This work was supported by the Joint Project &#8220;The use of plants for the production of an autoimmune diabetes edible vaccine (eDIVA)&#8221; (Project ID: 891854) funded by the University of Verona in the framework of the call 2014.

1. Red beet and spinach cultivation
<ol>
	<li>Grow red beet (B. vulgaris cv Moulin Rouge) and spinach (S. oleracea cv Industria) plants in a growth chamber, using 150 &#181;E of light intensity, 65% relative humidity, 12 h light/dark cycle at 23/21 &#176;C, respectively.</li>
	<li>After seed germination, fertilize the plants twice a week with a 1 g/L solution of a commercially available fertilizer (Table of Materials). For agroinfiltration use five-week-old spinach and six-week-old red beet plants.</li>
</ol>
2. Transient expression through the deconstructed plant virus-based technology
<ol>
	<li>Construction of plant expression vectors
	<ol>
		<li>Introduce the vectors - the 5&#8217; module (pICH20111), the 3&#8217; modules (pICH31070.GAD65mut, pICH31070.&#8710;87G65mut and pICH7410.eGFP), prepared as previously described1,2,7, and the integrase module (pICH14011) - in Agrobacterium tumefaciens GV3101 strain using standard techniques. Grow on LB medium containing 50 &#956;g/mL rifampicin and appropriate vector-specific antibiotics (50 &#956;g/mL carbenicillin for pICH20111, pICH14011 and pICH7410.eGFP, 50 &#956;g/mL kanamycin for pICH31070) for 2 days at 28 &#176;C.</li>
		<li>Screen the colonies by colony PCR using the following specific primers for each vector: 5&#8217;-ATCTAAGCTAGGGTACCTCG-3&#8217; and 5&#8217;-ACACCGTAAGTCTATCTCTTC-3&#8217; for both the 3&#8217; modules pICH31070.GAD65mut and pICH31070.&#8710;87G65mut, with an annealing temperature of 55 &#176;C and an elongation time of 110 s, 5&#8217;-TGAAGTTCATCTGCACCAC-3&#8217; and 5&#8217;-ACACCGTAAGTCTATCTCTTC-3&#8217; for the third 3&#8217; module pICH7410.eGFP, with an annealing temperature of 53 &#176;C and an elongation time of 30 s, 5&#8217;-AATGTCGATAGTCTCGTACG-3&#8217; and 5&#8217;-TCCACCTTTAACGAAGTCTG-3&#8217; for the 5&#8217; module, with an annealing temperature of 53 &#176;C and an elongation time of 20 s and 5&#8217;-GGCAACCGTTATGCGAATCC-3&#8217; and 5&#8217;-GATGCGTTCCGCAACGAACT-3&#8217; for the integrase module with an annealing temperature of 57 &#176;C and an elongation time of 45 s.</li>
		<li>Carry out the PCR reaction in a total volume of 20 &#956;L using the following specific reaction cycle: 5 min initial denaturation at 95 &#176;C, 35 cycles of 30 s at 95 &#176;C, 30 s at the annealing temperature and the elongation step at 72 &#176;C (annealing temperature and elongation time are specific for each primer couple) and a final elongation step of 7 min at 72 &#176;C.</li>
	</ol>
	</li>
	<li>Syringe agroinfiltration
	<ol>
		<li>Inoculate the three A. tumefaciens transformants in 50 mL of LB medium containing 50 &#956;g/mL rifampicin and the following appropriate vector-specific antibiotics: 50 &#956;g/mL carbenicillin for A. tumefaciens transformed with pICH20111, pICH14011 or pICH7410.eGFP, or 50 &#956;g/mL kanamycin for A. tumefaciens transformed with pICH31070. Grow by shaking overnight at 28 &#176;C.</li>
		<li>Pellet overnight bacterial cultures by centrifugation at 4,500 x g for 20 min and resuspend them in 100 mL (or two volumes of the initial bacterial culture) of infiltration buffer containing 10 mM 4-morpholineethanesulfonic acid (MES; pH 5.5) and 10 mM MgSO4, without considering the OD600. Incubate the suspensions at room temperature (RT) for 3 h.</li>
		<li>Mix equal volumes of bacterial suspensions containing one of the three modules, GAD65mut, &#8710;87GAD65mut or eGFP 3&#8217; module, with 5&#8217; module and integrase module. Use the suspension mix for syringe infiltration of red beet and spinach leaves.</li>
		<li>Place 5 mL of the suspension in a syringe without the needle. Press the tip of the syringe against the underside of the leaf for both spinach and red beet plants, and meanwhile apply a gentle counterpressure to the other side of the leaf.</li>
		<li>Infiltrate the first three completely expanded leaves starting from the apex for each plant. Label the agroinfiltrated leaves with a paper tag on the leaf stem. Return the plants in a growth chamber under standard conditions. 
		NOTE: For health and safety reasons, wear eye protection and gloves during infiltration process.</li>
		<li>Collect agroinfiltrated leaves from 4 to 14 days post infection (dpi) and freeze them in liquid nitrogen. Store plant tissue at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Vacuum agroinfiltration
	<ol>
		<li>Grow separately the three A. tumefaciens transformants in 50 mL of LB medium containing 50 &#956;g/mL rifampicin and appropriate vector-specific antibiotic by shaking overnight at 28 &#176;C.</li>
		<li>Pellet overnight bacterial cultures by centrifugation at 4,500 x g for 20 min. Resuspend the pellet in 1 L of infiltration buffer to an OD600 of 0.35 and incubate the suspensions at RT for 3 h.</li>
		<li>Add 0.01% v/v of the detergent (polysorbate 20) to each suspension. Mix equal volumes of bacterial suspensions containing one of the three modules, GAD65mut, &#8710;87GAD65mut or eGFP 3&#8217; module, with 5&#8217; module and integrase module.</li>
		<li>Insert one plant (six-week-old red beet plant, see section 1) in the holder. Invert the holder and place on top of a beaker containing the infiltration bath (2 L) to submerge the leaves in the infiltration suspension. 
		NOTE: Ensure that all the leaves are completely dipped in the bacterial suspension. Raise the level with the addition of extra infiltration suspension if required.</li>
		<li>Transfer the infiltration bath with the submerged plant to the infiltration chamber and close it. Turn on the vacuum pump and open the vacuum intake valve on the infiltration chamber.</li>
		<li>Once the pressure in the infiltration chamber has reduced to 90 mbar, keep the vacuum for 3 min. Release the vacuum for 45 s. Once the infiltration chamber has returned to atmospheric pressure, open the chamber and remove the infiltrated plant from the bacterial bath.</li>
		<li>Return the plants in a growth chamber under standard conditions.</li>
		<li>Collect agroinfiltrated leaves at the maximum expression dpi, depending on the recombinant protein, and freeze them in liquid nitrogen. Store plant tissue at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Recombinant protein expression analysis
<ol>
	<li>Total soluble protein (TSP) extraction
	<ol>
		<li>Grind the syringe or vacuum infiltrated red beet and spinach leaves, collected in steps 2.2.6 and 2.3.8, to fine powder in liquid nitrogen using mortar and pestle. Transfer the powder into 15 mL plastic tubes and store the material at -80 &#176;C.</li>
		<li>Add 900 &#181;L of extraction buffer (50 mM sodium phosphate pH 8.0, 20 mM sodium metabisulphite) to 300 mg of leaf powder. 
		NOTE: The selected ratio between plant tissue weight (mg) to buffer volume (&#181;L) is 1:3.</li>
		<li>Homogenize the mixture by vortexing for 1 min, then centrifuge at 30,000 x g for 40 min at 4 &#176;C.</li>
		<li>Collect the supernatant in a clean tube and store it at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Plant eGFP visualization and quantification
	<ol>
		<li>Load 100 &#181;L of each TSP extract obtained from eGFP expressing leaves, in three technical replicates, on a 96-well plate.</li>
		<li>Put the 96-well plate in a fluorescence reader and start the measurement. Use the 485/535 nm filter set required for eGFP fluorescence detection.</li>
		<li>For the absolute quantification, in the same plate prepare a calibration curve loading different quantities (62.5, 125, 500, 750 and 1,000 ng) of a purified eGFP.</li>
	</ol>
	</li>
	<li>Bicinchoninic acid (BCA) assay for TSP quantification
	<ol>
		<li>Mix 50 parts of Reagent A (Table of Materials) with 1 part of Reagent B (Table of Materials). Prepare sufficient volume of fresh BCA working solution for the samples to be assayed and the calibration standards. 
		NOTE: The volume of BCA working solution required for each sample is 1.9 mL. For the standard procedure with 9 standards (including a blank), 17.1 mL of BCA working solution is required.</li>
		<li>Pipette 0.1 mL of each standard (including a blank), and TSP extracts into a labelled tube. 
		NOTE: As calibration standards, prepare a fresh set of bovine serum albumin (BSA) standards in the 10-1,000 &#956;g/mL range, preferably using the same diluent as samples, such as water. The blank consists of 0.1 mL of the diluent used for calibration standard and sample preparation.</li>
		<li>Add 1.9 mL of BCA working solution and mix thoroughly. Cover the tubes and incubate at 37 &#176;C for 30 min.</li>
		<li>Cool the tubes to RT. Measure the absorbance at 562 nm of all the samples within 10 min. 
		NOTE: Even at RT, the color development continues. No significant error will be introduced if the absorbance measurements of all tubes are done within 10 min.</li>
		<li>Subtract the 562 nm absorbance value of the blank from the readings of the standards and the TSP extracts.</li>
		<li>Plot the blank-corrected 562 nm reading for each standard on its concentration. Determine the protein concentration of each TSP extract.</li>
	</ol>
	</li>
	<li>Perform Coomassie gel staining as previously described in Gecchele et al.8.</li>
	<li>Western blot analysis
	<ol>
		<li>Perform the western blot analysis as previously described in Gecchele et al.8.</li>
		<li>After the electrophoretic separation of proteins, transfer them onto a nitrocellulose membrane using standard techniques. Prepare the blocking solution by mixing 4% milk in phosphate-buffered saline (PBS) pH 7.4. Block the membrane with 10 mL of the blocking solution at RT for 1 h.</li>
		<li>Prepare the rabbit primary antibody at 1:10,000 for the anti-GAD65/67 and anti-LHCB2, and at 1:20,000 for the anti-eGFP in 5 mL of blocking solution with 0.1% detergent. Incubate the membrane with the prepared primary antibody solutions overnight at 4 &#176;C or for 4 h at RT with constant agitation.</li>
		<li>Discard the primary antibody and wash the membrane 3 times for 5 min each with blocking solution containing 0.1% detergent.</li>
		<li>Prepare the horseradish peroxidase (HRP)-conjugate anti-rabbit antibody at 1:10,000 in blocking solution with 0.1% detergent. Incubate the membrane for 1.5 h at RT with constant agitation.</li>
		<li>Discard the secondary antibody and wash the membrane 5 times for 5 min each with PBS-T (PBS supplemented with 0.1% detergent).</li>
		<li>Incubate the membrane with a commercially available luminol solution following the manufacturer&#8217;s instructions. Detect the signal using a chemiluminescence imaging system.</li>
	</ol>
	</li>
</ol>
4. Plant material processing
<ol>
	<li>Harvest the vacuum agroinfiltrated &#8710;87GAD65mut-expressing red beet leaves at the expression peak (11 dpi) and freeze them in liquid nitrogen.</li>
	<li>Lyophilize the frozen leaves for 72 h at -50 &#176;C and 0.04 mbar. Store them at -80 &#176;C.</li>
	<li>Grind the leaves to fine powder and store it at RT in a sealed container with silica gel to exclude the moisture.</li>
</ol>
5. Gastric digestion simulation and cell integrity analysis
<ol>
	<li>Gastric digestion simulation
	<ol>
		<li>Weigh 100 mg of grinded freeze-dried red beet leaves and resuspend it in 6 mL of PBS (pH 7.4).</li>
		<li>Adjust the sample pH to 2 with 6 M HCl.</li>
		<li>Add 4 mg/mL pepsin from porcine gastric mucosa in 10 mM HCl to obtain a final pepsin concentration of 1 mg/mL or a ratio of 1:20 to total soluble proteins. Shake the sample at 37 &#176;C for 120 min.</li>
		<li>Adjust the samples to pH 8 with 1 M NaOH to inactivate the pepsin.</li>
		<li>Centrifuge 750 &#181;L aliquots of each sample at 20,000 x g for 20 min at 4 &#176;C. Collect separately the supernatant and resuspend the pellet in one supernatant volume of loading buffer (1.5 M Tris HCl, pH 6.8, 3% SDS, 15% glycerol, and 4% 2-mercaptoethanol). 
		NOTE: For health and safety reasons, wear gloves and work under fume hood for sample preparation.</li>
		<li>Analyze both the supernatant and the resuspended pellet by western blot analysis8.</li>
	</ol>
	</li>
	<li>Cell integrity analysis
	<ol>
		<li>Prepare two samples of 100 mg of grinded freeze-dried red beet leaves and resuspend both in 6 mL of PBS (pH 7.4).</li>
		<li>Adjust the pH of only one sample to 2 with 6 M HCl. Shake both samples at 37 &#176;C for 120 min.</li>
		<li>Centrifuge 750 &#181;L aliquots of each sample at 20,000 x g for 20 min at 4 &#176;C. Collect separately the supernatant and resuspend the pellet in one supernatant volume of loading buffer.</li>
		<li>Analyze both the supernatant and the resuspended pellet by western blot analysis.</li>
	</ol>
	</li>
</ol>
6. Bioburden assay
<ol>
	<li>Weigh 100 mg of freeze-dried red beet leaves. Resuspend the powder in 8 mL of sterile PBS (pH 7.4) and vortex for 1 min.</li>
	<li>Prepare the LB medium without antibiotics or containing (i) 50 &#181;g/mL rifampicin, (ii) 50 &#181;g/mL each of rifampicin and carbenicillin, (iii) 50 &#181;g/mL each of rifampicin and kanamycin, or (iv) 50 &#181;g/mL each of rifampicin, carbenicillin, and kanamycin.</li>
	<li>Plate 1 mL of each freeze-dried leaf homogenate in one of the 5 selective LB media.</li>
	<li>Incubate all the plates for 3 days at 28 &#176;C.</li>
	<li>Count the Agrobacterium colonies grown on each plate.</li>
	<li>Calculate and define the residual bacterial charge as the number of colony forming units (CFU) per mL of the freeze-dried leaf homogenate (CFU/mL).</li>
</ol>
7. Metabolite extraction
<ol>
	<li>Primary metabolite extraction
	<ol>
		<li>Weigh 30 mg of -80 &#176;C stored, vacuum agroinfiltrated &#8710;87GAD65mut-expressing red beet leaf powder in a 2 mL plastic tube.</li>
		<li>Add 750 &#181;L of cold 70/30 (v/v) methanol/chloroform and vortex for 30 s. Incubate at -20 &#176;C for 2 h. 
		NOTE: All the solvents and additives must be LC-MS grade.</li>
		<li>Add 600 &#181;L of cold water and centrifuge at 17,900 x g at 4 &#176;C for 10 min. Collect and transfer the upper hydroalcoholic phase into a new 2 mL tube. Discard the lower chloroformic phase and the interphase.</li>
		<li>Put the samples into a vacuum concentrator for 3 h to evaporate the solvents.</li>
		<li>Dissolve the pellet obtained from step 7.1.4 in 300 &#181;L of 50/50 (v/v) acetonitrile/water and sonicate the samples for 3 min.</li>
		<li>Pass the solutions through 0.2 &#956;m membrane filters and put them into a transparent fixed 300 &#181;L insert glass tubes.</li>
	</ol>
	</li>
	<li>Secondary metabolite extraction
	<ol>
		<li>Weigh 300 mg of -80 &#176;C stored, vacuum agroinfiltrated &#8710;87GAD65mut-expressing red beet leaf powder in a 15 mL plastic tube.</li>
		<li>Add 3 mL of methanol, vortex for 30 s, and sonicate at 40 kHz for 15 min. 
		NOTE: All the solvents and additives must be LC-MS grade.</li>
		<li>Centrifuge the samples at 4,500 x g at 4 &#176;C for 10 min and transfer the supernatants into new 15 mL tubes.</li>
		<li>Dilute 100 &#181;L of extract 1:3 (v/v) with water and pass the solution through a 0.2 &#956;m membrane filter.</li>
		<li>Put the solution into a transparent fixed 300 &#181;L insert glass tube.</li>
	</ol>
	</li>
	<li>Polar lipid extraction
	<ol>
		<li>Weigh 200 mg of -80 &#176;C stored, vacuum agroinfiltrated &#8710;87GAD65mut-expressing red beet leaf powder in a 15 mL plastic tube.</li>
		<li>Add 200 &#181;L of water and then 2 mL of methanol, and then keep on ice for 1 h. 
		NOTE: All the solvents and additives must be LC-MS grade.</li>
		<li>Vortex for 30 s and sonicate at 40 kHz for 15 min.</li>
		<li>Centrifuge the samples at 4,500 x g at 4 &#176;C for 25 min. Collect and transfer the chloroform phase into 2 mL tubes. Discard the upper hydroalcoholic phase and the interphase.</li>
		<li>Put the samples into a vacuum concentrator for 3 h to evaporate the solvent.</li>
		<li>Dissolve the pellet obtained from step 7.3.5 with 600 &#181;L of methanol.</li>
		<li>Dilute 100 &#181;L of extract 1:5 (v/v) with methanol and pass the solution through a 0.2-&#956;m membrane filter.</li>
		<li>Put the solution into a transparent fixed 300 &#181;L insert glass tube.</li>
	</ol>
	</li>
</ol>
8. Liquid chromatography mass spectrometry analysis and data processing
<ol>
	<li>Set up the LC-MS system as recommended by the supplier. 
	NOTE: The LC section consists of an autosampler coupled with HPLC equipped with a C18 guard column (75 x 2.1 mm, particle size 5 &#181;m) in front of a C18 column (150 x 2.1 mm, particle size 3 &#181;m) for the analysis of secondary metabolites and polar lipids, whereas with a HILIC guard column (7.5 x 2.1 mm, 3 &#181;m) in front of an HILIC column (150 x 2.1 mm, particle size 2.7 &#181;m). The MS is an ion trap mass spectrometer provided with either an electrospray ionization (ESI) or an atmospheric pressure chemical ionisation (APCI) sources.</li>
	<li>Prepare the appropriate solvents for the HPLC gradients. For primary metabolite analysis, use 20 mM ammonium formate as solvent A; 95% acetonitrile, 5% water plus 10 mM ammonium formate as solvent B. For secondary metabolite analysis, use water plus 0.05% formic acid (A) and acetonitrile plus 0.05% formic acid (B). For polar lipid analysis, use water plus 0.05% formic acid (A) and 100% acetonitrile (B). 
	NOTE: The solvents and additives must be LC-MS grade.</li>
	<li>Use the gradients reported in Table 1 for metabolite elution. Set the flow rate at 0.2 mL/min. Inject 10 &#181;L of each sample for both secondary metabolites and polar lipids, whereas inject 5 &#181;L for primary metabolites.</li>
	<li>Prepare a quality control (QC) sample by mixing equal portions of different samples to have a representative mixture of each experimental condition. Analyse the QC sample during the experiment to monitor instrument efficiency. Specifically, insert a QC sample analysis after each 10 sample-batch.</li>
	<li>Randomize the samples to avoid instrument-driven effects.</li>
	<li>After 9 analyses, insert a column cleaning method and a blank analysis immediately after. 
	NOTE: Perform a slow gradient between the two solvents with an isocratic elution at high percentage of the strongest solvent. The blank consists of an injection of a pure methanol: water (50/50, v/v) to improve retention time reproducibility in the following analysis.</li>
	<li>Set up the instrument to acquire mass spectra in alternate positive and negative ionization modes using the parameters listed in Table 2. 
	NOTE: Other parameters depend on the specific platform.</li>
	<li>Perform the next data processing as explained in Dal Santo et al.9.</li>
</ol>

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D., Patel, A., Boddu, S. H. S., Mitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+for+enhancing+oral+bioavailability+of+peptides+and+proteins.">Approaches for enhancing oral bioavailability of peptides and proteins.</a> International Journal of Pharmaceutics. 447, 75-93 (2013).</li><li>. Encapsulation importance in pharmaceutical area, how it is done and issues about herbal extraction Available from: <a target="_blank" href="https://www.researchgate.net/publication/271702091_Encapsulation_importance_in_pharmaceutical_area_how_it_is_done_and_issues_about_herbal_extraction">https://www.researchgate.net/publication/271702091_Encapsulation_importance_in_pharmaceutical_area_how_it_is_done_and_issues_about_herbal_extraction</a> (2015)</li><li>Kamei, N., 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=Complexation+hydrogels+for+intestinal+delivery+of+interferon+beta+and+calcitonin.">Complexation hydrogels for intestinal delivery of interferon beta and calcitonin.</a> Journal of Controlled Release. 134, 98-102 (2009).</li><li>Tuesca, A., 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=Complexation+hydrogels+for+oral+insulin+delivery:+effects+of+polymer+dosing+on+in+vivo+efficacy.">Complexation hydrogels for oral insulin delivery: effects of polymer dosing on in vivo efficacy.</a> Journal of Pharmaceutical Sciences. 97, 2607-2618 (2008).</li><li>Twyman, R. M., Schillberg, S., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+yield+of+recombinant+pharmaceutical+proteins+in+plants.">Optimizing the yield of recombinant pharmaceutical proteins in plants.</a> Current Pharmaceutical Design. 19, 5486-5494 (2013).</li><li>Dhama, K., 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=Plant-based+oral+vaccines+for+human+and+animal+pathogens+&#8211;+a+new+era+of+prophylaxis:+current+and+future+perspectives.">Plant-based oral vaccines for human and animal pathogens &#8211; a new era of prophylaxis: current and future perspectives.</a> Journal of Experimental Biology and Agricultural Sciences. 447, 75-93 (2013).</li><li>Hefferon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconceptualizing+cancer+immunotherapy+based+on+plant+production+systems.">Reconceptualizing cancer immunotherapy based on plant production systems.</a> Future science. O3, (2017).</li></ol>

The authors have nothing to disclose.

In this study we showed preliminary analysis for the design of a candidate oral vaccine for autoimmune diabetes. The target protein for this experiment was a mutated form of the human 65 kDa Glutamate Decarboxylase, which production and functionality are easily detectable and measurable12. Its expression in different edible plant tissues was mediated by the vectors5, which mediate a high level of recombinant protein production in a very short time frame. The selection of th...

Since the plant molecular biology revolution in 1980s, plant-based systems for the production of biopharmaceuticals can be considered as an alternative to traditional systems based on microbial and mammalian cells1. Plants display several advantages over traditional platforms, with scalability, cost-effectiveness and safety being the most relevant2. The recombinant product can be purified from transformed plant tissue and then administered, either parenterally or orally and, moreover, transformed edible plant can be used directly for oral delivery. The oral route simultaneously promotes mucosal and systemic immunity, and it eliminates the need for needles and specialized medical personnel. Furthermore, oral delivery eliminates the complex downstream processing, which normally accounts for 80% of the total manufacturing cost of a recombinant protein3. All those advantages can be translated into savings in production, supplies and labor reducing the costs of each dose, making the drug affordable to most of the global population.
Several strategies, both for stable transformation and transient expression, were developed for the production of recombinant proteins in plants. Among them, a high-yield deconstructed plant virus-based expression system (e.g., magnICON) provides superior performance leading high yields of recombinant proteins over relatively short timescales4. Many examples of transient expression using the plant virus-based expression&#160;system in Nicotiana benthamiana plants are reported, being the gold standard production host. However, this model plant is not regarded as an edible species due to the alkaloids and other toxic metabolites that are accumulated in its leaves.
In this work, we describe the comparison between two edible plant systems, red beet (Beta vulgaris cv Moulin Rouge) and spinach (Spinacea oleracea cv Industria), for the expression of two candidate forms of the 65 kDa isoform of glutamic acid decarboxylase (GAD65), carried out by the plant virus-based vectors5. GAD65 is a major autoantigen associated to Type 1 Diabetes (T1D) and it is currently under investigation in human clinical trials to prevent or delay T1D by inducing tolerance6. The production of GAD65 in plants has been extensively studied in model plant species as Nicotiana tabacum and N. benthamiana4,5,6,7. Here, we describe the use of edible plant species for the production of the molecule in tissues that can be meant for a direct oral delivery. From a technical point of view, we studied and selected the system for plant agroinfiltration and the edible plant platform for GAD65 production by evaluating different parameters: the recombinant protein expression levels, the residual microbial charge in plant tissue meant for oral delivery, the resistance of GAD65 to the gastric digestion, and the bioequivalence of the transformed plants with the wild type.

<table>
	<tbody>
		<tr>
			<td>0.2-&#956;m Minisart RC4 membrane filters</td>
			<td>Sartorius-Stedim</td>
			<td>17764</td>
			<td></td>
		</tr>
		<tr>
			<td>2&#8211;mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td>Toxic; 4 % to make loading buffer with glycerol, SDS and Tris-HCl</td>
		</tr>
		<tr>
			<td>4-Morpholineethanesulfonic acid (MES)</td>
			<td>Sigma</td>
			<td>M8250</td>
			<td>pH 5.5</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sarstedt</td>
			<td>833924</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>27221</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>Acetonitrile LC-MS grade</td>
			<td>Sigma</td>
			<td>34967</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
			<td>Toxic &#8211; 0.1 M stock in DMSO</td>
		</tr>
		<tr>
			<td>Agar Bacteriological Grade</td>
			<td>Applichem</td>
			<td>A0949</td>
			<td>15 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Tryptone</td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Sigma</td>
			<td>70221</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-eGFP antibody</td>
			<td>ABCam</td>
			<td>ab290</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GAD 65/67 antibody</td>
			<td>Sigma</td>
			<td>G5163</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-LHCB2 antibody</td>
			<td>Agrisera</td>
			<td>AS01 003</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Blue R-250</td>
			<td>Sigma</td>
			<td>B7920</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 Column</td>
			<td>Grace</td>
			<td>&#160;&#160; -</td>
			<td>Alltima HP C18 (150 mm x 2.1 mm; 3 &#956;m) Column</td>
		</tr>
		<tr>
			<td>C18 Guard Column</td>
			<td>Grace</td>
			<td>&#160;&#160; -</td>
			<td>Alltima HP C18 (7.5 mm x 2.1 mm; 5 &#956;m) Guard&#160; Column</td>
		</tr>
		<tr>
			<td>CalMag Grower</td>
			<td>Peter Excel</td>
			<td>15-5-15</td>
			<td>Fertilizer</td>
		</tr>
		<tr>
			<td>Carbenicillin disodium</td>
			<td>Duchefa Biochemie</td>
			<td>C0109</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Chemiluminescence imaging system</td>
			<td>BioRad</td>
			<td>1708370</td>
			<td>ChemiDoc Touch Imaging System</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Detergent</td>
			<td>Sigma</td>
			<td>P5927</td>
			<td>Polysorbate 20</td>
		</tr>
		<tr>
			<td>Fluorescence reader</td>
			<td>Perkin-Elmer</td>
			<td>&#160;1420-011</td>
			<td>VICTOR Multilabel Counter</td>
		</tr>
		<tr>
			<td>Formic acid LC-MS grade</td>
			<td>Sigma</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>15 % to make loading buffer with Tris-HCl, SDS and 2&#8211;mercaptoethanol</td>
		</tr>
		<tr>
			<td>GoTaq G2 polymerase</td>
			<td>Promega</td>
			<td>M7841</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>HILIC Column</td>
			<td>Grace</td>
			<td>&#160;&#160; -</td>
			<td>Ascentis Express HILIC (150 mm x 2.1 mm; particles size 2.7 &#956;m) Column</td>
		</tr>
		<tr>
			<td>HILIC Guard Column</td>
			<td>Grace</td>
			<td>&#160;&#160; -</td>
			<td>Vision HT HILIC (7.5 mm x 2.1 mm; 3 &#956;m) Guard&#160; Column</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody</td>
			<td>Sigma</td>
			<td>A6154</td>
			<td>Do not freeze/thaw too many times</td>
		</tr>
		<tr>
			<td>HPLC Autosampler</td>
			<td>Beckman Coulter</td>
			<td>&#160;&#160; -</td>
			<td>System Gold 508 Autosampler</td>
		</tr>
		<tr>
			<td>HPLC System</td>
			<td>Beckman Coulter</td>
			<td>&#160;&#160; -</td>
			<td>System Gold 128 Solvent Module HPLC</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>24137</td>
			<td>Flamable</td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Sigma</td>
			<td>K4000</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>2 g/L with NaCl , Na2HPO4 and KH2PO4 to make PBS</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P9791</td>
			<td>2.4 g/L with NaCl , Na2HPO4 and KCl to make PBS</td>
		</tr>
		<tr>
			<td>Loading Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luminol solution</td>
			<td>Ge Healthcare</td>
			<td>RPN2232</td>
			<td>Prepare the solution using the ECL Prime Western Blotting System commercial kit</td>
		</tr>
		<tr>
			<td>Lyophilizator</td>
			<td>5Pascal</td>
			<td>LIO5P0000DGT</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectometer</td>
			<td>Bruker Daltonics</td>
			<td>&#160; -</td>
			<td>Bruker Esquire 6000; the mass spectrometer was equipped with an ESI source and the analyzer was an ion trap</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>32213</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Milk-blocking solution</td>
			<td>Ristora</td>
			<td>&#160;&#160; -</td>
			<td>3 % in PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td>Use with NaH2PO4 to make Sodium Phospate buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>80 g/L with KCl, Na2HPO4 and KH2PO4 to make PBS; 10 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, Tryptone and Agar Bacteriological Grade</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>&#160;Use with Na2HPO4 to make Sodium Phospate buffer; 14.4 g/L to make PBS</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulase membrane</td>
			<td>Ge Healthcare</td>
			<td>10600002</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin from porcine gastric mucosa</td>
			<td>Sigma</td>
			<td>P7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase substrate ECL</td>
			<td>GE Healthcare</td>
			<td>RPN2235</td>
			<td>Light sensitive material</td>
		</tr>
		<tr>
			<td>Pump Vacuum Press</td>
			<td>VWR</td>
			<td>111400000098</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent A</td>
			<td>Sigma</td>
			<td>B9643</td>
			<td>Use 50 parts of this reagent with 1 part of reagent B to prepare BCA working solution</td>
		</tr>
		<tr>
			<td>Reagent B</td>
			<td>Sigma</td>
			<td>B9643</td>
			<td>Use 1 part of this reagent with 50 parts of reagent A to prepare BCA working solution</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Duchefa Biochemie</td>
			<td>R0146</td>
			<td>Toxic &#8211; 25 mg/mL stock in DMSO</td>
		</tr>
		<tr>
			<td>SDS (Sodium dodecyl sulphate)</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td>Flamable, toxic, corrosive-10 % stock; 3 % to make loading buffer with Tris-HCl, Glycerol and 2&#8211;mercaptoethanol</td>
		</tr>
		<tr>
			<td>Sodium metabisulphite</td>
			<td>Sigma</td>
			<td>7681-57-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator system</td>
			<td>Soltec</td>
			<td>090.003.0003</td>
			<td>Sonica&#174; 2200 MH; frequency 40 khz</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Terumo</td>
			<td>&#160;&#160; -</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent fixed 300-&#181;L insert glass tubes</td>
			<td>Thermo Scientific</td>
			<td>11573680</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>Adjust pH with 1N HCl to make Tris-HCl buffer, use 1,5M Tris-HCl (pH 6.8) to make loading buffer with SDS, Glycerol and 2&#8211;mercaptoethanol</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Formedium</td>
			<td>TRP03</td>
			<td>10 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Agar Bacteriological Grade</td>
		</tr>
		<tr>
			<td>Vacuum concentrator</td>
			<td>Heto</td>
			<td>3878 F1-3</td>
			<td>Speed-vac System</td>
		</tr>
		<tr>
			<td>Water LC-MS grade</td>
			<td>Sigma</td>
			<td>39253</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma</td>
			<td>Y1333</td>
			<td>5 g/L to make LB medium (pH 7.5 with NaOH) with Tryptone, NaCl and Agar Bacteriological Grade</td>
		</tr>
	</tbody>
</table>

transient expression in red beet of a biopharmaceutical candidate vaccine for type-1 diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.

In this work, the workflow for the development of an oral vaccine in edible plant tissues is presented. The focus of this work is the expression of a target protein in an edible host plant species and the characterization of the potential oral vaccine.
The first step involved the evaluation of the suitability of the plant virus-based expression technology to produce recombinant proteins in edible plant systems. For this aim, we ...

Watch this Scientific Journal Video about Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes at JoVE.com

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the ...

transient-expression-red-beet-biopharmaceutical-candidate-vaccine-for

Research

JoVE Journal

Bioengineering

7.9K Views.  University of Verona. Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.

Video: Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

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 ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

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 ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

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., ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...