Name: automated counterflow centrifugal system for small-scale cell processing
Uploaded: 2019-12-12T12:00:00.000+00:00
Duration: 4 min 49 s
Description: Watch this Scientific Journal Video about Automated Counterflow Centrifugal System for Small-Scale Cell Processing at JoVE.com

This work is supported by the Victorian Government&#39;s Operational Infrastructure Support Program, and the Victorian Government Technology Voucher provided by the Department of Economic Development, Jobs, Transport and Resources. RL is the recipient of a National Health and Medical Research Council Career Development Fellowship. AL is the recipient of an Australian Postgraduate Award.

1. Preparation of reagents and cells for buffer exchange
<ol>
	<li>Prepare buffers (see Table of Materials) in a class 2 laminar flow hood. Using a syringe and needle assembly, remove 50 mL of saline solution from a 500 mL saline bag. Replace this with 50 mL of 20% human serum albumin (HSA) to make 2% HSA in saline, which will serve as the wash buffer.</li>
	<li>Remove the cells from the culture vessels and perform a cell count to determine the starting cell quantity and viability by trypan blue exclusion. Load the cells into a transfer bag. 
	NOTE: In this protocol Jurkat cells were cultured in RPMI and MSCs were cultured in DMEM:F12. Both media were supplemented with 10% fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic solution. Cells were cultured at 37 &#176;C with 5% CO2. For MSCs or adherent cells, add a digestion enzyme (e.g., trypsin) into the culture vessels in order to detach the cells, and quench with culture media once the cells are detached. A transfer bag was used in this protocol because the cells were expanded in culture vessels. However, if the cells are cultured in a cell culture bag, it can be directly connected to the single-use kit via sterile tube welding. In this protocol, either 3 x 107 Jurkat cells or 1 x 107 MSCs were used for each run, where cells were suspended in 40 mL of culture media.
	<ol>
		<li>Load cells into a transfer bag (see Table of Materials) using a syringe and needle assembly if a sterile tube welder is available to connect the transfer bag to the single use processing kit.</li>
		<li>Alternatively, use a pair of autoclaved scissors to cut the connecting tube with around 10 cm remainining attached to the transfer bag and add a female Luer connector to the end of the tubing. Use a syringe to aspirate cells and media, then connect the syringe to the Luer connector and load cells to the transfer bag.</li>
	</ol>
	</li>
	<li>Connect the bag containing the cells, wash buffer bag containing 500 mL of wash buffer prepared in step 1.1, waste bag, and collecting syringe to the single-use kit (Figure 1B). 
	NOTE: The connecting position of each bag depends on the settings in the program. In this protocol, we connected the waste bag at position A, wash buffer at position B, cell bag at positions D and F, and collection syringe at position H (Figure 1C).</li>
</ol>
2. Program for automated buffer exchange protocol
<ol>
	<li>Open the Graphic User Interface (GUI) of the device and press the &#34;START&#34; button on a laptop PC or tablet. Then &#34;Open&#34; an existing protocol or press &#34;New&#34; to create a new one.</li>
	<li>Use the &#34;Forward&#34; and &#34;Backward&#34; button to navigate through each step, select the valves to be opened/closed, centrifugal speed, pump speed, pump direction, and action triggers. The settings for each step are summarized in Table 1. Then save the protocol.</li>
</ol>
3. Setting up the machine
<ol>
	<li>Place the single-use processing kit on the machine and hang the connected bags accordingly. Press the red &#34;Stop/Reset&#34; button to reset all valves into the default closed position. 
	NOTE: The machine will perform a test when the door is closed to ensure the kit has been placed correctly and that all the valves are functioning appropriately.</li>
	<li>Connect the GUI to the processing device and press the &#34;Connect&#34; button. Then download the saved program. When the green &#34;Play&#34; button lights up, it indicates that the protocol is ready to start.</li>
	<li>Open the manual clamps for the transfer bag tubing. 
	NOTE: If the operator forgets to open the clamps, the device will stop and the pressure sensor warning will appear. Press the &#34;Stop&#34; button to reset the device and open the clamps to start again.</li>
</ol>
4. Automated buffer exchange
<ol>
	<li>Press &#34;Start&#34; to initiate the buffer exchange program. 
	NOTE: To manually alter the automated process, press the &#34;Next&#34; button to move to the next step before reaching the &#34;Trigger&#34; or press &#34;Pause&#34; to put the process on hold. This will recirculate the cells through the kit while keeping only valves J and K open (Figure 1C).</li>
	<li>At the end of automation step 6 (Table 1), the process will pause when air in the now emptied bag triggers the bubble sensor. Press &#34;Next&#34; to move the process to the next step if the bag is indeed empty, or press &#34;Pause&#34; to resume the processing if the sensor was triggered by a bubble in the line.</li>
</ol>
5. Collecting and sampling the cells
<ol>
	<li>When the automated process is finished, close all tubing clamps. Open the door and take the single-use kit out of the device. Disconnect the syringe to collect the cells for subsequent processes. Take a sample of the collected cells for a cell count and viability check (see step 6.2).</li>
</ol>
6. Process validation
<ol>
	<li>Perform control experiments using a manual buffer exchange protocol.
	<ol>
		<li>Centrifuge cell suspension at 200 x g for 5 min. Discard the supernatant and resuspend the cells with 30 mL of cell wash buffer. Centrifuge the cell suspension again at 200 x g for 5 min. Discard the supernatant and resuspend the cells with 10 mL of cell wash buffer.</li>
	</ol>
	</li>
	<li>Perform cell counting via the trypan blue exclusion assay to assess cell viability using an automated cell counter.</li>
	<li>Perform an MTS assay to quantify cell proliferation at 2, 24, and 48 h. 
	NOTE: The MTS assay was performed on a 96 well tissue culture plate according to the manufacturer&#39;s instructions. A 100 &#181;L aliquot of cell culture media (containing 10,000 Jurkat cells or 1,500) per well were plated after the buffer exchange process. At each time point, 20 &#181;L of MTS reagents were added into each well and incubated for 2 h at 37 &#176;C. The absorbance was assessed at 490 nm using a plate reader.</li>
	<li>Perform functional assays to compare the impact of the automated process versus the manual process on the Jurkat cells and MSC function.
	<ol>
		<li>Culture Jurkat cells in a 96-well plate at a density of 1 x 106 cells/mL stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 &#181;g/mL ionomycin (cell stimulation cocktail) in DMEM:F12 media containing 10% fetal bovine serum (FBS) for 24 h. Incubate the cells at 37 &#176;C with 5% CO2.</li>
		<li>Measure interleukin-2 production from culture supernatant using bead-based ELISA assay (see Table of Materials) according to the manufacturer&#39;s instructions.</li>
		<li>Culture MSCs in a 12 well plate at the density of 10,000 cells/cm2 in 1 mL of DMEM:F12 media containing 10% FBS supplemented with interferon-&#947; 50 units/mL for 48 h. Collect supernatant for the kynurenine concentration assay to measure indoleamine 2,3-dioxygenase (IDO) enzyme activity of MSCs as previously described21.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Lopes, A. G., Sinclair, A., Frohlich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost+Analysis+of+Cell+Therapy+Manufacture:+Autologous+Cell+Therapies,+Part+1.">Cost Analysis of Cell Therapy Manufacture: Autologous Cell Therapies, Part 1.</a> BioProcess International. 16 (3), (2018).</li><li>Hampson, B., Ceccarelli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factories+of+the+future:+Can+Patient-Specific+Cell+Therapies+Get+There+from+Here?.">Factories of the future: Can Patient-Specific Cell Therapies Get There from Here?.</a> BioProcess International. 14 (4), (2016).</li><li>Preti, R., Daus, A., Hampson, B., Sumen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+success+for+commercial+cell+therapy+manufacturing.">Mapping success for commercial cell therapy manufacturing.</a> BioProcess International. 13 (9), 33-38 (2015).</li><li>Heathman, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translation+of+cell-based+therapies:+clinical+landscape+and+manufacturing+challenges.">The translation of cell-based therapies: clinical landscape and manufacturing challenges.</a> Regenerative Medicine. 10 (1), 49-64 (2015).</li><li>Lipsitz, Y. Y., 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+roadmap+for+cost-of-goods+planning+to+guide+economic+production+of+cell+therapy+products.">A roadmap for cost-of-goods planning to guide economic production of cell therapy products.</a> Cytotherapy. 19 (12), 1383-1391 (2017).</li><li>Lopes, A. G., Sinclair, A., Frohlich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost+Analysis+of+Cell+Therapy+Manufacture:+Autologous+Cell+Therapies,+Part+2.">Cost Analysis of Cell Therapy Manufacture: Autologous Cell Therapies, Part 2.</a> BioProcess International. 16 (4), 12-19 (2018).</li><li>Hanley, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+manufacturing+of+therapeutic+mesenchymal+stromal+cells+with+the+use+of+the+Quantum+Cell+Expansion+System.">Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System.</a> Cytotherapy. 16 (8), 1048-1058 (2014).</li><li>Leong, W., Nakervis, B., Beltzer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automation:+what+will+the+cell+therapy+laboratory+of+the+future+look+like?.">Automation: what will the cell therapy laboratory of the future look like?.</a> Cell Gene Therapy Insights. 4 (9), 679-694 (2018).</li><li>Galipeau, J., Sensebe, L. <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:+Clinical+Challenges+and+Therapeutic+Opportunities.">Mesenchymal Stromal Cells: Clinical Challenges and Therapeutic Opportunities.</a> Cell Stem Cell. 22 (6), 824-833 (2018).</li><li>Salmikangas, P., Kinsella, N., Chamberlain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Antigen+Receptor+T-Cells+(CAR+T-Cells)+for+Cancer+Immunotherapy+-+Moving+Target+for+Industry?.">Chimeric Antigen Receptor T-Cells (CAR T-Cells) for Cancer Immunotherapy - Moving Target for Industry?.</a> Pharmaceutical Research. 35 (8), 152 (2018).</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 Gene Therapy Insights. 3 (4), 271-284 (2017).</li><li>Rafiq, Q. A., Thomas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+role+of+automation+in+process+development,+manufacture+of+cell,+gene-based+therapies.">The evolving role of automation in process development, manufacture of cell, gene-based therapies.</a> Cell Gene Therapy Insights. 2 (4), 473-479 (2016).</li><li>Rafiq, Q. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+Automated+Approaches+for+Cell+and+Gene+Therapy+Manufacture.">Emerging Automated Approaches for Cell and Gene Therapy Manufacture.</a> Cell Gene Therapy Insights. 4 (9), 911-914 (2018).</li><li>Contreras, T. J., Jemionek, J. F., French, J. E., Shields, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Granulocyte+Isolation+by+Continuous+Flow+Centrifugation+Leukapheresis+and+Counterflow+Centrifugation+Elutriation+(CFCL/CCE).">Human Granulocyte Isolation by Continuous Flow Centrifugation Leukapheresis and Counterflow Centrifugation Elutriation (CFCL/CCE).</a> Transfusion. 19 (6), 695-703 (1979).</li><li>Berger, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+elutriation+of+monocytes+within+a+closed+system+(Elutra&#8482;)+for+clinical-scale+generation+of+dendritic+cells.">Efficient elutriation of monocytes within a closed system (Elutra&#8482;) for clinical-scale generation of dendritic cells.</a> Journal of Immunological Methods. 298 (1), 61-72 (2005).</li><li>Chen, Y., Hoecker, P., Zeng, J., Dettke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+Cobe+AutoPBSC+and+Gambro+Elutra+as+a+platform+for+monocyte+enrichment+in+dendritic+cell+(DC)+therapy:+Clinical+study.">Combination of Cobe AutoPBSC and Gambro Elutra as a platform for monocyte enrichment in dendritic cell (DC) therapy: Clinical study.</a> Journal of Clinical Apheresis. 23 (5), 157-162 (2008).</li><li>Whitford, W. G., Subramanian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Continuous Processing in Pharmaceutical Manufacturing. , (2014).</li><li>. SMALL BATCH CELL SEPARATION, WASH &amp; CONCENTRATION Available from: <a target="_blank" href="https://www.scinogy.com/projects">https://www.scinogy.com/projects</a> (2019)</li><li>Yu, 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=Targeting+Jurkat+T+Lymphocyte+Leukemia+Cells+by+an+Engineered+Interferon-Alpha+Hybrid+Molecule.">Targeting Jurkat T Lymphocyte Leukemia Cells by an Engineered Interferon-Alpha Hybrid Molecule.</a> Cellular Physiology and Biochemistry. 42 (2), 519-529 (2017).</li><li>Moharram, S. A., Shah, K., Kazi, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+Acute+Lymphoblastic+Leukemia+Cells+Display+Activation+of+Different+Survival+Pathways.">T cell Acute Lymphoblastic Leukemia Cells Display Activation of Different Survival Pathways.</a> Journal of Cancer. 8 (19), 4124 (2017).</li><li>Ling, W., 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=Mesenchymal+stem+cells+use+IDO+to+regulate+immunity+in+tumor+microenvironment.">Mesenchymal stem cells use IDO to regulate immunity in tumor microenvironment.</a> Cancer Research. 74 (5), 1576-1587 (2014).</li><li>Tanzeglock, T., Soos, M., Stephanopoulos, G., Morbidelli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammalian+cell+death+by+simple+shear+and+extensional+flows.">Induction of mammalian cell death by simple shear and extensional flows.</a> Biotechnology and Bioengineering. 104 (2), 360-370 (2009).</li><li>Aguado, B. A., Mulyasasmita, W., Su, J., Lampe, K. J., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+viability+of+stem+cells+during+syringe+needle+flow+through+the+design+of+hydrogel+cell+carriers.">Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers.</a> Tissue engineering. Part A. 18 (7-8), 806-815 (2012).</li><li>Zhu, F., 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=Hydroxyethyl+starch+as+a+substitute+for+dextran+40+for+thawing+peripheral+blood+progenitor+cell+products.">Hydroxyethyl starch as a substitute for dextran 40 for thawing peripheral blood progenitor cell products.</a> Cytotherapy. 17 (12), 1813-1819 (2015).</li><li>Schwandt, S., Korschgen, L., Peters, S., Kogler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cord+blood+collection+and+processing+with+hydroxyethyl+starch+or+non-hydroxyethyl+starch.">Cord blood collection and processing with hydroxyethyl starch or non-hydroxyethyl starch.</a> Cytotherapy. 18 (5), 642-652 (2016).</li><li>Stroncek, D. F., 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=Counter-flow+elutriation+of+clinical+peripheral+blood+mononuclear+cell+concentrates+for+the+production+of+dendritic+and+T+cell+therapies.">Counter-flow elutriation of clinical peripheral blood mononuclear cell concentrates for the production of dendritic and T cell therapies.</a> Journal of Translational Medicine. 12, 241 (2014).</li><li>Mfarrej, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-clinical+assessment+of+the+Lovo+device+for+dimethyl+sulfoxide+removal+and+cell+concentration+in+thawed+hematopoietic+progenitor+cell+grafts.">Pre-clinical assessment of the Lovo device for dimethyl sulfoxide removal and cell concentration in thawed hematopoietic progenitor cell grafts.</a> Cytotherapy. 19 (12), 1501-1508 (2017).</li><li>Abonnenc, M., Pesse, B., Tissot, J. D., Barelli, S., Lion, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+washing+of+thawed+haematopoietic+progenitor+cell+grafts:+a+preclinical+evaluation.">Automatic washing of thawed haematopoietic progenitor cell grafts: a preclinical evaluation.</a> Vox Sanguinis. 112 (4), 367-378 (2017).</li><li>Panes, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+allogeneic+adipose-derived+mesenchymal+stem+cells+(Cx601)+for+complex+perianal+fistulas+in+Crohn's+disease:+a+phase+3+randomised,+double-blind+controlled+trial.">Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomised, double-blind controlled trial.</a> Lancet. 388 (10051), 1281-1290 (2016).</li><li>Lim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-In-Human+Administration+of+Allogeneic+Amnion+Cells+in+Premature+Infants+With+Bronchopulmonary+Dysplasia:+A+Safety+Study.">First-In-Human Administration of Allogeneic Amnion Cells in Premature Infants With Bronchopulmonary Dysplasia: A Safety Study.</a> Stem Cells Translational Medicine. 7 (9), 628-635 (2018).</li></ol>

SW, IF, and DJ are COO, CTO, and CEO of Scinogy Pty. Ltd. The access to the CFC device was also provided by Scinogy.

The automated buffer exchange protocol described is simple and user friendly. Nevertheless, there are a few key steps in this protocol that are critical and require particular attention. In our experience, when processing larger cells such as MSCs (average diameter 10&#8211;15 &#181;m) each run should include at least 1 x 107 cells to achieve optimal cell recovery (Figure 4B). Processing smaller cells, such as Jurkat cells (average ~10 &#181;m diameter), requires ...

The landscape of modern medicine has transformed rapidly through recent developments in gene and cell-based therapies (GCT). As one of the fastest growing fields in translational research, the GCT sector also faces unique and unprecedented challenges. In addition to robust clinical outcomes, efficient and cost-effective manufacturing processes are essential for the commercial success of GCT, which is particularly difficult to achieve in small-scale manufacturing1. The cost of time, labor, and quality assurances are magnified when each batch of cells only produces a few doses for one patient instead of hundreds or thousands. Unlike allogeneic cell therapies in which the manufacturing processes are more akin to the production of antibodies and recombinant proteins, autologous cell therapies are typically produced as small-scale operations1. As a relatively new phenomenon in biopharmaceutical manufacturing2, options for small-scale cell processing are currently quite limited.
Buffer exchange is essential to cell manufacturing. It is one of the downstream processes where cells are removed from culture media and concentrated for cryopreservation or infusion. Currently, small-scale cell manufacturing often applies processes similar to those in the academic research setting and relies on specialized clean rooms to maintain sterility3. Manual downstream processes often use benchtop centrifuges to pellet and resuspend cells for volume reduction and buffer exchange. These open processes are costly (i.e., labor and clean room maintenance) and have limited manufacturing capacity, which are not ideal for commercial production2,3.
Implementing automation has been proposed as a solution to improve manufacturing efficiency and achieving commercial scale productions2. Sterility cannot be achieved in cell-based products through traditional methods used for biologics, such as gamma irradiation or terminal end filtration. Instead, an automated closed system is deployed to reduce risks of contamination and operators relying on clean rooms to maintain sterility4. Process automation also addresses the issue of scalability by either having multiple systems running in parallel (scale-out) or increasing the processing capacity of an individual device (scale-up), which in turn minimizes the variability between operators. Furthermore, cost modelling analysis of autologous therapies suggests that automation may reduce the cost of manufacturing5,6. However, no cost benefit was found in an autologous stem cell clinical trial where an automated manufacturing platform was used7, suggesting that the cost benefit of automation may depend on the individual manufacturing process.
There are different strategies in which automation can be introduced into an existing manufacturing process. This can be achieved either by implementing a fully integrated platform or a modular-based processing chain. There are several fully integrated platforms commercially available for autologous cell manufacturing, such as CliniMACS Prodigy (Miltenyi Biotec), Cocoon (Octane Biotech), and Quantum (Terumo BCT). These integrated platforms, which are often described as &#34;GMP-in-a-box&#34;, have low demands on infrastructure and are easy to operate. However, the manufacturing capacity of a fully integrated setup may be restricted by the incubator attached to the system. For example, the culturing capacity of Prodigy is limited to its 400 mL chamber8 and the Quantum cartridge has a limiting surface area set to 2.1 m2 (equivalent to 120 T175 flasks)7, which may not be sufficient for patients requiring higher cell doses9,10. Additionally, Prodigy and Quantum have a common attribute that limits their use: the operational unit is occupied by a single batch of cells throughout the cell expansion period, thus limiting the number of batches that can be manufactured by each unit11. The modular approach to automation is to create a manufacturing chain with multiple modular units that simulates the commercial manufacturing process12,13. This approach, which separates the culture device from the cell washing device, can thereby maximize manufacturing efficiency. An ideal processing device would be one that is adaptable and scalable to manufacturing needs12.
Counterflow centrifugation (CFC) technology, which dates back to the 1970s, has had a long history in cell processing14. It achieves cell concentration and separation by balancing centrifugal force with a counterflow force. Typically, a cell suspension enters from the narrow end of a cell chamber under a constant flow rate while subjected to a centrifugal force (Figure 1A). The flow of the fluid is exerted in the opposite direction to the centrifugal force. This is referred to as the counterflow force, which forms a gradient within the cell chamber. The counterflow force then decreases as the cell chamber widens away from the tip of the cone-shaped cell chamber. Cells with higher density and larger diameter have a higher sedimentation rate, and thus they reach force equilibrium towards the tip of the cone-shaped cell chamber. Smaller particles may reach equilibrium towards the base of the chamber or be too small to be retained in the chamber and will be washed away. The CFC technology is mostly known for its application in processing blood apheresis products, such as isolating monocytes for dendritic cell therapies15,16. In terms of buffer exchange, the CFC technology has only been applied in large-scale manufacturing17 and has yet to be used for the smaller scale manufacturing of autologous cell therapies.
To address the need of a suitable device for small-scale cell manufacturing, an automated CFC device (See Table of Materials), was recently developed18. The automated cell processing device uses counterflow centrifugation technology to remove cell debris and facilitate buffer exchange. The device performs buffer exchange with a single-use kit that can be sterile-connected to a cell transfer bag, which allows the cells to be processed within a sterile, enclosed system. Here, we investigate the use of a counterflow centrifugal device to perform buffer exchange in mammalian cell cultures in automated protocols. In this study, we tested the buffer exchange protocol using Jurkat cells and mesenchymal stromal cells (MSCs) to model nonadherent and adherent cell types, respectively. Jurkat cells are immortalized T cells often used for the study of acute T cell leukemia19,20. MSCs are adult stem cells that have been studied in human clinical trials for a wide range of diseases9.

<table><tbody><tr><td>20 ml Luer lock syringes</td><td>BD</td><td>302830</td><td></td></tr><tr><td>20% Human serum albumin (HSA)</td><td>CSL Behring</td><td>AUST R 46283</td><td></td></tr><tr><td>4-(Dimethylamino)benzaldehyde</td><td>Sigma-Aldrich</td><td>156477-25g</td><td></td></tr><tr><td>500ml IV saline bag</td><td>Fresenius Kabi</td><td>K690521</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Thermo Fisher Scientific</td><td>15240112</td><td></td></tr><tr><td>Automated cell counter (Countess)</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>Cell counting chamber slides</td><td>Thermo Fisher Scientific</td><td>C10228</td><td></td></tr><tr><td>Cell stimulation cocktail (500x)</td><td>Thermo Fisher Scientific</td><td>00-4970-93</td><td></td></tr><tr><td>Cell transfer bags</td><td>Terumo</td><td>T1BBT060CBB</td><td></td></tr><tr><td>CellTiter AQueous One Solution Cell Proliferation Assay (MTS)</td><td>Promega</td><td>G3582</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>DMEM: F12 media</td><td>Thermo Fisher Scientific</td><td>11320082</td><td></td></tr><tr><td>EnVision plate Reader</td><td>Perkin Elmer</td><td>N/A</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific</td><td>10099141</td><td></td></tr><tr><td>Human Interleukin 2 (IL2) Kit</td><td>Perkin Elmer</td><td>Al221C</td><td></td></tr><tr><td>Luer (female) fittings</td><td>CPC</td><td>LF41</td><td></td></tr><tr><td>PC laptop or PC tablet device</td><td>ASUS</td><td>N/A</td><td></td></tr><tr><td>Plate reader (SpectraMax i3)</td><td>Molecular Device</td><td>N/A</td><td></td></tr><tr><td>Recombinant Human IFN-&amp;gamma;</td><td>PeproTech</td><td>300-02</td><td></td></tr><tr><td>Rotea counterflow centrifuge cell processing device</td><td>Scinogy</td><td>N/A</td><td></td></tr><tr><td>Rotea single-use processing kit</td><td>Scinogy</td><td>N/A</td><td></td></tr><tr><td>RPMI media</td><td>Thermo Fisher Scientific</td><td>11875119</td><td></td></tr><tr><td>Surgical scissors</td><td>ProSciTech</td><td>420SS</td><td></td></tr><tr><td>Trichloroacetic acide</td><td>Sigma-Aldrich</td><td>T6399-250g</td><td></td></tr><tr><td>Trypan Blue stain</td><td>Thermo Fisher Scientific</td><td>T10282</td><td></td></tr><tr><td>Trypsin digestion enzyme (TrypLE Express Enzyme)</td><td>Thermo Fisher Scientific</td><td>12604013</td><td></td></tr></tbody></table>

automated counterflow centrifugal system for small-scale cell processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

In this protocol, we used Jurkat cells and MSCs as representative examples to demonstrate the automated buffer exchange process. During the process, Jurkat cells and MSCs shared the same processing steps with differences in centrifugal force and pump speed that control the flow rate (Table 1). Figure 2 shows representative images captured by the camera of how the fluidized cell bed may appear during the buffer exchange process. Typically, the fluidized cell bed will resemble...

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Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

automated-counterflow-centrifugal-system-for-small-scale-cell

Research

JoVE Journal

Bioengineering

9.2K Views.  Hudson Institute of Medical Research. Dit protocol biedt een oplossing voor gesloten geautomatiseerde bufferuitwisseling en -concentratie voor kleinschalige celproductie. Belangrijk is dat de bufferuitwisseling kan worden uitgevoerd met een hoog herstel en een levering van een zeer klein volume geconcentreerd product. Nieuwe gebruikers kunnen moeite hebben om alle automatiseringsstappen te volgen.Het oefenen van het programmeren van deze stappen en het uitvoeren van testruns met medium zijn goede manieren om vertrouwd te r...

Video: Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Cancer Research

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Engineering

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Biochemistry

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.

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 ...

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

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