Name: a novel feeder-free system for mass production of murine natural killer cells in vitro
Uploaded: 2018-01-09T14:00:00.000+00:00
Duration: 10 min 36 s
Description: Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

This study was supported by the Research Grants Council of Hong Kong (GRF 468513, CUHK3/CRF/12R) and the Innovation and Technology Fund of Hong Kong (ITS/227/15, ITS InP/164/16, ITS-InP/242/16), Direct Grant for Research-CUHK (2016.035), and Hong Kong Scholar Program.
H.-Y.L. designed and supervised all experiments and contributed to manuscript preparation. P.M.-K.T. performed experiments, analyzed data and contributed to manuscript preparation. P.C.-T. T., J.Y.-F.C., J.S.-C.,H., Q.-M.W., and G.-Y.L. collected animal samples and participated in animal experiments. J.S., X.-R.H., and K.-F.T. contributed to manuscript preparation.

The protocol for obtaining and differentiating bone marrow-derived NK cells (BM-NK) is based on previously published methods20,21,22. All procedures with mice have been approved by the Animal Ethics Experimental Committee (AEEC) at the Chinese University of Hong Kong.
1. Preparation of OP9 Conditional Medium
<ol>
	<li>Culture the murine stroma cell line OP9 in alpha-MEM containing 20% FBS, 100 U/mL penicillin G and 100 &#181;g/mL streptomycin, at 37 &#176;C in a humidified atmosphere of air/CO2 (95%:5%).
	<ol>
		<li>Count the cells with a hemocytometer, then seed 2 x 106 of OP9 cells to each 75 cm2 culture flask with 15 mL of medium. Culture for 48 h.</li>
		<li>When the culture reaches 80% confluence, wash the flask with 10 mL of PBS and add 20 mL of plain alpha-MEM medium (without FBS and antibiotics) per flask.</li>
	</ol>
	</li>
	<li>Collect the OP9 condition medium from the culture flask at 24 h, clean up cell debris with 0.25 &#181;m polyethersulfone (PES) filter and preserve at 4 &#176;C (use within 2 weeks).</li>
</ol>
2. Isolation of Mouse Bone Marrow Cells
<ol>
	<li>Euthanize a 12-week-old C57BL/6J mouse with a pentobarbitone overdose (100 mg/kg, intraperitoneal injection). Confirm death by the lack of breathing, then harvest the femur bones with a surgical knife cutting at the junctions between bones and remove the remaining muscles with the blade by gentle scratching.</li>
	<li>Place the bones in a 50 mL centrifuge tube containing chilled sterile alpha-MEM, and then perform the following steps in a biosafety cabinet.</li>
	<li>Discard the alpha-MEM medium, and rinse the bones with 70% ethanol for 30 s.</li>
	<li>Wash the bones with ice-cold sterile PBS twice to clean up the remaining ethanol.</li>
	<li>Transfer the bones in a mortar containing 5 mL of ice-cold PBS, and gently fragmentize the bones with a pestle (do not pulverize them).</li>
	<li>Agitate the bones gently by swirling the pestle to release the bone marrow cells into the PBS. Collect the PBS containing bone marrow cells into a new 50 mL centrifuge tube.</li>
	<li>Repeat step 2.6 for four times until the bone fragments become solid white in color.</li>
	<li>Centrifuge the tube at 465 x g for 5 min at 4 &#176;C, then discard the supernatant.</li>
	<li>Resuspend the pellet with 18 mL of sterile distilled water and let it stand for 30 s to remove red blood cells.</li>
	<li>Add 2 mL of ice-cold 10X PBS to stop the reaction, then filter the mixture through a 70 &#181;m cell strainer to remove the lysed red blood cells.</li>
	<li>Centrifuge the tube at 465 x g for 5 min at 4 &#176;C. Resuspend the pellet with 20 mL of ice-cold PBS.</li>
	<li>Wash the cells by repeating step 2.11 a second time.</li>
	<li>Transfer the cell pellet into a 100-mm sterile Petri dish with 10 mL of OP9 conditional medium from step 1.2 supplemented with 20% FBS and a mixture of 0.5 ng/mL murine IL-7, 30 ng/mL mouse SCF, and 100 U/mL murine flt3L.</li>
	<li>Incubate the dish at 37 &#176;C with 5% CO2 for 2 h. Transfer the unattached cells into a new culture container at a density of 5 x 105 viable cells/mL (count cells by hemocytometer with trypan-blue exclusion) with the same medium formula as in step 2.13, and incubate at 37 &#176;C with 5% CO2.</li>
	<li>On day 4, change the culture condition to OP9 conditional medium supplemented with 20% FBS and 2,000 U/mL of murine IL-2.</li>
	<li>Refresh the culture medium every 3 days; mature NK cells can be obtained by Day 7 and qualified as in section 4.</li>
</ol>
3. siRNA-mediated Gene Silencing of Differentiating NK Cells
<ol>
	<li>Premix the siRNA or nonsense control (NC) with a transfection agent according to the product manual.</li>
	<li>Perform step 3.1 at any time point since day 0.</li>
	<li>Add the 50 nM of siRNA or NC mixture to the cells from step 2.14.</li>
	<li>Repeat step 3.1 on every medium refreshment (e.g., Day 0, 4, and 7) until the experimental end point.</li>
</ol>
4. Analysis of NK Cell Differentiation Using Flow Cytometry
<ol>
	<li>At an appropriate time point, collect the suspension of differentiating cells and wash with ice-cold PBS.</li>
	<li>Fix the cells with cell fixation buffer according to the product manual.</li>
	<li>Wash the fixed cells with ice-cold PBS, and then resuspend 1 x 106 cells in 100 &#181;L of Flow Cytometry Staining Buffer containing Cy3-conjugated anti-mouse NKp46 and PE-conjugated anti-mouse CD244 antibodies in a dilution ratio of 1:100.</li>
	<li>Stain the sample in the dark at room temperature for 2 h.</li>
	<li>Resuspend the stained samples with 300 &#181;L of PBS, then acquire FACS data and analyze with Cytobank platform20 (http://cytobank.org/).</li>
</ol>

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The authors have nothing to disclose.

In the present work, we have described a novel method for producing bone marrow-derived murine NK cells in vitro. The cell feeder in the original system21,22 is successfully replaced by the conditional medium of OP9 cells, which largely increased the stability of the differentiation system. In addition, the system can produce high quantity and purity of mature NK cells for in vitro as well as in vivo assays, which can facilitate the mec...

Cancer progression is largely dependent on the tumor microenvironment1,2, including host-derived immunocytes, e.g., NK cells. Several studies demonstrated that intratumoral NK cells are negatively correlated with the tumor progression3,4. In addition, clinical studies showed that NK cell adoptive therapy is a possible strategy for cancer5,6,7,8,9. NK cell-based cancer immunotherapy was recently suggested as a therapeutic option for solid tumors, but challenges exist due to the secretion of immunosuppressive cytokines and downregulation of activating ligands in the microenvironment of solid tumors10,11. Transforming growth factor-&#946; (TGF-&#946;) has been suggested to play a suppressive role in carcinogenesis, but paradoxically cancer cells also produce TGF-&#946;1 to support the tumor development12,13,14,15. TGF-&#946; signaling can suppress the cytolytic activity of NK cells via down-regulating interferon responsiveness and CD16-mediated interferon-gamma (IFN-&#947;) production in vitro16,17,18.
Although disruption of TGF-&#946; signaling in the tumor microenvironment may be a possible way for eliminating cancers, completely blocking TGF-&#946; signaling will cause autoimmune diseases due to its anti-inflammatory function, as evidenced by the development of adverse side effects including systemic inflammation, cardiovascular defects, and autoimmunity in mouse models19. Thus, understanding the working mechanism of TGF-&#946;-mediated immunosuppression will lead to the identification of an accessible therapeutic target for treating cancer.
To elucidate the molecular events necessary for NK cell development, Williams et al. established an in vitro system for differentiating murine bone marrow hematopoietic stem cells into NK cells20. This system largely facilitates the mechanistic study of NK cell development, including the identification of novel progenitors of NK cells21. However, the bone marrow progenitors should be cultured in the system with supporting OP9 stromal cells as a feeder layer20,21, and this heterogeneous cell population largely limits the further application of gene-disrupting tools (e.g., siRNA-mediated gene silencing) specifically applied to the differentiating NK cells.
Here, we describe a feeder-free system that has been developed by further modifying the in vitro system of Williams et al20. In our system, the OP9 stromal feeder cells are not required, and instead OP9 conditional medium is used without affecting the differentiation of NK cells in vitro, and this recently lead us to uncover that TGF-&#946; is able to promote cancer progression via suppressing E4bp4-dependent NK cell development in the tumor microenvironment22. This novel system successfully provides a background-free method for elucidating the molecular mechanism of NK cell development under specific conditions (e.g., high TGF- &#946;1, siRNA-mediated gene silencing, etc.) in vitro.

<table><tbody><tr><td>OP9 cell line</td><td>ATCC</td><td>ATCC&lt;sup&gt;&amp;reg;&lt;/sup&gt; CRL-2749&lt;sup&gt;&amp;trade;&lt;/sup&gt;</td><td></td></tr><tr><td>MEM &amp;alpha;, no nucleosides</td><td>Gibco</td><td>22561021</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10500064</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>Recombinant Murine IL-7</td><td>PEPROTECH</td><td>217-17</td><td></td></tr><tr><td>Recombinant Murine SCF</td><td>PEPROTECH</td><td>250-03</td><td></td></tr><tr><td>Recombinant Murine Flt3-Ligand</td><td>PEPROTECH</td><td>250-31L</td><td></td></tr><tr><td>Recombinant Murine IL-2</td><td>PEPROTECH</td><td>212-12</td><td></td></tr><tr><td>Lipofectamin RNAiMAX Transfection Reagent</td><td>Invitrogen</td><td>1377815</td><td></td></tr><tr><td>IC Fixation Buffer&amp;nbsp;</td><td>eBioscience</td><td>00-8222-49</td><td></td></tr><tr><td>Flow Cytometry Staining Buffer&amp;nbsp;</td><td>eBioscience</td><td>00-4222-26</td><td></td></tr><tr><td>PE-conjugated anti-mouse CD244</td><td>eBioscience</td><td>12-2441-83</td><td></td></tr><tr><td>Cy3-conjugated anti-mouse NKp46</td><td>Bioss</td><td>bs-2417R-cy3</td><td></td></tr><tr><td>Nonsense control (NC)</td><td>Ribobio</td><td>siN05815122147</td><td></td></tr><tr><td>siRNA against mouse E4BP4 mRNA</td><td>Ribobio</td><td>N/A</td><td>5&amp;prime;-GAUGAGGGUGUA&lt;br/&gt; GUGGGCAAGUCUU-3&amp;prime;</td></tr></tbody></table>

a novel feeder-free system for mass production of murine natural killer cells in vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

Representative results are obtained following the described protocol. Total bone marrow suspension cells were cultivated under the feeder-free differentiation system for 11 days; significant increase in proliferation rate was observed by day 7 compared with the number of total cells on day 0 (Figure 1A). Mature NK cells with high nuclear to cytoplasmic ratio and granule-rich cytoplasm morphology were found by day 6 in the system (<strong clas...

Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

a-novel-feeder-free-system-for-mass-production-murine-natural-killer

Research

JoVE Journal

Immunology and Infection

7.1K Views.  The Chinese University of Hong Kong. Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Video: A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

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, ...

Cancer Research

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Biomanufacturing and Characterization of NK-Extracellular Vesicles

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.

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 ...

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3&#8722;CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.

The present protocol shows how to differentiate CD3&#8722;/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular ...

Developmental Biology

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Streamlined Protocol for Mouse CAR-T Cell Generation in Syngeneic Models

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

Bioengineering

Isolating Invariant Natural Killer T Cells from a Mouse Model

Source: Meng, M., et al. <a href="https://app.jove.com/v/60048">Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice.</a>&#160;J. Vis. Exp.&#160;(2020)
This video illustrates a method for isolating invariant natural killer T cells, also known as iNKT cells. The procedure begins with injecting a synthetic glycolipid into a mouse to stimulate the activation and proliferation of iNKT cells in the spleen. Subsequently, single-cell suspensions are prepared from the spleen and incubated with iNKT-cell-recognizing complexes labeled with fluorophores, followed by magnetic bead-based separation, to isolate the iNKT cells.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Summary

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Chapters in this video

Murine Model of CD40-activation of B cells

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

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

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

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34⁺ Cells Injection, and Cytokine Delivery

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Isolating Invariant Natural Killer T Cells from a Mouse Model