We thank L. Brockmann for critical review of the manuscript. This work was supported by NIH 1R01EB030352 and UL1 TR001873.

All animal procedures were performed with approval from the Institutional Animal Care and Use Committee (Columbia University, protocols AC-AABQ5551 and AC-AAAZ4470) using 6-8-week-old female BALB/c or CF57BL/6 mice weighing between 20-25 g. The animals were obtained from a commercial source (see Table of Materials). This protocol is structured around the &#39;days post-activation&#39; of murine T cells, and viral production begins on Day -2. Retrovirus can be stored at -80 &#176;C following initial production, and for future use of this protocol, one may commence with step 2, T cell isolation and activation on Day 0.
1. Retroviral vector production
NOTE: The viral products have been made replication-defective by separation of the packaging genes into two separate plasmids (see Table of Materials), greatly reducing the likelihood of recombination events and inadvertent production of replication-competent virus.
<ol>
	<li>Prepare Phoenix Eco cells one day prior to transfection (Day -2 procedure).
	<ol>
		<li>Plate approximately 1 x 107 cells in a 15 cm TC-treated plate or T150 culture flask, using 30 mL of culture medium (Phoenix Eco culture medium, as detailed in Table 1).</li>
		<li>Incubate overnight at 37 &#176;C. After 18-24 h, the cells should be approximately 70% confluent and uniformly distributed to ensure a high viral yield without overgrowth. 
		NOTE: For optimal results, use cells with a low passage and passage them the day before plating for viral production. Do not allow Phoenix Eco cells to overgrow during routine culture.</li>
	</ol>
	</li>
	<li>Prepare the transfection mix containing the lipofection and enhancer reagents, pCL-Eco (Gag/Pol), and pMSCV expression plasmid (pMSCV_PGK_mGFP28z) in reduced-serum media (see Table of Materials) (Day -1 procedure). 
	NOTE: Co-transfection should be performed at a 1:1 ratio of pMSCV and pCL-Eco.
	<ol>
		<li>Prepare Tube A: Dilute 105 &#181;L of the transfection reagent in 3.75 mL of reduced serum medium per 15 cm plate and mix thoroughly by vortexing or pipetting up and down.</li>
		<li>Prepare Tube B: Dilute pMSCV expression plasmid (21 &#181;g) and pCL-Eco (21 &#181;g) with 90 &#181;L of enhancer reagent into 3.75 mL of reduced serum medium per 15 cm plate. Mix well by pipetting up and down. 
		NOTE: If generating multiple viral products, set up a master mix containing pCL-Eco and the enhancer reagent.</li>
		<li>Add Tube A to Tube B and mix thoroughly by pipetting. Incubate for 10-20 min at room temperature, resulting in a total volume of approximately 7.5 mL.</li>
		<li>Carefully remove 10 mL of cell culture media from the Phoenix Eco plate(s) and add the entire volume of the transfection mix to the remaining media by tilting the plate and pipetting dropwise. Return cells to a 37 &#176;C incubator.</li>
	</ol>
	</li>
	<li>Change the media on transfected Phoenix Eco cells 16-20 h post-transfection (Day 0 procedure).
	<ol>
		<li>Pre-warm serum and &#946;-mercaptoethanol (2-ME, see Table of Materials)-free murine T cell medium (mTCM-viral harvest, as listed in Table 1) to 37 &#176;C using a water or bead bath.</li>
		<li>Carefully remove the Phoenix Eco culture medium by tilting the plate and placing the pipette tip at the bottom corner, using a vacuum if possible. Avoid over-drying the cells.</li>
		<li>Gently add the warmed mTCM-viral harvest medium to the side of the tilted plate by pipetting 30 mL on the slowest setting. Return cells to a 37 &#176;C incubator. 
		NOTE: This step can cause Phoenix Eco cells to detach from the plate and reduce viral titers. Pre-warmed media and gentle pipetting will minimize disruption.</li>
	</ol>
	</li>
	<li>Harvest the viral supernatant 48 h post-transfection (Day 1 procedure).
	<ol>
		<li>Harvest the viral supernatant and filter it through 0.45 &#181;m PVDF filters (see Table of Materials) to remove cells and debris. Aliquot and freeze the virus at -80 &#176;C or proceed directly to Day 1 of step 3 if using fresh virus.</li>
		<li>Determine the viral titer (transducing units/mL) by flow cytometry, as previously described9. This step is optional.
		<ol>
			<li>Determine the viral titer by small-scale transduction of 1 x 105 activated murine T cells in non-tissue culture (TC)-treated 96-well plates, pre-coated with retronectin (see Table of Materials) and loaded with threefold serial dilutions of retroviral supernatant in a final volume of 100 &#181;L of mTCM-viral harvest medium. 
			NOTE: See step 2 and step 3 below for detailed instructions on T cell isolation, activation, and transduction.</li>
			<li>Determine CAR surface expression 4-5 days post-transduction by flow cytometry.</li>
			<li>Calculate the viral titer according to the formula10: (N x F x D)/V, where N is the number of cells transduced, F is the frequency of CAR-positive cells, D is the dilution factor, and V is the transduction volume in mL to obtain transducing units (TU)/mL. 
			NOTE: Viral titer may decrease upon freeze/thaw; therefore, viral titer is ideally determined on frozen and subsequently thawed virus.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Murine T cell isolation
<ol>
	<li>Isolate and activate murine T cells (Day 0 procedure). 
	NOTE: These steps can be performed at room temperature (RT) or 4 &#176;C and should be carried out in a sterile environment.
	<ol>
		<li>Harvest the spleen(s) from the mouse strain of interest (e.g., Balb/c, C57BL/6) as previously described11 and obtain a single-cell suspension through mechanical dissociation.</li>
		<li>Using the back of a sterile syringe, crush the spleen(s) through a 70-100 &#181;m cell strainer into a 50 mL conical tube.</li>
		<li>After setting the syringe aside, wash the strainer with 5 mL of T cell isolation buffer (phosphate-buffered saline, PBS, supplemented with 2% fetal bovine serum, FBS).</li>
		<li>Repeat the mashing step and wash the strainer once more with 5 mL of T cell isolation buffer. Bring the final volume to 50 mL with T cell isolation buffer.</li>
		<li>Count live cells using a manual hemocytometer or an automatic cell counter by diluting at a 1:20 ratio and then mixing at 1:1 with trypan blue.</li>
		<li>Pellet the cells by centrifugation for 10 min at 450 x g, at 4 &#176;C (or RT), and re-suspend spleenocytes at 1 x 108/mL if using the recommended T cell isolation kit (see Table of Materials). 
		NOTE: Spleenocytes can be re-strained through a 40-70 &#181;m strainer at this stage to remove clumps.</li>
	</ol>
	</li>
	<li>Isolate CD3+ T cells by negative selection following the manufacturer&#39;s instructions (see Table of Materials). After magnetic separation, transfer the isolate to a 15 mL conical tube and perform a final live cell count.</li>
	<li>Pellet the isolated T cells by centrifugation for 10 min at 450 x g, at 4 &#176;C (or RT), and re-suspend them at 1 x 106/mL in mTCM-activation medium (Table 1).</li>
	<li>Activate T cells by adding murine anti-CD3/anti-CD28 monoclonal antibody-coated magnetic beads (see Table of Materials) at a ratio of 25 &#181;L/1 x 106 T cells and 100 U/mL IL-2.</li>
	<li>Place the cells in an incubator at 37 &#176;C and leave them overnight. 
	NOTE: Due to the small size of T cells, a more accurate live T cell count may be achieved by diluting at a 1:10-1:20 ratio and mixing at 1:1 with trypan blue for manual counting using a hemocytometer. Purity may be determined by flow cytometry by staining cells with a fluorescently conjugated &#945;CD3 antibody. Each spleen will yield between 7-10 x 106 T cells depending on the age and strain of the mouse.</li>
</ol>
3. Murine T cell transduction
<ol>
	<li>Prepare plates for transduction (Day 0 procedure).
	<ol>
		<li>Pre-coat non-treated sterile 24-well plates with 0.5 mL of human fibronectin transduction enhancer reagent (see Table of Materials) at a final concentration of 20-40 &#181;g/mL by diluting it in sterile PBS and store at 4 &#176;C overnight. 
		NOTE: This step may also be performed on Day 1 by coating non-treated 24-well plates with the transduction reagent and incubating them at room temperature for 2 h.</li>
	</ol>
	</li>
	<li>Perform T cell transduction (Day 1 procedure).
	<ol>
		<li>Prepare the pre-coated plate for transduction. Remove the transduction reagent from each well of the pre-coated 24-well plate and block with an equivalent volume of sterile-filtered PBS + 2% bovine serum albumin (BSA) (0.5 mL) for 30 min at RT.</li>
		<li>Wash once with 0.5-1 mL of PBS.</li>
	</ol>
	</li>
	<li>Add 0.5-1 mL of neat retrovirus from step 1 or diluted based on viral titer to each pre-coated well and centrifuge for 90 min at 2,000 x g and 32 &#176;C.</li>
	<li>Add 1 mL of activated T cells to each virally loaded well and centrifuge for 10 min at 450 x g and 32 &#176;C. Return cells to a 37 &#176;C incubator overnight.</li>
	<li>24 h post-transduction, remove 1-1.5 mL of cell culture media and replace it with 1-1.5 mL of mTCM-complete and 10 ng/mL of recombinant human IL-7 and IL-15 (see Table of Materials). Return cells to a 37 &#176;C incubator (Day 2 procedure). 
	NOTE: During ex vivo culture, 10 ng/mL of cytokines must be added to the culture every 48 h, and T cells should not be diluted beyond 1 x 106/mL.</li>
	<li>48 h post-transduction, transfer cells from the transduction plate into a fresh 24-well or 6-well plate and return cells to a 37 &#176;C incubator (Day 3 procedure). 
	NOTE: T cell viability may improve by transferring cells on 24 h to 2 days post-transduction, depending on starting T cell viability and viral titer.</li>
	<li>De-bead the T cells and confirm CAR expression (Day 5-6 procedure).
	<ol>
		<li>Thoroughly re-suspend cells to dissociate activated T cells from the &#945;CD3/CD28-coated beads (see Table of Materials) and place the cell suspension on a magnet for 30 s. Transfer the cell suspension to the desired ex vivo culture vessel and return it to a 37 &#176;C incubator. 
		NOTE: T cells may be cultured in culture flasks or deep well culture plates. It is recommended to plate a minimum of 5 x 106 T cells in 30 mL of mTCM-complete medium per well in a deep well of a 6-well format plate (see Table of Materials).</li>
		<li>Determine CAR expression by flow cytometry8. 
		NOTE: GFP-CAR expression was determined by incubation with 100 ng/mL of purified GFP. Incorporation of an N-terminal epitope tag will facilitate the detection of alternative CAR constructs.</li>
	</ol>
	</li>
	<li>Perform ex vivo culture of CAR-T cells (Day 7-10 procedure).
	<ol>
		<li>During ex vivo culture, maintain the cells by removing 50% of the culture medium and replacing it with fresh mTCM-complete + 2x 10 ng/mL of IL-7 and IL-15 every 48 h. 
		NOTE: Murine CAR-T cells are ready for use in downstream applications 7 days post-activation and should not be cryopreserved due to poor viability post-thaw.</li>
	</ol>
	</li>
</ol>

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

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigating+CAR-T+cells+through+the+solid-tumour+microenvironment.">Navigating CAR-T cells through the solid-tumour microenvironment.</a> Nat Rev Drug Discov. 20 (7), 531-550 (2021).</li><li>Campesato, L. 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=Blockade+of+the+ahr+restricts+a+treg-macrophage+suppressive+axis+induced+by+l-kynurenine.">Blockade of the ahr restricts a treg-macrophage suppressive axis induced by l-kynurenine.</a> Nat Commun. 11 (1), 4011 (2020).</li><li>Kaneda, M. 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=Pi3kgamma+is+a+molecular+switch+that+controls+immune+suppression.">Pi3kgamma is a molecular switch that controls immune suppression.</a> Nature. 539 (7629), 437-442 (2016).</li><li>Hyrenius-Wittsten, A., Roybal, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paving+new+roads+for+cars.">Paving new roads for cars.</a> Trends Cancer. 5 (10), 583-592 (2019).</li><li>Giavridis, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR+T+cell-induced+cytokine+release+syndrome+is+mediated+by+macrophages+and+abated+by+il-1+blockade.">CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by il-1 blockade.</a> Nat Med. 24 (6), 731-738 (2018).</li><li>Lanitis, 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=Optimized+gene+engineering+of+murine+CAR-T+cells+reveals+the+beneficial+effects+of+il-15+coexpression.">Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of il-15 coexpression.</a> J Exp Med. 218 (2), e20192203 (2021).</li><li>Lambeth, C. R., White, L. J., Johnston, R. E., De Silva, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry-based+assay+for+titrating+dengue+virus.">Flow cytometry-based assay for titrating dengue virus.</a> J Clin Microbiol. 43 (7), 3267-3272 (2005).</li><li>Agarwal, S., Wellhausen, N., Levine, B. L., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+crispr-engineered+CAR-T+cells.">Production of human crispr-engineered CAR-T cells.</a> J Vis Exp. 169, e62299 (2021).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+Animal+Research.">Lab Animal Research.</a> Sterile Tissue Harvest. , (2023).</li><li>Giordano-Attianese, 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=A+computationally+designed+chimeric+antigen+receptor+provides+a+small-molecule+safety+switch+for+t-cell+therapy.">A computationally designed chimeric antigen receptor provides a small-molecule safety switch for t-cell therapy.</a> Nat Biotechnol. 38 (4), 426-432 (2020).</li><li>Kuhn, N. 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=Cd40+ligand-modified+chimeric+antigen+receptor+T+cells+enhance+antitumor+function+by+eliciting+an+endogenous+antitumor+response.">Cd40 ligand-modified chimeric antigen receptor T cells enhance antitumor function by eliciting an endogenous antitumor response.</a> Cancer Cell. 35 (3), 473-488.e6 (2019).</li><li>Jin, C., Ma, J., Ramachandran, M., Yu, D., Essand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR+T+cells+expressing+a+bacterial+virulence+factor+trigger+potent+bystander+antitumour+responses+in+solid+cancers.">CAR T cells expressing a bacterial virulence factor trigger potent bystander antitumour responses in solid cancers.</a> Nat Biomed Eng. 6 (7), 830-841 (2022).</li><li>Kurachi, 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=Optimized+retroviral+transduction+of+mouse+T+cells+for+in+vivo+assessment+of+gene+function.">Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function.</a> Nat Protoc. 12 (9), 1980-1998 (2017).</li><li>Jafarzadeh, L., Masoumi, E., Fallah-Mehrjardi, K., Mirzaei, H. 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No conflicts of interests declared.

This protocol describes the steps and reagents necessary for the retroviral transduction of murine T cells to generate CAR-T cells for in vivo studies. Optimizing retroviral transduction conditions achieves robust CAR expression without the need for viral concentration through ultracentrifugation or additional reagents. However, there are multiple modifications that can be applied to this methodology.
While this protocol describes the example generation of a GFP-specific CAR, these me...

Adoptive T cell therapies expressing chimeric antigen receptors (CARs) have revolutionized the field of cancer immunotherapy by harnessing the power of the adaptive immune system to specifically target and eliminate antigen-positive cancer cells1. While the success of CAR-T cell therapies targeting B cell malignancies has been clinically validated, preclinical studies performed in animal models remain vital for the development of new CARs targeting solid tumors. However, limited clinical efficacy has been demonstrated in solid tumor indications thus far, and it is becoming increasingly apparent that individual preclinical models do not accurately predict the pharmacodynamics and clinical efficacy of a living medicine2,3. Therefore, investigators have begun to expand the preclinical study of CAR-T cell products to include parallel assessments in xenograft and syngeneic models of human and murine cancers, respectively.
Unlike xenograft models, where human tumors and T cells are engrafted into immunodeficient mice, syngeneic models enable the examination of CAR-T cell responses in the context of a functional immune system. Specifically, immune-competent mice bearing syngeneic tumors provide a system to study the interaction between adoptively transferred T cells and context-specific milieus - including tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) known to suppress T cell function in the tumor microenvironment (TME)4,5,6. Moreover, syngeneic models offer an additional platform to assess on-target, off-tumor toxicity, and CAR-T cell interaction with host factors that may lead to additional toxicities, including cytokine release syndrome7.
Despite these advantages, the number of syngeneic CAR-T cell studies remains limited. Notably, syngeneic models require autologous engineering of CAR-T cells from the same mouse strain and thus present an additional challenge due to the lack of methodology for efficient murine T cell transduction and ex vivo expansion2,8. This protocol outlines the methods to achieve stable CAR expression through the production of retroviral vectors and optimized T cell transduction. A schematic of the entire process is shown in Figure 1. The use of this approach demonstrates efficient retroviral transduction of murine CAR-T cells and the achievement of high CAR expression without the need for viral concentration through ultracentrifugation. Strategies to change the antigen-specificity of the CAR construct are discussed in addition to the co-expression of additional transgenes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;m filters</td>
			<td>MilliporeSigma</td>
			<td>SLHVR33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>14-955-450</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>05-408-135</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe&#160;</td>
			<td>BD</td>
			<td>14-823-16E</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m strainer</td>
			<td>Corning</td>
			<td>07-201-432</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm TC treated cell culture dishes</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>130183</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes&#160;</td>
			<td>Falcon</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m strainer&#160;</td>
			<td>Corning</td>
			<td>07-201-430</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes&#160;</td>
			<td>Falcon</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m strainer</td>
			<td>Corning</td>
			<td>07-201-431</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT Flow Cytometer&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/C, 6-8 week old&#160;</td>
			<td>Jackson Laboratory</td>
			<td>651</td>
			<td></td>
		</tr>
		<tr>
			<td>B-Mercaptoethanol&#160;</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</td>
			<td>GOLDBIO</td>
			<td>A-420-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS), without Calcium and Magnesium&#160;</td>
			<td>Gibco</td>
			<td>14-190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet&#160;</td>
			<td>Invitrogen</td>
			<td>12-321-D</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet&#160;</td>
			<td>Stemcell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse T cell Isolation Kit</td>
			<td>Stemcell Technologies</td>
			<td>19851</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>BD</td>
			<td>BDB554657</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)&#160;</td>
			<td>Corning</td>
			<td>MT35011CV</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35-050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>G-Rex6</td>
			<td>Wilson Wolf</td>
			<td>80240M&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution&#160;</td>
			<td>Gibco</td>
			<td>15-630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant IL-15&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-765</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant IL-2</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-748</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant IL-7</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-363</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Invitrogen</td>
			<td>L3000008</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-CD3 BV421</td>
			<td>Biolegend</td>
			<td>100228</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-CD3/CD28 Dynabeads</td>
			<td>Gibco</td>
			<td>11-453-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-CD4 BV605</td>
			<td>BD</td>
			<td>563151</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-CD44 APC&#160;</td>
			<td>Biolegend</td>
			<td>103011</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-CD62L PE-Cy7</td>
			<td>Tonbo</td>
			<td>SKU 60-0621-U025</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-CD8 APC-Cy7</td>
			<td>Tonbo</td>
			<td>SKU 25-0081-U025</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti2 with Prime 95B camera&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non-treated 24 well plates&#160;</td>
			<td>CytoOne</td>
			<td>CC7672-7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31-985-062</td>
			<td></td>
		</tr>
		<tr>
			<td>pCL-Eco</td>
			<td>Addgene</td>
			<td>#12371</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin Solution</td>
			<td>Gibco</td>
			<td>15-070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phoenix Eco cells</td>
			<td>ATCC</td>
			<td>CRL-3214</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDG.2</td>
			<td>Addgene</td>
			<td>#12259</td>
			<td></td>
		</tr>
		<tr>
			<td>pMSCV_PGK_GFP28z</td>
			<td>N/A</td>
			<td>Produced by R.LV.</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified sfGFP</td>
			<td>N/A</td>
			<td>Produced by R.LV.</td>
			<td></td>
		</tr>
		<tr>
			<td>RetroNectin (&#39;transduction reagent&#39;)</td>
			<td>Takara Bio</td>
			<td>T100B</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>21875</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette 10 mL</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette 25 mL</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-678-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette 5 mL</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11-360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-treated 24 well plates&#160;</td>
			<td>Corning</td>
			<td>08-772-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue&#160;</td>
			<td>Gibco</td>
			<td>15-250-061</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The protocol described here aims to standardize the process of murine T cell transduction for the generation of mouse CAR-T cells. Figure 1 provides a detailed description of the steps involved. The process begins with the production of retroviral vectors via co-transfection of viral components into Phoenix Eco cells. Figure 2 provides an image of the optimal density of Phoenix Eco cells on the day of transfection. Isolated T cells are then activated 24...

Watch this Scientific Journal Video about Enhancing CAR-T Cell Function in Syngeneic Tumor Models at JoVE.com

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.

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 ...

efficient-generation-of-murine-chimeric-antigen-receptor-car-t-cells

Research

JoVE Journal

Bioengineering

2.9K Views.  Columbia University. This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

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

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug delivery and immune response. The present study stemmed from the observation that the effects of charge, size, and shape of solid particles on cell affinity are usually considered, but we seldom realize the essential role of softness, dynamic restructuring phenomenon, and complex interface interaction in cellular affinity. Here, we developed poly-lactic-co-glycolic acid (PLGA) nanoparticle-stabilized Pickering emulsion (PNPE) that overcame the shortcomings of rigid forms and simulated the flexibility and fluidity of pathogens. A method was set up to test the affinity of PNPE to cell surfaces and elaborate on the subsequent internalization by immune cells. The affinity of PNPE to bio-mimetic extracellular vesicles (bEVs)-the replacement for bone marrow dendritic cells (BMDCs)-was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed real-time monitoring of cell-emulsion adhesion. Subsequently, the PNPE was used to deliver the antigen (ovalbumin, OVA) and the uptake of the antigens by BMDCs was observed using confocal laser scanning microscope (CLSM). Representative results showed that the PNPE immediately decreased frequency (&#916;F) when it encountered the bEVs, indicating rapid adhesion and high affinity of the PNPE to the BMDCs. PNPE showed significantly stronger binding to the cell membrane than PLGA microparticles (PMPs) and AddaVax adjuvant (denoted as surfactant-stabilized nano-emulsion [SSE]). Furthermore, owing to the enhanced cellular affinity to the immunocytes through dynamic curvature changes and lateral diffusions, antigen uptake was subsequently boosted compared with PMPs and SSE. This protocol provides insights for designing novel formulations with high cell affinity and efficient antigen internalization, providing a platform for the development of efficient vaccines.

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug ...