The authors would like to acknowledge support from the Industry Alignment Fund Pre-Positioning (IAF-PP) funding (H18/01/a0/021 and H18/AH/a0/001) from A*STAR, Singapore.

<ol>
	<li>Zhou, 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=Challenges+and+advances+in+clinical+applications+of+mesenchymal+stromal+cells.">Challenges and advances in clinical applications of mesenchymal stromal cells.</a> Journal of Hematology &amp; Oncology. 14 (1), 24 (2021).</li><li>Garc&#237;a-Bernal, D., 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+current+status+of+mesenchymal+stromal+cells:+Controversies,+unresolved+issues+and+some+promising+solutions+to+improve+their+therapeutic+efficacy.">The current status of mesenchymal stromal cells: Controversies, unresolved issues and some promising solutions to improve their therapeutic efficacy.</a> Frontiers in Cell and Developmental Biology. 9, 650664 (2021).</li><li>Mastrolia, I., 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=Challenges+in+clinical+development+of+mesenchymal+stromal/stem+cells:+Concise+review.">Challenges in clinical development of mesenchymal stromal/stem cells: Concise review.</a> Stem Cells Translational Medicine. 8 (11), 1135-1148 (2019).</li><li>Jovic, D., 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+brief+overview+of+global+trends+in+MSC-based+cell+therapy.">A brief overview of global trends in MSC-based cell therapy.</a> Stem Cell Reviews and Reports. 18 (5), 1525-1545 (2022).</li><li>Lechanteur, C., Briquet, A., Bettonville, V., Baudoux, E., Beguin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC+manufacturing+for+academic+clinical+trials:+From+a+clinical-grade+to+a+full+GMP-compliant+process.">MSC manufacturing for academic clinical trials: From a clinical-grade to a full GMP-compliant process.</a> Cells. 10, 1320 (2021).</li><li>Fern&#225;ndez-Santos, M. E., 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=Optimization+of+mesenchymal+stromal+cell+(MSC)+manufacturing+processes+for+a+better+therapeutic+outcome.">Optimization of mesenchymal stromal cell (MSC) manufacturing processes for a better therapeutic outcome.</a> Frontiers in Immunology. 13, 918565 (2022).</li><li>Jossen, V., vanden Bos, C., Eibl, R., Eibl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manufacturing+human+mesenchymal+stem+cells+at+clinical+scale:+Process+and+regulatory+challenges.">Manufacturing human mesenchymal stem cells at clinical scale: Process and regulatory challenges.</a> Applied Microbiology and Biotechnology. 102 (9), 3981-3994 (2018).</li><li>Jayaraman, P., Lim, R., Ng, J., Vemuri, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+translational+mesenchymal+stromal+cell+therapy+through+consistent+quality+GMP+manufacturing.">Acceleration of translational mesenchymal stromal cell therapy through consistent quality GMP manufacturing.</a> Frontiers in Cell and Developmental Biology. 9, 648472 (2021).</li><li>Levy, O., 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=Shattering+barriers+toward+clinically+meaningful+MSC+therapies.">Shattering barriers toward clinically meaningful MSC therapies.</a> Science Advances. 6 (30), (2020).</li><li>Fri&#269;ov&#225;, D., Korchak, J. A., Zubair, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+translational+considerations+of+mesenchymal+stem/stromal+cell+therapy+for+Parkinson's+disease.">Challenges and translational considerations of mesenchymal stem/stromal cell therapy for Parkinson's disease.</a> npj Regenerative Medicine. 5 (1), 20 (2020).</li><li>Childs, P. G., Reid, S., Salmeron-Sanchez, M., Dalby, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hurdles+to+uptake+of+mesenchymal+stem+cells+and+their+progenitors+in+therapeutic+products.">Hurdles to uptake of mesenchymal stem cells and their progenitors in therapeutic products.</a> Biochemical Journal. 477 (17), 3349-3366 (2020).</li><li>James, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+short-term+gain+can+lead+to+long-term+pain.">How short-term gain can lead to long-term pain.</a> Cell &amp; Gene Therapy Insights. 3 (4), 271-284 (2017).</li><li>Ochs, J., Barry, F., Schmitt, R., Murphy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+automation+for+the+production+of+clinical-grade+mesenchymal+stromal+cells:+The+AUTOSTEM+robotic+platform.">Advances in automation for the production of clinical-grade mesenchymal stromal cells: The AUTOSTEM robotic platform.</a> Cell &amp; Gene Therapy Insights. 3 (8), 739-748 (2017).</li><li>Doulgkeroglou, M. 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=Automation,+monitoring,+and+standardization+of+cell+product+manufacturing.">Automation, monitoring, and standardization of cell product manufacturing.</a> Frontiers in Bioengineering and Biotechnology. 8, 811 (2020).</li><li>Chen, A. K. -. L., Reuveny, S., Oh, S. K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+human+mesenchymal+and+pluripotent+stem+cell+microcarrier+cultures+in+cellular+therapy:+Achievements+and+future+direction.">Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: Achievements and future direction.</a> Biotechnology Advances. 31 (7), 1032-1046 (2013).</li><li>Couto, P. S., Bersenev, A., Rafiq, Q. A., Fernandes, T. G., Diogo, M. M., Cabral, J. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Engineering Strategies for Regenerative Medicine. , 33-71 (2020).</li><li>Tsai, A. -. C., Pacak, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprocessing+of+human+mesenchymal+stem+cells:+From+planar+culture+to+microcarrier-based+bioreactors.">Bioprocessing of human mesenchymal stem cells: From planar culture to microcarrier-based bioreactors.</a> Bioengineering. 8 (7), 96 (2021).</li><li>Cherian, D. S., Bhuvan, T., Meagher, L., Heng, T. S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+considerations+in+scaling+up+therapeutic+cell+manufacturing.">Biological considerations in scaling up therapeutic cell manufacturing.</a> Frontiers in Pharmacology. 11, 654 (2020).</li><li>Mizukami, A., Swiech, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+From+discovery+to+manufacturing+and+commercialization.">Mesenchymal stromal cells: From discovery to manufacturing and commercialization.</a> Stem Cells International. 2018, 4083921 (2018).</li><li>Hassan, 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=Large-scale+expansion+of+human+mesenchymal+stem+cells.">Large-scale expansion of human mesenchymal stem cells.</a> Stem Cells International. 2020, 9529465 (2020).</li><li>Li, 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=Advances+in+automated+cell+washing+and+concentration.">Advances in automated cell washing and concentration.</a> Cytotherapy. 23 (9), 774-786 (2021).</li><li>Mehta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-use+centrifugation+solution+for+volume+reduction+and+cell+washing+process+in+cell+therapy+manufacturing.">Single-use centrifugation solution for volume reduction and cell washing process in cell therapy manufacturing.</a> Cytotherapy. 16, 101 (2014).</li><li>Giancola, R., Bonfini, T., Iacone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy:+cGMP+facilities+and+manufacturing.">Cell therapy: cGMP facilities and manufacturing.</a> Muscles Ligaments and Tendons Journal. 2 (3), 243-247 (2012).</li><li>Moutsatsou, P., Ochs, J., Schmitt, R. H., Hewitt, C. J., Hanga, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automation+in+cell+and+gene+therapy+manufacturing:+From+past+to+future.">Automation in cell and gene therapy manufacturing: From past to future.</a> Biotechnology Letters. 41 (11), 1245-1253 (2019).</li><li>Iancu, E. M., Kandalaft, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+advantages+of+cell+therapy+manufacturing+under+Good+Manufacturing+Practices+within+the+hospital+setting.">Challenges and advantages of cell therapy manufacturing under Good Manufacturing Practices within the hospital setting.</a> Current Opinion in Biotechnology. 65, 233-241 (2020).</li><li>Li, A., James, D., Lim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Gibco&#8482;+CTS&#8482;+Rotea&#8482;+system+story-A+case+study+of+industry-academia+collaboration.">The Gibco&#8482; CTS&#8482; Rotea&#8482; system story-A case study of industry-academia collaboration.</a> Gene Therapy. , (2021).</li><li>Li, 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=Improving+cell+viability+using+counterflow+centrifugal+elutriation.">Improving cell viability using counterflow centrifugal elutriation.</a> Cytotherapy. 24 (6), 650-658 (2022).</li><li>Li, 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=Automated+counterflow+centrifugal+system+for+small-scale+cell+processing.">Automated counterflow centrifugal system for small-scale cell processing.</a> Journal of Visualized Experiments. (154), e60423 (2019).</li><li>Shah, D., Naciri, M., Clee, P., Al-Rubeai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NucleoCounter-An+efficient+technique+for+the+determination+of+cell+number+and+viability+in+animal+cell+culture+processes.">NucleoCounter-An efficient technique for the determination of cell number and viability in animal cell culture processes.</a> Cytotechnology. 51 (1), 39-44 (2006).</li><li>Chan, A. K. C., Heathman, T. R. J., Coopman, K., Hewitt, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+flow+cytometry+for+the+characterisation+of+extracellular+markers+on+human+mesenchymal+stem+cells.">Multiparameter flow cytometry for the characterisation of extracellular markers on human mesenchymal stem cells.</a> Biotechnology Letters. 36 (4), 731-741 (2014).</li><li>Smith, D., 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=Towards+automated+manufacturing+for+cell+therapies.">Towards automated manufacturing for cell therapies.</a> Current Hematologic Malignancy Reports. 14 (4), 278-285 (2019).</li><li>Stroncek, D. F., Somerville, R. P. T., Highfill, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+cell+therapy+manufacturing;+it's+not+for+everyone.">Point-of-care cell therapy manufacturing; it's not for everyone.</a> Journal of Translational Medicine. 20 (1), 34 (2022).</li></ol>

P.J., A.B., R.L., and J.N. are employees of Thermo Fisher Scientific. A.L. and S.O. have no conflicts of interest.

In this work, we have shown the ability to close and semi-automate hMSC dissociation and wash and harvest on the bench using a counterflow centrifugation instrument. One of the critical steps in the entire workflow is making sure the tubings are connected according to the preset protocol defined in the counterflow centrifugation system protocol builder. The setup and operation are simple, and the time it took to process around 2 L of culture from a 10-layer flask from kit assembly to cell harvest was around 60 min. One of the limiting steps in this workflow is the fluid transfer from the multi-layered flask to the transfer bags connected to the counterflow centrifugation instrument. The high-flow single-use kit can only be run at a maximum flow rate of 165 mL/min, and this might be challenging for processing, for example, a 40-layer flask. To quicken the process of fluid transfer, external high-flow rate pumps can be used to transfer the trypsinized contents into a transfer bag first, followed by washing/concentrating and harvesting the cells from the transfer bag using the counterflow centrifugation system. Moreover, this protocol can also be applied for passaging cells from 4-layer to 10-layer multi-layered flasks. Further upstream, the counterflow centrifugation system could also be optimized for the washing of thawed hMSCs and direct harvest and medium formulation into multi-layered flasks to start the seed train. It should be noted that the minimum number of cells required to form the fluidized bed in the counterflow centrifugation chamber is approximately 30 million cells, and the maximum recommended volume to process per batch is 20 L.
Currently, the attachment of the custom tubing assembly to the multi-layered flask in the biosafety cabinet and the autoclaving of the parts of the components are not desirable in a cGMP setting. As an alternative, a custom gamma-sterilized tubing assembly could be outsourced to suppliers. Suppliers providing multi-layered flasks also offer the option of pre-fitting the flasks with desired tubing assemblies, including a 0.2 &#181;m filter, and gamma-sterilization of the entire outfit. This would ensure that the multi-layered flasks and attached tubing are truly closed, meaning the process could be completed on the bench in a Class C clean room setting.
This process utilizing the counterflow centrifugation system is not limited to adherent-based cultured cells in a multi-layered vessel and could be adapted to dynamic (stirred-tank or wave bioreactors) and static (gas-permeable) suspension-based cell expansion platforms. Specifically, for hMSCs expanded in 3D microcarrier cultures, protocols can be optimized on the counterflow centrifugation system to harvest, wash, and formulate the hMSCs dissociated from microcarriers. 
Overall, increasing interest in developing translational cellular therapies with improved process robustness and reliability has led to the development of closed, automated cell processing platforms. These systems are imperative, as they reduce the number of handling steps, prevent potential contamination by sterile connections, and reduce manufacturing costs by reducing labor and enhancing the effective use of clean room space21. In line with this, many of the cell therapy product developers who are seeking regulatory approval to translate their therapies are aware of the importance of closing the process and implementing full automation or semi-automation as early as the process development stage14,31,32. 
With the use of regulatory-friendly SFM XF medium, and along with ancillary reagents compliant with 21 CFR GMP Part 11 and international quality guidelines, this semi-automated process would be readily suitable for clinical manufacturing. We have shown the reproducibility of the closed process and maintenance of the quality of the WJ-MSCs. Improving the efficiency and safety of culturing adherent-based cells in multi-layered flasks would benefit not only the hMSC therapy field but also companies in cell line banking and adherent virus production.

Human mesenchymal stem cells (hMSCs) are a great candidate for clinical applications, both in tissue engineering and in cell therapies, given their therapeutic potential and high self-renewal potential to grow in vitro, which are critical for generating clinically-relevant dosages of cells1,2,3. According to ClinicalTrials.gov, there are over 1,000 clinical trials currently under investigation for various disease conditions4. Given the backdrop of increasing interest in using hMSCs, more clinical trials and market approvals are imminent in the near future5,6. However, the manufacturing of hMSCs has many inherent challenges in terms of batch-to-batch variability, the use of high-risk raw materials, concerns regarding contamination due to many open and manual processes, as the manufacturing involves multiple unit operations, higher labor costs, the cost of scaling out or scaling up, and regulatory hurdles6,7,8,9,10,11,12. These issues remain a significant barrier to current and future market access.
The development of closed, modular, automated manufacturing solutions and using low-risk ancillary reagents would address these challenges. This would also ensure consistent product quality, decrease the likelihood of batch failures due to human error, reduce labor costs, and improve process standardization and regulatory compliance, such as in terms of digital batch record-keeping8,12,13,14. To be able to obtain a clinically relevant dosage of cells, be it autologous or allogeneic, streamlined manufacturing that involves upstream cell expansion and downstream processing in a closed, automated manner is crucial.
For upstream hMSC expansion, the two most common manufacturing methods currently employed are scale-out (2D monolayer) and scale-up (3D microcarrier-based suspension system)15,16,17,18. The most traditional and widely adopted method for hMSC expansion is 2D monolayer-based culture due to the low production cost and ease of setup19.
Multi-layered flasks composed of flat surface trays stacked within a culture vessel are commonly utilized to scale out hMSC production. These systems typically come in 1-layer to 40-layer culture vessels20 and are handled manually inside biosafety cabinets. The processing steps during cell passaging and harvesting involve manually dispensing and decanting the expansion media, dissociation reagent, and wash buffer by pipetting or physically tilting the entire vessel. Besides, handling multiple units is challenging and time-consuming due to their sheer size and weight.
Subsequently, post-harvesting from multi-layered flasks, centrifugation for media exchange, cell wash, and volume reduction are essential steps across the entire cell manufacturing workflow21. Conventional benchtop centrifugation is a mostly open and manual process that involves a multitude of steps, such as transferring the cell suspension into capped tubes or bottles inside a biosafety cabinet, spinning down the cells, manually aspirating the supernatant, cell resuspension with the buffer, and repeated cell washes. This dramatically increases both the risk of contamination due to the opening and closing of the caps and the chances of losing the cell pellet during the manual aspiration/pipetting process22. In the context of handling multi-layered culture systems for adherent-based cells such as hMSCs, the operator would need to go through a laborious process of shuttling between the centrifuge and biosafety cabinet repeatedly and handling a heavy unit at the same time. These manual steps are laborious, pose risks in terms of human errors and contamination, and have to be conducted in a Class B clean room environment, which is costly23. In addition, the conventional manual centrifugation process is not scalable and could cause cellular shear and stress; thus, maximizing cell recovery, viability, and the wash-out efficiency of residual impurities are other major challenges22. Commercial cGMP scale manufacturing of cell therapies requires closed, modular automation solutions to reduce the risk of contamination, ensure consistent product quality, reduce labor and production costs, and increase process reliability24,25. Multi-layered flasks can be handled as a closed system by having a sterile 0.2 &#181;m filter in one of the ports to facilitate sterile gas exchange and a second port aseptically connected via connectors or tube-welded directly to an automated cell processing instrument for cell harvesting. We worked toward closing and automating most steps of WJ-hMSC passaging and harvesting by evaluating an innovative closed counterflow centrifuge intended for the manufacturing of cell, gene, or tissue-based products. This counterflow centrifuge also has the flexibility to perform a variety of cell processing applications, such as cell separation based on size, medium/buffer exchange, concentration, and harvesting for a variety of cell types8,26,27,28. The instrument uses a closed single-use kit that can be sterile-connected using tube welding or aseptic connectors to transfer bags or can be connected directly to any expansion platform of choice.
In this study, we designed a custom tubing assembly to allow closed sterile connections between the single-use counterflow centrifugation kit and the multi-layered flask. We optimized a protocol to enzymatically detach, wash, and harvest WJ-MSCs from the multi-layered flask in an entirely closed and semi-automated manner within a single run. The harvested WJ-hMSCs were characterized for purity (surface marker analysis) and potency (CFU-F, trilineage differentiation, and cytokine secretion profiles) to ensure that the final product met the critical quality attributes (CQAs) for lot release.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2L PVC transfer bag</td>
			<td>TerumoBCT</td>
			<td>BB*B200TM</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian blue solution, pH 2.5</td>
			<td>Merck</td>
			<td>101647</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin-Red Staining Solution</td>
			<td>Merck</td>
			<td>TMS-008-C</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-human CD73 Antibody</td>
			<td>Biolegend</td>
			<td>344015</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse IgG1, &#954; Isotype Ctrl (FC) Antibody</td>
			<td>Biolegend</td>
			<td>400121</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Plex MAGPIX Multiplex Reader</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counterflow Centrifugation System</td>
			<td>Thermo Fisher Scientific</td>
			<td>A47679</td>
			<td>Gibco CTS Rotea Counterflow Centrifugation System</td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Sigma-aldrich</td>
			<td>C0775</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS (L-alanyl-L-glutamine) GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1286001</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1285601</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>CTS Recombinant Human Vitronectin (VTN-N)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A27940</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS TrypLE Select Enzyme</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1285901</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom tubing assembly</td>
			<td>Saint-Gobain and Colder Product Company (CPC)</td>
			<td>N/A</td>
			<td>Gamma-sterilized 3/32&#8221; ID PVC line fitted with a sterile male MPC (1/8&#8221; barb) and sealed on the other end. Autoclave a short C-Flex line fitted with a sterile Cell Factory port connector on one end and a female MPC (3/8&#8221; barb) on the other. Connect the PVC and C-Flex lines in a biosafety cabinet</td>
		</tr>
		<tr>
			<td>Emflon II capsule (0.2um filter)</td>
			<td>Pall</td>
			<td>KM5V002P2G100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12662029</td>
			<td>Mesenchymal stem cell-qualified, USDA-approved regions</td>
		</tr>
		<tr>
			<td>FGF-basic</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0024</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD105 Antibody</td>
			<td>Biolegend</td>
			<td>323203</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD45 Antibody</td>
			<td>Biolegend</td>
			<td>304005</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD90 (Thy1) Antibody</td>
			<td>Biolegend</td>
			<td>328107</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1, &#954; Isotype Ctrl (FC) Antibody</td>
			<td>Biolegend</td>
			<td>400109</td>
			<td></td>
		</tr>
		<tr>
			<td>Hi-Flow Single Use Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A46575</td>
			<td>Gibco CTS Rotea Hi-flow single-use kit, flow rate of 30 &#8211; 165 mL/min</td>
		</tr>
		<tr>
			<td>Multi-layered systems&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>140360 (4-layers); 140410 (10-layers)</td>
			<td>Nunc Standard Cell Factory Systems</td>
		</tr>
		<tr>
			<td>NucleoCounter NC-3000</td>
			<td>Chemometec</td>
			<td>NC-3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil red O staining solution</td>
			<td>Merck</td>
			<td>102419</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP anti-human CD34 Antibody</td>
			<td>Biolegend</td>
			<td>343519</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP Mouse IgG1, &#954; Isotype Ctrl Antibody</td>
			<td>Biolegend</td>
			<td>400147</td>
			<td></td>
		</tr>
		<tr>
			<td>ProcartaPlex Multiplex Immunoassays</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Custom 19-Plex panel: FGF-2, HGF, IDO, IL-10, IL-1RA, IL-6, IL-8, IP-10, MCP-1, MCP-2 , MIP-1&#945;, MIP-1&#946;, MIP-3&#945;, PDGF-BB, RANTES, SDF-1&#945;, TGF&#945;, TNF-alpha, VEGF-A&#160;</td>
		</tr>
		<tr>
			<td>Sample port</td>
			<td>Thermo Fisher Scientific</td>
			<td>A50111</td>
			<td>Gamma-sterilized leur sample port with 2 PVC lines attached</td>
		</tr>
		<tr>
			<td>StemPro Adipogenesis Differentiation Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10070-01</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Chondrocyte Differentiation</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10071-01</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Custom MSC SF XF Medium Kit (SFM XF medium)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ME20236L1</td>
			<td>Contains StemPro MSC SFM Basal Medium and Custom MSC SF XF Supplement (100x)</td>
		</tr>
		<tr>
			<td>StemPro Osteogenesis Differentiation Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10072-01</td>
			<td></td>
		</tr>
		<tr>
			<td>T175 Nunc EasYFlask</td>
			<td>Thermo Fisher Scientific</td>
			<td>159910</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Nunc EasYFlask</td>
			<td>Thermo Fisher Scientific</td>
			<td>156472</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;1</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG9204</td>
			<td></td>
		</tr>
		<tr>
			<td>WJ MSCs&#160;</td>
			<td>PromoCell</td>
			<td>(#C12971; Germany)</td>
			<td>Human mesenchymal stem cells</td>
		</tr>
		<tr>
			<td>&#945;MEM media</td>
			<td>Thermo Fisher Scientific</td>
			<td>12571063</td>
			<td>With nucleosides</td>
		</tr>
	</tbody>
</table>

human mesenchymal stem cell processing for clinical applications using a closed semi-automated workflow

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market approvals for clinical use expected over the next few years. To facilitate this transition, addressing the bottlenecks of scale, lot-to-lot reproducibility, cost, regulatory compliance, and quality control is critical. These challenges can be addressed by closing the process and adopting automated manufacturing platforms. In this study, we developed a closed and semi-automated process for passaging and harvesting Wharton's jelly (WJ)-derived hMSCs (WJ-hMSCs) from multi-layered flasks using counterflow centrifugation. The WJ-hMSCs were expanded using regulatory compliant serum-free xeno-free (SFM XF) medium, and they showed comparable cell proliferation (population doubling) and morphology to WJ-hMSCs expanded in classic serum-containing media. Our closed semi-automated harvesting protocol demonstrated high cell recovery (~98%) and viability (~99%). The cells washed and concentrated using counterflow centrifugation maintained WJ-hMSC surface marker expression, colony-forming units (CFU-F), trilineage differentiation potential, and cytokine secretion profiles. The semi-automated cell harvesting protocol developed in the study can be easily applied for the small- to medium-scale processing of various adherent and suspension cells by directly connecting to different cell expansion platforms to perform volume reduction, washing, and harvesting with a low output volume.

The WJ-hMSC master cell bank (MCB) post-thawing was maintained for three successive passages (p1-p4) in classic serum-containing medium to produce enough working cell banks (WCBs) for the experiments. The p4 WCBs were thawed and expanded in both serum-containing medium and SFM XF medium for three more passages (p4-p7) in T-175 flasks. The WJ-MSCs adapted well when expanded in SFM XF medium and were able to maintain stable proliferation similar to that in serum-containing medium (Figure 2A). However, the cells expanded in SFM XF medium exhibited slightly longer fibroblast-like spindle-shaped morphology, resulting in a slightly larger cell size (Figure 2B) of an average of ~17 &#181;m compared to ~15 &#181;m in serum-containing medium. In both medium conditions across the three passages, the WJ-hMSCs consistently reached their maximum cell density of ~2.3 x 104 cells/cm2 and a population doubling time of ~34 h (Figure 2C,D).
For large-scale WJ-hMSC expansion in a closed system, we carried out a seed train expansion of WJ-hMSCs first in a 4-layer flask and, subsequently, in a 10-layer multi-layered flask. At around 80%-90% confluency after 4 days of culture, we harvested 9.6 x 107 &#177; 0.9 x 107 and 2.3 x 108 &#177; 0.2 x 108 cells for the 4-layer and 10-layer stacks, respectively. A higher cell density of 3.6 x 104-3.8 x 104 cells/cm2 was reached compared to in the T-175 flasks, meaning the stacks allowed better cell expansion by up to seven-fold.
Further, the WJ-hMSCs expanded in 10-layer culture vessels were harvested directly using counterflow centrifugation. The sterile connection to the single-use kit was easy to establish for direct fluid transfer at a maximum flow rate of 165 mL/min using the instrument&#39;s peristaltic pump. The semi-automated cell harvesting process was achieved by first harvesting the cells using enzymatic dissociation, loading the cells into the counterflow chamber for volume reduction and concentrating, and then washing with wash buffer, which was around 3x the volume of the counterflow centrifugation chamber. Further, the washed cells were then concentrated and harvested to the desired harvest volume preset in the protocol. The processing steps used for the semi-automated cell processing were designed to emulate the manual harvesting workflow. We achieved a 10-fold volume reduction, resulting in the generation of cell concentrations as high as 5.3 million cells/mL. The protocol was able to achieve high cell recovery at ~98% and high cell viability at ~99% consistently for all three independent runs (Figure 3A-C).
We carried out extensive cell characterization assays to determine the critical quality attributes of the cell harvest using counterflow centrifugation compared to manual centrifugation. To test the identity of the WJ-hMSCs, cell surface markers were analyzed by flow cytometry. As shown in Figure 4A, the WJ-hMSCs harvested using both methods displayed characteristic surface marker profiles according to the ISCT regulations, positive expression of CD73, CD90, and CD105, as well as negative expression of CD34 and CD45. Next, to evaluate the clonogenic potential of the WJ-hMSCs, CFU-F assays were carried out. As shown in Figure 4B, cells harvested from counterflow centrifugation displayed a similar CFU-F potential compared with cells harvested by manual centrifugation (21% &#177; 1% vs. 20% &#177; 1%, respectively). Furthermore, as shown in Figure 4C, the post-counterflow centrifugation-harvested cells retained the ability to differentiate into adipocytes, osteoblasts, and chondrocytes similar to the cells in the manual centrifugation method. Lastly, we investigated 18 different cytokine secretion profiles of the cells using multiplex immunoassays. As shown in Figure 4D, the cells washed and concentrated using the post-counterflow centrifugation maintained cytokine secretion profiles, and the profiles were comparable to those of the sample taken before washing/concentrating the cells (pre-counterflow centrifugation).
Overall, we have demonstrated efficient hMSC expansion in an SFM XF culture system, and the cells washed and concentrated using the closed, automated counterflow centrifugation system yielded high cell recovery and viability post-wash and could maintain their phenotype and functionality. The closed semi-automated process developed in this study can deliver product quality consistency in terms of final WJ-MSC recovery, as evidenced from three independent runs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64707/64707fig01.jpg" /> 
Figure 1:&#160;High-flow single-use kit configuration and assembly for the harvest, washing, and concentration of hMSCs. (A) Kit diagram after the bags have been connected in line with the respective tubing. (B) The 10-layer multi-layered flask connected to the high-flow single-use kit with the custom tubing assembly. (C) Visualization of the stable fluidized cell bed formed in the counterflow chamber via the camera function enabled in the graphical user interface of the counterflow centrifugation software. <a href="https://www.jove.com/files/ftp_upload/64707/64707fig01large.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/64707/64707fig02.jpg" /> 
Figure 2:&#160;Comparison of the cell morphology and expansion of hMSCs in serum-containing medium and SFM XF medium. (A) The representative cell morphology of hMSCs in classic serum medium and SFM XF medium. The SFM XF-expanded cells displayed a longer, spindle-shaped, characteristic fibroblast-like morphology, whereas the cells grown in serum-containing medium displayed a more flattened morphology. (B) The average MSC size between the serum-containing medium and SFM XF medium, measured by an automated cell counter (n = 3). It is clear to see that the SFM XF-expanded cells were generally larger than serum-expanded cells across different passages. The total cell yield in different passages (n= 3) (C) in terms of cells per culture surface area and (D) population doubling levels. Similar levels of cell yield were obtained between the SFM XF medium and serum-containing medium across different passages. Data are expressed as the mean &#177; standard deviation. <a href="https://www.jove.com/files/ftp_upload/64707/64707fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64707/64707fig03.jpg" /> 
Figure 3: Characterization of cells processed using the counterflow centrifugation system. (A) Total viable cells before and after the washing and concentration. (B) A 10x volume reduction was achieved post-counterflow centrifugation processing. (C) Total recovery and viability of the cells. Data are averaged over three biological replicates (n = 3) of washing and concentration runs. Data are expressed as mean &#177; standard deviation. <a href="https://www.jove.com/files/ftp_upload/64707/64707fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64707/64707fig04.jpg" /> 
Figure 4: Critical quality attributes analysis. (A) Representative data from flow cytometry. (B) Representative images showing the total CFU. (C) Representative microscopic images of the trilineage differentiation. (D) Cytokine expression analysis results before and after processing the cells on the counterflow centrifugation system (n = 3). Data are expressed as mean &#177; standard deviation. <a href="https://www.jove.com/files/ftp_upload/64707/64707fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Sequence of the hMSC harvest by trypsinization, washing, and concentration protocol on the counterflow centrifugation system, including the initial priming steps. <a href="https://www.jove.com/files/ftp_upload/64707/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow at JoVE.com

Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow

Here, we present a protocol to harvest adherent cells from multi-layered flasks in a closed semi-automated manner using a counterflow centrifugation system. This protocol can be applied for harvesting both adherent and suspension cells from other cell expansion platforms with few modifications to the existing steps.

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market ...

human-mesenchymal-stem-cell-processing-for-clinical-applications

Research

JoVE Journal

Biochemistry

1.6K Views.  Agency for Science, Technology and Research (A STAR). Here, we present a protocol to harvest adherent cells from multi-layered flasks in a closed semi-automated manner using a counterflow centrifugation system. This protocol can be applied for harvesting both adherent and suspension cells from other cell expansion platforms with few modifications to the existing steps.

Video: Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

Methods Article

Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow