The authors would like to acknowledge Drs. Simon Sauv&#233;, Roger Tam and Xu Zhang for their critical manuscript review. This work was supported by operating grants from the Genomics Research and Development Initiative (GRDI) Phase VII (2019-2025) from the Government of Canada obtained by JRL, LW, as well as operating grants from the Natural Sciences and Engineering Research Council RGPIN-2019-05220, Cancer Research Society/University of Ottawa 24064, the Canadian Institutes of Health (CIHR) Research Operating Grant 175177 obtained by LW, the CIHR MSc Scholarship obtained by MK, and the Queen Elizabeth II Scholarships in Science and Technology (QEII-GSST) obtained by FSDB.

Biomanufacturing and Characterization of NK-Extracellular Vesicles

<ol>
	<li>Cheng, M., Chen, Y., Xiao, W., Sun, R., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+cell-based+immunotherapy+for+malignant+diseases.">NK cell-based immunotherapy for malignant diseases.</a> Cell Mol Immunol. 10 (3), 230-252 (2013).</li><li>Sheridan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Industry+appetite+for+natural+killer+cells+intensifies.">Industry appetite for natural killer cells intensifies.</a> Nat Biotechnol. 41 (2), 159-161 (2023).</li><li>Shimasaki, N., Coustan-Smith, E., Kamiya, T., Campana, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+and+armed+natural+killer+cells+for+cancer+treatment.">Expanded and armed natural killer cells for cancer treatment.</a> Cytotherapy. 18 (11), 1422-1434 (2016).</li><li>Elsharkasy, O. 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=Extracellular+vesicles+as+drug+delivery+systems:+Why+and+how.">Extracellular vesicles as drug delivery systems: Why and how.</a> Adv Drug Deliv Rev. 159, 332-343 (2020).</li><li>St-Denis-Bissonnette, 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=Applications+of+extracellular+vesicles+in+triple-negative+breast+cancer.">Applications of extracellular vesicles in triple-negative breast cancer.</a> Cancers. 14 (2), 451 (2022).</li><li>Welsh, J. 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=Minimal+information+for+studies+of+extracellular+vesicles+(MISEV2023):+From+basic+to+advanced+approaches.">Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.</a> J Extracell Vesicles. 13 (2), e12404 (2024).</li><li>St-Denis-Bissonnette, 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=A+clinically+relevant+large-scale+biomanufacturing+workflow+to+produce+natural+killer+cells+and+natural+killer+cell-derived+extracellular+vesicles+for+cancer+immunotherapy.">A clinically relevant large-scale biomanufacturing workflow to produce natural killer cells and natural killer cell-derived extracellular vesicles for cancer immunotherapy.</a> J Extracell Vesicles. 12 (12), e12387 (2023).</li><li>Federici, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural-killer-derived+extracellular+vesicles:+Immune+sensors+and+interactors.">Natural-killer-derived extracellular vesicles: Immune sensors and interactors.</a> Front Immunol. 11, 262 (2020).</li><li>Lugini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+surveillance+properties+of+human+NK+cell-derived+exosomes.">Immune surveillance properties of human NK cell-derived exosomes.</a> J Immunol. 189 (6), 2833-2842 (2012).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+alternatives+to+extracellular+vesicle-based+immunotherapy+-+exosome+mimetics+derived+from+natural+killer+cells.">Novel alternatives to extracellular vesicle-based immunotherapy - exosome mimetics derived from natural killer cells.</a> Artif Cells Nanomed Biotechnol. 46 (sup3), S166-S179 (2018).</li><li>Cochran, A. 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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=Extracellular+vesicle-based+therapeutics:+natural+versus+engineered+targeting+and+trafficking.">Extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking.</a> Exp Mol Med. 51, 1-12 (2019).</li><li>Thery, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> J Extracell Vesicles. 7 (1), 1535750 (2018).</li><li>FiberCell-Systems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FiberCell systems user manual &amp; quick start guide. , (2024).</li><li>St-Denis-Bissonnette, 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=Evaluation+of+resazurin+phenoxazine+dye+as+a+highly+sensitive+cell+viability+potency+assay+for+natural+killer+cell-derived+extracellular+vesicle-based+cancer+biotherapeutics.">Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics.</a> J Extracell Biology. 3 (7), e166 (2024).</li><li>Herrmann, I. 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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=Natural+killer+(NK)+cell-derived+extracellular-vesicle+shuttled+microRNAs+control+T+cell+responses.">Natural killer (NK) cell-derived extracellular-vesicle shuttled microRNAs control T cell responses.</a> Elife. 11, e76319 (2022).</li><li>Geeurickx, 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=The+generation+and+use+of+recombinant+extracellular+vesicles+as+biological+reference+material.">The generation and use of recombinant extracellular vesicles as biological reference material.</a> Nat Commun. 10 (1), 3288 (2019).</li><li>Nathani, 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=Combined+role+of+interleukin-15+stimulated+natural+killer+cell-derived+extracellular+vesicles+and+carboplatin+in+osimertinib-resistant+H1975+lung+cancer+cells+with+EGFR+mutations.">Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in osimertinib-resistant H1975 lung cancer cells with EGFR mutations.</a> Pharmaceutics. 16 (1), 83 (2024).</li><li>Gobin, 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=Hollow-fiber+bioreactor+production+of+extracellular+vesicles+from+human+bone+marrow+mesenchymal+stromal+cells+yields+nanovesicles+that+mirrors+the+immuno-modulatory+antigenic+signature+of+the+producer+cell.">Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell.</a> Stem Cell Res Ther. 12 (1), 127 (2021).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+culture+system+improves+the+yield+of+MSCs-derived+extracellular+vesicles+and+enhances+their+therapeutic+efficacy+for+heart+repair.">A 3D culture system improves the yield of MSCs-derived extracellular vesicles and enhances their therapeutic efficacy for heart repair.</a> Biomed Pharmacother. 161, 114557 (2023).</li><li>Ryu, A. 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All authors declare no conflict of interest or disclosure.

Several studies suggest that NK-EVs possess vast potential as an anti-cancer therapeutic4,5,7,9,16,22,23,24,25,26,27,28,29,30. However, a scalable GMP-compliant biomanufacturing system capable of yielding large quantities of high-purity NK-EVs is required for further pre-clinical testing and future clinical applications. To address this issue, a previous study used a closed-looped HFB system to continuously produce NK cells and NK-EV-rich CM suitable for downstream experimentation. Due to their 3D design, HFB systems closely reflect the conditions of the vascular system and possess an incredibly high surface-area-to-volume ratio, permitting upwards of a billion cells to remain in culture, ultimately leading to improved EV production7,31,32. Importantly, this work was the first to ever report using an HFB system for culturing NK cells, likely due to the cell line IL-2 self-sufficiency7.
Additional steps must be taken to ensure the sterility of the HFB system and the production of high-purity NK-EVs. These precautions are especially crucial in the absence of a sterile, clean room, which may be the case for several research facilities. Before entering the biosafety cabinet, the HFB system is meticulously sprayed with 70% ethanol to disinfect all external surfaces. Additionally, wax film is wrapped around all Luer Lock connections to minimize the risk of contamination. This is particularly important as this biomanufacturing workflow does not use antibiotics, which are known to affect the biochemical profile of cell and cell-derived products33. Various metrics were used to assess cell health during cell product biomanufacturing. For example, daily assessments of the reservoir media&#39;s pH, glucose, and lactate levels were conducted as these are vital cell health surrogates for monitoring. In addition to quantitative assessments, qualitative observations of the HFB system (e.g., media color and visual signs of contamination such as turbidity) are also helpful for monitoring cell health. Cell counts on daily retrieved CM have not been found to be a representative metric of viability for the health of the culture (data not shown). This is likely a result of dead cells retrieved during CM sampling that were found within the tubing where media was not allowed to circulate (the small section between the ECS and the ECS syringe port), thereby undervaluing the viability of the overall cell culture. Only harvested NK cells produced by the HFB at the end of a production lot can provide a reliable metric of the culture&#39;s health. These cells consistently showed viability values above 70% across production lots7. Together, these quality assessment methods ensure the continuous production of high-purity NK-EVs.
Several isolation techniques have been developed to purify and isolate EVs34. One method, SEC, utilizes a column packed with a porous material - resin - allowing for molecule separation based on size discrimination. Here, the larger EVs are eluted through the column faster; this method is known as flow-through purification based on size exclusion. At the same time, smaller contaminants (dsDNA, free-floating proteins like endonuclease, salts, phenol red, etc.) are left behind and further retained within the resin by electrostatic forces (i.e., a bimodal resin was used). SEC-based processing removes non-EV-bound proteins while maintaining the original EV structure and functionality35,36. Furthermore, SEC-based purification is easily scalable without compromising the high yield and purity, making it a suitable choice for isolating NK-EVs for biotherapeutic uses. Despite these advantages, SEC has some drawbacks, such as the relatively diluted flow-through (eluent); hence, UF is required for product concentration, but it also permits buffer exchange. The non-sterile UF apparatus is rinsed with 70% ethanol and PBS and kept in the biosafety cabinet prior to use to ensure sterility. Typically, the flow-through can be concentrated to 35x-50x of the initial volume while removing small molecules that could have made their way into the eluent. Differential centrifugation and endonuclease treatment are performed before FPLC-SEC coupled with UF to remove residual cells, cellular debris, and long strands of antigenic dsDNA7.
Following NK-EV product isolation, characterization, and functional validation are performed per the guidelines in MISEV2018 and MISEV2023 to determine the product&#39;s suitability for further use6,18. Each isolation yields 1.0 - 1.5 mL of high-purity NK-EV product at a minimum concentration of 1 x 1012 EVs/mL, with an average concentration of 1.39 x 1012 particles/mL. Previously, Gupta et al. determined that the median EV dosage in vivo is 3.37 x 108 EVs/kg of body weight of mice37. Treating with the median dosage would require 8.43 x 106 EVs/mouse with a body weight of 25 g, a value far below the guaranteed minimum (1 x 1012 particles/mL) obtained through this workflow. Thus, the described biomanufacturing workflow can produce more than enough NK-EVs for pre-clinical experimentation or to meet dosing targets. Each isolation is tested for mycoplasma and microbial presence as part of the product&#39;s quality control assessment. In addition, a previous study demonstrated the absence of common viral entities and endotoxin in the final product and the absence of cellular components considered host cell contaminants (by western blot analysis)7,34. Lastly, functional assessment was performed using a validated highly sensitive resazurin-based cell viability assay to assess the NK-EVs&#39; functionality20. The described viability assay functions by reducing resazurin (weakly fluorescent) to resorufin (highly fluorescent) by metabolically active cells, allowing for the assessment of cell viability following NK-EV treatment. Compared to other alternative cell viability assays, the resazurin-based assay used in the study is highly sensitive to changes in cell viability (very low background noise) and allows for shortened incubation time to observe results (less than 30 min to obtain statistically significant results)20. Generally, the NK-EVs exhibit a dose-dependent effect upon K562 viability. Together, the results presented represent an NK-EV product that has met the product release criteria for pre-clinical evaluation and is suitable for downstream applications.
In conclusion, this protocol-based study describes the biomanufacturing of NK-EVs with clinical-grade potential. As discussed, the NK-EVs are produced using a closed-loop HFB system under serum-free, xeno-free, feeder-free, and antibiotic-free conditions7. A combination of FPLC-SEC/UF isolates and purifies the NK-EV product. Before releasing the products for downstream application, the NK-EVs must be characterized and functionally validated to ensure they are suitable for use. As demonstrated, following this biomanufacturing protocol can successfully generate a large quantity of high-purity NK-EVs that exhibit on-target cytotoxicity against cancer cells. Therefore, the described biomanufacturing protocol may be an asset for future studies that require the production of clinical-grade NK-EVs.

In the 21st century, remarkable advancements have been achieved in the battle against cancer. This is mainly due to the rise of cancer immunotherapeutics, a class of drugs that harnesses the immune system to fight cancer. Natural killer cell-derived extracellular vesicles (NK-EVs) represent promising contenders in the expanding realm of immunotherapy. Integrative to innate and adaptive immunity, NK cells play a crucial role in the body&#39;s defense against virus-infected, stressed, and malignant cells. They employ a comprehensive arsenal of anti-cancer machinery to eliminate abnormal cells through cytotoxic means1,2,3. Among these mechanisms is the production and secretion of EVs, nanoscale bilayer structures containing various biomolecules, such as proteins, RNAs, and DNAs, crucial for facilitating intercellular communication4,5,6. NK-EVs emerge as promising cell-free therapeutics due to their unique carrier properties. These include their small size, allowing filter-based sterilization, high biocompatibility, preferential accumulation within tumors, broad cargo delivery spectrum, capacity to overcome biological barriers such as the blood-brain barrier, and minimal toxicity profile. For several reasons, NK-EVs obviate the need for patient lymphodepletion via chemotherapy before administration: 1) conventionally, lymphodepletion is employed to create a more hospitable environment for cell-based therapy, enabling infused cells to proliferate and exert their therapeutic effects; 2) unlike cells, EVs lack the replication capacity and are substantially smaller in scale; 3) EVs operate through distinct mechanisms and exhibit diminished immunogenicity compared to cells5,6,7. Furthermore, NK-EVs consistently exhibited in vitro efficacy against various cancer models and have also shown immunomodulatory effects on immune cells that foster anti-cancer responses8,9. In vivo results corroborate these findings, showcasing cancer regression following NK-EV treatment and negligible toxicities10,11,12. Therefore, NK-EV-based therapeutics hold great promise to address the challenges of treating cold, immunologically inert solid tumors13,14,15,16,17.
Our recent study addresses a significant bottleneck to the clinical translation of NK-EVs through biomanufacturing7. The article presents a proof-of-concept for a cost-effective and scalable biomanufacturing workflow of NK-EVs meticulously designed to ensure in-process quality control testing. This approach continuously produced large quantities of high-purity NK-EV-based cancer biotherapeutics, with thorough product characterization conducted according to the MISEV2018 guidelines18. The scalability of the biomanufacturing workflow can be achieved by increasing cartridge size or having multiple bioreactors running in parallel. Similarly, the scalability of the EV isolation workflow can be easily attainable using techniques like Fast Protein Liquid Chromatography (FPLC) based size-exclusion chromatography (SEC), ultrafiltration (UF), and filter-based sterilization. The closed-loop hollow-fiber bioreactor (HFB) system grew the IL-2-self-sufficient NK cell line (NK92-MI cells) without requiring serum supplementation, a feeder system, and antibiotics. This was accomplished using a commercially available chemically defined and xeno-free medium (a GMP version is now commercially available). As a result, large quantities of NK cells (109 viable cells) and NK-EVs (1012 EVs) were successfully produced within 5 - 7 days using a single medium-size bioreactor cartridge, with both products extensively characterized. Throughout the biomanufacturing process, cell health was monitored daily using quantifiable metrics such as pH, glucose, and lactate levels, along with visual indicators such as media color and any sign of contamination, which are essential predictors of cell and EV quality. Post-harvest evaluation of NK cell viability and functionality generated in the HFB system, particularly cytotoxicity, revealed a significant enhancement compared to flask-based cultures7. Likewise, purified NK-EVs exhibited a high purity profile, devoid of bacteria, mycoplasma, common viral entities, and cellular components, and with negligible endotoxin levels. Importantly, purified NK-EVs constituted over 99.9% of all nanoparticles found in the final product7. Lastly, these purified NK-EVs retained key NK characteristics, including surface markers (CD2, CD45, CD56), cytokine payload (GzmB, PFN, IFN-g), and demonstrated potent cytotoxicity against leukemic K562 cells, the gold-standard line for assessing NK cell cytotoxicity7.
The present protocol details the scalable biomanufacturing workflow discussed above. It elucidates the methodology for isolating NK-EVs produced using FPLC-SEC coupled with UF and filter-based sterilization. Additionally, the protocol describes pivotal steps, including product characterization using nanoparticle tracking analysis (NTA), quality assessment using various tools (protein/dsDNA quantification and microbial testing), and functional validation of the purified NK-EV product against cancer cells by cell viability assay. Typically, this workflow yields 1.0 - 1.5 mL of NK-EV product with an average concentration of 1.18 x 1012 EVs/mL7, totaling a minimum of 1 x 1012 EVs based on approximately 40 mL of EV-rich CM. This process allows product release for various downstream applications, such as investigatory, preclinical, and multi-omics (proteomics, transcriptomics, genomics, metabolomics, lipidomics, and epigenomics) studies demanding high quantities of high-quality EVs while holding potential for clinical translation, with demonstrated reproducibility.

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		<tr>
			<td>0.1 &#181;m vacuum filtration unit Filtropur V50&#160;</td>
			<td>Sarstedt</td>
			<td>83,3941,002</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m Acrodisc Syringe Filter&#160;</td>
			<td>Pall Corporation</td>
			<td>PN4612</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>MB9204560TF-LAB</td>
			<td></td>
		</tr>
		<tr>
			<td>10 kDa Centricon Plus-70 Centrifugal Filter</td>
			<td>Sigma</td>
			<td>UFC701008</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL syringe&#160;</td>
			<td>BD Biosciences</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Flat Clear Bottom Black Polystyrene TC-treated Microplates&#160;</td>
			<td>Costar</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0491</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA Fast Protein Liquid Chromatograph&#160;</td>
			<td>GE Lifesciences</td>
			<td>29022094</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle - 18G</td>
			<td>BD Biosciences</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase Nuclease&#160;</td>
			<td>Sigma</td>
			<td>E1014-25KU</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy H1 Multimode Reader</td>
			<td>BioTek&#160;</td>
			<td>SH1M2G-SN</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Juice Gel Loading Buffer&#160;</td>
			<td>Invitrogen</td>
			<td>10816015</td>
			<td></td>
		</tr>
		<tr>
			<td>CaptoCore 700 resin&#160;</td>
			<td>Cytiva</td>
			<td>17548102</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer Auto 2000 Viability Counter&#160;</td>
			<td>Nexcelom BioScience LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS10 freezing medium&#160;</td>
			<td>Sigma</td>
			<td>C2874</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS&#8722;/&#8722;&#160;</td>
			<td>Fisher</td>
			<td>BP399-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual LED Blue/White Light Transilluminator&#160;</td>
			<td>Invitrogen</td>
			<td>LB0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Duet P3202 Flow Control Pump&#160;</td>
			<td>FiberCell Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Commercial Alcohols</td>
			<td>P006EAAN</td>
			<td></td>
		</tr>
		<tr>
			<td>Exosome-Depleted FBS&#160;</td>
			<td>Gibco</td>
			<td>A2720803</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorobrite DMEM&#160;</td>
			<td>Gibco</td>
			<td>A18967-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose meter</td>
			<td>AccuCheck</td>
			<td>Model 930</td>
			<td></td>
		</tr>
		<tr>
			<td>HiScale chromatography column 10/40&#160;</td>
			<td>Cytiva</td>
			<td>29360550</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoCult-XF (GMP medium alternative)</td>
			<td>StemCell Technologies</td>
			<td>100-0956</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoCult-XF T Cell Expansion Medium&#160;</td>
			<td>StemCell Technologies</td>
			<td>10981</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol&#160;</td>
			<td>EMD</td>
			<td>PX1834-1</td>
			<td></td>
		</tr>
		<tr>
			<td>K562 cells</td>
			<td>ATCC</td>
			<td>CCL-243</td>
			<td></td>
		</tr>
		<tr>
			<td>LB media&#160;</td>
			<td>BioBasic</td>
			<td>SD7002</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lactate Assay Kit&#160;</td>
			<td>Abcam</td>
			<td>ab65331</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium hollow-fibre cartridge&#160;</td>
			<td>FiberCell Systems</td>
			<td>C2011</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#160;</td>
			<td>Sigma</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycoplasma PCR detection kit&#160;</td>
			<td>Abcam</td>
			<td>ab289834</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NS300&#160;</td>
			<td>Malvern</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Supelco</td>
			<td>SX0607N-6</td>
			<td></td>
		</tr>
		<tr>
			<td>NK92-MI cells&#160;</td>
			<td>ATCC</td>
			<td>CRL-2408</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Strips-Mquant&#160;</td>
			<td>Sigma</td>
			<td>1,09533</td>
			<td></td>
		</tr>
		<tr>
			<td>PrestoBlue HS Cell Viability Reagent Assay&#160;</td>
			<td>Invitrogen</td>
			<td>P50200</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q33262</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q33231&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Flex Assay Tube Strips&#160;</td>
			<td>Invitrogen</td>
			<td>Q33252</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Flex Fluorometer&#160;</td>
			<td>Invitrogen</td>
			<td>Q33327</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Protein BR Assay Kit&#160;</td>
			<td>Invitrogen</td>
			<td>A50669</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Load 1Kb Plus DNA ladder&#160;</td>
			<td>NEB</td>
			<td>N0469S</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBRSafe DNA Gel Stain Invitrogen</td>
			<td>Invitrogen</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump&#160;</td>
			<td>Harvard Apparatus</td>
			<td>984730</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100&#160;</td>
			<td>Sigma</td>
			<td>T-9284</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure TAE Buffer&#160;</td>
			<td>Invitrogen</td>
			<td>15558042</td>
			<td></td>
		</tr>
		<tr>
			<td>ViaStain Acridine Orange and Propidium Iodide (AO/PI) Staining Solution&#160;</td>
			<td>ESBE Scientific</td>
			<td>CS2-0106</td>
			<td></td>
		</tr>
	</tbody>
</table>

scalable biomanufacturing workflow to produce and isolate natural killer cell-derived extracellular vesicle-based cancer biotherapeutics

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic nanovesicles targeting cancer cells and as immunomodulatory communicators. A scalable biomanufacturing workflow enables the production of large quantities of high-purity NK-EVs to meet the pre-clinical and clinical demands. The workflow employs a closed-loop hollow-fiber bioreactor, enabling continuous production of NK-EVs from the NK92-MI cell line under serum-free, xeno-free, feeder-free, and antibiotic-free conditions in compliance with Good Manufacturing Practices standards. This protocol-driven study outlines the biomanufacturing workflow for isolating NK-EVs using size-exclusion chromatography, ultrafiltration, and filter-based sterilization. Essential NK-EV product characterization is performed via nanoparticle tracking analysis, and their functionality is assessed through a validated cell viability-based potency assay against cancer cells. This scalable biomanufacturing process holds significant potential to advance the clinical translation of NK-EV-based cancer biotherapeutics by adhering to best practices and ensuring reproducibility.

NK-EVs possess inherent cytotoxic functions and have demonstrated high efficacy against various cancer models. However, there needs to be more standardization among current studies regarding a biomanufacturing workflow suitable for the large-scale production of NK-EVs6,21. Our previous study described the feasibility of a closed-looped hollow-fiber bioreactor (HFB) system to produce large quantities of high-purity NK-EV products7. As a follow-up, this protocol-based study details the biomanufacturing workflow and demonstrates its reproducibility by producing and isolating the NK-EV product (Figure 1). Furthermore, essential product characterization and validation are required before product release is performed, whereby new and original data are presented in this study.
The HFB system was selected for NK-EV production due to its ease of use, reliability, scalability, and GMP compliance7. In reference to the HFB system set-up, the NK cells are injected through the left ECS port and seeded into the bioreactor cartridge (Figure 2). At the same time, the media bottle is connected to the HFB through the side ports, and the media is allowed to flow throughout the system. The NK cells are cultured in serum-free, xeno-free, feeder-free, and antibiotic-free medium, where the media is replaced when the glucose content falls below 50% to maintain and maximize cell health over time. CM is collected daily, processed through differential centrifugations, and kept frozen (-80 &#176;C) until ready for further processing. Afterward, EV isolation is conducted through a combination of differential centrifugations and FPLC-SEC coupled with UF and filtration (Figure 3). This results in a concentrated and sterile NK-EV product with a final volume of approximately 1.0 - 1.5 mL. A representative chromatogram of the FPLC-SEC isolation of NK-EVs is provided (Figure 4). Before FPLC-SEC processing, the NK-EV-rich CM is treated with endonuclease, significantly reducing dsDNA levels, a potential host (NK) cell contaminant7. Thus, the described EV isolation workflow removes cellular debris and RNA/DNA contaminants from the NK-EV product, which is essential for ensuring a low and unwanted immunogenic potential and that the final product is suitable for downstream studies.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67227/67227fig04.jpg" /> 
Figure 4: NK-EV isolation chromatogram generated during Fast Protein Liquid Chromatography size Exclusion. The blue line represents the absorbance (mAU; maximum reading of 2000 mAU), the red line represents the conductivity, the red text represents the run log, and the gray shaded area represents the fractionated NK-EVs (denoted by fractions T2 - T7). <a href="https://www.jove.com/files/ftp_upload/67227/67227fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Following isolation, basic NK-EV characterization and quality assurance testing are used to evaluate if the NK-EV product can be released for further downstream experimentation. NK-EV product particle size range and concentration are measured using nanoparticle tracking analysis (NTA), with sizes ranging from 76.30 - 174.30 nm in diameter (D10 of 78.38 &#177; 2.07 nm, D50 of 106.72 &#177; 2.43 nm, and D90 of 169.80 &#177; 4.17 nm) and an average concentration of 1.39 x 1012 EVs/mL (Figure 5A-B). Additionally, fluorometer quantification showed a protein and dsDNA concentration of 298.90 &#177; 66.62 mg/mL and 225.60 &#177; 37.7 ng/mL for the final product, respectively (Figure 5C-D). This corresponds to an average ratio of 5.06 x 106 EV/&#181;g of protein and 6.16 x 1012 EV/&#181;g of DNA. Microbial and mycoplasma testing both returned negative results (data not shown). These results are consistent with the characterization of NK-EVs from previous work7. The earlier publication7 also provides a further in-depth characterization of the NK-EV products following the MISEV guidelines (i.e., TEM, western blot, endotoxin level, viral entities, and flow cytometry for surface antigens and cytokines).
Lastly, the NK-EV product&#39;s functionality (i.e., cytotoxicity against cancer cells) was assessed using a validated highly sensitive resazurin-based cell viability assay following NK-EV treatment against leukemic cell line K5627,20. K562 cell treatment with NK-EVs for 3 h produced a dose-dependent effect on cell viability, corresponding to an EC50 of 9.33 x 109 EVs/mL (i.e., the dosage that corresponds to the killing of 50% of the cell population; Figure 6A-B). Thus, following the outlined product release criteria, the NK-EV product is deemed suitable for further experimentation.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67227/67227fig05v3.jpg" /> 
Figure 5: Purified NK-EV product characterization. (A) NK-EV product size distribution measured by NTA, shown as mean from 5 independent experiments, each with 10 technical replicates (5 video captures x 2 dilutions). (B) NK-EV particle product concentration (particles/mL) measured by NTA, presented as mean &#177; SD from 5 independent experiments, each with technical duplicates. (C) NK-EV product protein concentration (mg/mL) was measured by using a fluorometer, presented as mean &#177; SD from 5 independent experiments, each with technical triplicates. (D) NK-EV product dsDNA concentration (ng/mL) measured by using a fluorometer, presented as mean &#177; SD from 5 independent experiments, each with technical triplicates. <a href="https://www.jove.com/files/ftp_upload/67227/67227fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67227/67227fig06v3.jpg" /> 
Figure 6: Purified NK-EV product functional validation. NK-EVs demonstrate a dose-dependent cytotoxicity against human K562 leukemia cells treated at various NK-EV concentrations for 3 hours using a highly sensitivity resazurin-based cell viability assay. (A) Normalized assay readouts (green line represents the untreated K562 leukemia cell control, and the black dashed line represents lysed K562 leukemia dead cell control; detergent-treated). Data are shown as mean &#177; SEM from 11 independent experiments with technical triplicates. (B) EC50 curve analysis with a variable slope for NK-EV treatment with 95% confidence interval/prediction bands (red dashed line represents 50% response). <a href="https://www.jove.com/files/ftp_upload/67227/67227fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-Derived Extracellular Vesicle-Based Cancer Biotherapeutics

Natural killer cell-derived extracellular vesicles (NK-EVs) hold promising potential as cancer biotherapeutics. This methodology-based study presents a scalable closed-loop biomanufacturing workflow designed to continuously produce and isolate large quantities of high-purity NK-EVs. In-process control testing is performed throughout the biomanufacturing workflow, ensuring the EVs meet quality standards for product release.

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic ...

scalable-biomanufacturing-workflow-to-produce-isolate-natural-killer

Research

JoVE Journal

Cancer Research

505 Views.  Biologic and Radiopharmaceutical Drugs Directorate. Natural killer cell-derived extracellular vesicles (NK-EVs) hold promising potential as cancer biotherapeutics. This methodology-based study presents a scalable closed-loop biomanufacturing workflow designed to continuously produce and isolate large quantities of high-purity NK-EVs. In-process control testing is performed throughout the biomanufacturing workflow, ensuring the EVs meet quality standards for product release. 

Video: Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-Derived Extracellular Vesicle-Based Cancer Biotherapeutics

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are available for studying the significant crosstalk; however, a two-dimensional transwell system for coculture remains a classic, reliable, and easy way for this crosstalk study. Here, we present a detailed protocol that shows the coculture of bone marrow adipocytes and melanoma cells. Nevertheless, such a coculture system could not only contribute to the study of cell signal transductions of cancer cells induced by bone marrow adipocytes, but also to the future mechanistic study of bone metastasis which may reveal new therapeutic targets for bone metastasis.

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of both synthetic lethal interactors leads to loss of cell viability or cell death. The best studied synthetic lethal interaction is between BRCA1/2 and PARP1, with PARP1 inhibitors being used in clinical practice to treat patients with BRCA1/2 mutated tumors. Large genetic screens in model organisms but also in haploid human cell lines have led to the identification of numerous additional synthetic lethal interaction pairs, all being potential targets of interest in the development of novel tumor therapies. One approach is to therapeutically target genes with a synthetic lethal interactor that is mutated or significantly downregulated in the tumor of interest. A second approach is to formulate drug combinations addressing synthetic lethal interactions. In this article, we outline a data integration workflow to evaluate and identify drug combinations targeting synthetic lethal interactions. We make use of available datasets on synthetic lethal interaction pairs, homology mapping resources, drug-target links from dedicated databases, as well as information on drugs being investigated in clinical trials in the disease area of interest. We further highlight key findings of two recent studies of our group on drug combination assessment in the context of ovarian and breast cancer.

Large genetic screens in model organisms have led to the identification of negative genetic interactions. Here, we describe a data integration workflow using data from genetic screens in model organisms to delineate drug combinations targeting synthetic lethal interactions in cancer.

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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Pancreatic Tissue Dissection to Isolate Viable Single Cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.