-SC and RBD-ST following the steps below.
	<ol>
		<li>Mix OMV-SC and RBD-ST in PBS at a 40:1 (w/w) ratio.</li>
		<li>Vertically rotate to blend the mixture overnight at 15 rpm at 4 &#176;C. 
		NOTE: Different antigens may react in different proportions. One could try different reaction ratios based on the characteristics of the antigen.</li>
	</ol>
	</li>
	<li>Verify the reaction efficiency.
	<ol>
		<li>Prepare 10 &#181;L of OMV-SC, RBD-ST, and the reaction product (step 4.2.). Add 2.5 &#181;L of 5x loading buffer (see Table of Materials), and heat the samples at 100 &#176;C for 5 min. Load the samples into the gel (10 &#181;L/well). Perform electrophoresis at 60 V for 20 min and change the condition to 120 V for 1 h.</li>
		<li>Transfer the protein from the gel to PVDF western blotting membranes (see Table of Materials) at 100 V for 70 min.</li>
		<li>Put the membrane into 5% non-fat powdered milk/TBST and shake for 1 h. Then, wash 3x with TBST for 5 min per wash with shaking.</li>
		<li>Put the membrane into 0.1% His-Tag antibody/TBST (see Table of Materials) and shake for 1 h, then wash 3x with TBST for 5 min per wash with shaking.</li>
		<li>Put the membrane into 0.02% anti-mouse IgG1 antibody (HRP)/TBST (see Table of Materials) and shake for 40 min, then wash 3x with TBST for 5 min per wash with shaking.</li>
		<li>Add enhanced chemiluminescence solution (see Table of Materials) and expose the membrane.</li>
	</ol>
	</li>
	<li>Perform purification of OMV-RBD.
	<ol>
		<li>Dilute 1 mL of reacted OMV-RBD solution to 10 mL, and centrifuge the dilution at 150,000 x g at 4 &#176;C for 2 h.</li>
		<li>Use a pipette to discard the supernatant and suspend the residue with 10 mL of PBS. Centrifuge the suspension at 150,000 x g at 4 &#176;C for 2 h.</li>
		<li>Discard the supernatant and suspend the residue with 1 mL of PBS. 
		&#8203;NOTE: If the solubility of the antigen is much lower, the same separation effect can be achieved by size-exclusion chromatography19.</li>
	</ol>
	</li>
</ol>
5. Characterization
<ol>
	<li>Perform dynamic light scattering (DLS).
	<ol>
		<li>Dilute the samples to a 100 &#181;g/mL concentration and add 1 mL of sample into the sample cell.</li>
		<li>Choose &#34;Size&#34; for &#34;Measurement type&#34;, &#34;Protein&#34; for &#34;Sample material&#34;, &#34;Water&#34; for &#34;Dispersant&#34;, &#34;25 &#176;C&#34; for &#34;Temperature&#34;, and then load samples and measure automatically23.</li>
	</ol>
	</li>
	<li>Capture images using transmission electron microscopy (TEM).
	<ol>
		<li>Dilute the samples to 100 &#181;g/mL. Take a 200-mesh copper grid, add 20 &#181;L of sample to it, and allow it to get absorbed for 10 min.</li>
		<li>Use filter paper to wick off the solution, add 20 &#181;L of 3% uranyl acetate to the support film, and stain for 30 s.</li>
		<li>Wick off the uranyl acetate and dry the film naturally at room temperature for 1 h.</li>
		<li>Load the samples to the TEM system (see Table of Materials) and capture images at 80 kV.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

To create a &#34;Plug-and-Display&#34; nanoparticle vaccine platform, SC-fused ClyA was expressed in BL21(DE3) strains, which is one of the most widely used models for recombinant protein production because of its advantages in protein expression24, so that there would be enough SC fragment displaying on the surface of the OMVs during the process of bacteria proliferation. At the same time, an ST-fused target antigen was prepared for the chemical coupling between the antigens and OMVs. This experi...

As a potential vaccine platform, outer membrane vesicles (OMVs) have attracted more and more attention in recent years1,2. OMVs, mainly secreted naturally by gram-negative bacteria3, are spherical nanoscale particles composed of a lipid bilayer, usually in the size of 20-300 nm4. OMVs contain various parental bacterial components, including bacterial antigens and pathogen-associated molecular patterns (PAMPs), which serve as solid immune potentiators5. Benefiting from their unique components, natural vesicle structure, and great genetic engineering modification sites, OMVs have been developed for use in many biomedical fields, including bacterial vaccines6, adjuvants7, cancer immunotherapy drugs8, drug delivery vectors9, and anti-bacterial adhesives10.
The SARS-CoV-2 pandemic, which has spread worldwide since 2020, has taken a heavy toll on global society. The receptor-binding domain (RBD) in spike protein (S protein) can bind with human angiotensin-converting enzyme 2 (ACE2), which then mediates the entry of the virus into the cell11,12,13. Thus, RBD seems to be a prime target for vaccine discovery14,15,16. However, monomeric RBD is poorly immunogenic, and its small molecular weight makes it difficult for the immune system to recognize, so adjuvants are often required17.
In order to increase the immunogenicity of RBD, OMVs displaying polyvalent RBDs were constructed. Existing studies using OMV to display RBD usually fuse RBD with OMV to be expressed in bacteria18. However, RBD is a virus-derived protein, and prokaryotic expression is likely to affect its activity. To solve this problem, the SpyTag (ST)/SpyCatcher (SC) system, derived from Streptococcus pyogenes, was used to form a covalent isopeptide with OMV and RBD in a conventional buffer system19. The SC domain was expressed with Cytolysin A (ClyA) as a fusion protein by bioengineered Escherichia coli, and ST was expressed with RBD via the HEK293F cellular expression system. OMV-SC and RBD-ST were mixed and incubated overnight. After purification by ultracentrifugation or size-exclusion chromatography (SEC), OMV-RBD was obtained.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ampicillin sodium</td>
			<td>Sangon Biotech</td>
			<td>A610028</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>Countstar</td>
			<td>BioTech</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein quantification Kit</td>
			<td>cwbio</td>
			<td>cw0014s</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touching Imaging System</td>
			<td>Bio-rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Danamic Light Scattering</td>
			<td>Malvern</td>
			<td>Zetasizer Nano S90</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Cavoy</td>
			<td>Power BV</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Buffers H 10X TBST Buffer</td>
			<td>Sangon Biotech</td>
			<td>C520009</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat pAb to mouse IgG1</td>
			<td>Abcam</td>
			<td>ab97240</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed freezing centrifuge</td>
			<td>Bioridge</td>
			<td>H2500R</td>
			<td></td>
		</tr>
		<tr>
			<td>His-Tag mouse mAb</td>
			<td>Cell signaling technology</td>
			<td>2366s</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sangon Biotech</td>
			<td>A600277</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl beta-D-thiogalactopyranoside</td>
			<td>Sangon Biotech</td>
			<td>A600118</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA His-Bind Superflow</td>
			<td>Qiagen</td>
			<td>70691</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat powdered milk</td>
			<td>Sangon Biotech</td>
			<td>A600669</td>
			<td></td>
		</tr>
		<tr>
			<td>OPM-293 cell culture medium</td>
			<td>Opm biosciences</td>
			<td>81075-001</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1 RBD-ST plasmid</td>
			<td>Wuhan genecreat biological techenology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>ZSGB-bio</td>
			<td>ZLI-9061</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine Linear</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Prestained protein ladder</td>
			<td>Thermo</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>pThioHisA ClyA-SC plasmid</td>
			<td>Wuhan genecreat biological techenology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Western Blotting Membranes</td>
			<td>Roche</td>
			<td>03010040001</td>
			<td></td>
		</tr>
		<tr>
			<td>Quixstand benchtop systems (100 kD hollow fiber column)</td>
			<td>GE healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE loading buffer (5x)</td>
			<td>Beyotime</td>
			<td>P0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sangon Biotech</td>
			<td>A100241</td>
			<td></td>
		</tr>
		<tr>
			<td>Supersignal west pico PLUS (enhanced chemiluminescence solution)</td>
			<td>Thermo</td>
			<td>34577</td>
			<td></td>
		</tr>
		<tr>
			<td>Suspension instrument</td>
			<td>Life Technology</td>
			<td>Hula Mixer</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Hitachi</td>
			<td>HT7800</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman coulter</td>
			<td>XPN-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet spectrophotometer</td>
			<td>Hitachi</td>
			<td>U-3900</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sangon Biotech</td>
			<td>A610961</td>
			<td></td>
		</tr>
	</tbody>
</table>

a "plug-and-display" nanoparticle vaccine platform based on outer membrane vesicles displaying sars-cov-2 receptor-binding domain

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derived&#160;OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a &#34;Plug-and-Display&#34; vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.

The flowchart for this protocol is shown in Figure 1. This protocol could be a general approach to utilizing OMVs as a vaccine platform; one only needs to choose the appropriate expression systems based on the type of antigens.
Figure 2&#160;provides a feasible plasmid design scheme. The SC gene is connected with the ClyA gene via a flexible linker, while ST connects to the 5&#39; terminal of the RBD gene with a His-tag gene for purif...

Watch this Scientific Journal Video about A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain at JoVE.com

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane ...

a-plug-display-nanoparticle-vaccine-platform-based-on-outer-membrane

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

2.4K Views.  Third Military Medical University. The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

Video: A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...