Name: automating citrus budwood processing for downstream pathogen detection through instrument engineering
Uploaded: 2023-04-21T13:10:00
Description: Watch this Scientific Journal Video about Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering at JoVE.com

The authors acknowledge the Cahuilla people as the Traditional Custodians of the Land on which the experimental work was completed. We are grateful to Professor Norman Ellstrand at the University of California, Riverside, for providing lab space to carry out research activities for this project under the UCR California Agriculture and Food Enterprise (CAF&#201;) Initiative. This research was supported by the CDFA - Specialty Crop Block Grant Program (grant no. 18-0001-055-SC). Additional support was also provided by the CRB project 6100; USDA National Institute of Food and Agriculture, Hatch project 1020106; and the National Clean Plant Network-USDA Animal and Plant Health Inspection Service (AP17PPQS&#38;T00C118, AP18PPQS&#38;T00C107, AP19PPQS&#38;T00C148, &#38; AP20PPQS&#38;T00C049) awarded to Georgios Vidalakis.

1. Collecting the citrus budwood samples to ship
<ol>
	<li>Send Technology Evolving Solutions a spreadsheet of tree information for them to load into their web server (eventually, the user will create new trees).</li>
	<li>Use the phone app TES tracker to select a tree, and hold a near-field communication (NFC) collar tag to the phone to load the tree information to the tag.</li>
	<li>Insert three to four citrus budwood samples into the BTE-compatible plastic bag carrier, and close wit...

<ol>
	<li>Verni&#232;re, C., 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=Interactions+between+citrus+viroids+affect+symptom+expression+and+field+performance+of+clementine+trees+grafted+on+trifoliate+orange.">Interactions between citrus viroids affect symptom expression and field performance of clementine trees grafted on trifoliate orange.</a> Phytopathology. 96 (4), 356-368 (2006).</li><li>Verni&#232;re, C., 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=Citrus+viroids:+Symptom+expression+and+effect+on+vegetative+growth+and+yield+of+clementine+trees+grafted+on+trifoliate+orange.">Citrus viroids: Symptom expression and effect on vegetative growth and yield of clementine trees grafted on trifoliate orange.</a> Plant Disease. 88 (11), 1189-1197 (2004).</li><li>Zhou, C., Talon, M., Caruso, M., Gmitter, F. G., 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=Chapter+19+-+Citrus+viruses+and+viroids.">Chapter 19 - Citrus viruses and viroids.</a> The Genus Citrus. , 391-410 (2020).</li><li>Trends and issues facing the U.S. citrus industry. Choices Magazine Online Available from: <a target="_blank" href="https://www.choicesmagazine.org/choices-magazine/theme-articles/trends-and-challenges-in-fruit-and-tree-nut-sectors/trends-and-issues-facing-the-us-citrus-industry">https://www.choicesmagazine.org/choices-magazine/theme-articles/trends-and-challenges-in-fruit-and-tree-nut-sectors/trends-and-issues-facing-the-us-citrus-industry</a> (2021)</li><li>Fruit and Tree Nuts Outlook. United States Department of Agriculture-Economic Research Service Available from: <a target="_blank" href="https://www.ers.usda.gov/webdocs/outlooks/98171/fts-370.pdf?v=5697">https://www.ers.usda.gov/webdocs/outlooks/98171/fts-370.pdf?v=5697</a> (2020)</li><li>Forsyth, J., Fruits Damiani, J. C. i. t. r. u. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrus+Fruits.+Types+on+the+market.">Citrus Fruits. Types on the market.</a> Encyclopedia of Food Sciences and Nutrition. , 1329-1335 (2003).</li><li>Bostock, R. M., Thomas, C. S., Hoenisch, R. W., Golino, D. A., Vidalakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+health:+How+diagnostic+networks+and+interagency+partnerships+protect+plant+systems+from+pests+and+pathogens.">Plant health: How diagnostic networks and interagency partnerships protect plant systems from pests and pathogens.</a> California Agriculture. 68 (4), 117-124 (2014).</li><li>Osman, F., Dang, T., Bodaghi, S., Vidalakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+multiplex+RT-qPCR+detects+three+citrus+viroids+from+different+genera+in+a+wide+range+of+hosts.">One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.</a> Journal of Virological Methods. 245, 40-52 (2017).</li><li>Wang, J., 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=Past+and+future+of+a+century+old+Citrus+tristeza+virus+collection:+A+California+citrus+germplasm+tale.">Past and future of a century old Citrus tristeza virus collection: A California citrus germplasm tale.</a> Frontiers in Microbiology. 4, 366 (2013).</li><li>Gergerich, R. C., 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=Safeguarding+fruit+crops+in+the+age+of+agricultural+globalization.">Safeguarding fruit crops in the age of agricultural globalization.</a> Plant Disease. 99 (2), 176-187 (2015).</li><li>Moreno, P., Ambr&#243;s, S., Albiach-Mart&#237;, M. R., Guerri, J., Pe&#241;a, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrus+tristeza+virus:+A+pathogen+that+changed+the+course+of+the+citrus+industry.">Citrus tristeza virus: A pathogen that changed the course of the citrus industry.</a> Molecular Plant Pathology. 9 (2), 251-268 (2008).</li><li>Yokomi, R. 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=Identification+and+characterization+of+Citrus+tristeza+virus+isolates+breaking+resistance+in+trifoliate+orange+in+California.">Identification and characterization of Citrus tristeza virus isolates breaking resistance in trifoliate orange in California.</a> Phytopathology. 107 (7), 901-908 (2017).</li><li>Selvaraj, V., Maheshwari, Y., Hajeri, S., Yokomi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+detection+tool+for+VT+isolates+of+Citrus+tristeza+virus+by+immunocapture-reverse+transcriptase+loop-mediated+isothermal+amplification+assay.">A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture-reverse transcriptase loop-mediated isothermal amplification assay.</a> PLoS One. 14 (9), 0222170 (2019).</li><li>Babcock, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+impact+of+California's+citrus+industry+in+2020.">Economic impact of California's citrus industry in 2020.</a> Journal of Citrus Pathology. 9, (2022).</li><li>Gottwald, T. R., Polek, M., Riley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History,+present+incidence,+and+spatial+distribution+of+Citrus+tristeza+virus+in+the+California+central+valley.">History, present incidence, and spatial distribution of Citrus tristeza virus in the California central valley.</a> International Organization of Citrus Virologists Conference Proceedings (1957-2010). 15, (2002).</li><li>Yokomi, R., 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=Molecular+and+biological+characterization+of+a+novel+mild+strain+of+citrus+tristeza+virus+in+California.">Molecular and biological characterization of a novel mild strain of citrus tristeza virus in California.</a> Archives of Virology. 163 (7), 1795-1804 (2018).</li><li>Fuchs, M., 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=Economic+studies+reinforce+efforts+to+safeguard+specialty+crops+in+the+United+States.">Economic studies reinforce efforts to safeguard specialty crops in the United States.</a> Plant Disease. 105 (1), 14-26 (2021).</li><li>The real cost of HLB in Florida. Citrus Industry Magazine Available from: <a target="_blank" href="https://citrusindustry.net/2019/07/30/the-real-cost-of-hib-in-florida/">https://citrusindustry.net/2019/07/30/the-real-cost-of-hib-in-florida/</a> (2019)</li><li>McRoberts, 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=Using+models+to+provide+rapid+programme+support+for+California's+efforts+to+suppress+Huanglongbing+disease+of+citrus.">Using models to provide rapid programme support for California's efforts to suppress Huanglongbing disease of citrus.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 374 (1776), 20180281 (2019).</li><li>Albrecht, C., 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=Action+plan+for+Asian+citrus+psyllid+and+huanglongbing+(citrus+greening)+in+California.">Action plan for Asian citrus psyllid and huanglongbing (citrus greening) in California.</a> Journal of Citrus Pathology. 7 (1), (2020).</li><li>Navarro, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Citrus+Variety+Improvement+Program+in+Spain+in+the+period+1975-2001.">The Citrus Variety Improvement Program in Spain in the period 1975-2001.</a> International Organization of Citrus Virologists Conference Proceedings (1957-2010). 15 (15), (2002).</li><li>Vidalakis, G., Gumpf, D. J., Polek, M. L., Bash, J. A., Ferguson, L., Grafton-Cardwell, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+California+Citrus+Clonal+Protection+Program.">The California Citrus Clonal Protection Program.</a> Citrus Production Manual. , 117-130 (2014).</li><li>Dang, T., Rao, A. L. N., Lavagi-Craddock, I., Vidalakis, G., 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=High-throughput+RNA+extraction+from+citrus+tissues+for+the+detection+of+viroids.">High-throughput RNA extraction from citrus tissues for the detection of viroids.</a> In Viroids: Methods and Protocols. 2316, (2022).</li><li>Osman, F., Vidalakis, G., Rao, A. L. N., Lavagi-Craddock, I., Vidalakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+detection+of+viroids+using+singleplex+and+multiplex+quantitative+polymerase+chain+reaction.">Real-time detection of viroids using singleplex and multiplex quantitative polymerase chain reaction.</a> Viroids: Methods and Protocols. 2316, (2022).</li><li>Li, R., 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=A+reliable+and+inexpensive+method+of+nucleic+acid+extraction+for+the+PCR-based+detection+of+diverse+plant+pathogens.">A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens.</a> Journal of Virological Methods. 154 (1-2), 48-55 (2008).</li><li>Saponari, M., Manjunath, K., Yokomi, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+detection+of+Citrus+tristeza+virus+in+citrus+and+aphids+by+real-time+reverse+transcription-PCR+(TaqMan).">Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan).</a> Journal of Virological Methods. 147 (1), 43-53 (2008).</li><li>Damaj, M. B., 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=Reproducible+RNA+preparation+from+sugarcane+and+citrus+for+functional+genomic+applications.">Reproducible RNA preparation from sugarcane and citrus for functional genomic applications.</a> International Journal of Plant Genomics. 2009, 765367 (2009).</li><li>Dang, T., 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=First+report+of+citrus+leaf+blotch+virus+infecting+Bearss+lime+tree+in+California.">First report of citrus leaf blotch virus infecting Bearss lime tree in California.</a> Plant Disease. 104 (11), 3088 (2020).</li><li>Manchester, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+UV+methods+for+measurement+of+protein+and+nucleic+acid+concentrations.">Use of UV methods for measurement of protein and nucleic acid concentrations.</a> BioTechniques. 20 (6), 968-970 (1996).</li><li>Teare, J. M., 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=Measurement+of+nucleic+acid+concentrations+using+the+DyNA+QuantTM+and+the+GeneQuantTM.">Measurement of nucleic acid concentrations using the DyNA QuantTM and the GeneQuantTM.</a> BioTechniques. 22 (6), 1170-1174 (1997).</li><li>Imbeaud, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+of+RNA+quality+assessment+using+user-independent+classifiers+of+microcapillary+electrophoresis+traces.">Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.</a> Nucleic Acids Research. 33 (6), 56-56 (2005).</li><li>Menzel, W., Jelkmann, W., Maiss, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+four+apple+viruses+by+multiplex+RT-PCR+assays+with+coamplification+of+plant+mRNA+as+internal+control.">Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control.</a> Journal of Virological Methods. 99 (1-2), 81-92 (2002).</li><li>Vidalakis, G., Rao, A. L. N., Lavagi-Craddock, I., Vidalakis, G., 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=SYBR+Green+RT-qPCR+for+the+universal+detection+of+citrus+viroids.">SYBR Green RT-qPCR for the universal detection of citrus viroids.</a> Viroids: Methods and Protocols. , 211-217 (2022).</li><li>Arredondo Vald&#233;s, R., 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=A+review+of+techniques+for+detecting+Huanglongbing+(greening)+in+citrus.">A review of techniques for detecting Huanglongbing (greening) in citrus.</a> Canadian Journal of Microbiology. 62 (10), 803-811 (2016).</li><li>Li, S., Wu, F., Duan, Y., Singerman, A., Guan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrus+greening:+Management+strategies+and+their+economic+impact.">Citrus greening: Management strategies and their economic impact.</a> HortScience. 55 (5), 604-612 (2020).</li><li>. CDFA California Citrus Pest and Disease Prevention Program Operations Subcomittee Meeting. Meeting Minutes Available from: <a target="_blank" href="https://www.cdfa.ca.gov/citrus/docs/minutes/2019/OpsSubcoMinutes-11062019.pdf">https://www.cdfa.ca.gov/citrus/docs/minutes/2019/OpsSubcoMinutes-11062019.pdf</a> (2019)</li></ol>

With the advent of HLB citrus disease, to reduce losses, the citrus industry, regulatory agencies, and diagnostic laboratories have been urged to rely on high-throughput nucleic acid extraction methods combined with low-throughput manual sample processing and pathogen detection assays such as qPCR34 for the testing of individual trees, in combination with disease management practices35. California's HLB positivity rate has gone from 0.01% in 2012 to 1.2% in 2020. Even thoug...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65159">Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering</a>. The Authors section was updated from:
Deborah Pagliaccia1,6 
Douglas Hill2 
Emily Dang1 
Gerardo Uribe1 
Agustina De Francesco1 
Ryan Milton2 
Anthony De La Torre2 
Axel Mounkam2 
Tyler Dang1 
Sohrab Botaghi1 
Irene Lavagi-Craddock1 
Alexandra Syed1 
William Grover3 
Adriann Okamba4,5 
Georgios Vidalakis2 
1Department of Microbiology and Plant Pathology, University of California Riverside 
2Technology Evolving Solutions (TES) 
3Department of Bioengineering, University of California Riverside 
4Ecole Sup&#233;rieure d&#39;Ing&#233;nieurs L&#233;onard de Vinci ESILV 
5University of California Riverside 
6California Agriculture and Food Enterprise (CAF&#201;), University of California Riverside
to:
Deborah Pagliaccia1,6 
Douglas Hill2 
Emily Dang1 
Gerardo Uribe1 
Agustina De Francesco1 
Ryan Milton2 
Anthony De La Torre2 
Axel Mounkam2 
Tyler Dang1 
Sohrab Bodaghi1 
Irene Lavagi-Craddock1 
Alexandra Syed1 
William Grover3 
Adriann Okamba4,5 
Georgios Vidalakis1 
1Department of Microbiology and Plant Pathology, University of California Riverside 
2Technology Evolving Solutions (TES) 
3Department of Bioengineering, University of California Riverside 
4Ecole Sup&#233;rieure d&#39;Ing&#233;nieurs L&#233;onard de Vinci ESILV 
5University of California Riverside 
6California Agriculture and Food Enterprise (CAF&#201;), University of California Riverside

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65159">Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering</a>. The Authors section was updated.

Erratum: Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

Graft-transmissible phloem-limited pathogens of citrus, such as viroids, viruses, and bacteria, have caused devastating epidemics and serious economic losses in every citrus-producing area of the world. Citrus viroids are limiting production factors because of the exocortis and cachexia diseases they cause in economically important citrus types, such as trifoliate, trifoliate hybrids, mandarins, clementines, and tangerines1,2,3. In California, these viroid-sensitive citrus types are the basis of the growing and profitable market of &#34;easy-peelers&#34;, following the shifting trend in consumers&#39; preference for fruits that are easy to peel, segmented, and seedless4,5,6. Thus, citrus viroids are regulated under the California Department of Food and Agriculture (CDFA) &#34;Citrus Nursery Stock Pest Cleanliness Program-Senate Bill 140&#34;, and the laboratories of CDFA&#39;s Plant Pest Diagnostics Branch perform thousands of citrus viroid tests annually7,8,9,10. Citrus tristeza virus (CTV) has been responsible for the death of over 100 million citrus trees since the beginning of the global epidemic in the 1930s3,9,10,11. In California, stem pitting and trifoliate breaking resistance isolates of the virus pose a serious threat to the $3.6 billion California citrus industry12,13,14. Consequently, CDFA classifies CTV as a regulated class-A plant pest, and the laboratory of the Central California Tristeza Eradication Agency (CCTEA) performs extensive field surveys and thousands of virus tests every year15,16. The bacterium &#34;Candidatus Liberibacter asiaticus&#34; (CLas) and the huanglongbing (HLB) disease are estimated to have caused close to $9 billion of economic damage to Florida as a result of a 40% reduction of citrus acreage, a 57% decrease in citrus operations, and a loss of almost 8,000 jobs17,18. In California, a hypothetical 20% reduction in citrus acreage due to HLB was predicted to result in more than 8,200 job losses and a reduction of over half a billion dollars in the state&#39;s gross domestic product. Therefore, the Citrus Pest and Disease Prevention Program spends over $40 million annually on surveys to test, detect, and eradicate CLas from California14,17,19,20.
A key element of the management of citrus viroids, viruses, and bacteria is the use of pathogen-tested propagative materials (i.e., budwood) for tree production. Pathogen-tested citrus budwood is produced and maintained within comprehensive quarantine programs that employ advanced pathogen elimination and detection techniques10,21. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, tests thousands of budwood samples every year from citrus varieties newly imported into the state and the USA, as well as citrus budwood source trees, to protect California&#39;s citrus and support the functions of the National Clean Plant Network for Citrus10,17,22. To handle the large volume of citrus testing, high-throughput, reliable, and cost-effective pathogen detection assays are a fundamental component for the success of programs such as the CCPP7,10,22.
While molecular-based pathogen detection assays such as polymerase chain reaction (PCR) have allowed for significant increases in throughput in plant diagnostic laboratories, in our experience, one of the most critical bottlenecks in the implementation of high-throughput protocols is the plant tissue sample processing step. This is particularly true for citrus because the currently available protocols for the processing of phloem-rich tissues such as leaf petioles and budwood bark are labor-intensive, time-consuming, and require expensive and specialized laboratory equipment. These protocols require hand-chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation8,23,24. For example, at the CCPP diagnostic laboratory, sample processing includes (i) hand-chopping (6-9 samples/h/operator), (ii) freeze-drying (16-24 h), (iii) pulverization (30-60 s), and (iv) centrifugation (1-2 h). The process also requires specialized supplies (e.g., heavy-duty safe-lock tubes, stainless steel grinding balls, adapters, blades, gloves) and multiple pieces of costly lab equipment (e.g., ultra-low freezer, freeze-dryer, tissue pulverizer, liquid nitrogen cryostation, refrigerated centrifuge).
As in any industry, equipment engineering and the automation of processes are key to lowering costs, increasing throughput, and providing high-quality, uniform product and services. The citrus industry needs low-cost tissue-processing instruments that require minimum skill to operate and, as such, are easy to transfer to diagnostic laboratories and field operations to allow high sample-processing capacity for rapid downstream pathogen detection. Technology Evolving Solutions (TES) and the CCPP developed (i.e., design and fabricate) and validated (i.e., tested with citrus samples and compared to standard laboratory procedures) a low-cost (i.e., eliminated the need for specialized laboratory equipment) instrument for the rapid processing of phloem-rich citrus tissues (i.e., budwood), named the budwood tissue extractor (BTE). As seen in Figure 1, the BTE includes a base component for power and controls, plus a removable chamber for the processing of citrus budwood. The BTE chamber is composed of a grinding wheel specifically designed to strip the phloem-rich bark tissues from the citrus budwood. The shredded bark tissue is ejected rapidly through a slide port into a syringe containing extraction buffer, filtered, and made ready for nucleic acid extraction and purification without any additional handling or preparation (Figure 1). The BTE system also includes a paperless sample tracking application and an integrated weighing application, which record the sample processing information in an online database in real time.
The BTE system has increased the CCPP&#39;s lab diagnostic capacity by over 100% and has consistently produced citrus tissue extracts suitable for the purification of high-quality nucleic acids and the downstream detection of graft-transmissible pathogens of citrus using PCR assays. More specifically, BTE has reduced the time for tissue processing from over 24 h to ~3 min per sample, replaced laboratory instruments costing over $60,000 (Figure 2, steps 2-4), and allowed for the processing of larger sample sizes.
This paper presents the BTE high-throughput citrus bark tissue processing, nucleic acid extraction, and pathogen detection validation data with citrus budwood samples from source trees, including all the appropriate positive and negative controls from the CCPP Rubidoux Quarantine Facility and Lindcove Foundation Facility, respectively. We also present the throughput and processing time changes compared to the current laboratory procedure (Figure 2). In addition, this work provides a detailed, step-by-step protocol for citrus pathogen testing laboratories and demonstrates how the BTE can support the functions of pathogen-clean nursery stock, survey, and eradication programs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65159/65159fig01.jpg" /> 
Figure 1: Budwood tissue extractor. The BTE includes a base component for power and controls, plus a removable chamber for the processing of citrus budwood. The BTE chamber is composed of a grinding wheel specifically designed to strip the phloem-rich bark tissues from citrus budwood. The shredded bark tissue is ejected rapidly through a slide port into a syringe, filtered, and made ready for nucleic acid extraction and purification without any additional handling or preparation. Abbreviation: BTE = budwood tissue extractor. <a href="https://www.jove.com/files/ftp_upload/65159/65159fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65159/65159fig02.jpg" /> 
Figure 2: Step-by-step comparison between the conventional hand-chopping lab procedure and BTE processing. BTE processing involves high-throughput citrus bark tissue processing, nucleic acid extraction, and pathogen detection. The time for each step is indicated in parentheses. <a href="https://www.jove.com/files/ftp_upload/65159/65159fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.08&#34; Hex Trimmer line</td>
			<td>PowerCare</td>
			<td>FPRO07065</td>
			<td>Needed to replace blades.</td>
		</tr>
		<tr>
			<td>1 Hp, 8 gal air compressor</td>
			<td>California Air Tools</td>
			<td>8010</td>
			<td>Quickly dry chambers after rinsed</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Globe Scientific</td>
			<td>111558B</td>
			<td>Store sample in after swishing with syinges</td>
		</tr>
		<tr>
			<td>10 mL Syringe Set</td>
			<td>Technology Evolving Solutions</td>
			<td>TE006-F1-10A-G1000-E1</td>
			<td>Syringe material is cut into. 1 L bottle with guanidine thiocyanate buffer. WARNING - contains guanidine thiocyanate, hazardous waste service required - do not mix with bleach</td>
		</tr>
		<tr>
			<td>12&#34; Ruler</td>
			<td>Westcott</td>
			<td>&#8206;16012</td>
			<td>To measure trimmer line before cutting</td>
		</tr>
		<tr>
			<td>12% Sodium Hypochlorite</td>
			<td>Hasa</td>
			<td>1041</td>
			<td>Disinfects chambers after processing</td>
		</tr>
		<tr>
			<td>-20 C Freezer</td>
			<td>Insignia</td>
			<td>NS-CZ70WH0</td>
			<td>Store sample after processing</td>
		</tr>
		<tr>
			<td>4&#34; x 12&#34; plastic bags</td>
			<td>Plymor</td>
			<td>FP20-4x12-10</td>
			<td>Bags to hold branches during shipping. O-rings attach bag to BTE chamber to seal</td>
		</tr>
		<tr>
			<td>6&#34; Cotton Swab</td>
			<td>Puritan</td>
			<td>806-PCL</td>
			<td>Swab to remove clogs</td>
		</tr>
		<tr>
			<td>7 Gallon Storage Tote</td>
			<td>HDX</td>
			<td>206152</td>
			<td>Holds sodium hypochlorite solution to disinfect chambers and water to rinse chambers</td>
		</tr>
		<tr>
			<td>Air blow gun</td>
			<td>JASTIND</td>
			<td>&#8206;JTABG103A</td>
			<td>Directs air into the chambers at high pressure</td>
		</tr>
		<tr>
			<td>Black Sharpie</td>
			<td>Sharpie</td>
			<td>&#160;S-19421</td>
			<td>Mark 1.5 mL tubes so you can identify sample later</td>
		</tr>
		<tr>
			<td>Bottle Top Dispensor</td>
			<td>Brand</td>
			<td>Z627569</td>
			<td>Adjustable bottle top dispensor to dispense guandine into syringe</td>
		</tr>
		<tr>
			<td>BTE Chamber</td>
			<td>Technology Evolving Solutions</td>
			<td>TE002BB-A05-E1</td>
			<td>Used to process budwood. Includes O-rings, BTE Slide, slide plunger, drain valve, lid, blade set, and blade set removal tool</td>
		</tr>
		<tr>
			<td>Dish Soap</td>
			<td>Dawn</td>
			<td>57445CT</td>
			<td>Surfectant to improve sodium hypochlorite penetration into chamber</td>
		</tr>
		<tr>
			<td>Fume hood with hepa filter</td>
			<td>Air Science</td>
			<td>P5-36XT-A</td>
			<td>Fume hood with hepa filter (ASTS-030)&#160; to limit possible contamination and protect against chemical spills</td>
		</tr>
		<tr>
			<td>Insulated foam shipping container</td>
			<td>PolarTech</td>
			<td>261/J50C</td>
			<td>Insulated shipping container to ship samples on ice after they are collected</td>
		</tr>
		<tr>
			<td>Lab coat</td>
			<td>Red Kap</td>
			<td>KP14WH LN 46</td>
			<td>Lab coat to limit possible contamination and protect against chemical spills</td>
		</tr>
		<tr>
			<td>Laptop</td>
			<td>Microsoft</td>
			<td>Surface</td>
			<td>Wifi capable laptop to run TES GUI. Needed for initial setup and provides more indepth information about the tissue processing base</td>
		</tr>
		<tr>
			<td>NFC Capable Phone</td>
			<td>Samsung</td>
			<td>Galaxy S9</td>
			<td>Phone to download and use TES phone app</td>
		</tr>
		<tr>
			<td>NFC clip tag</td>
			<td>Technology Evolving Solutions</td>
			<td>TE005-Clip-E1</td>
			<td>Sample tag that can be linked with trees. Made to function with TES phone app</td>
		</tr>
		<tr>
			<td>NFC Collar Tag</td>
			<td>Technology Evolving Solutions</td>
			<td>TE005-Collar-E1</td>
			<td>Tag that is attached to a tree. Made to function with TES phone app</td>
		</tr>
		<tr>
			<td>Nitrile Gloves</td>
			<td>Usa Scientific</td>
			<td>3915-4400</td>
			<td>Gloves to limit possible contamination and protect against chemical spills</td>
		</tr>
		<tr>
			<td>Noise-Reducing Earmuff</td>
			<td>3M</td>
			<td>90565-4DC-PS</td>
			<td>Protect ears while operating air compressor and tissue processing base</td>
		</tr>
		<tr>
			<td>Polyurethane Recoil Air Hose</td>
			<td>FYPower</td>
			<td>&#8206;510019</td>
			<td>Attaches air gun to compressor</td>
		</tr>
		<tr>
			<td>Saftey glasses</td>
			<td>Solidwork</td>
			<td>SW8329-US</td>
			<td>Protect eyes for chemical and physical hazards</td>
		</tr>
		<tr>
			<td>Spray bottle</td>
			<td>JohnBee</td>
			<td>B08QM81BJV</td>
			<td>Spray bleach to deconatinate surfaces</td>
		</tr>
		<tr>
			<td>Tissue Extractor Base</td>
			<td>Technology Evolving Solutions</td>
			<td>TE001-A-E1</td>
			<td>System to process plant tissue. Needs BTE or LTE chambers to function. Includes power cable, blade adapter, and 8/32&#34; allen wrench</td>
		</tr>
		<tr>
			<td>Tissue Processing Base Weight Scale</td>
			<td>Technology Evolving Solutions</td>
			<td>TE003-A05-200g-01-E1</td>
			<td>200 g, 0.01 resolution weight scale that connects to tissue processing base to enforce weight ranges and/or link weights with sample. Includes scale, power cable, connection cable, 5ml syringe holder, tower air shield&#160;</td>
		</tr>
		<tr>
			<td>Vermiculite</td>
			<td>EasyGoProducts</td>
			<td>B07WQDZGRP</td>
			<td>Needed to transport hazardous waste (guanidine thiocyanate) using a hazardous waste disposal service</td>
		</tr>
		<tr>
			<td>Wire Cutter</td>
			<td>Boenfu</td>
			<td>&#8206;BOWC-06002-US</td>
			<td>Wire cutters to cut trimmer line</td>
		</tr>
	</tbody>
</table>

automating citrus budwood processing for downstream pathogen detection through instrument engineering

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious economic losses worldwide. For example, the citrus tristeza virus killed over 100 million citrus trees globally, while &#34;Candidatus Liberibacter asiaticus&#34; has cost Florida $9 billion. The use of pathogen-tested citrus budwood for tree propagation is key for the management of such pathogens. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, uses polymerase chain reaction (PCR) assays to test thousands of samples from citrus budwood source trees every year to protect California&#39;s citrus and to provide clean propagation units to the National Clean Plant Network. A severe bottleneck in the high-throughput molecular detection of citrus viruses and viroids is the plant tissue processing step.
Proper tissue preparation is critical for the extraction of quality nucleic acids and downstream use in PCR assays. Plant tissue chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation is time-intensive and labor-intensive and requires expensive and specialized laboratory equipment. This paper presents the validation of a specialized instrument engineered to rapidly process phloem-rich bark tissues from citrus budwood, named the budwood tissue extractor (BTE). The BTE increases sample throughput by 100% compared to current methods. In addition, it decreases labor and the cost of equipment. In this work, the BTE samples had a DNA yield (80.25 ng/&#181;L) that was comparable with the CCPP&#39;s hand-chopping protocol (77.84 ng/&#181;L). This instrument and the rapid plant tissue processing protocol can benefit several citrus diagnostic laboratories and programs in California and become a model system for tissue processing for other woody perennial crops worldwide.

RNA extraction, purification, and quality using BTE-processed budwood citrus tissue and assessment of time for tissue processing 
We used budwood samples from 255 representative citrus trees for this test to compare the RNA quality from the BTE versus the standard procedure. Samples were processed by the budwood tissue extractor (BTE) (protocol steps 4.1-4.6 and Figure 2, right side, step 1, step 5, and step 6) or prepared following the regulatorily approved citrus budw...

Watch this Scientific Journal Video about Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering at JoVE.com

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

We engineered, fabricated, and validated an instrument that rapidly processes phloem-rich bark citrus budwood tissues. Compared to current methods, the budwood tissue extractor (BTE) has increased sample throughput and decreased the required labor and equipment costs.

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious ...

The demonstrated technique uses a low-cost instrument to rapidly process phloem-rich citrus tissue, eliminating the need for labor intensive protocols and specialized laboratory equipment. This allows for rapid downstream detection and management of citrus viroids, viruses, and bacteria. This method could increase throughput and improve the quality and uniformity of testing services benefiting the citrus industry and potentially aiding in diagnosing and treating diseases in nursery and field operations.Citrus plants are infected by pathogen, causes significant economic losses worldwide. Advanced pathogen detection techniques, like the Budwood Tissue Extractor, or BTE, are necessary to produce pathogen tested propagative materials for large scale tree production. Although tested and validated with citrus samples, the method can be applied in diagnostic laboratories and field operations for many other crops.Beginners performing this technique must familiarize themselves with the protocol, follow instructions precisely, and be patient. To begin, use the phone app Test Tracker to select a tree and hold a Near-Field Communication, or NFC, collar tag to the phone to load the tree information to the tag. Insert three to four citrus budwood samples into the Budwood Tissue Extractor, or BTE, compatible plastic bag carrier, and close it with the clip.After ensuring the quality of the sample by following the steps described in the text, use the phone app Test Tracker to scan the NFC collar tag of the tree, and link it with the NFC clip tag on the sample bag. Inside a properly disinfected fume hood, flip the switch on the back of the tissue extractor base. Ensure the switch on the left side of the box is pressed down on the top, and wait til the blinking green LED indicates the chamber is ready.To prepare the BTE chamber, attach an empty sample bag to the back of the chamber by securing it to the back nozzle using an O-ring. Inspect the blade for signs of wear, or damage, such as cuts on the blade continuing into the plastic, or cuts on the tip. Ensure the arrow on the blade lines up with the single lock symbol, and the air release on the bottom of the chamber is turned toward the O symbol.Place the clear lid over the chamber opening. Using the track on the right of the chamber, slide the clear BTE slide onto the chamber, pushing the lock on the bottom of the chamber as far as it can go into the slide. Ensure the plunger cap is mounted on the top of the slide.To load the BTE chamber onto the BTE base, place the prepared chamber on the base with the BTE base nozzle protruding into the back of the chamber. Load the sample onto the BTE base by moving the white sticker on the NFC clip tag in a slow circular motion to a Z on the right side of the box until the yellow light starts blinking. After attaching the sample to the base, ensure the sample bag has no holes in it.Then place the O-ring over the sample bag to secure it to the front of the BTE nozzle. To load the unused syringe set, first, tear the syringe set. Then place the unused syringe set onto the weigh scale tower.Remove the syringe set when a red light begins flashing, or the scale displays zero. Remove the plunger with the filter from the syringe while ensuring the fluid remains in the bottom syringe. Place the plunger on a paper towel, or the tower, where it is not touching any surface.Finally, attach the syringe to the slide exit port by pressing the exit port into the syringe and turning it 90 degrees. To begin processing the budwood sample, press the top black button to start the machine. Grab one budwood stick from the bag, and put it through the top of the BTE nozzle.Grab the budwood stick on the other side of the chamber with the other hand and slowly inch down into the blade. Slowly move the budwood back and forth while rotating it. When a slight buzzing is heard, move the branch deeper into the machine to focus on the phloem.After completing the same for all the branches, press the top black button to stop the processing, and wait for the light to begin flashing yellow again. To verify the sample weight, rotate the syringe 90 degrees and pull it down. Place the plunger back on the syringe and place the syringe on the tower.After the scale auto-detects the sample, a green LED blinking indicates the sample weight is within the proper range. If the red light blinks, indicating too low a sample weight, repeat the sample processing. If the weight is too high, remove some samples, and the blinking of the yellow LED will become slower.To homogenize the sample, remove the plunger from the syringe containing the sample. Push the fluid into the syringe and insert the plunger back. Push the plunger to pass the buffer and plant sap through the mesh filter via the rubber tubing into the empty syringe.Mix the sample by pushing the buffer and plant sap back and forth three to four times from one syringe to another until the sample turns into a homogeneous green liquid. Once well homogenized, push the plant sap sample into the syringe without the mesh filter, and detach the rubber tubing and the syringe with the filter from it. Then expel the plant sap sample from the syringe into a two milliliter sterile microcentrifuge tube.After labeling the sample with the bag number using a permanent marker, store them at minus 20 degrees Celsius until further use. Sanitize the BTE removable chamber after the 10th sample is processed and the green LED continues blinking instead of changing to blue. To disassemble the chamber and clean all contaminants, remove the clear plastic cover, the clear chamber slide, and the clog port on the chamber slide.Then turn the air release valve on the bottom of the chamber to the open padlock mark. After placing the chamber components into the setup ultrasonic cleaner, place the chamber in the bath with the lid underneath it to prevent the lid from floating. Run the ultrasonic cleaner for 15 minutes.Rinse the chamber components in the water bath for at least 30 seconds before moving the chamber components to the drying station. To begin drying, position the air gun in line with the groove of the upper opening of the chamber, and press the trigger of the gun to dispense air for about 30 seconds. Turn the air gun towards the top nozzles of the chamber, and after pressing the trigger, slowly trace three full circles of each nozzle entrance.Position the air gun in the center of the BTE, and starting from the innermost point hold the trigger down and move until the air gun points toward the slide. Quickly run air over the entire chamber front and back to get the surface water off the outside. Comparison of the BTE and conventional laboratory protocol for citrus tissue processing indicated that the extracted nucleic acid concentrations determined by measuring absorbance at 260 nanometers in both cases were comparable.The purity of the nucleic acids extracted by the BTE determined as the ratio of the absorbances at 260 and 280 nanometers was high with low protein contamination. The nucleic acid integrity analyzed by RT-qPCR targeting the mRNA of the citrus NADH dehydrogenase gene was very similar for both BTE and the standard manual protocol. The conventional lab procedure required almost seven to 10 minutes for hand chopping per sample, and more time for tissue processing, including freeze drying, grinding, and centrifuging.However, the BTE processed each sample for a nucleic acid extraction in three minutes, including preparing, weighing, and cleaning steps. None of the 72 healthy samples from the first BTE sample processing produced amplification curves for the citrus pathogens tested, thereby validating the BTE processing of citrus tissue. In the second BTE sample processing, with two introduced mix infected samples, the extracted nucleic acids showed the presence of different citrus viruses and viroids in batches one and five.However, no cross-contamination between heads, or false-positive, or negative results were detected. While processing, leaving the branch in one place for too long might cut too deep into the branch, increasing the chances of clogging, and collecting acellular fibery material. Once processed, the material is ready for most DNA and RNA extraction and purification protocol, and can be tested with any molecular detection assay for plant pathogens.Developing the Budwood Tissue Extractor opened the door to high-throughput area and grove-wide screening. Along with the Leaf Tissue Extractor, it allows for processing at a large scale of plant tissues that they were previously deemed difficult to process, such as roots and peduncles.

automating-citrus-budwood-processing-for-downstream-pathogen

Research

JoVE Journal

Bioengineering

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

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

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

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

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...