Name: human mesenchymal stem cell processing for clinical applications using a closed semi-automated workflow
Uploaded: 2023-03-17T14:40:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow at JoVE.com

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>
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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. 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(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. 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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 ...

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